Modified oncolytic viruses, compositions, and their use

Abstract

A modified oncolytic virus having a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immunoactivator is provided, as are pharmaceutical compositions comprising the modified oncolytic virus and methods of treating cancer comprising administering the modified oncolytic virus or the pharmaceutical composition to a subject. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] This disclosure generally relates to modified oncolytic viruses, compositions comprising modified oncolytic viruses, and their use in the treatment of tumors. [Background technology]

[0002] Globally, more than 14 million people are diagnosed with tumors each year. Despite frequent advances in medical research, tumors still account for approximately 16% of all deaths.

[0003] Malignant tumors often present significant therapeutic challenges due to their resistance to conventional treatments. For example, micrometastases can develop very early in the development of a primary tumor. Therefore, at the time of diagnosis, many tumor patients already have micrometastases. Tumor-responsive T cells locate and destroy these micrometastases while preserving surrounding healthy tissue. However, the naturally occurring T cell response to malignant tumors is often insufficient to induce remission of the primary or metastatic tumor.

[0004] Oncolytic viruses have shown potential as antitumor agents. Unlike conventional gene therapy, oncolytic viruses can spread through tumor tissue through viral replication and simultaneous cell lysis. However, oncolytic viruses themselves are insufficient to treat either primary or metastatic tumors.

[0005] Therefore, there is an urgent need to enhance the efficacy of oncolytic viruses and eliminate metastatic tumor cells. [Overview of the Initiative]

[0006] In one aspect, the present disclosure relates to a modified oncolytic virus comprising a viral genome having a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immuno activator.

[0007] In certain embodiments, the oncolytic virus is selected from the group consisting of vaccinia, adenovirus, reovirus, measles, herpes simplex, Semliki Forest virus, Venezuelan equine encephalitis, parvovirus, fowl anemia virus, measles virus, coxsackievirus, vesicular stomatitis virus, Seneca Valley virus, Maraba virus, and Newcastle disease virus. In certain embodiments, the oncolytic virus is derived from the Western Reserve strain.

[0008] In certain embodiments, the modified oncolytic virus is attenuated and capable of replicating in tumor cells. In certain embodiments, the viral genome comprises at least one deletion or disruption that enables the virus to selectively replicate within tumor cells. In certain embodiments, the deletion or disruption is in an open reading frame (ORF) encoding at least a portion of an enzyme that is essential for viral replication and is preferentially expressed in tumor cells over non-tumor cells. In certain embodiments, the enzyme is a kinase. In certain embodiments, the enzyme is thymidine kinase.

[0009] In certain embodiments, the immune checkpoint inhibitor is a first antibody or an antigen-binding fragment thereof that can specifically bind to an immune checkpoint protein. In certain embodiments, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1 / 2, CTLA-4, B7-H3 / 4, LAG3, TIM-3, VISTA, and CD160.

[0010] In certain embodiments, the first antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO: 1.

[0011] In certain embodiments, the first antibody or antigen-binding fragment thereof comprises a first heavy chain comprising SEQ ID NOs: 2, 3, and 4. In certain embodiments, the first heavy chain comprises a variable region having the nucleic acid sequence of SEQ ID NO: 5 or a homologous sequence having at least 80% sequence identity thereto. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 6 or a homologous sequence having at least 80% sequence identity thereto.

[0012] In certain embodiments, the first heterologous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7 or a homologous sequence having at least 80% sequence identity thereto. In certain embodiments, the first heterologous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8 or a homologous sequence having at least 80% sequence identity thereto.

[0013] In certain embodiments, the first antibody or antigen-binding fragment thereof further comprises a first light chain comprising SEQ ID NOs: 9, 10, and 11. In certain embodiments, the first light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 12 or a homologous sequence having at least 80% sequence identity thereto. In certain embodiments, the first light chain comprises the amino acid sequence of SEQ ID NO: 13 or a homologous sequence having at least 80% sequence identity thereto.

