TNFSF-L fusion proteins and uses thereof

Abstract

The present disclosure provides a composition comprising a nucleic acid encoding a fusion protein of a tumor necrosis factor superfamily ligand fused to an oligomerization domain. Viral and non-viral vectors for delivering such compositions are further provided. The present disclosure also provides a composition comprising a fusion protein of a tumor necrosis factor superfamily ligand fused to an oligomerization domain. The oligomerization domain is in closer sequence proximity with the neck domain in the fusion protein.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 211,766, filed June 17, 2021, which is incorporated by reference herein in its entirety. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jun. 14, 2022, is named 199249-715601_SL.txt and is 45,505 bytes in size. [Background technology]

[0003] background The TNF superfamily (TNFSF) is composed of proteins important for the development and function of many mammalian systems, including the immune, hematologic, and skeletal systems. TNFSF proteins are ligands for a corresponding set of receptors in the TNF receptor superfamily (TNFS). Members of the TNFSF are generally expressed as type II membrane proteins, with the exception of lymphotoxin alpha, which is produced as a secreted protein.

[0004] To produce soluble forms of TNFSF proteins, membrane proteins are generally expressed in cell lines that often have proteases capable of separating the TNFSF extracellular domain from the transmembrane domain, or truncated forms of TNFSF proteins are produced that have the extracellular domain plus the signal sequence. In either case, certain soluble forms of TNFSF ligand (TNFSF-L) proteins are often unstable in solution as homotrimers composed only of the extracellular domain. Thus, there is a need for more stable forms of these important signaling proteins. Summary of the Invention [Means for solving the problem]

[0005] Abstract A composition is provided herein, comprising a nucleic acid, the nucleic acid encoding a fusion protein, the fusion protein comprising a TNF-superfamily ligand (TNFSF-L) or a functional variant thereof and a plurality of domains from a collectin family protein, the plurality of domains from the collectin family protein comprising an oligomerization domain or a functional variant thereof and a neck domain or a functional variant thereof, the oligomerization domain or a functional variant thereof and the neck domain or a functional variant thereof being in closer sequence proximity compared to their positions in the collectin family protein.Furthermore, in the composition provided, the fusion protein comprises, in order from N-terminus to C-terminus, an oligomerization domain or a functional variant thereof, a neck domain or a functional variant thereof, optionally a linker sequence, and a TNFSF-L or a functional variant thereof. In further provided compositions, the TNFSF-L comprises lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. In further provided compositions, the TNFSF-L is a CD40 ligand. In further provided compositions, the CD40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:1. In further provided compositions, the TNFSF-L is an OX40 ligand. In further provided compositions, the OX40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:2 or SEQ ID NO:3. Further provided compositions include TNFSF-L is 4-1BBL. Further provided compositions include 4-1BBL comprises a sequence comprising at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5. Further provided compositions include TNFSF-L is LIGHT.Further provided compositions, LIGHT comprises a sequence comprising at least 85% sequence identity to SEQ ID NO:6. Further provided compositions, TNFSF-L is TNF superfamily member 18 ligand (GITRL). Further provided compositions, GITRL comprises a sequence comprising at least 85% sequence identity to SEQ ID NO:7 or SEQ ID NO:8. Further provided compositions, the collectin family protein is SP-A, SP-D, mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1 or CL-46. Further provided compositions, the collectin family protein is SP-D. Further provided compositions, the oligomerization domain comprises a sequence comprising at least 85% sequence identity to SEQ ID NO:9. Further provided compositions, the oligomerization domain and the neck domain both comprise a sequence comprising at least 85% sequence identity to SEQ ID NO:10. Further provided compositions, the oligomerization domain and the neck domain both comprise SEQ ID NO:10. Further provided are compositions, wherein the fusion protein comprises a linker sequence, the linker sequence comprising GSG (glycine-serine-glycine) (SEQ ID NO: 12). Further provided are compositions, wherein the nucleic acid comprises RNA. Further provided are compositions, wherein the nucleic acid comprises DNA.

[0006] The compositions provided herein include a nucleic acid, the nucleic acid comprising a sequence that comprises at least 85% sequence identity to SEQ ID NO:21, 22, 23, 24, 25, 26, or 27.

[0007] The compositions provided herein include fusion proteins, which comprise a sequence that comprises at least 85% sequence identity to SEQ ID NO:14, 15, 16, 17, 18, 19, or 20.

[0008] The composition provided herein comprises a vector and a nucleic acid encoding a TNF-superfamily ligand (TNFSF-L) or a functional variant thereof, wherein the TNFSF-L or a functional variant thereof is fused to an oligomerization domain. In the composition further provided herein, the nucleic acid encodes a fusion protein, wherein the fusion protein comprises a TNF-superfamily ligand (TNFSF-L) or a functional variant thereof and a plurality of domains from a collectin family protein, wherein the plurality of domains from a collectin family protein comprises an oligomerization domain or a functional variant thereof and a neck domain or a functional variant thereof, wherein the oligomerization domain or a functional variant thereof and the neck domain or a functional variant thereof are in closer sequence proximity compared to their positions in the collectin family protein. In the composition further provided herein, the vector is a viral vector or a non-viral vector. In the composition further provided herein, the non-viral vector comprises a nanoparticle carrier. In the composition further provided herein, the non-viral vector comprises a lipid nanoparticle carrier. In the composition further provided herein, the nanoparticle carrier comprises gold, silica, carbon nanotube, water-soluble fullerene, silicon nanowire, quantum dot, or any combination thereof.In the composition further provided herein, the vector comprises bacteriophage, virus-like particle (VLP), erythrocyte ghost, bactofection, exosome, or any combination thereof.In the composition further provided herein, the fusion protein comprises, in order from N-terminus to C-terminus, an oligomerization domain or a functional variant thereof, a neck domain or a functional variant thereof, optionally a linker sequence, and a TNFSF-L or a functional variant thereof.In the compositions further provided herein, TNFSF-L comprises lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. In the compositions further provided herein, TNFSF-L is CD40 ligand. In the compositions further provided herein, CD40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:1. In the compositions further provided herein, TNFSF-L is OX40 ligand. Further provided herein is a composition, wherein OX40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:2 or SEQ ID NO:3. Further provided herein is a composition, wherein TNFSF-L is 4-1BBL. Further provided herein is a composition, wherein 4-1BBL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5. Further provided herein is a composition, wherein TNFSF-L is LIGHT. Further provided herein is a composition, wherein LIGHT comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:6. Further provided herein is a composition, wherein TNFSF-L is TNF superfamily member 18 ligand (GITRL). Further provided herein is a composition, wherein GITRL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:7 or SEQ ID NO:8. In the composition further provided herein, the collectin family protein comprises SP-A, SP-D, mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1 or CL-46. In the composition further provided herein, the collectin family protein is SP-D. In the composition further provided herein, the oligomerization domain comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:9.Further provided herein is a composition, wherein the oligomerization domain and said neck domain both comprise a sequence comprising at least 85% sequence identity to SEQ ID NO: 10. Further provided herein is a composition, wherein the oligomerization domain and said neck domain both comprise SEQ ID NO: 10. Further provided herein is a composition, wherein the fusion protein comprises said linker sequence, said linker sequence comprising GSG (glycine-serine-glycine) (SEQ ID NO: 12). Further provided herein is a composition, wherein the nucleic acid comprises RNA. Further provided herein is a composition, wherein the nucleic acid comprises DNA.

[0009] A composition is provided herein, the composition comprising a vector and a nucleic acid, the nucleic acid comprising a sequence comprising at least 85% sequence identity to SEQ ID NO:21, 22, 23, 24, 25, 26, or 271.

[0010] A composition is provided herein, the composition comprising a vector and a nucleic acid, the nucleic acid encoding a protein comprising a sequence comprising at least 85% sequence identity to SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20.

[0011] Provided herein is a cell, which comprises the composition described herein. Further provided herein is a cell, which is an immune cell. Further provided herein is a cell, which is an immune cell, which is a lymphoid cell.

[0012] Provided herein is a fusion protein, which comprises a TNF-superfamily ligand (TNFSF-L) or a functional variant thereof and a plurality of domains derived from a collectin family protein, which comprises an oligomerization domain or a functional variant thereof and a neck domain or a functional variant thereof, and the oligomerization domain or a functional variant thereof and the neck domain or a functional variant thereof are in closer sequence proximity compared to their positions in the collectin family protein.Further provided herein is a fusion protein, which comprises, in the order of N-terminus to C-terminus, an oligomerization domain or a functional variant thereof, a neck domain or a functional variant thereof, and optionally a linker sequence and a TNFSF-L or a functional variant thereof. Further provided herein is a fusion protein, wherein TNFSF-L comprises lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. Further provided herein is a fusion protein, wherein TNFSF-L is CD40 ligand. Further provided herein is a fusion protein, wherein CD40 ligand comprises at least 85% sequence identity with SEQ ID NO:1. Further provided herein is a fusion protein, wherein TNFSF-L is OX40 ligand. In the fusion protein further provided herein, OX40 ligand comprises at least 85% sequence identity with SEQ ID NO:2 or SEQ ID NO:3. In the fusion protein further provided herein, TNFSF-L is 4-1BBL. In the fusion protein further provided herein, 4-1BBL comprises at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5.In the fusion protein further provided herein, TNFSF-L is LIGHT. In the fusion protein further provided herein, LIGHT comprises at least 85% sequence identity with SEQ ID NO:6. In the composition further provided herein, TNFSF-L is TNF superfamily member 18 ligand (GITRL). In the composition further provided herein, GITRL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:7 or SEQ ID NO:8. In the fusion protein further provided herein, the collectin family protein comprises SP-A, SP-D, mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1, CL-46, or any combination thereof. In the fusion protein further provided herein, the collectin family protein is SP-D. In the fusion protein further provided herein, the oligomerization domain comprises at least 85% sequence identity with SEQ ID NO:9. Further provided herein in the fusion protein, the oligomerization domain and the neck domain together comprise at least 85% sequence identity to SEQ ID NO: 10. Further provided herein in the fusion protein, the oligomerization domain and the neck domain together comprise SEQ ID NO: 10. Further provided herein in the fusion protein, the fusion protein comprises said linker sequence, said linker sequence comprises GSG (glycine-serine-glycine) (SEQ ID NO: 12).

[0013] Oncolytic viruses are provided herein, and the oncolytic viruses comprise an exogenous nucleic acid encoding a TNF-superfamily ligand (TNFSF-L) or its functional variant, and the TNFSF-L or its functional variant is fused to an oligomerization domain. In the oncolytic viruses further provided herein, the oncolytic viruses are Newcastle disease virus (NDV), reovirus (RV), myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV), poliovirus (PV), Sendai virus, flavivirus, lentivirus, poxvirus, retrovirus, adeno-associated virus, or adenovirus. In the oncolytic viruses further provided herein, the nucleic acid encoding TNFSF-L or its functional variant is inserted into the viral genome. In the oncolytic virus further provided herein, the nucleic acid encoding TNFSF-L or its functional variant is inserted into the thymidine kinase gene.In the oncolytic virus further provided herein, the TNFSF-L comprises lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof.In the oncolytic virus further provided herein, the TNFSF-L is a CD40 ligand.In the oncolytic virus further provided herein, the CD40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:1. In the oncolytic virus further provided herein, TNFSF-L is OX40 ligand.In the oncolytic virus further provided herein, OX40 ligand comprises the sequence of SEQ ID NO:2 or SEQ ID NO:3 and at least 85% sequence identity.In the oncolytic virus further provided herein, the TNFSF-L is 4-1BBL. In the oncolytic virus further provided herein, the 4-1BBL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5. In the oncolytic virus further provided herein, the TNFSF-L is LIGHT. In the oncolytic virus further provided herein, the LIGHT comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:6. In the composition further provided, the TNFSF-L is TNF superfamily member 18 ligand (GITRL). In the composition further provided, the GITRL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:7 or SEQ ID NO:8. In the oncolytic virus further provided herein, the oligomerization domain comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:9. In the oncolytic virus further provided herein, the nucleic acid comprises RNA. In the oncolytic virus further provided herein, the nucleic acid comprises DNA.

[0014] Provided herein is a vaccinia virus, which comprises an exogenous nucleic acid encoding TNF-superfamily ligand (TNFSF-L) or its functional variant, and said TNFSF-L or its functional variant is fused to an oligomerization domain.Further provided herein is a vaccinia virus, which is Western Reserve vaccinia virus (ATCC VR-1354), Copenhagen strain, vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), recombinant vaccinia virus Ankara (MVA), NYVAC strain, vaccinia virus strain Wyeth (ATCC VR-1536), vaccinia virus Wyeth (ATCC VR-325), Wyeth (NYCBOH) strain, Tian Tan strain, Lister strain, USSR strain, and modified strain of Evans strain. In the vaccinia virus further provided herein, the exogenous nucleic acid encoding TNFSF-L or its functional variant is inserted into the viral genome.In the vaccinia virus further provided herein, the exogenous nucleic acid encoding TNFSF-L or its functional variant is inserted into the thymidine kinase gene.In the vaccinia virus further provided herein, the TNFSF-L comprises lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof.In the vaccinia virus further provided herein, the TNFSF-L is CD40 ligand. Further provided herein is a vaccinia virus, wherein the CD40 ligand comprises a sequence comprising at least 85% sequence identity to SEQ ID NO:1.In the vaccinia virus further provided herein, TNFSF-L is OX40 ligand. In the vaccinia virus further provided herein, OX40 ligand comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:2 or SEQ ID NO:3. In the vaccinia virus further provided herein, TNFSF-L is 4-1BBL. In the vaccinia virus further provided herein, 4-1BBL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5. In the vaccinia virus further provided herein, TNFSF-L is LIGHT. In the vaccinia virus further provided herein, LIGHT comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:6. In the composition further provided herein, TNFSF-L is TNF superfamily member 18 ligand (GITRL). In the composition further provided herein, GITRL comprises a sequence comprising at least 85% sequence identity with SEQ ID NO:7 or SEQ ID NO:8. In the vaccinia virus further provided herein, the oligomerization domain comprises a sequence comprising at least 85% sequence identity to SEQ ID NO: 9. In the vaccinia virus further provided herein, the nucleic acid comprises RNA. In the vaccinia virus further provided herein, the nucleic acid comprises DNA.

