Recombinant vaccinia virus

Abstract

overview Provided are a genetically modified vaccinia virus, a pharmaceutical composition comprising the same, and a method of using the same to treat a subject in need thereof. The genetically modified vaccinia virus comprises a mutated viral sequence and a heterologous sequence. The mutated viral sequence is used for selective replication in tumor cells and / or activation of immune cells. The heterologous sequence encodes an immune costimulatory pathway activating molecule, an immunomodulatory factor gene, a truncated viral envelope gene, and / or a tumor suppressor. The heterologous sequence is stably integrated into the genome of the genetically modified vaccinia virus. The pharmaceutical composition comprises an effective amount of the genetically modified vaccinia virus and a pharmaceutically acceptable vehicle. The method of using the same to treat a subject in need thereof comprises administering the genetically modified vaccinia virus to the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 873,221, filed July 12, 2019, the entire contents of which are incorporated herein by reference. FIELD OF THEINVENTION

[0002] The present disclosure relates to recombinant vaccinia viruses for the treatment of cancer. 2. Background of the Invention

[0003] Cancer is a serious and dangerous disease. Nearly 5 million new cases of cancer occur in China every year, and the number of patients who die from cancer is close to 3 million. These figures are increasing year by year. This indicates that traditional cancer treatments, such as surgery, chemotherapy, and radiotherapy, are ineffective in saving most cancer patients. Therefore, cancer patients are in great need of more effective treatments. Although new cancer immunotherapy has made some progress, how to effectively treat patients with solid tumors remains one of the biggest challenges. In recent years, oncolytic viruses, a type of cancer immunotherapy, have attracted attention in the industry.

[0004] Oncolytic viruses are viruses that selectively infect or replicate in tumor cells, thereby targeting and killing tumor cells. In addition, oncolytic viruses can effectively provide the risk signals required to induce and amplify the host's anti-tumor immune response, thereby enabling the body's immune system to generate a strong and specific anti-tumor immune response.

[0005] Vaccinia virus (VV) is a double-stranded DNA virus, and its unique properties make it an advantageous choice among oncolytic viruses. 1) VV has demonstrated good safety in the process of preventing smallpox as an infectious disease vaccine, and its safety as an oncolytic virus is guaranteed. In addition, many clinical trials have confirmed that VV can be safely used as an oncolytic virus. 2) It rapidly replicates itself in cells and produces new virus particles in about 6 to 8 hours, so it can rapidly lyse infected cells. 3) The genome of VV is about 200 Kbp, and this large genome has a large capacity to carry foreign genes. 4) Vaccinia virus has a wide affinity for various types of tumors because it does not require a specific receptor to infect cells. 5) It can be administered in various ways, such as local intratumoral injection, intraperitoneal injection, intrathoracic injection, and systemic intravenous injection. This diversity of administration methods makes it possible to treat tumors in any part of the body. 6) In addition, the hypoxic microenvironment commonly found in solid tumors negatively impacts the replication and efficacy of many types of oncolytic viruses. However, VVs can replicate efficiently even in a hypoxic environment (Hiley et al, Gene Therapy 17, 281-287). (2010).7) There are a variety of promoters, both natural and synthetic, that can be applied to VVs and are suitable for carrying transgenes. In recent years, clinical results using vaccinia virus have demonstrated that VVs have good antitumor effects and are safe (Haddad et al, Annals of Surgical Oncology 19 Suppl 3, S665-674 (2012); Park et al, Lancet Oncol 9: 533-542, (2008); Breitbach et al, Nature 477: 99-102, (2011)).

[0006] Various deletion mutants of vaccinia virus have been reported. The Western Reserve strain mutant, which has deletions of the thymidine kinase (TK) gene and viral growth factor (VGF) gene, can efficiently elicit immune responses against tumor antigens (McCart et al, Cancer Res 61, 8751-8757 (2001)). In addition, viruses with heterologous genes inserted, such as genes encoding cytokines, can further activate antitumor immune responses.

[0007] After infecting tumor cells, VV is released by lysis of the infected cells, allowing it to infect tumor cells where the virus is localized or to infect tumor cells distant from the site of infection via the circulating blood. Vaccinia virus has two types of infectious virus particle forms, mainly intracellular mature virus (IMV) and extracellular enveloped virus (EEV) (Appleyard et al., J. Gen.Virology 13, 9-17 (1971)). IMV is the major infectious form of virus particles. Because EEV is enveloped by the host cell membrane, it can antagonize the innate (complement) and adaptive (neutralizing) immune attacks of the host system, and VV spreads widely and long distances within the host (Smith, GL & Vanderplasschen, A and Law, M J. Gen.Virol.83, 2915-2931 (2002); Payne, LG & Kristensson, K, J. Gen.Virol.66 (c), 643-646 (1985)). However, most VV strains produce only small amounts of EEV (less than 1% of the total infectious virus).

[0008] VV has genes encoding proteins specific to EEV. They are A33R, A34R, A36R, F13L, B5R and A56R. The B5R gene encodes a 42 kDa glycoprotein containing four copies of a 50-70 amino acid repeat called a short consensus repeat (SCR). Deletion of the B5R gene results in smaller plaques and a significant reduction (≦10-fold) in EEV formation (Blasco, R. & Moss, B., J. Virol. 65, 5910-5920 (1991); Engelstad, M. & Smith, GL, Virology 194, 627-637 (1993)). The transmembrane and cytoplasmic tail sequences of B5R are important for targeting packaging cells of viral proteins (Katz et al, J. Virol. 71, 3178-3187 (1997)). VVs carrying B5R gene mutants lacking SCR4, SCR3, 4 or SCR2, 3, 4 produce smaller plaques but produce infectious EEVs several tens of times higher than wild-type virus, forming a comet-tail plaque distribution (Sanderson et al., J. Gen Virol. 79 (f), 1415-1425 (1998); Mathew et al., J. Virol. 72, 2429-2438 (1998)).

[0009] Although there have been advances in the field of oncolytic virus research in the past decade, no vaccinia virus-based therapeutics have yet been introduced into the clinic, and the challenge remains how to scientifically modify vaccinia virus to enhance its antitumor effects, so there is an urgent need to develop better oncolytic vaccinia virus formulations to meet the needs of cancer treatment.

[0010] There are various vaccinia strains with different degrees of toxicity to humans and animals. Many different strains were used around the world in the smallpox eradication program in the 1970s. For example, the New York City Department of Health (NYCBOH) strain and its derivative, the Wyeth strain, are predominant in the United States, while the Copenhagen (CPN) and Lister strains are predominant in Europe. Previous studies have shown that the Lister strain has excellent antitumor effects. In this project, the Lister strain was selected for development as an oncolytic virus. Summary of the Invention

[0011] The present disclosure relates to oncolytic viruses, and more particularly to recombinant oncolytic vaccinia viruses. The recombinant oncolytic vaccinia viruses of the present disclosure can selectively infect cancer cells and can be beneficially used in the treatment of cancer.

[0012] The present disclosure provides a recombinant vaccinia virus comprising a mutated viral sequence and a heterologous sequence, wherein the mutated viral sequence promotes selective replication in tumor cells and / or activation of immune cells, and the heterologous ... / or activation of immune co-stimulatory pathway activating molecules, immunomodulatory molecules, factor Genes, truncated viral envelope genes, and / or tumor suppressors factor and the heterologous sequence is stably integrated into the genome of the recombinant vaccinia virus.

[0013] In recombinant vaccinia virus embodiments, the heterologous sequence is stably integrated into the mutant viral sequence of the recombinant vaccinia virus.

[0014] In one embodiment of the recombinant vaccinia virus, the mutated viral sequence comprises at least one of the following:

[0015] (a) mutations in L025, TK, A46R, or any combination thereof;

[0016] (b) partial deletions L025, TK, A46R, or any combination thereof;

[0017] (c) deleted L025, TK, A46R, or any combination thereof;

[0018] (d) Part or all of L025, TK, or A46R is Replaced by one of the sequences set forth in sequence numbers 2, 4, 6, 8, 10, 12, or 14. attitude R ;

[0019] (e) Part or all of L025, TK, or A46R, Replaced by tumor target genes attitude R ;

[0020] (f) Part or all of L025, TK, or A46R, Displaced by T cell-targeting ligands or antibodies attitude R ;

[0021] (g) Part or all of L025, TK, or A46R, Replaced by a therapeutic gene or a variant thereof attitude R ; or

[0022] (h) Part or all of L025, TK, or A46R, Replaced by therapeutic antibodies attitude R 。

[0023] In one embodiment of the recombinant vaccinia virus, the heterologous sequence comprises at least one of the following:

[0024] (a) the sequence set forth in SEQ ID NO:2;

[0025] (b) the sequence set forth in SEQ ID NO:4;

[0026] (c) the sequence set forth in SEQ ID NO:6;

[0027] (d) the sequence set forth in SEQ ID NO:8;

[0028] (e) the sequence set forth in SEQ ID NO:10;

[0029] (f) the sequence set forth in SEQ ID NO:12; or

[0030] (g) The sequence set forth in SEQ ID NO:14.

[0031] In one embodiment of the recombinant vaccinia virus, the heterologous sequence encodes at least one of the following:

[0032] (a) the sequence set forth in SEQ ID NO:1;

[0033] (b) the sequence set forth in SEQ ID NO:3;

[0034] (c) the sequence set forth in SEQ ID NO:5;

[0035] (d) the sequence set forth in SEQ ID NO:7;

[0036] (e) the sequence set forth in SEQ ID NO:9;

[0037] (f) the sequence set forth in SEQ ID NO:11; or

[0038] (g) The sequence set forth in SEQ ID NO:13.