[0014] In certain embodiments, the first heterologous polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 14 or a homologous sequence having at least 80% sequence identity thereto. In certain embodiments, the first heterologous polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 15 or a homologous sequence having at least 80% sequence identity thereto.

[0015] In a particular embodiment, the immunoactivator is a co-stimulatory activator. In a particular embodiment, the immunoactivator is a second antibody or its antigen-binding fragment that binds to a co-stimulatory molecule.

[0016] In a particular embodiment, the co-stimulatory molecule is selected from the group consisting of CD137(4-1BB), CD27, CD70, CD86, CD80, CD28, CD40, CD122, TNFRS25, OX40, GITR, Neutrophilin, and ICOS.

[0017] In a particular embodiment, the second antibody or its antigen-binding fragment specifically binds to SEQ ID NO: 16.

[0018] In a particular embodiment, the second antibody or its antigen-binding fragment comprises a double helix containing SEQ ID NOs. 17, 18, and 19. In a particular embodiment, the double helix contains a variable region having the amino acid sequence of SEQ ID NO. 20 or a homologous sequence having at least 80% sequence identity. In a particular embodiment, the double helix contains the amino acid sequence of SEQ ID NO. 21 or a homologous sequence having at least 80% sequence identity.

[0019] In a particular embodiment, the second heterologous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 22 or a homologous sequence having at least 80% sequence identity. In a particular embodiment, the second heterologous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 23 or a homologous sequence having at least 80% sequence identity.

[0020] In a particular embodiment, the second antibody or its antigen-binding fragment further comprises a second light chain comprising SEQ ID NOs: 24, 25, and 26. In a particular embodiment, the second light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 27 or a homologous sequence thereof having at least 80% sequence identity. In a particular embodiment, the second heterologous polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 28 or a homologous sequence thereof having at least 80% sequence identity.

[0021] In a particular embodiment, the second light chain includes the amino acid sequence of SEQ ID NO: 29 or a homologous sequence having at least 80% sequence identity.

[0022] In a particular embodiment, the second heterologous polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 30 or a homologous sequence having at least 80% sequence identity.

[0023] In a particular embodiment, the immunoactivator is an NK activator that stimulates NK cell activity. In a particular embodiment, the NK activator is a second antibody or its antigen-binding fragment that binds to an NK molecule. In a particular embodiment, the NK molecule is selected from the group consisting of Siglec, TIGIT, KIRs, and NKG2A / D.

[0024] In a particular embodiment, the immunoactivator is a macrophage activator that stimulates macrophage cell activity. In a particular embodiment, the macrophage activator is a secondary antibody or its antigen-binding fragment that binds to a macrophage molecule. In a particular embodiment, the macrophage molecule is selected from the group consisting of CSF1R, CSF1 kinase, PS, and CD47.

[0025] In a particular embodiment, the immune checkpoint inhibitor is an antibody or its antigen-binding fragment that specifically binds to PD-1, and the immunoactivator is an antibody or its antigen-binding fragment that specifically binds to CD137.

[0026] In a particular embodiment, the first heterologous polynucleotide and the second heterologous polynucleotide are inserted at the site of the deletion.

[0027] In a particular embodiment, the first heterologous polynucleotide is located immediately upstream or immediately downstream of the second heterologous polynucleotide.

[0028] In a particular embodiment, the first heterologous polynucleotide encodes the first heavy chain and the first light chain of the first antibody. In a particular embodiment, the first heterologous polynucleotide further comprises a first promoter capable of driving the expression of the first heavy chain and a second promoter capable of driving the expression of the first light chain, wherein the first and second promoters are head-to-head oriented.

[0029] In a particular embodiment, the second heterologous polynucleotide encodes the double hemisphere and the second light chain of the second antibody. In a particular embodiment, the second heterologous polynucleotide further comprises a third promoter capable of driving the expression of the double hemisphere and a fourth promoter capable of driving the expression of the second light chain, wherein the third and fourth promoters are oriented head-to-head.