[0015] Provided herein is a method for treating cancer, comprising administering to a subject an amount of a pharmaceutical composition sufficient for treating cancer, wherein the pharmaceutical composition comprises the composition, cell, fusion protein, oncolytic virus, or vaccinia virus described herein.Further provided herein is a method for treating cancer, wherein the cancer comprises blood cancer or solid cancer.Further provided herein is a method for treating cancer, wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric cancer, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. In the method for treating cancer further provided herein, the cancer comprises bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer.In the method for treating cancer further provided herein, administering comprises systemic administration or local administration.In the method for treating cancer further provided herein, administering comprises intratumoral administration, intravenous administration, local administration, intraperitoneal administration, parenteral administration, intramuscular administration, subcutaneous administration, intraarterial administration, or any combination thereof.In the method for treating cancer further provided herein, administering comprises intratumoral administration.In the method for treating cancer further provided herein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In the method for treating cancer further provided herein, the pharma- ceutically acceptable carrier comprises a buffer, an emulsion, a bioabsorbable polymer, a gel, or any combination thereof. In the method for treating cancer further provided herein, the composition, cell, or fusion protein is administered at a dose of about 0.01 μg / dose to about 1 g / dose. In the method for treating cancer further provided herein, the oncolytic virus or vaccinia virus is administered at a dose of about 10 3 ~about 10 12In the method for treating cancer further provided herein, the pharmaceutical composition is administered in a treatment cycle that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. In the method for treating cancer further provided herein, the pharmaceutical composition is administered in each dose and is administered for about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day or more. In the method for treating cancer further provided herein, each dose is independent of any other dose. In the method for treating cancer further provided herein, two or more doses in a treatment cycle are separated by an administration interval in which no dose is administered. In the method for treating cancer further provided herein, the administration interval is about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or more.In the method for treating cancer further provided herein, each administration interval is independent of any other administration interval.

[0016] Provided herein is a method for reducing tumor cell growth, comprising administering to a tumor cell a composition, cell, fusion protein, oncolytic virus, or vaccinia virus as described herein in an amount sufficient to reduce tumor cell growth. Further provided herein are methods for reducing tumor cell growth, wherein the tumor comprises a liquid tumor or a solid tumor. Further provided herein are methods for reducing tumor cell growth, wherein the tumor comprises melanoma, hepatocellular carcinoma, breast tumor, lung tumor, peritoneal tumor, prostate tumor, bladder tumor, ovarian tumor, leukemia, lymphoma, renal carcinoma, pancreatic carcinoma, epithelial carcinoma, gastric tumor, colon carcinoma, duodenal tumor, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal tumor, intestinal type gastric cancer, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein is a method for reducing tumor cell growth, the tumor comprises a bladder tumor, a blood tumor, a bone tumor, a bone marrow tumor, a brain tumor, a breast tumor, a colon tumor, an esophageal tumor, a gastrointestinal tumor, a gum tumor, a head tumor, a kidney tumor, a liver tumor, a lung tumor, a nasopharyngeal tumor, a neck tumor, an ovarian tumor, a prostate tumor, a skin tumor, a stomach tumor, a testicular tumor, a tongue tumor, or a uterine tumor. Further provided herein is a method for reducing tumor cell growth, the administering comprises systemic administration or local administration. Further provided herein is a method for reducing tumor cell growth, the administering comprises intratumoral administration, intravenous administration, local administration, intraperitoneal administration, parenteral administration, intramuscular administration, subcutaneous administration, intraarterial administration, or any combination thereof. Further provided herein is a method for reducing tumor cell growth, the administering comprises intratumoral administration. Further provided herein is a method for reducing tumor cell growth, the composition further comprises a pharma- ceutically acceptable carrier. Further provided herein is a method for reducing tumor cell growth, wherein the pharma- ceutically acceptable carrier comprises a buffer, an emulsion, a bioabsorbable polymer, a gel, or any combination thereof. Further provided herein is a method for reducing tumor cell growth, wherein the composition, cell, or fusion protein is administered at a dose of about 0.01 μg / dose to about 1 g / dose.Further provided herein is a method for reducing tumor cell growth, wherein the oncolytic virus or vaccinia virus is about 10. 3 ~about 10 12 In the method for reducing tumor cell growth further provided herein, the administration is in a treatment cycle that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. In the method for reducing tumor cell growth further provided herein, each dose is administered and administered for about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day or more. In the method for reducing tumor cell growth further provided herein, two or more doses are separated by an administration interval in which no dose is administered. Further provided herein is a method for reducing tumor cell growth, the administration interval is about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or more. Further provided herein is a method for reducing tumor cell growth, each administration interval is independent of any other administration interval. [Brief description of the drawings]

[0017] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0018] [Figure 1] FIG. 1 shows a diagram of an exemplary domain structure with an oligomerization domain, a neck domain, an optional linker, and a TNFSF-L domain.

[0019] [Diagram 2]Figure 2 shows expression of CD40L scaffold protein in cells infected with a modified oncolytic vaccinia virus transgene expressing the CD40L scaffold protein compared to cells infected with an oncolytic vaccinia virus not expressing the transgene or uninfected cells. The Y-axis represents mCD40L in ng / mL and the X-axis represents samples (uninfected cells, vaccinia virus with A52R deletion and thymidine kinase deletion, and vaccinia virus with thymidine kinase deletion and variant CD40L insertion).

[0020] [Diagram 3] Figure 3 shows the results of a mOX40L ELISA assay, where the supernatants are diluted 100-fold after accounting for the dilution factor and screened for mOX40L expression. The Y-axis represents mOX40L in pg / mL, and the X-axis represents samples (uninfected cells, vaccinia virus with A52R deletion and thymidine kinase deletion, vaccinia virus with thymidine kinase deletion and native OX40L, and vaccinia virus with thymidine kinase deletion and insertion of variant OX40L).

[0021] [Figure 4] Figure 4 shows the results of a 41BBL ELISA assay in which undiluted supernatants were screened for 41BBL expression. The Y-axis represents m41BBL in ng / mL and the X-axis represents samples (uninfected cells, vaccinia virus with an A52R deletion and a thymidine kinase deletion, vaccinia virus with a thymidine kinase deletion and native 41BBL, and vaccinia virus with a thymidine kinase deletion and an insertion of variant 41BBL).

[0022] [Diagram 5]Figure 5 shows the results of the CD40L reporter cell assay. The Y axis is OD and the X axis is the volume of supernatant used. The samples are uninfected cells, cells infected with vaccinia viruses with A52R deletion and thymidine kinase deletion, and cells infected with vaccinia viruses containing thymidine kinase deletion and variant CD40L.

[0023] [Figure 6] FIG. 6 shows the survival probability in mice treated with virus harboring a TK deletion (TK-), virus harboring a TNFSFL fusion construct (s3CD40L), or buffer control.

[0024] [Figure 7] FIG. 7 shows luciferase expression levels (RLU) in uninfected control cells (buffer), cells infected with a virus expressing 41BBL, and cells infected with a virus expressing trimeric OX40L (3sOX40L).

[0025] [Figure 8] Figure 8 shows luciferase expression levels (RLU) in uninfected control cells (buffer), cells infected with a virus expressing trimeric GITRL as a negative control, a virus expressing 41BBL monomer, and a virus expressing trimeric 41BBL (3s41BBL).

[0026] [Figure 9] FIG. 9 shows IL-2 expression levels in CD8 T cells stimulated with CD3 alone, or CD3 and CD28, recombinant 41BBL, recombinant OX40L, virus expressing 3s41BBL, or virus expressing 3xOX40L.

[0027] [Figure 10]Figure 10 shows the average tumor volume in BALB / c mice bearing subcutaneous RENCA tumors, with volume (mm3) on the y-axis measured in days after treatment on the x-axis. Volumes were measured 0, 5, 8, and 12 days after treatment with buffer control, control vaccinia virus with thymidine kinase deletion (HCCTKM), virus expressing 3s41BBL, or virus expressing 3sOX40L. Tumor volumes in treatment groups containing virus expressing ligand 3s41BBL or 3sOX40L showed smaller tumor volumes compared to the negative control after 5, 8, and 12 days.

[0028] [Figure 11] 11A shows a scatter plot of tumor volumes (mm3) in BALB / c mice bearing subcutaneous Lewis Lung Carcinoma (LLC) tumors after 30 days of treatment with buffer control, control vaccinia virus with a thymidine kinase deletion (HCCTKM), or virus expressing 3sGITRL. The mean tumor volumes for each group after 30 days are shown, showing approximately 1400 mm3 for the buffer control group, approximately 750 mm3 for the HCCTKM group, and <100 mm3 for the 3sGITRL group.

[0029] FIG. 11B shows tumor volume (mm ) in BALB / c mice bearing subcutaneous RENCA tumors 17 days after treatment with a buffer control, a control vaccinia virus with a thymidine kinase deletion (HCCTKM), or a virus expressing 3sGITRL. 3 The average tumor volume of each group after 17 days is shown, with the buffer control group at approximately 800 mm 3 , and approximately 350 mm in the HCCTTKM group. 3 , and about 250 mm in the 3sGITRL group. 3 This shows that. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in implementing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0031] Provided herein is a composition and its use for treating cancer, comprising a nucleic acid encoding a fusion protein of TNF superfamily ligand (TNFSF-L) or its functional variant fused to a polymerization domain. In one exemplary arrangement, referring to FIG. 1, TNFSF-L is fused to an oligomerization domain at the N-terminus (optionally via a linker) and optionally fused to a neck domain at the C-terminus of the oligomerization domain. In the illustration of FIG. 1, the oligomerization domain and the neck domain are functional domains from collectin family protein SP-D. The further oligomerization domain, and the optional neck domain and / or linker domain, allow polymerization of the fusion protein, providing a structure with increased activity in eliciting an immune response, or a marker thereof. Described herein in more detail are TNFSF-L, domains for fusion to TNFSF-L, viral and non-viral vectors for delivery of the nucleic acid encoding the fusion protein, conditions for use of the composition, and methods of administration. Further provided herein are compositions and methods for treating cancer, generally comprising an oncolytic virus comprising a nucleic acid encoding a TNFSF-L fusion protein. Further provided herein is a cell comprising a nucleic acid that expresses a fusion protein described herein. definition

[0032] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" can include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "contains," "containing," "including," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0033] The term "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. When a particular value is described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean an acceptable error range of the particular value, e.g., ±10% of the value modified by the term "about."

[0034] The term "heterologous nucleic acid sequence," or "exogenous nucleic acid sequence," or "transgene," as used herein with respect to a particular virus, can refer to a nucleic acid sequence derived from a source other than the designated virus.

[0035] The term "mutation" as used herein can refer to a deletion, an insertion of a heterologous nucleic acid, an inversion or a substitution, including an open reading frame removal mutation as generally understood in the art.

[0036] As used herein, the term "gene" can refer to a segment of nucleic acid that encodes a particular protein or RNA (also called a "coding sequence" or "coding region"), along with associated regulatory regions, such as promoters, operators, terminators, etc., which can be located upstream or downstream of the coding sequence, as appropriate.

[0037] A "promoter" as used herein may be a regulatory sequence that is a region of a nucleic acid sequence where the initiation and rate of transcription is controlled. In some embodiments, a promoter may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" may mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence and controls the transcription initiation and / or expression of that sequence. In some embodiments, a promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0038] The term "homology" as used herein may refer to the calculation of "homology" or "percent homology" between two or more nucleotide or amino acid sequences, which may be determined by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced into the sequence of the first sequence). The nucleotides at corresponding positions may then be compared, and the percent identity between the two sequences may be a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100). For example, a position in a first sequence may be occupied by the same nucleotide as the corresponding position in a second sequence, in which case the molecules are identical at that position. The percent homology between two sequences may be a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. In some embodiments, the length of sequences aligned for comparison purposes may be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. A BLAST® search may determine the homology between two sequences. The homology may be between the full length of the two sequences or between a portion of the full length of the two sequences. The two sequences may be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences may be accomplished by well-known methods, for example, using a mathematical algorithm. When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) may be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or can be varied (e.g., W=5 or W=20). Other examples include the Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA algorithms.

[0039] The term "subject" can refer to animals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms "subject" and "patient" are used interchangeably herein in reference to a mammalian subject, such as a human subject.

[0040] The terms "treat," "treating," and "treatment" can be meant to include alleviating or arresting a disorder, disease, or condition; one or more symptoms associated with a disorder, disease, or condition; or alleviating or eradicating the cause of the disorder, disease, or condition itself.

[0041] The term "effective amount" or "therapeutically effective amount" can refer to the amount of a compound that, when administered, may be sufficient to prevent the onset of, or alleviate to some extent, one or more symptoms of the disorder, disease, or condition being treated.

[0042] The term "oncolytic" as used herein can refer to the killing of cancer or tumor cells by an agent, such as an oncolytic poxvirus, such as an oncolytic vaccinia virus, for example, by direct lysis of the cells, stimulating an immune response against the cells, apoptosis, expression of toxic proteins, blocking autophagy and protein synthesis, inducing anti-tumor immunity, or any combination thereof. Direct lysis of cancer or tumor cells infected with an agent, such as an oncolytic vaccinia virus, can be the result of viral replication within the cells. In certain instances, the term "oncolytic" can refer to the killing of cancer or tumor cells without lysis.

[0043] As used herein, the term "oncolytic virus" can refer to a virus that preferentially infects and kills tumor cells.