[0039] In one embodiment of the recombinant vaccinia virus, the recombinant vaccinia virus comprises a sequence of the following formula: 5'-A1-X-A2-B1-Y-B2-C1-Z-C2-3' (In the formula, A1 and A2 are respectively The left arm of the first viral gene and right arm And B1 and B2 teeth each The left arm of the second viral gene and right arm and 、C 1 and C2 are each The left arm of the third viral gene and right arm and C1 and C2 are the left arms of the third viral gene, where X, Y and Z teeth,Each is selected from one of an immunomodulatory gene, a cytokine, a therapeutic gene, a truncated viral envelope gene, a tumor suppressor gene, a gene encoding a therapeutic antibody, and a gene encoding a ligand of a therapeutic antibody. It is a foreign gene .)

[0040] In one embodiment of the genetically modified vaccinia virus, the first viral gene is L025, the second viral gene is TK, and the third viral gene is A46R.

[0041] In one embodiment of the genetically modified vaccinia virus, X is a hybrid gene of IL-21 and modified B5R, Y is 4-1BBL, and Z is HIC1.

[0042] In one embodiment of the recombinant vaccinia virus, the mutant viral sequence comprises deletion mutations at L025, TK and A46R, and the heterologous sequence comprises IL-21 and 4-1BBL.

[0043] In an embodiment of the recombinant vaccinia virus, the mutant viral sequence comprises deletion mutations at L025, TK and A46R, and the heterologous sequence comprises IL-21 and modified B5R. Hybrid genes with , and 4-1BB L include.

[0044] In one embodiment of the genetically modified vaccinia virus, the immunomodulatory gene is IL-12, IL-21, IL-2, IL-15, IL-8, or a modification thereof. body It is a cytokine gene that encodes.

[0045] In one embodiment of the genetically modified vaccinia virus, the immune costimulatory pathway activating molecule is CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27, Flt3 ligand, or modifications thereof. body It consists of:

[0046] In one embodiment of the genetically modified vaccinia virus, the tumor suppressor gene is HIC1.

[0047] In one embodiment of the recombinant vaccinia virus, the recombinant vaccinia virus is selected from the group consisting of Lister, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA 382, ​​Modified Vaccinia Virus Ankara (MVA), Dairen I, LC16m8, LC16M0, LIVP, ACAM 2000, WR65-16, Connaught, New York City Department of Health (NYCBH), EM-63 and NYVAC strains.

[0048] In one embodiment of the genetically modified vaccinia virus, the truncated viral envelope gene is B5R, which contains a deletion of the short consensus repeat (SCR)2, SCR3, and SCR4 domains.

[0049] In one embodiment of the genetically modified vaccinia virus, the tumor suppressor is HIC1.

[0050] The present disclosure further provides a pharmaceutical composition comprising an effective amount of the genetically modified vaccinia virus of any one of the embodiments of the present disclosure and a pharma- ceutically acceptable carrier.

[0051] In one embodiment of the pharmaceutical composition, the pharmaceutical composition is formulated for oral, topical, parenteral delivery, or interventional therapy.

[0052] In one embodiment of the pharmaceutical composition, the pharmaceutical composition is formulated for local intratumoral, intraperitoneal, intrathoracic, systemic intravenous, intramuscular, subcutaneous, intrathecal, direct intraventricular, intracardiac, or intranasal injection.

[0053] In one embodiment of the pharmaceutical composition, the genetically modified vaccinia virus is used as a monotherapy; or in combination with one or more anti-cancer agents, immunosuppressants, and / or oncolytic virus enhancing agents.

[0054] In one embodiment of the pharmaceutical composition, the recombinant vaccinia virus is used in combination with 5-fluorouracil (FU), folinic acid (FA), methotrexate, capecitabine, oxaliplatin, bevacizumab, cetuximab, immune checkpoint inhibitors (such as anti-PD1, anti-PDL1, anti-CTLA4), other types of oncolytic viruses, pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, adenovirus, or combinations thereof.

[0055] The present disclosure provides a method for use in treating cancer in a subject in need thereof, the method further comprising administering to the subject an effective amount of a genetically modified vaccinia virus described in any one of the embodiments of the present disclosure or a pharmaceutical composition described in any one of the embodiments of the present disclosure.

[0056] In one embodiment of this method, HIC1 is inactivated, underexpressed, or lost in the cancer.

[0057] In one embodiment of this method, the cancer is selected from the group consisting of lung cancer, melanoma, pancreatic cancer, liver cancer, colon cancer, breast cancer, glioblastoma, sarcoma, gastric cancer, ovarian cancer, mesothelioma, and leukemia.

[0058] A method for increasing tumor-specific infectivity of a vaccinia virus, comprising administering to a subject a genetically modified vaccinia virus described in any one of the embodiments of the present disclosure, wherein the genetically modified vaccinia virus is administered in an amount effective to elicit an anti-tumor immune response in the subject.

[0059] A method of conferring sustained immunity against tumor recurrence in a subject in need thereof, comprising administering to the subject a genetically modified vaccinia virus of any one of the embodiments of the present disclosure, comprising administering to the subject a genetically modified vaccinia virus of any one of the embodiments of the present disclosure.

[0060] A method for screening a patient based on the percentage of GFP positive cells 48 hours after infection of the patient's cancer cells with a genetically modified vaccinia virus according to any one of the embodiments of the present disclosure.

[0061] A genetically modified vaccinia virus for use in inducing cancer cell death, modulating the biological activity of cancer cells, modulating the immune response, enhancing T cell proliferation, and / or T cell cytotoxicity, comprising a recombinant tumor solubility 5. A recombinant vaccinia virus as provided in any one of the embodiments of the present disclosure.

[0062] A genetically modified vaccinia virus for use in inducing cancer cell death, modulating the biological activity of cancer cells, modulating an immune response, enhancing T cell proliferation, and / or enhancing T cell cytotoxicity, wherein the biological activity of cancer cells comprises inhibiting the replication of cancer cells, inhibiting the division of cancer cells, inhibiting DNA repair of cancer cells, inhibiting the proliferation ... Yusou Inhibition of, or cancer cell Promoting death of a recombinant tumor solubility is provided in any one of the embodiments of the present disclosure. It is as follows , genetically engineered vaccinia virus.

[0063] A genetically modified vaccinia virus for use in the manufacture of a medicament for treating lung cancer, melanoma, pancreatic cancer, liver cancer, colon cancer, breast cancer, glioblastoma, sarcoma, gastric cancer, ovarian cancer, mesothelioma, and leukemia, comprising a recombinant tumor solubility As provided in any one of the embodiments of the present disclosure, a genetically modified vaccinia virus.

[0064] A recombinant vaccinia virus for use in the manufacture of a medicament for inhibiting the proliferation of cancer cells, inducing the death of cancer cells, and / or modulating the biological activity of cancer cells, comprising a recombinant oncolytic sex However, any one of the embodiments of the present disclosure Offered be provided It is as follows , genetically engineered vaccinia virus. [Brief description of the drawings]

[0065] In order to more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments are briefly introduced below. Note that the accompanying drawings in the following description are only a part of the embodiments of the present invention. Based on the accompanying drawings, a person skilled in the art can obtain other drawings without making creative efforts.

[0066] FIG. 1A is a schematic diagram of a product carrying mB5R.

[0067] FIG. 1B is a schematic diagram of a product carrying sB5R.

[0068] FIG. 2A is a schematic diagram of a shuttle vector carrying the TK gene-deficient mouse 4-1BBL.

[0069] FIG. 2B is a schematic diagram of a shuttle vector carrying TK gene-deleted human 4-1BBL.

[0070] FIG. 3A shows a shuttle vector carrying mouse IL-21 and modified B5R with L025 gene deletion (mB5R / sB5R).

[0071] FIG. 3B shows the shuttle vector carrying human IL-21 and modified B5R with the L025 gene deletion (mB5R / sB5R).

[0072] FIG. 4 shows a shuttle vector carrying the A46R gene-deleted HIC1 gene.

[0073] FIG. 5 is a diagram showing the structure of the mB5R / sB5R gene.

[0074] FIG. 6 shows the recombinant virus carrying the L025 gene deletion.

[0075] FIG. 7 shows the recombinant virus in which the TK gene was deleted and the hSPD-41BBL gene was inserted.

[0076] FIG. 8 shows a recombinant virus in which the A46R gene was deleted and the inserted gene HIC1 was expressed by the virus.

[0077] 9A-9B show killing of a panel of solid tumor cell lines by KM1 with increasing doses of virus over a 2-day period in 6-well plates. The amount of virus used to infect individual wells was 0.01 pfu / cell, 0.1 pfu / cell, and 1 pfu / cell. At the end of the experiment, the remaining cells in the wells were stained with crystal violet.

[0078] Figures 10A-10C are examples of EC50 assays in 17 cell lines.

[0079] FIG. 11 shows EC50 values ​​for different cell lines.

[0080] FIG. 12 shows the killing of leukemia cell lines by vaccinia virus and is an example of an EC50 assay in two cell lines.

[0081] FIG. 13 shows an example of leukocytes from a leukemia patient infected with KM1.

[0082] FIG. 14 shows live cells / dead cells 3 days after infection with KM1.

[0083] FIG. 15 shows the antitumor effect of KM1 in a Syrian hamster pancreatic cancer HPD 1NR model.

[0084] FIG. 16 shows the treatment of human breast cancer cells MDA MB-231 with HIC1-armed vaccinia virus and its corresponding control HY3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] With reference to the accompanying drawings, in which like reference numerals represent like components, the following description is based on specific embodiments of the present disclosure as illustrated and should not be construed as a limitation on other embodiments of the present application not described in detail herein.