[0044] The term "modified oncolytic virus" as used herein can refer to an oncolytic virus that includes modifications to its components, such as, but not limited to, modifications in the native genome ("backbone") of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral proteins, and introduction of exogenous or modified viral proteins into the viral capsid. In general, oncolytic viruses can be modified (also known as "engineered") to obtain improved therapeutic effects against tumor cells. Fusion constructs

[0045] A nuclear construct encoding a fusion protein is provided herein. In some embodiments, the fusion protein described herein optionally comprises a TNFSF-L region and an oligomerization region from a collectin protein. Such a fusion protein can be encoded by a nucleic acid and inserted into an oncolytic virus. The resulting expressed fusion protein can further comprise a linker region between the TNFSF-L region and the oligomerization region. Examples of TNF superfamily members included in the TNFSF-L region include, but are not limited to, lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), TNF-related apoptosis weak inducer, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. In further embodiments, the TNFSF-L domain can be linked at the N-terminus to a linker peptide. In some examples, the linker peptide comprises up to 10 amino acids in length and is optionally glycine and / or serine rich.

[0046] In some embodiments, the fusion protein itself is provided herein. In some embodiments, the fusion protein optionally comprises a TNFSF-L region and an oligomerization region from a collectin protein. Such a fusion protein can be encoded by a nucleic acid and inserted into an oncolytic virus. The resulting expressed fusion protein can further comprise a linker region between the TNFSF-L region and the oligomerization region. Examples of TNF superfamily members included in the TNFSF-L region include, but are not limited to, lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. In further embodiments, the TNFSF-L domain can be linked at the N-terminus to a linker peptide. In some instances, the linker peptide comprises up to 10 amino acids in length and is optionally glycine and / or serine rich.

[0047] The amino acid sequences of exemplary tumor necrosis superfamily ligands that may be included in whole or in part in the fusion constructs described herein are shown in Table 1. Such exemplary TNFSF-L proteins include the ectodomains from mouse CD40L, human OX40L, mouse OX40L, mouse 4-1BBL, human 4-1BBL, human GITRL, mouse GITRL, mouse 3sCD40L, mouse 3sOX40L, mouse 3s4-1BBL, mouse 3sGITRL, human 3sOX40L, human 3s4-1BBL, and mouse 3sGITRL. Table 1. [Table 1]

[0048] Many TNF superfamily receptors blind their cognate ligands as homotrimers. The ligands are natively expressed as transmembrane tethered proteins on the surface of cells. Fusion constructs are provided herein that include a TNFSF ligand fused to an oligomerization-promoting fragment, such as from human collectin protein. In some embodiments, the oligomerization region includes a collectin protein polypeptide fragment optionally linked to the N-terminus of the TNFSF-L region or to the N-terminus of a linker peptide, the C-terminus of the linker peptide being linked to the N-terminus of the TNFSF-L region. Typically, collectins include a C-terminal carbohydrate section, a neck section, a collagenous section, and an N-terminal section. The C-terminal carbohydrate region of a lectin is also known as a lectin domain or CRD, which typically contains cystine residues that aid in oligomerization. The neck section initiates trimerization, forming a zipper-like fashion along the collagenous tail of the neck section toward the N-terminal section. The N-terminal section itself contains a cystine-rich portion that aids in oligomerization by disulfide bridging. Oligomerization Domain

[0049] Provided herein are TNFSF-L fusion constructs that include an oligomerization region. Such a region allows for improved stability and functionality of the expressed TNFSF-L, and thus increased activation of host immune system activity. In some embodiments, the oligomerization region includes at least one domain from a collectin family protein or a functional variant thereof. Collectin proteins are known to form stable oligomers. Collectins are one of 18 group members that make up the protein lectin superfamily, which includes a structural protein fold called the C-type lectin domain. Some members have been shown to include additional structural features. Thus, they include the following components: i) an N-terminal collagen domain bound to an α-helical segment, also called the neck region, and iii) a C-terminal CRD. Examples of collectins include surfactant protein A (SP-A), surfactant protein D (SP-D), mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1, and CL-46. SP-A and SP-D contain an N-terminal cysteine ​​that participates in disulfide-mediated oligomerization of preformed trimers. In some embodiments, nucleic acids encoding the above peptide sequences are also contemplated. In some embodiments, the fusion constructs described herein comprise a collectin oligomerization domain or a functional variant thereof. In some embodiments, the fusion constructs described herein comprise a collectin oligomerization domain or a functional variant thereof and a collectin neck domain or a functional variant thereof. Exemplary sequences are provided in Table 2 (amino acids) and Table 3 (nucleic acids). Table 2 [Table 2] Table 3 [Table 3]

[0050] In some embodiments, the collectin polypeptide fragment is linked to the N-terminus of the TNFSF-L region of the fusion protein or the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the TNFSF-L region of the fusion protein. Linker

[0051] Fusion constructs are provided herein that include a flexible linker element located between the oligomerization domain and the TNFSF-L domain. In some embodiments, the flexible linker element has a length of 25 amino acids or less. In some embodiments, the linker element has a length of 3-30 amino acids, in particular 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 30 amino acids. In some embodiments, the length of the linker comprises 3-10 or 5-10 amino acids. In some embodiments, the linker element is constructed with small hydrophilic uncharged amino acids. In some embodiments, the linker element according to the invention may comprise an amino acid selected from G, S, A and T. The linker element is preferably a glycine / serine linker, i.e. a peptide linker comprising the amino acids glycine and serine. In some embodiments, the linker comprises the amino acid sequence (GSG) (SEQ ID NO: 12). In some embodiments, the linker comprises the amino acid sequence (GSS)a(SSG)b(GSG)c (SEQ ID NO: 13), where a, b, c are each repeated 0, 1, 2, 3, 4, 5 or 6 times. If TNFSF-L ends or begins with a serine or glycine amino acid, such residue may also form the first residue of the linker. A component of a linker (liker) element may be composed of 1, 2, 3, 4, 5 or more amino acids. In general, a linker element as used herein may be composed of a component or may be composed of a sequence of amino acids. Delivery Vector

[0052] Provided herein is a system for delivering a nucleic acid encoding a fusion construct described herein, which allows the nucleic acid construct to enter a target cell while reducing nuclease degradation of the nucleic acid. Such delivery systems can include viral or non-viral vectors. Oncolytic viruses

[0053] Provided herein is a vector in the form of a virus. In some embodiments, the viral delivery vector comprises an oncolytic virus. Provided herein is a composition comprising an oncolytic virus, wherein the oncolytic virus comprises the TNFSF-L fusion construct described herein. An oncolytic virus used herein kills cancer or tumor cells by mechanisms such as direct lysis of the cells, by stimulating immune response against the cells, apoptosis, expression of toxic proteins, autophagy and shutting down protein synthesis, induction of anti-tumor immunity, or any combination thereof. Exemplary oncolytic viruses included in the compositions described herein include, but are not limited to, poxvirus, Newcastle disease virus (NDV), reovirus (RV), myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV), poliovirus (PV), Sendai virus, flavivirus, lentivirus, retrovirus, retrovirus, adeno-associated virus, and adenovirus. In some embodiments, oncolytic virus can be poxvirus.In some embodiments, poxvirus comprises β-entomopoxvirus, yatapoxvirus, cervidopoxvirus, γ-entomopoxvirus, leporipoxvirus, suipoxvirus, molluscpoxvirus, krokodilidopoxvirus, α-entomopoxvirus, capripoxvirus, avipoxvirus, parapoxvirus, canarypoxvirus, or fowlpoxvirus.These oncolytic viruses tend to target cancer cells specifically, and cause significant cell death and tumor regression during viral replication.

[0054] In some embodiments, the oncolytic virus can be a modified oncolytic virus that can have one or more modifications that can provide a greater therapeutic effect on tumor cells compared to an otherwise identical virus that does not include modifications.Some non-limiting examples of greater therapeutic effect can include: enhanced immune evasion of the virus, enhanced tumor-targeted systemic delivery of the virus, enhanced intratumoral and intertumoral spread of the virus, and enhanced tumor-specific replication of the virus, or release of immunomodulatory and antitumor agents into the extracellular matrix, or any combination thereof.The modified oncolytic virus of the present disclosure can be utilized as a platform vector for systemic delivery in some cases.

[0055] In some embodiments of the present disclosure, modified oncolytic viruses are provided that include modifications that can enhance tumor-targeted systemic delivery of the virus. Typically, oncolytic viruses are (a) administered systemically, or (b) inoculated locally onto the tumor, or in many cases, can be directly injected into the tumor ("intratumoral delivery"). It is believed that systemic delivery of oncolytic viruses can provide an opportunity to simultaneously treat both the primary tumor and overt or undiagnosed metastatic deposits. As a result, this delivery method can be a very attractive option for treating patients with advanced / metastatic disease, or those with inaccessible disease, such as those with pancreatic or brain cancer, where access is difficult due to physiological barriers, such as the blood-brain barrier. However, there can be barriers to successful systemic delivery of many oncolytic viruses. For example, in some cases, as described above, host defenses limit the ability of most oncolytic viruses to infect tumors after systemic administration. Insufficient viral escape from blood cells, complement, antibodies, and antiviral cytokines, as well as non-specific uptake by other tissues such as lung, liver and spleen, tissue-resident macrophages, and even vascular compartments, is one of the main barriers to systemic delivery of oncolytic viruses. In some embodiments of the present disclosure, the disclosed oncolytic viruses may contain modifications that can promote the persistent presence of the virus in the circulatory system, at least through enhanced immune evasion as described above. On the other hand, enhanced tumor-targeted delivery of the virus may be desirable under certain circumstances, since it not only increases therapeutic efficacy against cancer, but also can limit non-tumor infection and avoid undesirable side effects of viral infection, thus reducing safety concerns related to virus-mediated tumor therapy. Some embodiments herein relate to oncolytic viruses that contain modifications that can promote tumor-targeted delivery of the virus.

[0056] In some embodiments of the present disclosure, modified oncolytic viruses are provided that include modifications that can enhance intratumoral and intertumoral spread of the virus. Enhanced intratumoral and intertumoral spread of the oncolytic virus can enhance therapeutic efficacy by increasing the number of cancer cells infected with the virus. In some embodiments, modified oncolytic viruses are provided herein that can include exogenous nucleic acid. In some embodiments, modified oncolytic viruses are provided herein that can include modifications in the genome of the virus. In some embodiments, modified oncolytic viruses are provided herein that can include exogenous nucleic acid, as well as modifications in the genome of the virus.

[0057] In some embodiments, oncolytic viruses may include, but are not limited to, (i) viruses that naturally replicate preferentially in cancer cells and are often non-pathogenic in humans due to increased susceptibility to innate antiviral signaling or dependency on oncogenic signaling pathways; and (ii) viruses that are genetically engineered for use. In some embodiments, oncolytic viruses may be measles viruses, polioviruses, poxviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vesicular stomatitis viruses, reoviruses, Newcastle disease viruses, sencena viruses, retroviruses, dengue viruses, or myxoma viruses. In some embodiments, oncolytic viruses may be poxviruses. In some embodiments, poxviruses may be vaccinia viruses. In some cases, modified poxviruses may be attenuated canarypox viruses. In some cases, modified poxviruses may be fowlpox viruses.

[0058] In some embodiments, modified oncolytic viruses are used.Generally, such viruses include modifications to their components, such as, but not limited to, modifications in the native genome ("backbone") of the virus, such as mutation or deletion of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral protein, and introduction of exogenous protein or modified viral protein into viral capsid.

[0059] In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode LIGHT. In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode IL15. In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode IL15 and an exogenous nucleic acid that can encode CCL5. In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode IL15 and an exogenous nucleic acid that can encode IL15-Rα. In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode ITAC (CXCL11) and an exogenous nucleic acid that can encode fractalkine (CX3CL1). In some embodiments, the modified oncolytic virus may include an exogenous nucleic acid that can encode ITAC (CXCL11), an exogenous nucleic acid that can encode fractalkine (CX3CL1), an exogenous nucleic acid that can encode IL15, and an exogenous nucleic acid that can encode IL15-Rα.

[0060] In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode a cytokine, such as IL15. In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode a cytokine, such as IL15, and an exogenous nucleic acid that may encode a chemokine, such as CCL5. In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode a cytokine, such as IL15, and an exogenous nucleic acid that may encode a receptor for the cytokine, such as IL15Rα. In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode LIGHT. In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode ITAC (CXCL11). In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may include an exogenous nucleic acid that may encode fractalkine (CX3CL1). In some embodiments, the modified oncolytic virus may include a mutation or deletion of the K7R gene and may further include an exogenous nucleic acid that may encode ITAC (CXCL11), and an exogenous nucleic acid that may encode fractalkine (CX3CL1).

[0061] In some embodiments, the modified oncolytic virus may comprise a mutation or deletion of the A52R gene and may further comprise an exogenous nucleic acid that may encode a cytokine, such as IL15. In some embodiments, the modified oncolytic virus may comprise a mutation or deletion of the A52R gene and may further comprise an exogenous nucleic acid that may encode a chemokine, such as IL15, and an exogenous nucleic acid that may encode a chemokine, such as CCL5. In some embodiments, the modified oncolytic virus may comprise a mutation or deletion of the A52R gene and may further comprise an exogenous nucleic acid that may encode a cytokine, such as IL15, and an exogenous nucleic acid that may encode a receptor for the cytokine, such as IL15Rα. In some embodiments, the modified oncolytic virus may comprise a mutation or deletion of the A52R gene and may further comprise an exogenous nucleic acid that may encode LIGHT. In some embodiments, the modified oncolytic virus may include a mutation or deletion of the A52R gene and may further include an exogenous nucleic acid that may encode ITAC (CXCL11). In some embodiments, the modified oncolytic virus may include a mutation or deletion of the A52R gene and may further include an exogenous nucleic acid that may encode fractalkine (CX3CL1). In some embodiments, the modified oncolytic virus may include a mutation or deletion of the A52R gene and may further include an exogenous nucleic acid that may encode ITAC (CXCL11), and an exogenous nucleic acid that may encode fractalkine (CX3CL1).

[0062] In some cases, co-expression of a cytokine (e.g., IL15) and its receptor (e.g., IL15-Rα) from a modified oncolytic virus may result in enhanced immunomodulatory effects of the oncolytic virus, in some cases, for example, due to improved ability of the complex formed by IL15 and IL15-Rα (IL15:IL15-R complex) to activate natural killer cells and promote T cell responses. Without being bound by any particular theory, it is believed that IL15 in the IL15:IL15-Rα complex is presented to IL15-Rβγ (IL15 receptor beta gamma complex) that is presented on the surface of T cells and natural killer (NK) cells, thereby conferring a strong immunomodulatory effect on NK cells and T cells.

[0063] In some embodiments, modified oncolytic viruses are provided that can have the A52R gene mutated or deleted and can further include an exogenous nucleic acid that can encode a secreted hyaluronidase (e.g., HysA). In some embodiments, modified oncolytic viruses are provided that can have the A52R gene mutated or deleted and can further include an exogenous nucleic acid that can encode a chemokine receptor, such as CXCR4.

[0064] In some embodiments, the modified oncolytic virus may include a complete or partial deletion of the viral thymidine kinase (TK) gene. In some embodiments, in the genome of the modified oncolytic virus disclosed herein, one or more exogenous nucleic acids are inserted at the locus of the deleted TK gene.

[0065] In some embodiments, in modified oncolytic viruses, such as oncolytic vaccinia viruses, the viral TK gene may be replaced with the TK gene from herpes simplex virus (HSV-TK). HSV TK may function as a replacement for the deleted TK and may have multiple benefits. For example, (i) HSV TK may be used as an additional therapeutic prodrug-converting enzyme to convert ganciclovir (GCV) to its cytotoxic metabolites in tumors. In addition to the added therapeutic effect, this modification may also function as a suicide gene, e.g., vaccinia-expressing cells may be efficiently killed by the addition of GCV, thereby halting the virus in the event of an adverse event or uncontrolled replication. Thus, in some examples, the modified oncolytic viruses of the present disclosure may act as a safety switch). In an additional example, a mutant form of HSV TK may be used to enable PET imaging of labeled substrates with significantly increased sensitivity. Thus, in some cases, modified oncolytic viruses containing HSV TK that can be used in PET imaging can act as reporters of viral replication in vivo to determine therapeutic activity early after treatment.

[0066] In some cases, modified oncolytic virus can comprise the above-mentioned full-length viral backbone gene or viral backbone protein, or its truncated form, or its functional domain, or its fragment, or its variant.In various examples, modified oncolytic virus can comprise one or more mutations or deletions of viral backbone gene or viral backbone protein as described above.Mutation of viral backbone gene and viral backbone protein can include insertion, deletion, substitution or modification of nucleotides in nucleic acid sequence and amino acids in protein sequence.Deletion can include complete or partial deletion of viral backbone gene or protein in some examples. Vaccinia virus

[0067] In some embodiments, the oncolytic virus is a vaccinia virus.Exemplary vaccinia viruses include, but are not limited to, wild-type or attenuated vaccinia virus strains, such as Western Reserve vaccinia virus (ATCC VR-1354), Copenhagen strain, vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), recombinant vaccinia virus Ankara (MVA), NYVAC strain, vaccinia virus strain Wyeth (ATCC VR-1536), vaccinia virus Wyeth (ATCC VR-325), Wyeth (NYCBOH) strain, Tian Tan strain, Lister strain, USSR strain, and Evans strain, for example, modified by including fusion constructs as described herein. The base vaccinia virus strain modified as described herein may itself include one or more mutations relative to its parental strain, such as, but not limited to, one or more of the following: deletion of TK ("TK-") and deletion of A52R (also referred to herein as "A52R-"). The vaccinia virus may be recombinant or selected to have low toxicity and accumulate in target tissues. In some embodiments, the modification in the viral backbone / viral genome is a modification that renders the vaccinia virus non-replicative or includes poor replicative capacity. Non-limiting examples of such modifications may include mutations in the following viral genes: A1, A2, VH1, A33, and I7.In some embodiments, the viral backbone mutation is selected from the group consisting of: A52R gene complete or partial deletion; TK gene complete or partial deletion; B15R gene complete or partial deletion; K7R gene complete or partial deletion; B14R gene complete or partial deletion; N1L gene complete or partial deletion; K1L gene complete or partial deletion; M2L gene complete or partial deletion; A49R gene complete or partial deletion; VH1 gene complete or partial deletion; A33 gene complete or partial deletion; A1 complete or partial deletion; A2 gene complete or partial deletion; I7 gene complete or partial deletion, and A46R gene complete or partial deletion.As used herein, reference to a viral gene can be made by reference to the protein encoded by the gene (e.g., A33 gene can mean the gene encoding the A33 protein). In some embodiments, viral backbone mutations, including any combination of substitutions, insertions, and deletions, can result in sequences with 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less sequence homology with the wild-type sequence of the viral gene or viral protein encoded by the gene. In some embodiments, the viral gene and the protein encoded thereby are selected from the group consisting of B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. In some embodiments, the viral backbone can include 1, 2, 3, 4, 5, or more mutations in the amino acid sequence of the viral protein (e.g., viral antigen). The viral antigens are in some examples selected from the group consisting of B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. The present disclosure provides, in some embodiments, a recombinant vaccinia virus that contains one more mutation in the genome of the virus (viral backbone), such that the mutation increases the T cell arm of the immune response. The mutation can be an addition, deletion, or substitution of one or more nucleic acids in the viral genome (wild type or attenuated native strain of vaccinia virus).In a non-limiting example, the mutation can be a complete or partial deletion of a gene known to inhibit cytokines involved in Th1 immune responses. As a non-limiting example, the mutation can be a deletion of a nucleic acid encoding B8R (interferon gamma (IFN-g) binding protein), C12L (interleukin-18 (IL-18) binding protein). In a further non-limiting example, the mutation can be a complete or partial deletion of a gene in innate immune signaling. As non-limiting examples, the mutations can be deletions of nucleic acids encoding (B18R (type I interferon (IFN) binding protein); A52R (nuclear factor kappa B (NF-κB) inhibitor protein); E3L (protein kinase (PKR) inhibitor); C4, C16 (STING pathway inhibitors). In further non-limiting examples, the mutations can be full or partial deletions of genes encoding proteins for inhibition of other components of the immune response. As non-limiting examples, the mutations can be full or partial deletions of nucleic acids encoding B15, K7, B14, N1, K1, M2, A49, VH1, A46, or combinations thereof. Viral backbone mutations can also include replacing vaccinia virulence genes with genes of substantially equivalent function from other poxviruses. As provided herein, The vaccinia virus to be modified comprises additional insertions, mutations, deletions or substitutions in the viral genome. The vaccinia virus may comprise one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more of chemokine receptors, TRIF protein or its functional domains, or leptin, interleukin-2 (IL2), interleukin-15 / interleukin-15Ra (IL15 / IL15Ra), interleukin-7 (IL-7), leptin-interleukin fusion proteins (e.g., leptin-IL2 fusion protein shown as L2 in Example 1). Modifications such as the insertion of chemokine receptors are the insertion of wild-type and / or mutant CXCR4, CCR2, CCL2.The vaccinia virus may further comprise one or more additional deletions or partial deletions of one or more genes from A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, VH1 and their functional domains or fragments or variants, or any combination thereof. In some cases, the vaccinia virus provided herein may comprise a full or partial deletion of the A52R gene and an insertion of a chemokine receptor, such as CCR2. In some cases, the vaccinia virus provided herein may comprise at least one full or partial deletion of the A52R or TK viral gene and an insertion of an exogenous nucleic acid encoding a fusion protein (e.g., a metabolic regulatory protein fused to a cytokine, such as a leptin-IL2 fusion protein). Additional Vectors

[0068] The vector for delivery of the nucleic acid constructs described herein may include physical, chemical or biological means of transfection. In some embodiments, the vector provides for the delivery of the proteins described herein. In some embodiments, the vector is a lipid nanoparticle carrier.

[0069] In some embodiments, physical transfection includes electroporation, heat-assisted gene transfer, biolistic (or gene gun), microinjection, laser-assisted transfection, ultrasound-assisted gene transfer, hydrodynamic gene transfer, magnetofection, or mechanical massage, or any combination thereof. In some embodiments, laser-assisted transfection includes photoinjection, laser-induced stress waves, photochemical internalization, or selective cell targeting via light-absorbing particles. In some embodiments, microinjection includes a single needle or an array.

[0070] In some embodiments, chemical transfection includes calcium phosphate-mediated transfection; diethylaminoethyl (DEAE) dextran-mediated transfection; cationic lipid-mediated transfection; liposomes; polymers; other nanoparticles, such as gold, silica, carbon nanotubes, water-soluble fullerenes, silicon nanowires, or quantum dots; or any combination thereof.

[0071] In some embodiments, transfection is mediated by cationic, ionizable, or other types of lipids. In some embodiments, the lipid described herein is tetrakis(8-methylnonyl) 3,3',3'',3''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)) tetrapropionate (306O i10);Decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9);Ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-iso5-2DC18);((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);2-[(polyethylene glycol)-2000]-N,N-ditetradecyl (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13 dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol (β-sitosterol);Bis(2-(dodecyldisulfanyl)ethyl)-3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6 -diazahexacosyl)azanediyl)dipropionate (BAME-O16B); 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide ( BHEM-cholesterol;1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol);(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA); 1,2-Dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE); 2,3-Dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro acetate (DOSPA); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine (DSPC); hexa(octan-3-yl)9,9',9'',9'''',9'''',9'''' -((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Lipid H(SM-102));(((3,6-dioxopiperazine-2,5-diyl) bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin);1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(PEG2000-DMG);N; 1 ,N 3 ,N 5 - tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), dioctadecyl-amidolys-spermine, or any combination thereof.

[0072] In some embodiments, the liposomes described herein are charged or neutral. In some embodiments, the liposomes are unilamellar or multilamellar vesicles. In some embodiments, the liposomes are inverse liposomes.

[0073] In some embodiments, the polymers described herein include cationic peptides and derivatives thereof, such as polyornithine or polylysine. In some embodiments, the cationic polymers include linear or branched synthetic polymers including polyethyleneimine or polybrene. In some embodiments, the cationic polymers include polysaccharide-based delivery compounds including chitosan or cyclodextrin. In some embodiments, the cationic polymers include natural polymers including collagen or histone.

[0074] In some embodiments, the carbon nanotubes (CNTs) described herein include single-walled carbon nanotubes (SWNTs), multi-walled carbon nanotubes (MWNTs), or larger carbon nanotubes. In some embodiments, the CNTs described herein include concentric sheets rolled into a cylinder.

[0075] In some embodiments, the quantum dots described herein include nanoparticles made of semiconductor materials such as cadmium, selenide, silicon, indium arsenide, cadmium sulphide, or any combination thereof.

[0076] In some embodiments, the biological transfection comprises a bacteriophage, a virus-like particle (VLP), an erythrocyte ghost, a bactofection, an exosome, or any combination thereof. nucleic acid

[0077] Provided herein is a nucleic acid encoding the fusion construct described herein. In some embodiments, the nucleic acid encodes a fusion protein. In some embodiments, the fusion protein comprises a full-length protein, a truncated version of a full-length protein, a functional domain of a full-length protein, a fragment of a full-length protein, a variant of a full-length protein, or any combination thereof. A variant of a full-length protein may, in some instances, comprise amino acid substitutions (conservative or non-conservative), deletions, additions, modifications, or any combination thereof. In some embodiments, the nucleic acid encodes a chemokine receptor. In some embodiments, the nucleic acid encodes a trimerization fusion protein comprising a tumor necrosis superfamily member ligand (TNFSF-L). In some embodiments, the nucleic acid encodes lymphotoxin alpha ligand, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, a fragment thereof, or any combination thereof.

[0078] Provided herein is a nucleic acid encoding fusion constructs that include TNFSF-L and oligomerization domain.Exemplary TNFSF-Ls include lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), TNF-related apoptosis weak inducer, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. Exemplary oligomerization domains include, but are not limited to, collectin regions from surfactant protein A (SP-A), surfactant protein D (SP-D), mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1, or CL-46. Sequences of exemplary fusion constructs containing combinations of such features are provided in Table 4 (amino acids) and Table 5 (nucleic acids). Table 4. [Table 4-1] [Table 4-2] Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

[0079] In some embodiments, the nucleic acid described herein is combined with a vector. In some embodiments, the nucleic acid is integrated into a genome. In some embodiments, the genome is a viral genome, a bacterial genome, or a cellular genome. In some embodiments, the cell is a mammalian cell. In some embodiments, the target cell is an immune cell. In some embodiments, the mammalian cell is a tumor cell.

[0080] Provided herein is a modified oncolytic virus comprising one or more exogenous nucleic acids encoding a fusion protein, the fusion protein comprising a full-length protein, a truncated version of a full-length protein, a functional domain of a full-length protein, a fragment of a full-length protein, a variant of a full-length protein, or any combination thereof. A variant of a full-length protein may, in some instances, comprise an amino acid substitution (conservative or non-conservative), a deletion, an addition, a modification, or any combination thereof. In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid (also referred to herein as a transgene) capable of encoding a chemokine receptor. In some cases, the exogenous nucleic acid may be a therapeutic transgene. In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid capable of encoding a trimerization fusion protein comprising a tumor necrosis superfamily member ligand (TNFSF-L).

[0081] In some embodiments, the modified oncolytic virus comprises an exogenous nucleic acid encoding lymphotoxin alpha ligand, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, a fragment thereof, or any combination thereof. Each of these proteins or any combination thereof may contribute to the greater therapeutic benefit of the scaffold oncolytic virus.