[0086] Please refer to Figures 1A-1B. Figure 1A is a schematic diagram of a product carrying mB5R, and Figure 1B is a schematic diagram of a product carrying sB5R.

[0087] The vaccinia viruses and viral vectors of the present disclosure comprise sequences of the following formula: 5'-A1-X-A2-B1-Y-B2-C1-Z-C2-3' where A1 and A2 are the left and right arms, respectively, of a first viral gene, B1 and B2 are the left and right arms, respectively, of a second viral gene, C1 and C2 are the left and right arms, respectively, of a third viral gene, where X, Y, and Z are , exemption Immunoregulatory genes, cytokines, therapeutic genes, and therapeutic antibodies, Therapeutic antibodies ligands). Exemplary structures of the resulting products are shown in Figures 1A and 1B. The first viral gene is L025, the second viral gene is TK, and the third viral gene is A46R. X is a hybrid gene of IL-21 and modified B5R, Y is 4-1BBL, and Z is HIC1.

[0088] The gaps between "A1-X-A2" and "B1-Y-B2" and between "B1-Y-B2" and "C1-Z-C2" can contain other viral genes known from vaccinia virus.

[0089] Product design and construction:

[0090] 1. Deletion of the TK gene and integration of the 4-1BBL gene into the TK gene site

[0091] Please refer to Figures 2A-2B. Figure 2A is a schematic diagram of a shuttle vector carrying TK gene-deficient mouse 4-1BBL. Figure 2B is a schematic diagram of a shuttle vector carrying TK gene-deficient human 4-1BBL.

[0092] In Figure 2A, the left arm of TK targets the left side of the TK gene (L089), and the right arm of TK targets the right side of the TK gene (L091). The Loxp site is flanked by the H5 promoter and red fluorescent protein RFP, and is located between the TK left arm and the promoter H5 of the mouse 4-1BBL gene.

[0093] In Figure 2B, the left arm of TK targets the left side of the TK gene (L089), and the right arm of TK targets the right side of the TK gene (L091). The Loxp site is flanked by the H5 promoter and red fluorescent protein RFP, and is located between the left arm of TK and the promoter H5 of the human 4-1BBL gene.

[0094] The expression cassette is designed as follows (see schematic diagrams in Figures 2A and 2B).

[0095] TK Left Arm -- loxp-H5-RFP-loxp-H5-m / h4-1BBL-TK Right Arm

[0096] To delete two or more genes with the same reporter protein (RFP), it is important to delete the reporter gene using an appropriate homologous recombination system, such as Cre-Lox, or the Flp / FRT system. Gene insertion elements can be added to the expression cassette around the nucleic acid sequence encoding the protein and upstream of the promoter region that drives the expression of the protein.

[0097] The Loxp sites are located upstream of H5 and downstream of RFP, respectively, and C5 recombinase can be used to excise H5-RFP in new recombinant viruses by acting on the FRET sites.

[0098] The H5 promoter drives expression of the 4-1BBL gene. The amino acid sequence of SPD-m4-1BBL is set forth in SEQ ID NO:5, and the nucleotide sequence of SPD-m4-1BBL of SPD-m4-1BBL is set forth in SEQ ID NO:6. The amino acid sequence of SPD-h4-1BBL is set forth in SEQ ID NO:7, and the nucleotide sequence of SPD-h4-1BBL of SPD-h4-1BBL is set forth in SEQ ID NO:8.

[0099] To perform targeted homologous recombination at the TK gene (L090) site of vaccinia virus, the recombination region used to prepare the vector of the present disclosure must additionally be provided with sequences complementary to the TK gene and / or genes adjacent to the TK gene. The TK left arm (L-arm) targets the left side of the TK gene (L089), and the TK right arm (R-arm) targets the right side of the TK gene (L091). The actual expression cassettes containing the 4-1BBL gene and the reporter gene RFP can then be placed between the TK-L arm and the TK-R arm, and inserted into the targeted TK region of vaccinia virus. The expression cassettes can be inserted into the recombination vector before transforming the vaccinia virus vector.

[0100] 2. The L025 gene is deleted and interleukin 21 (IL-21) and mB5R (or sB5R) are integrated into the L025 region.

[0101] Please refer to Figures 3A-3B. Figure 3A is a diagram showing a shuttle vector carrying mouse IL-21 and modified B5R with the L025 gene deleted (mB5R / sB5R). Figure 3B is a diagram showing a shuttle vector carrying human IL-21 and modified B5R with the L025 gene deleted (mB5R / sB5R).

[0102] In Figure 3A, the left arm of L025 targets the left side of the L025 gene (L024), and the right arm of L025 targets the right side of the L025 gene (L026). The FRT sites are located on either side of the H5 promoter and red fluorescent protein RFP, between the left arm of L025 and the right arm of L025.

[0103] In Figure 3B, the left arm of L025 targets the left side of the L025 gene (L024), and the right arm of L025 targets the right side of the L025 gene (L026). The FRT sites are located on either side of the H5 promoter and red fluorescent protein RFP, between the left arm of L025 and the right arm of L025.

[0104] The expression cassette was designed as follows (schematic diagrams are shown in Figs. 3A and 3B).

[0105] L025 Left Arm - FRT-H5 - RFP - FRT-H5-m / s B5R-H5-m / h IL-21 - L025 Right Arm

[0106] Red fluorescent protein (RFP) is used as a reporter protein to facilitate screening of new recombinant viruses. A promoter is provided upstream of the reporter protein to drive the expression of the reporter protein, here we use the H5 promoter. FRT is the recognition site for Flipase, which can excise RFP.

[0107] The amino acid sequence of mouse IL-21 is set forth in SEQ ID NO: 9, and the nucleotide sequence of mouse IL-21 is set forth in SEQ ID NO: 10. The amino acid sequence of human IL-21 is set forth in SEQ ID NO: 11, and the nucleotide sequence of human IL-21 is set forth in SEQ ID NO: 12.

[0108] Please refer to Figure 5. Figure 5 shows the structure of the mB5R / sB5R gene. mB5R is composed of a signal peptide, a stalk, a transmembrane region (TM), and an intracellular tail segment (CT) gene fragment, while sB5R is composed of a signal peptide, a short consensus repeat 1 (SCR1), a stalk, a transmembrane region (TM), and an intracellular tail segment (CT) gene fragment.

[0109] The H5 promoter drives expression of the immunotherapeutic gene IL21 and expression of mB5R or sB5R. For a schematic structural diagram of mB5R and sB5R, see Figure 5. The amino acid sequence of mB5R is set forth in SEQ ID NO:1, and the nucleotide sequence of mB5R is set forth in SEQ ID NO:2. The amino acid sequence of sB5R is set forth in SEQ ID NO:3, and the nucleotide sequence of sB5R is set forth in SEQ ID NO:4.

[0110] The recombination region needs to be inserted into the L025 gene of vaccinia virus, where the insertion is performed using the method of homologous recombination, and the integration site is between the left and right arms of L025.

[0111] In order to perform targeted homologous recombination at the L025 gene site of vaccinia virus, it is necessary to provide additional sequences complementary to the L025 gene and / or genes adjacent to the L025 gene in the recombination region used to prepare the vector of the present disclosure. The L025 left arm (L-arm) targeted the left side of the L025 gene (L024), and the L025 right arm (R-arm) targeted the right side of the L025 gene (L026). The actual expression cassettes containing the HIC1 gene and the reporter gene GFP can then be placed between the L025-L arm and the L025-R arm and inserted into the targeted L025 region of vaccinia virus. The expression cassettes can be inserted into the recombination vector before transforming the vaccinia virus vector.

[0112] 3. The HIC1 gene is inserted into the A46R region.

[0113] Please refer to Figure 4. Figure 4 shows the shuttle vector carrying the HIC1 gene with A46R gene deletion. The left arm of A46R targets the left side of the A46R gene (L163) and the right arm of A46R targets the right side of the A46R gene (L165). The H5 promoter drives the expression of the green fluorescent protein GFP. The H5 promoter drives the expression of the HIC1 gene.

[0114] The expression cassette is designed as follows (see Figure 4 for a schematic):

[0115] A46R left arm-H5-GFP-H5-HIC1-A46R right arm

[0116] In order to perform targeted homologous recombination at the A46R gene (L164) site of vaccinia virus, it is necessary to additionally provide sequences complementary to the A46R gene and / or genes adjacent to the A46R gene for the recombination region used to prepare the vector of the present disclosure. The A46R left arm (L-arm) targets the left side of the A46R gene (L163), and the A46R right arm (R-arm) targets the right side of the A46R gene (L165). The actual expression cassettes containing the HIC1 gene and the reporter gene GFP can then be placed between the A46R-L arm and the A46R-R arm and inserted into the target A46R region of vaccinia virus. The expression cassettes can be inserted into the recombination vector before transforming the vaccinia virus vector.

[0117] Materials and Methods

[0118] Cell lines: All tumor cell lines used were from ATCC. All human cancer cell lines were genotyped by STR assay. The murine tumor cell lines used in this study are as follows: colon cancer cell line MC38 was derived from C57B / 6 mice. CV1 is an African green monkey "normal" kidney cell line obtained from ATCC, Virginia, USA, and used as a stock cell line to facilitate large-scale production of virus and all virus titration assays.

[0119] Virus: Wild-type VV Lister is identified under ATCC accession number VR-1549. TM It is commercially available from the American Type Culture Collection (ATCC) at 10:00 am-7:00 pm. Wild-type VV Lister has also been deposited at the China Type Culture Collection (CCTCC) under CTCCC accession number: V201937. The date of deposit was July 4, 2019, and the strain designation is Lister vaccinia virus 01 (VV 01). ATCC VR-1549 TM Further information is also available from the product sheet. The recombinant VV Lister has been deposited with the CCTCC under CCTCC accession number V201938. The date of deposit was July 4, 2019, and the designated strain is vaccinia virus Lister KM1 strain. Both wild-type VV Lister and recombinant VV Lister have been deposited under the Budapest Treaty. The wild-type VV Lister and recombinant VV Lister have been deposited at the China Center for Typical Culture Preservation (CCTCC), School of Life Sciences, Wuhan University, 430072, China. The depositor of the wild-type VV Lister and recombinant VV Lister is Shenzhen Hua Yao Kang Ming Biopharmaceutical Co., Ltd. c al Co. Ltd., 14 Jinhui road, Pingshan District, Shenzhen, China (the Applicant).

[0120] Construction of TK shuttle vector:

[0121] The TK shuttle vector contains a left arm (L089) that targets the TK gene and a right arm (L091) that targets the right side of the TK gene. The Loxp site is flanked by the H5 promoter and red fluorescent protein RFP, and is located between the TK left arm and the promoter H5 of the 4-1BBL gene. All of the above sequences were compiled by us, synthesized by a certain company, and cloned into the PUC57 vector.

[0122] Construction of the L025 shuttle vector: A schematic diagram of the L025 shuttle vector is shown in Figures 3A and 3B.

[0123] The L025 shuttle vector contains the L025 left arm (L024) that targets the left side of the L025 gene and the L025 right arm (L026) that targets the right side of the L025 gene. The FRT sites are located on both sides of the H5 promoter and the red fluorescent protein RFP, and are located between the left arm of L025 and the promoter H5 of the mB5R / sB5R gene. All of the above sequences were spliced ​​and synthesized by our company, and cloned into the PUC57 vector.

[0124] Construction of the A46R shuttle vector:

[0125] A schematic diagram of the A46R shuttle vector is shown in Figure 4. The A46R shuttle vector contains the A46R left arm (L163) that targets the left side of the A46R gene and the A46R right arm (L165) that targets the right side of the A46R gene. The H5 promoter drives the expression of the green fluorescent protein GFP. The H5 promoter drives the expression of the HIC1 gene. All of the above sequences were compiled by our company, synthesized by a certain company, and cloned into the PUC57 vector.

[0126] Obtaining the mB5R gene:

[0127] mB5R is composed of a signal peptide, a stalk, a transmembrane (TM), and a intracellular tail (CT) gene segments. The H5 promoter drives the expression of the sB5R gene.

[0128] Obtaining the sB5R gene:

[0129] sB5R is composed of signal peptide, SCR1, stalk, transmembrane (TM), and intracellular tail (CT) gene segments.

[0130] Cas9-mediated homologous recombination:

[0131] One day before transfection, 3 × 10 5 CV-1 cells were seeded in one well of a 6-well plate. gRNA vectors (L025gRNA targeting the L025 region, TKgRNA targeting the TK region, and A46RgRNA targeting the A46R region) were co-transfected with Cas9 and introduced into CV-1 cells in the 6-well plate. The next day, the wells transfected with the gRNA vector and Cas9 gene were infected with 0.01 PFU / cell of the backbone virus. The shuttle vector for homologous recombination was transfected into the infected well 2 hours after virus infection. After 24 hours, the cells were harvested and frozen at -80°C for purification of the recombinant virus.

[0132] Purification of recombinant viruses:

[0133] The cell lysate recovered by Cas9-mediated homologous recombination was thawed, and 0.5 μl of this lysate was used to infect all wells of a 6-well plate containing CV1 cells grown to 80-90% confluence. 48 hours after infection, each well was observed under a fluorescent microscope to see if there was any virus that emitted red or green fluorescence. After confirming the positive infection spots, the underside of the plate was marked with a marker. Next, after aspirating the medium from the wells in a fume hood for culturing cells, plaques were carefully selected with a 20 μl tip. The tip was then immersed in a cryotube containing 200 μl of cell culture medium. After one freeze-thaw cycle, 5-20 μl of this virus solution was added to each well of a new 6-well plate containing CV1 cells. This process was repeated until each plaque expressed red or green fluorescence, i.e., until all plaques were formed by the recombinant virus. Usually, 3-5 rounds of plaque purification are required to obtain a pure recombinant virus. After confirming that the virus was purified, the infected cells were scraped off and centrifuged to obtain a cell pellet. Some of the cells were then harvested and viral DNA was extracted. The target gene was amplified by PCR from the extracted viral DNA to confirm the purity of the virus.

[0134] Viral amplification

[0135] Once the desired recombinant virus was confirmed, 50 μl of the virus lysate was added to a T175 flask containing CV1 cells and grown to 80-90% confluence in approximately 30 ml of cell culture medium. After 48 hours, the cells and medium were scraped off and the "primary virus amplification" was saved.

[0136] Validation of recombinant viruses with deleted L025 genes

[0137] CV-1 cells were infected with the purified virus. Two days after infection, infected cells were harvested and VV DNA was extracted. To confirm the deletion of the L025 gene, a DNA fragment spanning the L025 and L026 genes was amplified by PCR using a forward primer (SEQ ID NO: 15: 5'-TATCTAGCAATGACCGT-3') (within the L025 gene) and a reverse primer (SEQ ID NO: 16: 5'-CCGAAGGTAGTAGCATGGA-3') (within the L026 gene). Amplification of a control gene A DNA fragment spanning the A46R and A47L genes was obtained by PCR amplification using a forward primer (SEQ ID NO: 17: 5'-TTGGCTATTAAACAGTATGGA-3') and a reverse primer (SEQ ID NO: 18: 5'-GATCCCGATAACAAATG-3'). The PCR products were analyzed by 1% agarose gel electrophoresis.

[0138] Validation of recombinant viruses expressing mB5R or sB5R genes:

[0139] To verify the incorporation of the mB5R gene into the recombinant virus, mB5R was amplified by PCR using the forward primer (SEQ ID NO: 19: 5'-ATGAAAACGATTCCGTTTTTTACGT-3') and reverse primer (SEQ ID NO: 20: 5'-TCACGGT AGCAATTTATGGAACTT-3').

[0140] To verify the integration of the sB5R gene into the recombinant virus, the sB5R gene was amplified by PCR using the forward primer (SEQ ID NO: 21: 5'-ATGAAAACGATTTCCGTTGTTACGT-3') and reverse primer (SEQ ID NO: 22: 5'-TCACGGTAGCAATTTATGGAACTT-3').

[0141] Excision of RFP using Flp recombinase:

[0142] pCAG-Flpe (from Addgene) was transfected into one well of a 6-well plate of CV-1 cells. CV-1 cells were infected with 100–200 recombinant viruses Flp-RFP VV 24 h after transfection of pCAG-Flpe. After 2 days, RFP-negative plaques were picked and used to infect CV-1 cells in a 6-well plate to purify RFP-negative plaques. RFP-negative plaques were then picked every 2 days and infected into CV-1 cells until no RFP-positive plaques were observed under a fluorescent microscope.

[0143] Validation of recombinant viruses with deleted TK gene:

[0144] CV-1 cells were infected with purified virus. Two days after infection, infected cells were harvested and VV DNA was extracted. To verify the deletion of the TK gene, the gene was amplified by PCR using a forward primer (SEQ ID NO:23: 5'-GTTATAGTAGCCGCACTCGA-3') (within the TK gene) and a reverse primer (SEQ ID NO:24: 5'-ATTTCAGCTGAATGAAGGA-3') (within the L091 gene). A DNA fragment spanning the TK and L091 genes: Amplification of a control gene A DNA fragment spanning the A46R and A47L genes was obtained by PCR amplification using a forward primer (SEQ ID NO:17: 5'-TTGGCTATTAAACAGTATGGA-3') and a reverse primer (SEQ ID NO:18: 5'-GATCCCGATAACAAATG-3'). The PCR products were analyzed by 1% agarose gel electrophoresis. The PD1 antibody gene was confirmed by amplifying its gene fragment by PCR. The primers used were: (SEQ ID NO:25: 5'-TCATAAATACCCGAGCCACC-3') and (SEQ ID NO:26: 5'-ACCCATTCAAGACCCTTTCC-3').

[0145] Excision of RFP using Cre recombinase:

[0146] pCAG-Cre (from Addgene) was transfected into CV-1 cells in one well of a 6-well plate. CV-1 cells were infected with 100-200 Cre-RFP viruses 24 hours after transfection of pCAG-Cre. After 2 days, RFP-negative plaques were selected and used to infect CV-1 cells in a 6-well plate and purify RFP-negative plaques. RFP-negative plaques were then picked every 2 days and infected into CV-1 cells until no RFP-positive plaques were observed under a fluorescent microscope.

[0147] Validation of the recombinant virus with the A46R gene deleted:

[0148] CV-1 cells were infected with purified virus. Two days after infection, infected cells were harvested and VV DNA was extracted. To verify the deletion of the A46R gene, the gene was amplified by PCR using a forward primer (SEQ ID NO:17: 5'-TTGGCTATTAAACAGTATGGA-3') (within the A46R gene) and a reverse primer (SEQ ID NO:18: 5'-GGATCCCGATAACAAATG-3') (within the A47R gene). For the DNA fragment spanning the A46R and A47R genes: control gene, a DNA fragment of the L09 gene was amplified by PCR using a forward primer (SEQ ID NO:27: 5'-TGTTGTTCGCTGCTATGA-3') and a reverse primer (SEQ ID NO:28: 5'-TGGCACAACCATATCTTGTA-3'). PCR products were analyzed by 1% agarose gel electrophoresis. The expression of HIC1 was detected by extracting proteins from cells infected with the recombinant virus and performing Western blotting.