[0082] In some embodiments, the modification of the oncolytic virus increases the efficacy of tumor-targeted systemic delivery of the virus by at least about 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 800, 1000, 2500, 5000, 1x10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 7.5×10 5 , 1×10 6 , 2.5×10 6 , 5×10 6 , 7.5×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , 7.5×107 , 1×10 8 , 2.5×10 8 , 5×10 8 , 7.5×10 8 , 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 This can result in a fold or greater increase in the number of cells in the tumor. In some embodiments, the efficacy of tumor-targeted systemic delivery of the virus can be measured by quantifying the virus that infects tumor cells, as opposed to the virus that infects non-tumor cells in the body, if desired. In some embodiments, the virus can be quantified by staining viral particles in tissue sections, or blood smears in the case of leukemia, lymphoma, or myeloma. In some embodiments, the quantification can be performed by reporter molecules engineered to be expressed by the virus, such as luciferase and fluorescent proteins. In some cases, such quantification can be performed by quantifying the viral genome in the tumor. Without limitation, tumor-targeted systemic delivery of the virus can be measured by quantifying certain downstream effects of viral infection in tumor cells, such as cytokines in response to viral infection or lymphocyte accumulation. In some embodiments, the oncolytic virus comprises an exogenous nucleic acid that can encode an oligomerized fusion protein designed for extracellular release that includes a tumor necrosis superfamily member ligand (TNFSF-L) domain, and the presence of the exogenous nucleic acid can result in about a 5-fold to 10-fold increase in efficacy of tumor-targeted systemic delivery of the virus compared to an otherwise identical oncolytic virus that does not contain the exogenous nucleic acid.

[0083] In some embodiments, the modified oncolytic virus comprises an exogenous nucleic acid encoding a trimerization fusion protein designed for extracellular release comprising a TNFSF-L domain and an oligomerization domain, and the expression of this fusion protein by the modified oncolytic virus can result in an enhanced immune response to the infected tumor. As a result, the immunosuppressive microenvironment in the tumor can be altered, resulting in enhanced immunotherapeutic activity of the modified oncolytic virus compared to an otherwise identical virus that comprises a nucleic acid encoding the fusion protein. In some embodiments, the increased immunotherapeutic activity is at least about 1.1, 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 800, 1000, 2500, 5000, 1x10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 2.5×10 5 , 5×10 5 , 1×10 6 The increase in immunotherapy activity can be 1-fold or more.Without being limited, the increase in immunotherapy activity can be reflected by the increase in B cell accumulation in tumor, the increase in T cell response to tumor-associated immunogen, or both.B cell accumulation can be measured, for example, by quantifying B cells in tumor, and T cell immune activity can be measured, for example, by interferon-gamma (interferon-gamma) secretion in ELISPOT assay.

[0084] Provided herein is an oncolytic virus comprising an exogenous nucleic acid sequence that codes for fusion constructs that comprise TNFSF-L and oligomerization domain.Exemplary TNFSF-Ls include lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), TNF-related apoptosis weak inducer, A proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, and functional domains derived from any combination thereof. Exemplary oligomerization domains include, but are not limited to, collectin regions from surfactant protein A (SP-A), surfactant protein D (SP-D), mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1, or CL-46. Exemplary fusion constructs containing combinations of such features are provided in Table 4 (amino acids) and Table 5 (nucleic acids). Symptoms and Administration

[0085] Provided herein is a fusion construct for use in an expression system for the treatment of cancer. In some embodiments, the fusion construct is expressed in cancer cells. In some embodiments, the cancer is a blood cancer (also known as hematopoietic cancer) or solid cancer. In some embodiments, the cancer includes, but is not limited to, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric cancer, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. In some embodiments, the cancer comprises bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer.

[0086] In some embodiments, the fusion construct is expressed in an immune cell. In some embodiments, the cell is a lymphoid cell or a myeloid cell. In some embodiments, the lymphoid cell is a B cell, a natural killer cell, or a T cell. In some embodiments, the T cell is an effector cell. In some embodiments, the effector cell is a cytotoxic T cell or a CD8+ cell. In some embodiments, the effector cell is a helper cell or a CD4+ cell. In some embodiments, the effector cell is a regulatory T cell. In some embodiments, the T cell is a memory T cell. In some embodiments, the myeloid cell is a neutrophil, an eosinophil, or a monocyte.

[0087] Provided herein is a fusion construct for use in an expression system for reducing tumor cell growth.In some embodiments, the tumor is a liquid tumor or a solid tumor.In some embodiments, the tumor comprises melanoma, hepatocellular carcinoma, breast tumor, lung tumor, peritoneal tumor, prostate tumor, bladder tumor, ovarian tumor, leukemia, lymphoma, kidney carcinoma, pancreatic carcinoma, epithelial carcinoma, gastric tumor, colon carcinoma, duodenal tumor, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal tumor, intestinal type gastric cancer, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. In some embodiments, the tumor comprises a bladder tumor, a blood tumor, a bone tumor, a bone marrow tumor, a brain tumor, a breast tumor, a colon tumor, an esophageal tumor, a gastrointestinal tumor, a gum tumor, a head tumor, a kidney tumor, a liver tumor, a lung tumor, a nasopharyngeal tumor, a cervical tumor, an ovarian tumor, a prostate tumor, a skin tumor, a stomach tumor, a testicular tumor, a tongue tumor, or a uterine tumor.

[0088] In some embodiments, the present disclosure provides a method of treating a subject by administering one or more modified oncolytic viruses disclosed herein. "Individual" or "Subject": As used interchangeably herein, refers to a human or non-human subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, poultry, horses, cows, goats, sheep, crustaceans, etc. In some embodiments, the subject is a human.

[0089] In some embodiments, the present disclosure provides a method for producing a toxic effect on cancer cells, comprising administering a therapeutically effective amount of a modified virus, such as the oncolytic vaccinia virus described above, or a pharmaceutical composition comprising the same to cancer cells.The present disclosure also provides a method for inhibiting at least one of the growth and proliferation of a second cancer cell, comprising administering the modified oncolytic virus described above to a first cancer cell, so that the first cancer cell is infected with the virus.Therefore, in some embodiments of the method disclosed herein, it is believed that administering a therapeutically effective amount of the oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising the same, does not infect all cancer or tumor cells, and can inhibit the proliferation of non-infected cells without directly infecting them.

[0090] In some embodiments, the methods and compositions of the present disclosure can be used to contact cancer cells or tumors with a therapeutically effective dose of the exemplary oncolytic vaccinia virus described herein or a pharmaceutical composition comprising the same to induce oncolysis, kill cells, inhibit proliferation, inhibit metastasis, reduce tumor size, or otherwise reverse or reduce the malignant phenotype of tumor cells. In some embodiments, the modified oncolytic virus of the present disclosure, such as an effective amount of the oncolytic vaccinia virus described herein or a pharmaceutical composition thereof, can comprise an amount sufficient to induce oncolysis, destruction or lysis of cancer cells, or inhibition or reduction of the proliferation or size of cancer cells. Reducing the growth of cancer cells can be manifested, for example, by cell death, or a slower replication or growth rate of tumors comprising the cells, or an increased survival time of subjects comprising the cancer cells.

[0091] In some embodiments, a method of treating a subject having cancer or tumor is provided, comprising administering an effective amount of the nucleic acid, fusion protein, or modified virus to the subject. The effective amount in such a method may include an amount that reduces the growth rate or spread of cancer, or an amount that extends the subject's survival time. The present disclosure provides a method of reducing tumor growth, which may include administering an effective amount of the nucleic acid, fusion protein, or modified oncolytic virus to the tumor. In some embodiments, the effective amount of the nucleic acid, fusion protein, or modified virus, or pharmaceutical composition thereof, may include an amount sufficient to induce a delay, inhibition, or reduction in tumor growth or size, including eradication of the tumor. The reduction in tumor growth may be manifested, for example, by a reduction in the growth rate or an extension of the survival time of the subject containing the tumor.

[0092] In some embodiments, the nucleic acid described herein is administered to a subject in an amount of about 0.01 μg / dose to about 1 g / dose or about 0.5 μg / dose to about 500 mg / dose. In some embodiments, the nucleic acid is administered to a subject in an amount of about 0.01 μg / dose, about 0.05 μg / dose, about 0.1 μg / dose, about 0.5 μg / dose, about 1 μg / dose, about 5 μg / dose, about 10 μg / dose, about 50 μg / dose, about 100 μg / dose, about 500 μg / dose, about 1 mg / dose, about 5 mg / dose, about 10 mg / dose, about 50 mg / dose, about 100 mg / dose, about 500 mg / dose, about 1 g / dose.

[0093] In some embodiments, the cells described herein are administered to a subject in an amount of about 0.01 μg / dose to about 1 g / dose or about 0.5 μg / dose to about 500 mg / dose. In some embodiments, the cells are administered to a subject in an amount of about 0.01 μg / dose, about 0.05 μg / dose, about 0.1 μg / dose, about 0.5 μg / dose, about 1 μg / dose, about 5 μg / dose, about 10 μg / dose, about 50 μg / dose, about 100 μg / dose, about 500 μg / dose, about 1 mg / dose, about 5 mg / dose, about 10 mg / dose, about 50 mg / dose, about 100 mg / dose, about 500 mg / dose, about 1 g / dose.

[0094] In some embodiments, the fusion protein described herein is administered to a subject in an amount of about 0.01 μg / dose to about 1 g / dose or about 0.5 μg / dose to about 500 mg / dose. In some embodiments, the fusion protein is administered to a subject in an amount of about 0.01 μg / dose, about 0.05 μg / dose, about 0.1 μg / dose, about 0.5 μg / dose, about 1 μg / dose, about 5 μg / dose, about 10 μg / dose, about 50 μg / dose, about 100 μg / dose, about 500 μg / dose, about 1 mg / dose, about 5 mg / dose, about 10 mg / dose, about 50 mg / dose, about 100 mg / dose, about 500 mg / dose, or about 1 g / dose.

[0095] In some embodiments, the amount of a modified oncolytic virus of the present disclosure, e.g., an oncolytic virus or a vaccinia virus, administered to a subject is about 10 3 ~10 12 Infectious viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8 PFU, or about 10 8 ~10 10 In some embodiments, the amount of the modified oncolytic virus of the present disclosure, e.g., an oncolytic virus or a vaccinia virus, administered to a subject is about 10 3 ~10 12 Viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8 PFU, or about 10 8 ~10 10 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, is about 10 PFU. 3 PFU / dose~about 10 4 PFU / dose, approximately 10 4 PFU / dose~about 10 5 PFU / dose, approximately 10 5 PFU / dose~about 10 6 PFU / dose, approximately 107 PFU / dose~about 10 8 PFU / dose, approximately 10 9 PFU / dose~about 10 10 PFU / dose, approximately 10 10 PFU / dose~about 10 11 PFU / dose, approximately 10 11 PFU / dose~about 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, is administered at a dose that may include about 2×10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, approximately 10 4 PFU / dose, approximately 2 x 10 4 PFU / dose, approximately 3 x 10 4 PFU / dose, approximately 4 x 10 4 PFU / dose, approximately 5 x 10 4 PFU / dose, approximately 6 x 10 4 PFU / dose, approximately 7 x 10 4 PFU / dose, approximately 8 x 10 4 PFU / dose, approximately 9 x 10 4 PFU / dose, approximately 10 5 PFU / dose, 2×10 5 PFU / dose, approximately 3 x 10 5 PFU / dose, approximately 4 x 10 5 PFU / dose, approximately 5 x 10 5 PFU / dose, approximately 6 x 10 5 PFU / dose, approximately 7 x 10 5 PFU / dose, approximately 8 x 10 5 PFU / dose, approximately 9 x 105 PFU / dosage, approximately 10 6 PFU / dosage, 2×10 6 PFU / dosage, approximately 3×10 6 PFU / dosage, approximately 4×10 6 PFU / dosage, approximately 5×10 6 PFU / dosage, approximately 6×10 6 PFU / dosage, approximately 7×10 6 PFU / dosage, approximately 8×10 6 PFU / dosage, approximately 9×10 6 PFU / dosage, approximately 10 7 PFU / dosage, 2×10 7 PFU / dosage, approximately 3×10 7 PFU / dosage, approximately 4×10 7 PFU / dosage, approximately 5×10 7 PFU / dosage, approximately 6×10 7 PFU / dosage, approximately 7×10 7 PFU / dosage, approximately 8×10 7 PFU / dosage, approximately 9×10 7 PFU / dosage, approximately 10 8 PFU / dosage, 2×10 8 PFU / dosage, approximately 3×10 8 PFU / dosage, approximately 4×10 8 PFU / dosage, approximately 5×10 8 PFU / dosage, approximately 6×10 8 PFU / dosage, approximately 7×10 8 PFU / dosage, approximately 8×10 8 PFU / dosage, approximately 9×10 8 PFU / dosage, approximately 10 9 PFU / dosage, 2×10 9 PFU / dosage, approximately 3×10 9 PFU / dosage, approximately 4×10 9 PFU / dosage, approximately 5×10 9 PFU / dosage, approximately 6×10 9 PFU / dosage, approximately 7×10 9 PFU / dosage, approximately 8×10 9 PFU / dosage, approximately 9×10 9 PFU / dosage, approximately 10 10 PFU / dosage, 2×10 10 PFU / dosage, approximately 3×10 10 PFU / dosage, approximately 4×10 10PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 10 PFU / dose, 2×10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 11 PFU / dose, 2×10 11 PFU / dose, approximately 3 x 10 11 PFU / dose, approximately 4 x 10 11 PFU / dose, approximately 5 x 10 11 PFU / dose, approximately 6 x 10 11 PFU / dose, approximately 7 x 10 11 PFU / dose, approximately 8 x 10 11 PFU / dose, approximately 9 x 10 11 PFU / dose, approximately 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure, such as oncolytic vaccinia viruses, may be administered at doses that may include 5×10 PFU / dose. 9 In some embodiments, the modified oncolytic viruses of the present disclosure, such as oncolytic vaccinia viruses, may be administered at doses that may include 5×10 PFU / dose. 9 The antibodies may be administered in doses that may include up to PFU / dose.