[0149] Enzyme-linked immunosorbent assay

[0150] Expression of mIL-21 and hIL-21 was detected by enzyme-linked immunosorbent assay ELISA according to the reagent manufacturer's instructions.

[0151] Large-scale virus production:

[0152] The primary viral amplification from above was flash frozen, thawed once, and diluted to the volume required for cell culture to infect 36 T175 flasks containing CV1 cells (80-90% confluence). After 48 h, the infected CV1 cells were scraped and harvested by repeated centrifugation at 2,000 rpm (4 °C) several times. The precipitate was washed with PBS, resuspended in 12 ml of 10 mM Tris-HCl (pH 9) buffer, and stored at -80 °C for later purification.

[0153] Virus purification:

[0154] The concentrated virus lysate suspension from above was thawed once and vortexed for a few seconds. After centrifugation at 2,000 rpm for 5 min at 4°C, the supernatant (containing the released virions) was collected and diluted to a total volume of 30 ml with 10 mM Tris-HCl buffer. An average of 30 ml was placed into four Beckman ultracentrifuge tubes, after which 17 ml of 36% sucrose solution was gently added to the virus solution and centrifuged at 13,500 rpm for 80 min at 4°C. The final pellet was resuspended in 1-4 ml of virus resuspension buffer (PBS; 10% glycerol; 138 mM NaCl; pH 7.4) and stored at -80°C.

[0155] Determination of viral replication:

[0156] Depending on the proliferation rate, place 2–4 × 10 cells in 3 wells of a 6-well plate containing cell culture medium. 5 The cells were seeded at 1000 cells / well and infected with vaccinia virus at 1 PFU / cell the next day. The infected cells and their culture medium were harvested 24, 48, and 72 hours after infection. The virus concentration was then measured.

[0157] In vitro viral cytotoxicity assessment:

[0158] Depending on the growth rate, cells may grow up to 1 x 10 in a 96-well plate. 3 pcs and 1×10 4 Cells were seeded at 1000 / well and infected with the virus 16-18 hours later. Cell viability was measured by MTS assay 6 days after virus infection, and EC50 values ​​were calculated as described previously (50% of tumor cells killed by virus administration). All assays were performed at least in triplicate.

[0159] Assessing the susceptibility of solid tumor cells to viral infection in vitro:

[0160] 3 x 10 in a 6-well plate 5Cells were seeded at 100 / well depending on the growth rate and infected with virus 16–18 h later. Cell viability was measured by crystal violet staining 2 days after virus infection, and the results were scanned as described previously.

[0161] Assessment of susceptibility of leukemia cell lines to viral infection in vitro:

[0162] 3 x 10 in a 6-well plate 5 Cells were seeded in triplicate at 1000 cells / well depending on the proliferation rate, and infected with the virus after seeding. Cell viability was measured on the 1st, 2nd, and 3rd days after virus infection by counting cells after differentiation of live and dead cells by trypan blue staining.

[0163] In vitro assessment of susceptibility of leukemia patient specimens to viral infection:

[0164] With the consent of leukemia patients, 5 ml of blood was collected in an anticoagulant tube. After spun at 2000 rpm for 5 min and removing the plasma, 5 ml of red blood cell lysis buffer was added and incubated at room temperature for 10 min. After red blood cell lysis, the cells were transferred to a 15 ml tube containing 8 ml of sterile PBS and reacted at 2000 rpm for 5 min. After centrifugation, the supernatant was removed and the cell pellet was resuspended using 5 mls of RPMI1640 containing 10% FBS and antibiotics. 1 x 10 cells were plated in a 24-well plate. 6 Two wells were seeded with 100 cells / well, one well was infected with 1 pfu KM1 virus / cell after cell seeding, and the other well was used as a control. Three days after virus infection, the infected cells were observed and photographed under a fluorescent microscope, and then the infected and control cells were collected in a 15 ml tube containing 10 ml of PBS and spun at 2000 rpm for 5 minutes. 100 ml of antibody mixture containing antibodies against L / D, CD14, CD16, and CD33 dissolved in PBS was added to the cell pellet, and stained for 15 minutes at room temperature in the dark. After staining, the cells were washed once with PBS, resuspended in PBS at 300 puls, and analyzed by FAC.

[0165] In vivo efficacy experiment for cancer treatment using KM1

[0166] 5 x 10 6 Ten mice per treatment group were given subcutaneous tumors in their dorsum, measuring 0.4-0.5 cm in diameter, by subcutaneous injection of 1 x 10 cancer cells. They were then regrouped by tumor size and given 1 x 10 8 PFU (immunodeficient mice) or PBS was administered on days 1, 2, 3, 4, and 5. Tumor volumes (volume = (length x width 2 x π) / 6) were measured twice a week until the mice were sacrificed when the tumor area reached 1.69 cm2. Animals used were 4-5 week old male Syrian hamsters.

[0167] statistical analysis

[0168] Comparative statistical analysis was performed using Graphpad Prism 5 unless otherwise stated. Two-condition comparisons were performed using independent t-tests. For additional variables of more than two conditions, 1 or 2 ANOVAs are performed separately. Survival data are presented as Kaplan-Meier plots with log-rank analysis to plot whether differences between groups were statistically significant.

[0169] Oncolytic vaccinia viruses and viral vectors thereof are provided, which include one or more of the following features:

[0170] (a) Generate a combination of viral gene deletions and introduce immune modulating genes (e.g., IL21 and 4-1BBL), tumor suppressor genes (e.g., HIC1) and a novel viral spread gene mB5R / sB5R into infected target cells.

[0171] (b) Deletion of the TK gene allows vaccinia virus to target replication in tumor cells. Vaccinia virus thymidine kinase (TK) allows quiescent cells (such as the majority of normal cells in the body) to produce thymidine for replication. TK-deficient vaccinia virus depends on thymidine kinase produced by the host cell. Thymidine kinase is naturally produced in tumor cells but is not produced or is produced in low amounts in normal cells. Thus, TK-deficient vaccinia virus can selectively replicate in tumor cells, especially those that have activated the EGFR / Ras / ERK pathway.

[0172] (c) Deletion of the L025 gene The protein encoded by the L025 gene can suppress immune responses by inhibiting the activation of the NF Kappa B signaling pathway in immune cells. Immune system inhibition by said gene products This can eliminate tumor growth factor receptors and improve antitumor immune responses.

[0173] (d) Deletion of the A46R gene The protein encoded by the A46R gene acts upstream of the NF Kappa B signaling pathway to suppress immune cell activation, and deletion of this gene improves antitumor immune responses.

[0174] (e) The virus is The B5R-partial region deletion gene (mB5R or sB5R) of vaccinia virus was added. Have .

[0175] (f) The virus is Interleukin 21 (IL-21) gene Have Interleukin 21 also activates NK cells and killer T cells. Have The virus can stimulate an immune response against the tumor. The therapeutic gene is inserted into the L025 region.

[0176] (g) The virus expresses the 4-1BBL gene Have 4-1BB is expressed on activated T cells, and 4-1BB binds to its ligand 4-1BBL to enhance T cell proliferation and cytotoxicity. To make this product more effective, the 4-1BBL gene is incorporated into the virus. The therapeutic gene is inserted into the TK region.

[0177] (h) The virus encodes the tumor suppressor gene HIC1. Have HIC1 is inactivated or lost in many tumors. In vivo studies have shown that expression of this gene in tumors that have lost it can suppress tumor progression. This gene is inserted into the A46R gene region.

[0178] (i) The virus expresses the interleukin 12 (IL-12) gene. Have Interleukin 12 controls natural killer cells (NK cells) and killer T cells. and It is known that it activates NK cells and T cells. Interleukin 12 contained in this drug has been shown in preliminary efficacy studies to activate NK cells and T cells. The therapeutic gene is inserted into the A46R region.

[0179] (j) Provide a new strategy to delete multiple viral genes. In the process of constructing a virus, the present inventors first introduced the CRISPR Cas 9 system to delete fluorescent proteins for genetic screening. Various gRNA sequences designed for the CRISPR Cas 9 system can be used to repeatedly delete fluorescent proteins, so that vaccinia viruses with any combination of genes can be deleted.

[0180] According to feature (e) of the present disclosure, there is provided a vaccinia virus vector comprising a nucleic acid sequence encoding a domain-deleted B5R gene, the sequence being integrated into and replacing the L025 gene of the vaccinia virus. Yes , allowing the virus to spread better, thereby increasing its ability to kill tumor cells.

[0181] The B 5 R gene of vaccinia virus contains an open reading frame (ORF) that encodes a membrane protein essential for the formation of EEV. Deletion of the B5R ORF significantly reduces EEV production, and the virus forms small cell plaques in vitro and has a significantly reduced ability to spread in vivo. The extracellular portion of B5R is composed of four domains similar to short consensus repeats (SCRs) found mainly in complement control proteins.

[0182] The partial mB5R gene or sB5R can be driven to be expressed by its upstream promoter. Thus, the nucleic acid sequence of the promoter can be part of an expression cassette. The expression cassette can be part of a vector that includes a promoter, an open reading frame (and a 3' untranslated region). A promoter is a region of DNA that has a specific sequence that initiates the transcription of a downstream gene. Promoters for expressing heterologous genes in vaccinia include promoters that control early and late transcriptional activity, such as mH5, H5, P7.5, and pE / L. A heterologous gene is a gene that is not normally present in a virus.