[0096] In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, is 3 Virus particles / dose ~ approx. 10 4 Viral particles / dose, approximately 10 4 Virus particles / dose ~ approx. 10 5 Viral particles / dose, approximately 10 5 Virus particles / dose ~ approx. 10 6 Viral particles / dose, approximately 10 7 Virus particles / dose ~ approx. 10 8 Viral particles / dose, approximately 10 9 Virus particles / dose ~ approx. 10 10 Viral particles / dose, approximately 10 10 Virus particles / dose ~ approx. 10 11 Viral particles / dose, approximately 10 11 Virus particles / dose ~ approx. 10 12 Viral particles / dose, approximately 10 12 Virus particles / dose ~ approx. 10 13 Viral particles / dose, approximately 10 13 Virus particles / dose ~ approx. 10 14 viral particles / dose, or approximately 10 14 Virus particles / dose ~ approx. 10 15 It may be administered in a dose that may contain viral particles / dose.

[0097] In some embodiments, the modified oncolytic virus of the present disclosure is 3 PFU / kg ~ approx. 10 4 PFU / kg, approximately 10 4 PFU / kg ~ approx. 10 5 PFU / kg, approximately 10 5 PFU / kg ~ approx. 10 6 PFU / kg, approximately 10 7 PFU / kg ~ approx. 10 8 PFU / kg, approximately 10 9 PFU / kg ~ approx. 10 10 PFU / kg, approximately 10 10 PFU / kg ~ approx. 10 11 PFU / kg, approximately 10 11 PFU / kg ~ approx. 10 12 PFU / kg, approximately 10 12PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may be administered at a dose that may include about 2×10 PFU / kg. 3 PFU / kg, 3×10 3 PFU / kg, 4×10 3 PFU / kg, 5×10 3 PFU / kg, 6×10 3 PFU / kg, 7×10 3 PFU / kg, 8×10 3 PFU / kg, 9×10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2×10 5 PFU / kg, 3×10 5 PFU / kg, 4×10 5 PFU / kg, 5×10 5 PFU / kg, 6×10 5 PFU / kg, 7×10 5 PFU / kg, 8×10 5 PFU / kg, 9×10 5 PFU / kg, approximately 10 6 PFU / kg, 2×10 6 PFU / kg, 3×10 6 PFU / kg, 4×10 6 PFU / kg, 5×10 6 PFU / kg, 6×10 6 PFU / kg, 7×10 6 PFU / kg, 8×106 PFU / kg, 9×10 6 PFU / kg, approximately 10 7 PFU / kg, 2×10 7 PFU / kg, 3×10 7 PFU / kg, 4×10 7 PFU / kg, 5×10 7 PFU / kg, 6×10 7 PFU / kg, 7×10 7 PFU / kg, 8×10 7 PFU / kg, 9×10 7 PFU / kg, approximately 10 8 PFU / kg, 2×10 8 PFU / kg, 3×10 8 PFU / kg, 4×10 8 PFU / kg, 5×10 8 PFU / kg, 6×10 8 PFU / kg, 7×10 8 PFU / kg, 8×10 8 PFU / kg, 9×10 8 PFU / kg, approximately 10 9 PFU / kg, 2×10 9 PFU / kg, 3×10 9 PFU / kg, 4×10 9 PFU / kg, 5×10 9 PFU / kg, 6×10 9 PFU / kg, 7×10 9 PFU / kg, 8×10 9 PFU / kg, 9×10 9 PFU / kg, approximately 10 10 PFU / kg, 2×10 10 PFU / kg, 3×10 10 PFU / kg, 4×10 10 PFU / kg, 5×10 10 PFU / kg, 6×10 10 PFU / kg, 7×10 10 PFU / kg, 8×10 10 PFU / kg, 9×10 10 PFU / kg, approximately 10 10 PFU / kg, 2×10 10 PFU / kg, 3×10 10 PFU / kg, 4×10 10 PFU / kg, 5×1010 PFU / kg, 6×10 10 PFU / kg, 7×10 10 PFU / kg, 8×10 10 PFU / kg, 9×10 10 PFU / kg, approximately 10 11 PFU / kg, 2×10 11 PFU / kg, 3×10 11 PFU / kg, 4×10 11 PFU / kg, 5×10 11 PFU / kg, 6×10 11 PFU / kg, 7×10 11 PFU / kg, 8×10 11 PFU / kg, 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may be administered at a dose that may include 5×10 PFU / kg. 9 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may be administered at a dose that may include 5×10 PFU / kg. 9 The antibody may be administered in a dose that may include up to PFU / kg.

[0098] In some embodiments, the modified oncolytic virus of the present disclosure is 3 Virus particles / kg ~ approx. 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg ~ approx. 10 5 Virus particles / kg, approximately 10 5 Virus particles / kg ~ approx. 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg ~ approx. 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg ~ approx. 1010 Virus particles / kg, approximately 10 10 Virus particles / kg ~ approx. 10 11 Virus particles / kg, approximately 10 11 Virus particles / kg ~ approx. 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg ~ approx. 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg ~ approx. 10 14 Virus particles / kg, or approximately 10 14 Virus particles / kg ~ approx. 10 15 It may be administered in a dose that may contain viral particles / kg.

[0099] The liquid dosage forms of the oncolytic vaccinia viruses described herein, in some embodiments, contain about 10 3 PFU / mL~about 10 4 PFU / mL, approximately 10 4 PFU / mL~about 10 5 PFU / mL, approximately 10 5 PFU / mL~about 10 6 PFU / mL, approximately 10 7 PFU / mL~about 10 8 PFU / mL, approximately 10 9 PFU / mL~about 10 10 PFU / mL, approximately 10 10 PFU / mL~about 10 11 PFU / mL, approximately 10 11 PFU / mL~about 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may contain a viral load of about 2×10 PFU / mL. 3 PFU / mL, 3×10 3 PFU / mL, 4×10 3 PFU / mL, 5×10 3PFU / mL, 6×10 3 PFU / mL, 7×10 3 PFU / mL, 8×10 3 PFU / mL, 9×10 3 PFU / mL, approximately 10 4 PFU / mL, approximately 2×10 4 PFU / mL, approximately 3×10 4 PFU / mL, approximately 4×10 4 PFU / mL, approximately 5×10 4 PFU / mL, approximately 6×10 4 PFU / mL, approximately 7×10 4 PFU / mL, approximately 8×10 4 PFU / mL, approximately 9×10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6 PFU / mL, 2×10 6 PFU / mL, 3×10 6 PFU / mL, 4×10 6 PFU / mL, 5×10 6 PFU / mL, 6×10 6 PFU / mL, 7×10 6 PFU / mL, 8×10 6 PFU / mL, 9×10 6 PFU / mL, approximately 10 7 PFU / mL, 2×10 7 PFU / mL, 3×10 7 PFU / mL, 4×10 7 PFU / mL, 5×10 7 PFU / mL, 6×10 7 PFU / mL, 7×10 7 PFU / mL, 8×10 7 PFU / mL, 9×10 7 PFU / mL, approximately 10 8 PFU / mL, 2×108 PFU / mL, 3×10 8 PFU / mL, 4×10 8 PFU / mL, 5×10 8 PFU / mL, 6×10 8 PFU / mL, 7×10 8 PFU / mL, 8×10 8 PFU / mL, 9×10 8 PFU / mL, approximately 10 9 PFU / mL, 2×10 9 PFU / mL, 3×10 9 PFU / mL, 4×10 9 PFU / mL, 5×10 9 PFU / mL, 6×10 9 PFU / mL, 7×10 9 PFU / mL, 8×10 9 PFU / mL, 9×10 9 PFU / mL, approximately 10 10 PFU / mL, 2×10 10 PFU / mL, 3×10 10 PFU / mL, 4×10 10 PFU / mL, 5×10 10 PFU / mL, 6×10 10 PFU / mL, 7×10 10 PFU / mL, 8×10 10 PFU / mL, 9×10 10 PFU / mL, approximately 10 10 PFU / mL, 2×10 10 PFU / mL, 3×10 10 PFU / mL, 4×10 10 PFU / mL, 5×10 10 PFU / mL, 6×10 10 PFU / mL, 7×10 10 PFU / mL, 8×10 10 PFU / mL, 9×10 10 PFU / mL, approximately 10 11 PFU / mL, 2×10 11 PFU / mL, 3×10 11 PFU / mL, 4×10 11 PFU / mL, 5×10 11 PFU / mL, 6×10 11 PFU / mL, 7×10 11PFU / mL, 8×10 11 PFU / mL, 9×10 11 PFU / mL, or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may be administered at a dose that may include 5×10 PFU / mL. 9 In some embodiments, the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus, may be administered at a dose that may include 5×10 PFU / mL. 9 The antibody may be administered in doses that may contain up to PFU / mL.

[0100] In some instances, when the modified oncolytic virus is administered by injection, the dosage is about 10 mg / injection. 3 10 viral particles per injection 4 10 viral particles per injection 5 10 viral particles per injection 6 10 viral particles per injection 7 10 viral particles per injection 8 10 viral particles per injection 9 10 viral particles per injection 10 10 viral particles per injection 11 10 viral particles per injection 12 2 x 10 viral particles per injection 12 10 viral particles per injection 13 10 viral particles per injection 14 viral particles, or 10 per injection 15 In a further example, when the modified oncolytic virus is administered by injection, the dosage can include about 10 viral particles per injection.3 10 infectious viral particles per injection 4 10 infectious viral particles per injection 5 10 infectious viral particles per injection 6 10 infectious viral particles per injection 7 10 infectious viral particles per injection 8 10 infectious viral particles per injection 9 10 infectious viral particles per injection 10 10 infectious viral particles per injection 11 10 infectious viral particles per injection 12 infectious viral particles, 2 x 10 per injection 12 10 infectious viral particles per injection 13 10 infectious viral particles per injection 14 infectious viral particles, or 10 per infection 15 In some embodiments, the virus can be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor.

[0101] In some embodiments, a single dose of the pharmaceutical composition described herein can refer to the amount administered to a subject or tumor over 1, 2, 5, 10, 15, 20 or 24 hours. In some embodiments, the dose can be spread over time or by separate injections. In some embodiments, multiple doses (e.g., 2, 3, 4, 5, 6 or more doses) of the pharmaceutical composition described herein can be administered to a subject, for example, a second treatment can be administered within 1, 2, 3, 4, 5, 6, 7 days or within 1, 2, 3, 4, 5, 6, 7 weeks of a first treatment. In some embodiments, multiple doses of the pharmaceutical composition described herein can be administered to a subject over a period of 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments, the pharmaceutical compositions described herein or the pharmaceutical compositions described herein can be administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or more. The frequency of administration of the oncolytic vaccinia virus or pharmaceutical compositions described herein can be, in some cases, once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months). In some embodiments of the methods disclosed herein, the oncolytic vaccinia virus or pharmaceutical composition can be administered independently at an initial dose for a first period, an intermediate dose for a second period, and a high dose for a third period. In some embodiments, the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose.In some embodiments, the first, second, and third periods are independently from about 1 week to about 2 weeks, from about 2 weeks to about 3 weeks, from about 3 weeks to about 4 weeks, from about 4 weeks to about 5 weeks, from about 6 weeks to about 7 weeks, from about 7 weeks to about 8 weeks, from about 8 weeks to about 9 weeks, from about 9 weeks to about 10 weeks, from about 10 weeks to about 11 weeks, from about 11 weeks to about 12 weeks, from about 12 weeks to about 24 weeks, from about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or more.

[0102] One or more doses of the pharmaceutical composition comprise a treatment cycle. Two or more doses in a treatment cycle can be separated by a dosing interval in which the pharmaceutical composition is not administered. One or more treatment cycles comprise a series of treatments. Two or more treatment cycles in a series of treatments can be separated by a treatment interval in which no treatment is administered.

[0103] In some embodiments of the methods described herein, one or more doses of the compositions, cells, fusion proteins, oncolytic viruses, or vaccinia viruses described herein comprise a treatment cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of the compositions, cells, fusion proteins, oncolytic viruses, or vaccinia viruses described herein comprise a treatment cycle. In some embodiments, the doses are administered for a period of about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day, or more. In some embodiments, all doses in a treatment cycle are approximately the same in dose amount and duration. In some embodiments, each dose in a treatment cycle is independent of any other dose. In some embodiments, two or more doses in a treatment cycle are separated by an administration interval during which no dose is administered. In some embodiments, the dosing interval is about 1 minute, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or more. In some embodiments, all dosing intervals are about the same. In some embodiments, each dosing interval is independent of any other dosing interval.

[0104] In some embodiments of the methods described herein, one or more treatment cycles include a series of treatments. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten or more treatment cycles include a series of treatments. The treatment cycles described herein are 1-24 hours, 1-6 days, 1-4 weeks, 1-12 months or more in duration. In some embodiments, the treatment cycles are 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more in duration. In some embodiments, all treatment cycles are approximately the same. In some embodiments, each treatment cycle is independent of any other treatment cycle. In some embodiments, two or more treatment cycles are separated by a treatment interval during which no treatment is administered. In some embodiments, the treatment interval is 1-6 days, 1-4 weeks, 1-12 months, 1-5 years, or more. In some embodiments, the treatment interval is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more. In some embodiments, all treatment intervals are about the same. In some embodiments, each treatment interval is independent of any other treatment interval.

[0105] In some embodiments of the methods described herein, one or more treatments with the compositions, cells, fusion proteins, oncolytic viruses, or vaccinia viruses described herein comprise the methods described herein. In some embodiments, one, two, three, four, five, six, seven, eight, or more courses of treatment comprise the methods. In some embodiments, the course of treatment is 1-6 days, 1-4 weeks, 1-12 months, 1-5 years, or more. In some embodiments, the course of treatment is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more. In some embodiments, all the courses of treatment are the same. In some embodiments, each course of treatment is independent of any other course of treatment. In some embodiments, each course of treatment is followed by a course interval during which no treatment is administered. In some embodiments, the course interval is 1-6 days, 1-4 weeks, 1-12 months, 1-5 years, or more. In some embodiments, the course interval is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more. In some embodiments, the duration of the course interval depends on the incidence or recurrence of the disease. In some embodiments, all course intervals are about the same. In some embodiments, each course interval is independent of any other course interval.

[0106] In some examples, the subject may be placed on a reduced carbohydrate diet, e.g., a ketogenic diet, prior to, concurrently with, and after administration of a modified oncolytic virus described herein, e.g., an oncolytic vaccinia virus, or a pharmaceutical composition comprising same, according to any of the methods of treatment described herein. In some embodiments, the subject is placed on a diet that may include consuming less than 500 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 400 grams of carbohydrates per day, less than 350 grams of carbohydrates per day, less than 300 grams of carbohydrates per day, less than 250 grams of carbohydrates per day, less than 200 grams of carbohydrates per day, less than 150 grams of carbohydrates per day, less than 100 grams of carbohydrates per day, less than 90 grams of carbohydrates per day, less than 80 grams of carbohydrates per day, less than 70 grams of carbohydrates per day, less than 60 grams of carbohydrates per day, less than 50 grams of carbohydrates per day, less than 40 grams of carbohydrates per day, less than 30 grams of carbohydrates per day, less than 20 grams of carbohydrates per day, less than 10 grams of carbohydrates per day.

[0107] An exemplary method for delivering the modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus described herein or pharmaceutical compositions comprising the same, to cancer or tumor cells can be by intratumoral injection. However, alternative administration methods can also be used. The route of administration can vary depending on the location and nature of the tumor. In some embodiments, administration can be local or systemic. In some embodiments, the route of administration is intravenous, local (e.g., in close proximity to the tumor, particularly the vasculature of the tumor or adjacent vasculature), intraperitoneal, parenteral, intramuscular, subcutaneous, intraarterial, percutaneous, intrathecal, intratracheal, intravesical, transdermal, intradermal, inhalation, irrigation, intranasal, intraurethral, ​​intravaginal, lavage, oral, intradermal, rectal, or any combination thereof. The injectable dose of the oncolytic virus can be administered as a bolus injection or slow infusion. In some embodiments, the modified oncolytic virus can be administered to the patient from a source implanted in the patient. In some embodiments, the administration of the modified oncolytic virus can be carried out by continuous infusion over a selected period of time.In some examples, the oncolytic vaccinia virus described herein or the pharmaceutical composition comprising the same can be administered at a therapeutically effective dose by infusion over a period of about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or more. The oncolytic virus or pharmaceutical composition of the present disclosure can be administered as a liquid dosage having a total dosage of about 1 mL to about 5 mL, about 5 mL to about 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to about 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to about 500 mL, about 500 mL to about 750 mL, or about 750 mL to about 1000 mL. Pharmaceutical Compositions

[0108] Provided herein is a pharmaceutical composition comprising a fusion construct or a nucleic acid encoding such a construct and a pharma- ceutically acceptable carrier. As used herein, "pharma- ceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to which it is administered. Non-limiting examples of pharma- ceutically acceptable carriers include buffers, water, emulsions, various types of wetting agents, sterilizing solutions, preservatives, stabilizers, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, and any combination thereof. In some embodiments, the buffer comprises a citrate buffer, a phosphate buffer, an acetate buffer, or any combination thereof. In some embodiments, the emulsion is an oil-in-water (O / W) emulsion, a water-in-oil emulsion (W / O), or a multiple emulsion. Further non-limiting examples of pharma- ceutically acceptable carriers may include gels, bioabsorbable polymers, embedded elements containing the fusion construct or nucleic acid, or any other suitable vehicle, delivery or dispensing means or material. In some embodiments, the bioabsorbable polymer comprises polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxone (PDO), or any combination thereof. In some embodiments, the gel comprises a protein, a polysaccharide, a cellulose derivative, a synthetic polymer, or any combination thereof. In some embodiments, the protein comprises gelatin, collagen, or any combination thereof. In some embodiments, the polysaccharide comprises pectin, gellum gum, alginic acid, agar, xanthin, cassia tora, tragacanth, carrageenan, guar gum, or any combination thereof. In some embodiments, the cellulose derivative comprises methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, or any combination thereof. In some embodiments, the synthetic polymer comprises carbomer, polyacrylamide, poloxamer, polyvinyl alcohol, polyethylene or its copolymers, or any combination thereof.Such carriers can be formulated in a conventional manner and administered to a subject in an effective amount.

[0109] In some embodiments, the pharmaceutical compositions described herein are formulated as aqueous solution, oily solution, dispersion in glycerol, liquid polyethylene glycol, solid, inhalable form, intranasal form, liposome, nanoparticle, microparticle, polymer, or any combination thereof.In some embodiments, the pharmaceutical compositions described herein may include stabilizers and buffers.In some embodiments, the pharmaceutical compositions described herein may include solubilizers such as sterile water or buffers. Manufacturing method

[0110] In some embodiments, the modified oncolytic virus of the present disclosure is packaged in a cell line. In some embodiments, the modified oncolytic virus can be grown in a suitable host cell, such as HeLa, 293 or Vero cells, isolated from the host cell, and stored under conditions that promote viral stability and integrity, thereby minimizing loss of infectivity over time. In certain exemplary methods, the modified oncolytic virus is grown in the host cell using cell stacks, roller bottles, or perfusion bioreactors. In some examples, downstream methods for purifying the modified oncolytic virus can include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, or chromatographic capture. The modified oncolytic virus can be stored, for example, by freezing or drying, such as lyophilization. In some embodiments, prior to administration, the stored modified oncolytic virus can be reconstituted (if dried for storage) and diluted with a pharma- ceutically acceptable carrier for administration. In some embodiments, the modified oncolytic viruses described herein exhibit higher titers in HeLa and 293 cells compared to otherwise identical viruses that do not contain modifications in the modified oncolytic viruses. In some cases, higher titers in HeLa and 293 cells are seen for the modified oncolytic viruses. kit

[0111] In embodiments, the present disclosure provides a kit for administering the modified oncolytic virus described herein.In some embodiments, the kit of the present disclosure may include a modified oncolytic virus or a pharmaceutical composition comprising the modified oncolytic virus described above.In some embodiments, the kit of the present disclosure may further include one or more components such as instructions, devices and additional reagents, as well as components such as tubes, containers and syringes for carrying out the methods disclosed above.In some embodiments, the kit of the present disclosure may further include one or more agents that can be administered in combination with the modified virus, such as at least one of anticancer agents, immunomodulators, or any combination thereof.

[0112] In some embodiments, the kits of the present disclosure may include one or more containers that contain the modified viruses disclosed herein. For example, but not by way of limitation, the kits of the present disclosure may include one or more containers that contain the modified oncolytic viruses of the present disclosure.

[0113] In some embodiments, the kit of the present disclosure may include instructions for use, a device for administering the modified oncolytic virus to a subject, or a device for administering an additional agent or compound to a subject. For example, but not limited to, the instructions may include a description of the modified oncolytic virus and other components included in the kit as needed, as well as administration methods, including methods for determining the appropriate condition of the subject, the appropriate dosage, and the appropriate administration method for administering the modified virus. The instructions may also include guidance for monitoring the subject over the course of treatment time.

[0114] In some embodiments, the kit of the present disclosure may include a device for administering modified oncolytic viruses to a subject.Any of the various devices known in the art for administering medicaments and pharmaceutical compositions can be included in the kit provided herein.For example, but not limited to, such devices include hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers such as eyedroppers.In some embodiments, the modified oncolytic viruses that are delivered systemically, for example, by intravenous injection, intratumoral injection, or intraperitoneal injection, can be included in the kit with hypodermic needles and syringes. Exemplary embodiments

[0115] Provided herein is a fusion protein, which comprises a TNF (tumor necrosis factor)-superfamily ligand (TNFSF-L) or a functional variant thereof and a plurality of domains derived from a collectin family protein, which comprises an oligomerization domain or a functional variant thereof and a neck domain or a functional variant thereof, and the oligomerization domain or a functional variant thereof and the neck domain or a functional variant thereof are in closer sequence proximity compared to their positions in the collectin family protein.Further provided herein is a fusion protein, which comprises, in the order of N-terminus to C-terminus, an oligomerization domain or a functional variant thereof, a neck domain or a functional variant thereof, and optionally a linker sequence and a TNFSF-L or a functional variant thereof. In the fusion protein further provided herein, TNFSF-L is lymphotoxin alpha, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa-beta ligand (RANKL), weak inducer of TNF-related apoptosis, A-proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18, ectodysplasin A. In the fusion protein further provided herein, TNFSF-L is CD40 ligand. In the fusion protein further provided herein, CD40 ligand comprises at least 85% sequence identity with SEQ ID NO:1. In the fusion protein further provided herein, TNFSF-L is OX40 ligand. In the fusion protein further provided herein, OX40 ligand comprises at least 85% sequence identity with SEQ ID NO:2 or SEQ ID NO:3. In the fusion protein further provided herein, TNFSF-L is 4-1BBL. In the fusion protein further provided herein, 4-1BBL comprises at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5.In the fusion protein further provided herein, TNFSF-L is LIGHT. In the fusion protein further provided herein, LIGHT comprises at least 85% sequence identity with SEQ ID NO:6. In the fusion protein further provided herein, the collectin family protein is SP-A, SP-D, mannose-binding lectin (MBL), conglutinin, CL-43, CL-L1, CL-K1, CL-P1, or CL-46. In the fusion protein further provided herein, the collectin family protein is SP-D. In the fusion protein further provided herein, the oligomerization domain comprises at least 85% sequence identity with SEQ ID NO:9. In the fusion protein further provided herein, the oligomerization domain and the neck domain together comprise at least 85% sequence identity with SEQ ID NO:11. In the fusion protein further provided herein, the oligomerization domain and the neck domain together comprise SEQ ID NO:11. Further provided herein is a fusion protein, wherein the fusion protein comprises said linker sequence, wherein said linker sequence comprises GSG (glycine-serine-glycine) (SEQ ID NO: 12).

[0116] Provided herein are fusion proteins comprising a sequence having at least 85% sequence identity to SEQ ID NO:14, 15, 16, 18, or 19.

[0117] Oncolytic viruses are provided herein, comprising a TNF (tumor necrosis factor)-superfamily ligand (TNFSF-L) or a functional variant thereof fused to an oligomerization domain. In oncolytic viruses further provided herein, the oncolytic viruses are Newcastle disease virus (NDV), reovirus (RV), myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV) poliovirus (PV). In oncolytic viruses further provided herein, TNFSF-L or a functional variant thereof is inserted into the viral genome. In oncolytic viruses further provided herein, TNFSF-L or a functional variant thereof is inserted into the thymidine kinase gene.

[0118] Provided herein is a vaccinia virus, which comprises a TNF (tumor necrosis factor)-superfamily ligand (TNFSF-L) or a functional variant thereof fused to an oligomerization domain. Provided herein is a vaccinia virus, which is a Western Reserve vaccinia virus (ATCC VR-1354), an Ankara vaccinia virus (ATCC VR-1508), an Ankara vaccinia virus (ATCC VR-1566), a Wyeth vaccinia virus (ATCC VR-1536), or a modified strain of a Wyeth vaccinia virus (ATCC VR-325). Provided herein is a vaccinia virus, which is TNFSF-L or a functional variant thereof inserted into the viral genome. Provided herein is a vaccinia virus, which is TNFSF-L or a functional variant thereof inserted into the thymidine kinase gene.

[0119] Provided herein is an oncolytic virus, which comprises the fusion protein described herein.

[0120] Provided herein is a vaccinia virus, which comprises the fusion protein described herein.

[0121] Provided herein is a method for the treatment of cancer, comprising administering to a subject a vector comprising a nucleic acid in an amount sufficient to treat the cancer, wherein the nucleic acid encodes a fusion protein described herein.

[0122] Provided herein is a method for the treatment of cancer, the method comprising administering to a subject an oncolytic virus described herein in an amount sufficient to treat the cancer.

[0123] Provided herein is a method for the treatment of cancer, the method comprising administering to a subject a vaccinia virus described herein in an amount sufficient to treat the cancer.

[0124] Provided herein is a method for reducing tumor cell growth, comprising administering to a tumor cell in an amount sufficient to reduce tumor cell growth a vector comprising a nucleic acid, wherein the nucleic acid encodes a fusion protein described herein.

[0125] Provided herein is a method for reducing tumor cell growth, the method comprising administering to a tumor cell an oncolytic virus described herein in an amount sufficient to reduce tumor cell growth.

[0126] Provided herein is a method for the reduction of tumor cell growth, the method comprising administering to a tumor cell a vaccinia virus described herein in an amount sufficient to reduce tumor cell growth. EXAMPLES

[0127] The following examples further illustrate the described embodiments without limiting the scope of the disclosure. Example 1: Expression of native and TNFSF-L fusion constructs

[0128] The following fusion TNFSF-L constructs were designed: 3sCD40L (mouse); 3sOX40L (mouse); 3s41BBL (mouse); 3sOX40L (human); and 3s41BBL (human). The sequences of these constructs are listed in Tables 4 and 5.

[0129] Additionally, the following native monomer producing constructs were designed: mOX40L (mouse, native) and m41BBL (mouse, native). The sequences of these constructs are provided in Table 6 (amino acids) and Table 7 (nucleic acids). Table 6. [Table 6] Table 7. [Table 7-1] [Table 7-2]

[0130] Expression of TNFSF-L native monomer or variant fusion constructs from oncolytic viruses was performed. Briefly, HeLa cells were infected at an MOI of 5.0 with viruses expressing monomer or scaffold-TNFSF-L fusions (for OX40L or 41BBL), or with control viruses not expressing TNFSF-L, or with viruses expressing scaffold-TNFSF-L fusions (for CD40L). Media was collected at 24 hours and run with a commercial ELISA kit according to the manufacturer's instructions (samples were undiluted or diluted as indicated). Samples were diluted 1:100 and binding was recorded and amounts charted taking into account the dilution factor.