[0183] One embodiment of the disclosure provides an array comprising at least one of the following:

[0184] (a) the sequence set forth in SEQ ID NO:1;

[0185] (b) the sequence set forth in SEQ ID NO:3;

[0186] (c) the sequence set forth in SEQ ID NO:5;

[0187] (d) the sequence set forth in SEQ ID NO:7;

[0188] (e) the sequence set forth in SEQ ID NO:9;

[0189] (f) the sequence set forth in SEQ ID NO:11;

[0190] (g) the sequence set forth in SEQ ID NO: 13;

[0191] (h) Therapeutic genes or variants thereof

[0192] (i) the 4-1BBL gene or a variant thereof; or

[0193] (j) T cell-targeting ligands or antibodies

[0194] In one embodiment of the disclosure, a virus is provided, comprising at least one of the following:

[0195] (a) the sequence set forth in SEQ ID NO:1;

[0196] (b) the sequence set forth in SEQ ID NO:3;

[0197] (c) the sequence set forth in SEQ ID NO:5;

[0198] (d) the sequence set forth in SEQ ID NO:7;

[0199] (e) the sequence set forth in SEQ ID NO:9;

[0200] (f) the sequence set forth in SEQ ID NO:11;

[0201] (g) the sequence set forth in SEQ ID NO: 13;

[0202] (h) Therapeutic genes or variants thereof

[0203] (i) the 4-1BBL gene or a variant thereof; or

[0204] (j) T cell-targeting ligands or antibodies

[0205] The sequence is immunomodulatory Factor Gene, immune co-stimulatory pathway activating molecules, checkpoint inhibitors, cytotoxic genes, tumor suppressor genes, anti-angiogenic genes, and the like.

[0206] The immune regulator gene may include a cytokine gene. Examples of such cytokines include lymphokines, monokines, growth factors, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and -β; Mullerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; and integrins. Thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factors; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF; granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; These include IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-24, LIF, G-CSF, GM-CSF, M-CSF, EPO, Kit ligand, or FLT-3. Most preferred are IL-12, IL-21, IL-2, IL-15, IL-8, or any of their variants.

[0207] Immune costimulatory pathway activating molecules may include genes encoding CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 or Flt3 ligand, or variants of any of these.

[0208] The checkpoint inhibitor may comprise a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a variant of any of these.

[0209] The tumor suppressor genes may include HIC1, etc., or variants of any of these.

[0210] One embodiment of the disclosure provides an array comprising at least one of the following:

[0211] (a) mutations in L025, TK, A46R, or any combination thereof;

[0212] (b) partial deletions L025, TK, A46R, or any combination thereof;

[0213] (c) deleted L025, TK, A46R, or any combination thereof;

[0214] (d) part or all of L025, TK, or A46R is replaced with one of the sequences set forth in SEQ ID NOs:1-2;

[0215] (e) all or part of L025, TK, or A46R is replaced with one of the sequences set forth in SEQ ID NOs:3-4;

[0216] (f) some or all of L025, TK, or A46R is replaced by 4-1BBL or a variant thereof;

[0217] (g) part or all of L025, TK, or A46R is replaced by HIC1 or a variant thereof;

[0218] (h) all or part of L025, TK, or A46R is replaced by a tumor-targeting gene;

[0219] (i) part or all of L025, TK, or A46R is replaced by a ligand or antibody that targets T cells;

[0220] (j) part or all of L025, TK, or A46R is replaced by a therapeutic gene or a variant thereof; and

[0221] (k) some or all of L025, TK, or A46R is replaced with a therapeutic antibody;

[0222] In one embodiment of the disclosure, a virus is provided, comprising at least one of the following:

[0223] (a) L025, TK, or A46R mutation;

[0224] (b) partial deletion L025, TK, or A46R;

[0225] (c) deletion of L025, TK, or A46R;

[0226] (d) part or all of L025, TK, or A46R is replaced with one of the sequences set forth in SEQ ID NOs:1-2;

[0227] (e) all or part of L025, TK, or A46R is replaced with one of the sequences set forth in SEQ ID NOs:3-4;

[0228] (f) some or all of L025, TK, or A46R is replaced by 4-1BBL or a variant thereof;

[0229] (g) part or all of L025, TK, or A46R is replaced by HIC1 or a variant thereof;

[0230] (h) all or part of L025, TK, or A46R is replaced by a tumor-targeting gene;

[0231] (i) part or all of L025, TK, or A46R is replaced by a ligand or antibody that targets T cells;

[0232] (j) part or all of L025, TK, or A46R is replaced by a therapeutic gene or a variant thereof; and

[0233] (k) some or all of L025, TK, or A46R is replaced with a therapeutic antibody;

[0234] In one embodiment of the disclosure, an expression vector or host cell is provided comprising any of the sequences described above.

[0235] In one embodiment of the disclosure, there is provided a virus for use in a method of treating the human or animal body, comprising at least one of the following:

[0236] (a) Used alone as monotherapy; and

[0237] (b) Used in combination with one or more anticancer drugs.

[0238] In one embodiment of the present disclosure, a virus is provided for use in the manufacture of a medicament for treating the human or animal body.

[0239] In one embodiment of the present disclosure, a virus is provided for use in inducing the death of cancer cells, modulating the biological activity of cancer cells, modulating the immune response, enhancing T cell proliferation and / or cytotoxicity.

[0240] In one embodiment of the present disclosure, a method for the manufacture of a medicament for inhibiting the growth of, inducing the death of, and / or modulating the biological activity of, a cancer cell is provided. Used forThe present invention provides a virus for use in the present invention.

[0241] The biological activity of cancer cells is inhibition of cancer cell replication, inhibition of cancer cell division, inhibition of DNA repair of cancer cells, Yusou Inhibition of, or cancer cell Including facilitation of death.

[0242] In one embodiment of the disclosure, an article of manufacture is provided that includes a virus in a sterile vial, ampoule, or syringe.

[0243] In one embodiment of the present disclosure, a pharmaceutical composition is provided comprising a virus according to an embodiment of the present disclosure.

[0244] In one embodiment of the present disclosure, the pharmaceutical composition further comprises an anti-cancer agent and / or an antibody.

[0245] In one embodiment of the present disclosure, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent, and / or excipient.

[0246] In one embodiment of the disclosure, there is provided a method for treating a disease comprising administering an effective amount of a sequence, expression vector, host cell, virus, pharmaceutical composition, or medicament.

[0247] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0248] Here, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0249] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" may be used interchangeably and refer to a carrier or diluent that does not significantly irritate the living body and does not impair the biological activity and properties of the administered compound.

[0250] Techniques for formulating and administering drugs are described in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. (Remington. The Science and Practice of Pharmacy, Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa., 20th ed, 2000), which is incorporated herein by reference in its entirety.

[0251] The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, eg, by conventional mixing, dissolving, granulating, dragee-making, filling, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0252] Thus, pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate processing of the active ingredient into a pharma- ceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0253] In an embodiment of the present disclosure, the pharmaceutical composition further comprises an antioxidant that can protect cells or macromolecules (e.g., polysaccharides) from oxidative stress (oxidative damage caused by free radicals). Thus, the antioxidant can extend the viability of macromolecules by preventing oxidative depolymerization of the macromolecules. Non-limiting examples of suitable antioxidants include molecules such as glutathione, vitamin C (sodium ascorbate), vitamin E (tocopherol and tocotrienol), N-Ac-L-cysteine, hydroquinone, glutamic acid, or enzymes such as catalase, superoxide dismutase, glutathione peroxidase, glucose 6-phosphate dehydrogenase (G6PD) (see Osman I., Naziroglu M., Okutan R. Comparative study of antioxidant enzymes in tissues surrounding implant in rabbits. Cell. Biochem. Funct. 24:275-281, 2006).

[0254] Pharmaceutical compositions for potential administration include aqueous solutions of the active formulation in water-soluble form. Additionally, suspensions of the active ingredient can be prepared as appropriate oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.

[0255] The compositions typically contain one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants, flavors, carriers, excipients, buffers, stabilizers, solubilizers, commercial adjuvants, and / or other additives known in the art.

[0256] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile, pyrogen-free, aqueous solution, before use.

[0257] The compositions, pharmaceutical compositions of the present disclosure may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, or article of manufacture (including packaging materials) which may contain one or more unit dosage forms containing the active ingredient. Blister The composition may be constructed of a metal or plastic foil such as a pack. The pack or dispenser device may be accompanied by instructions for administration, implantation and / or treatment of a subject. The pack or dispenser may also be contained with a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects the approval by the government agency of the composition's form or for human or veterinary administration. Such notice may be, for example, that of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. The compositions, matrices or hydrogels of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container and labeled for treatment of an indicated condition, as further detailed above.

[0258] Suitable routes of administration include, for example, oral, rectal, transmucosal, particularly nasal, intestinal or parenteral delivery, and may include intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intracerebroventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal or intraocular injections.

[0259] As used herein, the phrase "chemotherapeutic agent" refers to any chemical agent that has therapeutic utility in the treatment of cancer. As used herein, chemotherapeutic agents include chemical and biological agents. These agents function to inhibit cellular activities that cancer cells depend on to remain viable. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and other antineoplastic agents. Most, if not all, of these agents are directly toxic to cancer cells and do not require immune stimulation. Suitable chemotherapeutic agents are described, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal medicine, 14th edition; Perry et al., Chemotherapeutic, Ch 17 in Abeloff, Clinical Oncology 2nd ed., 2000 ChrchillLivingstone, Inc.; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Luois, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): Cancer Chemotherapy Handbook, 4th ed. St. Luois, Mosby-Year Handbook.

[0260] Chemotherapeutic agents of the present invention include, but are not limited to, cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), asprin, sulindac, curcumin, alkylating agents; nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU); ethyleneimines / methylmelamines such as triethyleneamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkylsulfonates such as busulfan; and triazines such as dacarbazine (DTIC). Antimetabolites such as folate analogues such as methotrexate and trimetrexate, pyrimidine analogues such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (Arac, cytarabine), 5-azacytidine, 2. Purine analogues such as 2-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, 2-chlorodeoxyadenosine (cladribine, 2-CdA), etc., vinca alkaloids such as paclitaxel, vinblastine (VLB), vincristine, vinorelbine, and anticancer drugs such as taxotere, estramustine, and estramustine phosphate. Epipodophyllotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; inhibitors of FGF or VEGF, such as enzymes such as L-asparaginase, cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β, GM-CSF, anti-angiogenic factors such as angiostatin, endostatin, soluble forms of receptors for angiogenic factors such as soluble VGF / VEGF receptors.Platinum complexes such as cisplatin, carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives such as N-methylhydrazine (MIH) and procarbazine, mitotane (ο,ρ´-DDD), adrenal system suppressants such as aminoglutethimide, etc. Hormones and antagonists such as prednisone and equivalents, corticosteroid antagonists such as dexamethasone and aminoglutethimide, progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate, estrogens such as diethylstilbestrol and ethinyl estradiol equivalents, antiestrogens such as tamoxifen, androgens such as testosterone propionate and fluoxymesterone / equivalents, antiandrogens such as flutamide, gonadotropin releasing hormone analogues and leuprolide, nonsteroidal antiandrogens such as flutamide. Kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, Stat inhibitors and receptor tyrosine kinase inhibitors (imatinib mesylate (sold as Glibac or Glivac) and erlotinib (now sold as Tarbecca)); etc. Antivirals (oseltamivir phosphate, amphotericin B, palivizumab, etc.).

[0261] In some embodiments, the daily dosage of a chemotherapeutic agent (e.g., cytarabine) of the present invention or a pharmaceutical composition comprising same ranges between 1-10 g per square meter volume, between 1.5-5 g per square meter volume, or between 2-4 g per square meter volume.

[0262] Various embodiments and aspects of the present invention as delineated herein and claimed below find experimental support in the following examples.

[0263] Reference is now made to the following examples which, together with the above description, illustrate the invention in a non-limiting manner.

[0264] Example 1: Viruses carrying the L025 gene deletion.

[0265] Please refer to Figure 6. Figure 6 shows the recombinant virus with the L025 gene deleted. M: molecular weight marker, 1: control virus, 2: recombinant virus with the L025 gene deleted. A46R is the amplified control gene fragment. L025 is the fragment where the deletion of the L025 gene was detected.

[0266] Using the VVLister virus as the female parent, a shuttle vector for deleting the L025 gene was used for homologous recombination to obtain a virus with the L025 gene deleted. After obtaining pure fluorescent infection spots by red fluorescence screening, a small-scale virus amplification was performed, and the viral DNA was extracted and the deletion of the L025 gene was identified by PCR. As shown in Figure 6, a novel virus with the L025 gene deleted was obtained.

[0267] Example 2: Viruses carrying the L025 gene deletion and the TK gene deletion.

[0268] The L025 gene-deleted novel virus obtained in Example 1 contains FRT sites on both sides of the red fluorescent protein RFP. The FRT sites were cut with Flipase to delete the RFP gene, and an RFP-negative new type VV with the L025 gene deleted was obtained. This virus was used as a backbone virus, and homologous recombination was performed using a TK gene-deleted shuttle vector to obtain a virus in which the TK gene was deleted and the PD1 antibody gene was inserted into this region at the same time. After fluorescent screening, small-scale virus amplification was performed to obtain pure virus-infected plaques, and then viral DNA was extracted and the TK gene deletion was identified by PCR. A novel virus in which both the L025 gene and the TK gene were deleted was obtained, and the PD1 antibody gene was inserted into the TK region (Figure 7).

[0269] Example 3: Virus with a deletion of the L025 gene, a deletion of the TK gene, and a deletion of the A46R gene.

[0270] The novel virus having the L025 gene deletion and the TK gene deletion obtained in Example 2 contains Loxp sites on both sides of the red fluorescent protein RFP. The Loxp sites were deleted by Cre recombinase to delete the RFP gene, and a new RFP-negative virus having the L025 gene deletion and the TK gene deletion was obtained. This virus was used as a backbone virus, and the A46R gene deletion shuttle vector was subjected to homologous recombination to obtain a virus having the A46R gene deletion and the HIC1 gene inserted into this region at the same time. After green fluorescent screening, small-scale virus amplification was performed to obtain pure virus-infected plaques, and then viral DNA was extracted and the A46R gene deletion was identified by PCR. A novel virus having the L025 gene, the TK gene, and the A46R gene deletion was obtained, and the PD1 antibody gene was inserted into the TK region and the HIC1 gene was inserted into the A46R region to obtain a virus expressing the green fluorescent protein GFP.

[0271] Example 4: A virus was obtained in which the L025 gene was deleted and IL21 and mB5R / sB5R were inserted into this region; a virus in which the TK gene was deleted and 4-1BBL gene was inserted into this region; and a virus in which the A46R gene was deleted and HIC1 gene was inserted into this region.

[0272] Please refer to Figure 14-15. Figure 7 shows the recombinant viruses with TK gene deletion and hSPD-41BBL gene insertion. mV: modified virus, V: wild type virus, TK: amplified TK gene (negative indicates that the gene has been deleted in the genome of the recombinant virus detected), hSPD-41BBL: inserted human SPD-41BBL, positive amplification indicates that the gene has been inserted in the genome of the recombinant virus under test), L09: amplified control gene fragment.

[0273] Figure 8. Recombinant virus with deleted A46R gene and viral expression of inserted gene HIC1. A: PCR was used to confirm deletion of A46R gene. M: molecular weight marker, 1: control virus, 2: recombinant virus with deleted A46R gene. L09 is the amplified control gene fragment. B: Western blot analysis confirmed HIC1 protein expression. C is control virus, T is new recombinant virus with deleted A46R gene and HIC1 gene. HIC1 is the result of antibody detection, β-actin is protein loading control.

[0274] The novel virus lacking the L025 gene, TK gene, and A46R gene obtained in Example 3 was used as a backbone virus, and the shuttle vector lacking the L025 gene was homologously recombined to simultaneously insert the IL21 and mB5R / sB5R genes into this region to obtain a virus lacking the L025 gene. After obtaining a pure recombinant virus by fluorescent screening, the small-scale virus was amplified, viral DNA was extracted, and the L025 gene deletion was identified by PCR. A novel virus lacking the L025 gene, TK gene, and A46R gene was obtained, and the 4-1BBL gene was inserted into the TK region, the HIC1 gene (Figure 8) into the A46R region, and the IL21 and mB5R / sB5R genes into the L025 region to obtain a virus lacking the L025 gene.

[0275] Example 5: Panel of solid tumor cells infected with KM1 virus.

[0276] See Figures 9A-9B. Figures 9A-9B show KM1 killing of a panel of solid tumor cell lines with increasing doses of virus over a 2-day period in 6-well plates. The amount of virus used to infect individual wells was 0.01 pfu / cell, 0.1 pfu / cell, and 1 pfu / cell. At the end of the experiment, the remaining cells in the wells were stained with crystal violet.

[0277] Two days after infection with 0.01 pfu, 0.1 pfu, or 1 pfu / cell, the cells were stained with crystal violet, and the stained plates were scanned as shown in Figures 9A-9B.

[0278] Example 6: Sensitivity of solid tumor cells to KM1. A panel of solid tumor cells infected with KM1 virus in an EC50 assay.

[0279] See Figures 10A-11. Figures 10A-10C show examples of EC50 assays in 17 cell lines, where EC50s were performed in triplicate in each cell line. Figure 11 shows the EC50 values ​​for different cell lines.

[0280] Cell viability was measured by MTS 6 days after virus infection. After incubation with MTS, plates were read (Figures 10A-10C and 11).

[0281] Example 7: Sensitivity of leukemia cell lines to KM1 A panel of leukemia cell lines was infected with KM1.

[0282] This is shown in Figure 12. Figure 12 shows the killing of leukemia cell lines by vaccinia virus. EC50 assays performed on three cell lines (K562, C8166, THP-1) are provided as illustrative examples.

[0283] Panels A, B, and C show cell counts at the time of seeding, 48 hours and 72 hours post-infection in control wells (no virus infection, solid line) and virus-infected wells (donation). Sub-panel D shows an image of infected THP1 cells 48 hours post-infection. Green fluorescent cells represent intracellular virus replication / proliferation.

[0284] The vaccinia virus of the present disclosure infected and killed the majority of leukemia cells at 72 hours. Live / dead cells were counted at 24 hours, 48 ​​hours, and 73 hours after virus infection. The results of selectively sensitive cells are presented (Figure 12).

[0285] Example 8: Sensitivity of leukemia patient cells to KM1.

[0286] Please refer to Figures 13-14. Figure 13 shows an example of leukocytes from a leukemia patient infected with KM1. Figure 14 shows live / dead cells on day 3 after KM1 infection.

[0287] Figure 13A: a sample from a patient with CMML; Figure 13B: a sample from a patient with M5b; and Figure 13C: a sample from a patient with M4.

[0288] Figure 14 (above) : FACs profiles of live / dead cells in control wells (without KM1 infection, top panel) and KM1-infected wells of leukemia patients 0518#, 6#, 7#, 8# and 2#. Figure 14B: Percentage of dead cells in control wells (without KM1 infection, top panel) and KM1-infected wells of leukemia patients 0518#, 6#, 7#, 8# and 2#.

[0289] White blood cells from a leukemia patient were infected with the virus. 72 hours after infection, the cells were examined using a fluorescent microscope and FAC (Figure 13-14).

[0290] Example 9. KM1 treatment of pancreatic cancer in Syrian golden hamsters.