[0131] Referring to Figure 2, uninfected HeLa cells did not show detectable levels of mCD40L. Similarly, HeLa cells infected with oncolytic vaccinia virus with A52R and TK deletion also did not show detectable levels of mCD40L. HeLa cells infected with oncolytic vaccinia virus with TK deletion and fusion construct 3sCD40L (mouse) with oligomerization domain inserted into TK gene show very high levels of expression of about 400ng / mL.

[0132] Referring to FIG. 3, uninfected HeLa cells did not show detectable levels of mOX40L. Similarly, HeLa cells infected with oncolytic vaccinia virus with A52R and TK deletions also did not show detectable levels of mOX40L. HeLa cells infected with oncolytic vaccinia virus with TK deletions and native mOX40L inserted into the TK gene showed a slight increase in mOX40L expression, with approximately 4500 pg / mL detected. HeLa cells infected with oncolytic vaccinia virus with TK deletions and fusion construct 3sOX40L (mouse) containing an oligomerization domain inserted into the TK gene showed a much higher level of mOX40L expression, with approximately 265,000 pg / mL of mOX40L detected.

[0133] Referring to FIG. 4, uninfected HeLa cells did not show detectable levels of m41BBL. HeLa cells infected with oncolytic vaccinia virus with A52R and TK deletions also did not show detectable levels of m41BBL. HeLa cells infected with oncolytic vaccinia virus with TK deletions and native m41BBL inserted into the TK gene showed minimal expression, with 1.4 ng / mL detected. HeLa cells infected with oncolytic vaccinia virus with TK deletions and fusion construct 3s41BBL (mouse) containing an oligomerization domain inserted into the TK gene showed much higher levels of 41BBL expression, with approximately 1500 ng / mL of 41BBL detected.

[0134] Example 2: Expression of native and scaffold structures

[0135] The functional effect of modified oncolytic vaccinia virus expressing a fusion construct of TNFSF-L with an oligomerization domain was assayed. A SEAP reporter assay of CD40L activity was used, and the amount of SEAP production correlates with CD40L functional activity. Briefly, HeLa cells were infected with oncolytic vaccinia virus (without CD40L) or oncolytic vaccinia virus expressing fusion construct 3sCD40L (containing [oligomerization domain]-[neck domain]-[mCD40L]) at an MOI of 1. Supernatants from infected and non-infected cells were collected 24 hours later. In a 96-well plate, 20 μl, 10 μl or 1 μl of supernatant was added in triplicate to wells containing 20 μl of medium. Recombinant CD40L at a concentration of 10 ng / ml was used as a positive control. 180 μl of reporter cell line was added to the wells to reach approximately 50,000 cells per well. The cells were incubated overnight in a CO2 incubator. The next day, 1 ml of Quanti-Blue solution was added to 1 ml of QB buffer and 98 ml of sterile water, and the prepared reagent was dispensed into a 96-well plate at 180 μl / well. 20 μl of supernatant from the experimental plate was added to the plate containing Quanti Blue solution. After 3 hours of incubation at 37°C, the relative levels of SEAP were determined by measuring the optical density (OD) at 625 nM using a plate reader / spectrophotometer. The results are shown in Table 8 below and visualized in Figure 5. Table 8. [Table 8]

[0136] Unexpectedly, these results from both Examples 1 and 2 show that over 1000-fold more functionally active TNFSF-L is produced in the extracellular environment. Notably, the monomeric sequence did not produce functionally active trimers even in cell culture, and fusion to the scaffold domain resulted in a significant increase in production. Example 3: Analysis of in vivo activity of s3CD40L

[0137] The activity of the TNFSF-L fusion construct 3sCD40L (mouse) described in Example 1 in mice was measured. Balb / cCr mouse renal cortical adenocarcinoma-derived cells (RENCA) were subcutaneously transplanted into BALB / c mice. Tumors were grown to 50-100 mm 3 The cells were grown to 1 × 10 expressing the 3sCD40L fusion construct with a TK deletion (TK-). 7 Treatment was delivered intratumorally (IT) in a single dose. Tumor growth was followed until tumors reached 1000 mm 3 Mice were removed from the study if their blood pressure measured more than 1000 mm. 3 Overall survival in tumor-bearing mice up to 10 days is shown in Figure 6. The data show that mice treated with TNFSF-L fusions showed a higher probability of survival compared to mice treated with TK-virus or buffer control. Example 4: Functional activity of s3OX40L

[0138] The in vitro activity of the TNFSF-L fusion construct 3sOX40L (human) was measured as described in Example 1. HeLa cells were cultured at 1 × 10 6 Cells were seeded with 1000 cells / well. Cells were inoculated with a virus expressing the 3sOX40L fusion construct, a virus expressing m41BBL as described in Example 1 as a negative control or a buffer control at an MOI of 5.0. Cells were incubated with the virus for 3 hours at 37°C and 5% CO2. The medium was removed from the wells and the cells were washed with DMEM. DMEM+10% FBS was added and the plate was incubated for 60 hours at 37°C and 5% CO2. The supernatant was collected 60 hours after medium change. Luciferase expression in infected cells was measured using luciferase detection and expressed in relative light units (RLU). Figure 7 shows higher expression in the virus with the s3OX40L fusion construct. Example 5: Functional activity of 3s41BBL

[0139] The in vitro activity of the TNFSF-L fusion construct 3s41BBL (human sequence) was measured as described in Example 1. HeLa cells were cultured at 4 × 10 5 Cells were seeded at 1000 x 1000 cells / well. Cells were infected with virus expressing 3s41BBL fusion construct, virus expressing 41BBL monomer, irrelevant TNFSF-L, trimeric GITRL, or buffer control at an MOI of 5.0. Cells were incubated with virus for 48 hours at 37°C, 5% CO2. Supernatants were harvested 48 hours post-infection. Luciferase expression in infected cells was measured using luciferase detection and expressed in relative light units (RLU). Figure 8 shows that only the virus expressing fusion construct 3s41BBL, but not the virus expressing 41BBL monomer, is active. Example 6: IL-2 secretion after stimulation with mouse sequences s3OX40L and s3 41BBL

[0140] The change in IL-2 secretion in cells infected with viruses containing TNFSF-L fusion constructs was measured. CD8 T cells from mouse spleens were purified using the EasySep™ Mouse CD8+ T Cell Isolation Kit (#19853, STEMCELL Technologies, Inc., Vancouver, Canada). Cells were labeled with carboxyfluorescein succinimidyl ester (CFSE). Cells were then unstimulated, stimulated with CD3 alone, or stimulated with CD3 and CD28, recombinant 41BBL trimer, recombinant OX40L trimer, virus expressing 3s41BBL fusion construct, virus expressing 3sOX40L fusion construct, or virus negative control. Cells were incubated for 3 days. IL-2 secretion in the supernatant was analyzed using a mouse IL-2 ELISA kit. The resulting detected IL-2 levels are shown in Figure 9. The graph shows that virally expressed TNFSF-L fusion constructs 3s41BBL and 3sOX40L are functionally active in inducing IL-2 expression. Example 7: Tumor growth inhibition in mice using 3s41BBL and 3sOX40L scaffold constructs

[0141] To demonstrate tumor growth inhibition (TGI) in vivo, BALB / c mice in which subcutaneous RENCA tumors had previously been induced as in Example 3 were treated with a single IT dose of buffer control or 1×10 6 of a control vaccinia virus with thymidine kinase deletion only (HCCTKM). 7 PFU, virus expressing 3s41BBL or virus expressing 3sOX40L. Preliminary results show that by 12 days after treatment, the mean tumor volume in mice receiving either virus expressing 3s41BBL or 3sOX40L was lower than tumors treated with buffer alone or HCCTKM, as shown in Figure 10. Example 8: Tumor growth inhibition in mice using GITRL SCAFFOLD constructs

[0142] To demonstrate tumor growth inhibition (TGI) in vivo, BALB / c mice in which subcutaneous LLC or RENCA tumors had previously been induced as in Example 3 were treated with a single IT dose of buffer control (VFB), 1×10 6 of control vaccinia virus with thymidine kinase deletion only (HCCTKM), or 1×10 7 of vaccinia virus with thymidine kinase deletion only (HCCTKM). 7 PFU, or virus expressing 3sGITRL. LLC tumors were measured 30 days later, as shown in FIG. 11A. RENCA tumors were measured 17 days later, as shown in FIG. 11B. The results show that the mean tumor volume of mice receiving virus expressing 3sGITRL was lower than that of tumors treated with buffer alone or control HCCTKM.

[0143] The foregoing description and accompanying drawings set forth certain representative embodiments at the present time. Various modifications, additions, and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope of the invention, which is set forth by the following claims rather than the foregoing description. All changes and variations that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. A oncolytic virus, wherein the oncolytic virus contains an exogenous nucleic acid encoding a fusion protein, the fusion protein contains a TNF-superfamily ligand (TNFSF-L) or a functional variant thereof, and the TNFSF-L or a functional variant thereof is fused to an oligomerization domain derived from a collectin family protein. Oncolytic virus.

2. The oncolytic virus according to claim 1, wherein the fusion protein further contains a neck domain derived from a collectin family protein or a functional variant thereof, and the oligomerization domain and the neck domain have a closer sequence proximity compared to their positions in the collectin family protein.

3. The oncolytic virus according to claim 1, wherein the oncolytic virus is Newcastle disease virus (NDV), reovirus (RV), myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV), poliovirus (PV), Sendai virus, flavivirus, lentivirus, poxvirus, retrovirus, adeno-associated virus, or adenovirus.

4. The oncolytic virus according to claim 3, wherein the oncolytic virus is vaccinia virus.

5. The oncolytic virus according to claim 4, wherein the vaccinia virus is a modified strain of Western Reserve vaccinia virus (ATCC VR-1354), Copenhagen strain, vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), recombinant vaccinia virus Ankara (MVA), NYVAC strain, vaccinia virus strain Wyeth (ATCC VR-1536), vaccinia virus Wyeth (ATCC VR-325), Wyeth (NYCBOH) strain, Tian Tan strain, Lister strain, USSR strain, and Evans strain. **Claim 6**: The oncolytic virus according to claim 1, wherein the oncolytic virus contains a viral genome, and the nucleic acid encoding the TNF-SF-L or a functional variant thereof is inserted into the viral genome. **Claim 7** The oncolytic virus according to claim 4, wherein the nucleic acid encoding the TNF-SF-L or a functional variant thereof is inserted into the thymidine kinase gene of the viral genome. **Claim 8** The oncolytic virus according to claim 1, wherein the TNF-SF-L includes lymphotoxin α, OX40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor κ-β ligand (RANKL), TNF-related apoptosis weak inducer, A proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF), LIGHT, vascular endothelial growth inhibitor (VEGI), TNF superfamily member 18 ligand (GITRL), ectodysplasin A, or any combination thereof. **Claim 9** The oncolytic virus according to claim 6, wherein the CD40 ligand includes a sequence having at least 85% sequence identity with SEQ ID NO:

1. **Claim 10** The oncolytic virus according to claim 6, wherein the OX40 ligand includes a sequence having at least 85% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

3. **Claim 11** The oncolytic virus according to claim 6, wherein the 4-1BBL includes a sequence having at least 85% sequence identity with SEQ ID NO: 5 or SEQ ID NO:

4. **Claim 12** The oncolytic virus according to claim 6, wherein the LIGHT includes a sequence having at least 85% sequence identity with SEQ ID NO:

6. **Claim 13** The oncolytic virus according to claim 6, wherein the GITRL comprises a sequence having at least 85% sequence identity with SEQ ID NO: 8 or SEQ ID NO:

7.

14. The fusion protein, in the order from the N-terminus to the C-terminus, the oligomerization domain or a functional variant thereof, the neck domain or a functional variant thereof, optionally a linker sequence, and the TNF-SF-L or a functional variant thereof The oncolytic virus according to claim 2, comprising:

15. The oncolytic virus according to claim 1, wherein the oligomerization domain comprises a sequence having at least 85% sequence identity with SEQ ID NO:

9.

16. The oncolytic virus according to claim 12, wherein both the oligomerization domain and the neck domain comprise a sequence having at least 85% sequence identity with SEQ ID NO:

10.

17. The oncolytic virus according to claim 12, wherein the nucleic acid encoding both the oligomerization domain and the neck domain comprises SEQ ID NO:

11.

18. The oncolytic virus according to claim 13, wherein the fusion protein comprises the linker sequence, and the linker sequence comprises GSG (glycine-serine-glycine) (SEQ ID NO: 12).

19. The oncolytic virus according to claim 1, wherein the nucleic acid comprises RNA.

20. The oncolytic virus according to claim 1, wherein the nucleic acid comprises DNA.

21. A pharmaceutical composition for use in the treatment of cancer, comprising: the oncolytic virus according to any one of claims 1 to 20, and a pharmaceutically acceptable carrier A pharmaceutical composition comprising and characterized in that the pharmaceutical composition is administered to a subject in an amount sufficient for the treatment of cancer.

22. The pharmaceutical composition according to claim 21, wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, cholangiocarcinoma, squamous cell carcinoma of the head and neck, colorectal cancer, intestinal-type gastric cancer, squamous cell carcinoma of the cervix, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

23. The pharmaceutical composition according to claim 21, wherein the administration includes systemic administration or local administration.

24. The pharmaceutical composition according to claim 21, wherein the pharmaceutically acceptable carrier includes a buffer solution, an emulsion, a bioadhesive polymer, a gel, or any combination thereof.

25. The pharmaceutical composition according to claim 21, characterized in that the composition is administered at a dose of about 0.01 μg / dose to about 1 g / dose.

26. The pharmaceutical composition according to claim 21, wherein the oncolytic virus or vaccinia virus is administered at a dose of about 10 ３ to about 10 １２ PFU / dose.

Images