[0291] Please refer to Figure 15. Figure 15 shows the antitumor effect of KM1 in the Syrian hamster pancreatic cancer HPD 1NR model. The left panel shows the change in tumor volume after treatment with KM1 and in the control group without KM1 treatment. The right panel shows the change in tumor volume in individual animals of the KM1 administration group.

[0292] Example 10. Treatment of human breast cancer cells MDA MB-231 with HIC1-armed vaccinia virus.

[0293] Please refer to Figure 16. Figure 16 shows the treatment of human breast cancer cells MDA MB-231 with HIC1-armed vaccinia virus and its corresponding control HY3. The top panel shows the treatment of human breast cancer cells MDA MB-231 with HIC1-armed vaccinia virus (HIC1) and its corresponding control HY3 in a dose-escalating manner in a 6-well plate. The bottom panel shows the calculated EC50 from the experiment in the top panel.

[0294] Thus, the present disclosure provides a genetically modified vaccinia virus, a pharmaceutical composition comprising the same, and a method of using the same to treat a subject in need thereof. The genetically modified vaccinia virus comprises a combination of deletions of viral genes and introduction of immune co-stimulatory pathway activating molecules, immune regulator genes, truncated viral envelope genes, and / or tumor suppressors into infected target cells. The oncolytic vaccinia virus of the present disclosure can selectively infect cancer cells, selectively replicate in tumor cells, and selectively target tumor cells with activated EGFR / Ras / ERK pathways. The oncolytic vaccinia virus can also suppress tumor-induced immune suppression and activate the NF-kappa B signaling pathway in immune cells. The oncolytic vaccinia virus can also trigger anti-tumor immune responses and enhance T cell proliferation and cytotoxicity. It can also suppress tumor progression. The oncolytic vaccinia virus can incorporate tumor suppressor genes into the genome of cancer cells that do not express or are deficient in tumor suppressor genes.

[0295] As used herein, the term "about" means ±10%.

[0296] As used herein, the term "method" refers to methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, methods known to those skilled in the art of chemistry, pharmacy, biology, biochemistry, and medicine, or readily developed from known methods, means, techniques, and procedures.

[0297] The term "treating" refers to preventing the occurrence of pathology (disease, disorder or condition) and / or reducing, ameliorating, or regressing pathology. Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess reduction, amelioration, or regression of pathology. It will be understood that treatment can be performed alone or in combination with other treatments.

[0298] As used herein, the term "prevent" refers to preventing a disease, disorder or condition from occurring in a subject who may be at risk for the disease but who has not yet shown symptoms of the disease disorder or condition or has not yet been diagnosed as having the disease disorder or condition. Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess the occurrence of a pathological condition.

[0299] As used herein, the term "subject" or "subject in need thereof" includes mammals, preferably humans of any age or sex. The subject may exhibit preliminary signs of pathology, e.g., a disease, disorder or condition associated with a mutant or non-functional HIC1 protein, e.g., a hyperproliferative disease.

[0300] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular biological, biochemical, microbiological and recombinant DNA techniques, which are fully explained in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al.(eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in US Pat.Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat.Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.(1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds.(1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0301] It will be understood that any sequence number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or an RNA sequence, even if the SEQ ID NO is expressed only in DNA sequence format or RNA sequence format, depending on the context in which the SEQ ID NO is referred to. For example, SEQ ID NO:3 is expressed in DNA sequence format (e.g., reciting T for thymine), but can refer to either a DNA sequence corresponding to a WNT3A nucleic acid sequence, or an RNA sequence of an RNA molecule nucleic acid sequence. Similarly, some sequences are expressed in the form of an RNA sequence (e.g., reciting U for uracil), but can refer to either a sequence of an RNA molecule that constitutes a dsRNA, or a sequence of a DNA molecule that corresponds to the depicted RNA sequence, depending on the type of actual molecule being described. In any event, both DNA and RNA molecules having the disclosed sequences with any substitutions are envisioned.

[0302] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention as appropriate. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0303] In summary, a person skilled in the art may make various changes and modifications according to the technical solutions and technical concepts of the present application, and all such changes and modifications shall fall within the scope of protection of the claims appended hereto.

Claims

1. Mutated viral sequences for selective replication in tumor cells and / or activation of immune cells, and A genetically modified vaccinia virus comprising a heterologous sequence encoding an immune co-stimulatory pathway activating molecule and an immune regulator gene, the mutant viral sequence comprises the deletions L025, TK, and A46R; The immune costimulatory pathway activating molecule is a 4-1BB ligand or a variant thereof; the immunomodulatory factor is IL-21 or a variant thereof; The heterologous sequence is stably integrated into the genome of the recombinant vaccinia virus. The recombinant vaccinia virus.

2. The genetically modified vaccinia virus of claim 1, wherein the heterologous sequence comprises at least one of the following: (a) the sequence set forth in SEQ ID NO:6; (b) the sequence set forth in SEQ ID NO:8; (c) the sequence set forth in SEQ ID NO: 10; or (d) The sequence set forth in SEQ ID NO:

12.

3. The genetically modified vaccinia virus of claim 1, wherein the heterologous sequence encodes at least one of the following: (a) the sequence set forth in SEQ ID NO:5; (b) the sequence set forth in SEQ ID NO:7; (c) the sequence set forth in SEQ ID NO:9; or (d) The sequence set forth in SEQ ID NO:

11.

4. The recombinant vaccinia virus of claim 1, wherein the recombinant vaccinia virus comprises a sequence of the formula: 5'-A1-X-A2-B1-Y-B2-C1-Z-C2-3' where A1 and A2 are the left and right arms, respectively, of a first viral gene, B1 and B2 are the left and right arms, respectively, of a second viral gene, C1 and C2 are the left and right arms, respectively, of a third viral gene, and X, Y and Z are heterologous genes selected from one of an immunomodulatory gene, a cytokine, a therapeutic gene, a truncated viral envelope gene, a tumor suppressor gene, a gene encoding a therapeutic antibody, and a gene encoding a ligand of a therapeutic antibody.

5. 5. The genetically modified vaccinia virus of claim 4, wherein the first viral gene is L025, the second viral gene is TK, and the third viral gene is A46R.

6. the mutant viral sequence has the mutations L025, TK, and A46R deletion; 2. The genetically modified vaccinia virus of claim 1, wherein the heterologous sequences comprise IL-21 and 4-1BBL.

7. the heterologous sequence further encodes a truncated viral envelope gene and / or a tumor suppressor gene; the truncated viral envelope gene is B5R, which contains a deletion of the short consensus repeat (SCR) 2, SCR3, and SCR4 domains; 2. The recombinant vaccinia virus of claim 1.

8. the mutant viral sequence has deletion mutations at L025, TK, and A46R; The heterologous sequence comprises a hybrid gene of IL-21 and modified B5R, and 4-1BBL; The recombinant vaccinia virus of claim 7.

9. The genetically modified vaccinia virus of claim 7, wherein the tumor suppressor gene is HIC1.

10. The genetically modified vaccinia virus of claim 1, wherein the genetically modified vaccinia virus is selected from the group consisting of Lister, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA 382, ​​Modified Vaccinia Uncara (MVA), Dairen I, LC16m8, LC16M0, LIVP, ACAM2000, WR 65-16, Connaught, New York City Board of Health (NYCBH), EM-63 and NYVAC strains.

11. A pharmaceutical composition comprising the recombinant vaccinia virus of claim 1.

12. 12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is formulated for oral, topical, parenteral delivery, or interventional therapy.

13. 12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is formulated for local intratumoral injection, local intra-arterial (blood vessel supplying the tumor) injection, intraperitoneal injection, intrathoracic injection, systemic intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection, direct intraventricular injection, intracardiac injection, or intranasal injection.

14. The pharmaceutical composition of claim 11, wherein the recombinant vaccinia virus is used as a monotherapy or in combination with an anti-cancer drug, an immunosuppressant, and / or an oncolytic virus enhancer.

15. 12. The pharmaceutical composition of claim 11, wherein the recombinant vaccinia virus is used in combination with 5-fluorouracil (FU), folinic acid (FA), methotrexate, capecitabine, oxaliplatin, bevacizumab, cetuximab, immune checkpoint inhibitors, other types of oncolytic viruses, or any combination thereof.

16. 12. The pharmaceutical composition of claim 11, for use in treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition.

17. 17. The pharmaceutical composition of claim 16, wherein the cancer is selected from the group consisting of lung cancer, melanoma, pancreatic cancer, liver cancer, colon cancer, breast cancer, glioblastoma, sarcoma, gastric cancer, ovarian cancer, mesothelioma, and leukemia.

18. A method for screening cancer tissues or cancer cells using an effective amount of the recombinant vaccinia virus according to claim 1.

19. The pharmaceutical composition according to claim 11, for enhancing tumor-specific infectivity of vaccinia virus, wherein the pharmaceutical composition is administered to a subject in an amount effective to induce an anti-tumor immune response.

20. The pharmaceutical composition of claim 11 for conferring sustained immunity against tumor recurrence in a subject in need thereof.

21. The pharmaceutical composition according to claim 11, for screening a patient based on the proportion of GFP-positive cells 48 hours after infection of the patient's cancer cells.

22. 12. The pharmaceutical composition of claim 11 for use in inhibiting the proliferation of cancer cells, inducing the death of cancer cells, modulating the biological activity of cancer cells, modulating the immune response, enhancing the proliferation of T cells, and / or for T cell cytotoxicity.

23. The pharmaceutical composition described in claim 22, wherein the modulation of the biological activity of cancer cells includes inhibiting cancer cell replication, inhibiting cancer cell division, inhibiting cancer cell migration or promoting cancer death.

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