[0030] In a particular embodiment, the first heterologous polynucleotide and the second heterologous polynucleotide are configured to be expressed at the same or different stages in the replication cycle of the modified oncolytic virus.

[0031] In a particular embodiment, the first promoter and the second promoter are the same or different. In a particular embodiment, both the first promoter and the second promoter are late promoters. In a particular embodiment, the late promoter is a pSL.

[0032] In certain embodiments, the third promoter and the fourth promoter are the same or different. In certain embodiments, both the third and the fourth are the early promoter and the late promoter. In certain embodiments, the early promoter and the late promoter are pSE / L.

[0033] In a particular embodiment, the modified oncolytic virus includes, in the 5' to 3' direction of the sense strand, the following elements in frame: a polynucleotide encoding the light chain of an antibody that binds to CD137 - first early and late promoters - second early and late promoters - a polynucleotide encoding the heavy chain of an antibody that binds to CD137 - a polynucleotide encoding the heavy chain of an antibody that binds to PD-1 - first late promoter - second late promoter - a polynucleotide encoding the light chain of an antibody that binds to PD-1.

[0034] In a particular embodiment, the immune checkpoint inhibitor expressed from the first heterologous polynucleotide and the immunoactivator expressed from the second heterologous polynucleotide are expressed as separate proteins.

[0035] In another embodiment, the Disclosure relates to a pharmaceutical composition comprising a modified oncolytic virus and a pharmaceutically acceptable carrier.

[0036] In another embodiment, the Disclosure relates to a method for treating a tumor, comprising administering an effective amount of the modified oncolytic virus or the pharmaceutical composition of the Disclosure to the target.

[0037] In a particular embodiment, the subject is a human being.

[0038] In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, squamous cell carcinoma of the head and neck, bladder cancer, colorectal cancer, or hepatocellular carcinoma.

[0039] In certain embodiments, the route of administration is local. In certain embodiments, the route of administration is intratumoral injection. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 shows the structure of WR-GS-600 with thymidine kinase (TK) deletion, anti-PD-1, and anti-4-1BB antibody insertion. [Figure 2] Figure 2 shows the structure of WR-GS-620 with TK deletion and anti-PD-1 antibody insertion. [Figure 3] Figure 3 is a schematic diagram of the recombination steps for WR-GS-600. [Figure 4] Figure 4 is a schematic diagram of the recombination steps for WR-GS-620. [Figure 5] Figure 5 shows the primer positions relative to the GS-600 virus genome. [Figure 6] Figure 6 shows the primer positions relative to the GS-610 virus genome. [Figure 7] Figure 7 shows the primer positions relative to the GS-620 virus genome. [Figure 8] Figure 8 shows the alignment of WR-GS-600 with the WR wild-type strain. [Figure 9] Figure 9 shows the alignment of WR-GS-610 with the WR wild-type strain. [Figure 10] Figure 10 shows the alignment of WR-GS-620 with the WR wild-type strain. [Figure 11]Figure 11 shows the results of immunofluorescence detection of human IgG expression. Figure 11a shows a phase-contrast image of U2OS cells. Figure 11b shows background staining. Figure 11c shows an image of U2OS cells infected with WR-GS-600. Figure 11d shows an image of U2OS cells infected with WR-GS-620. [Figure 12] Figure 12 shows the results of Western blotting of human antibodies expressed by recombinant viruses (WR-GS-600, WR-GS-610, and WR-GS-620) using cell lysates, where P600, P610, and P620 refer to WR-GS-600, WR-GS-610, and WR-GS-620, respectively. This Western blotting experiment detects two bands with molecular weights of approximately 50 kDa and 25 kDa, corresponding to the heavy and light chains of the human antibodies, respectively. [Figure 13] Figure 13 shows the results of Western blotting of human antibodies expressed by recombinant viruses (WR-GS-600, WR-GS-610, and WR-GS-620) using the supernatant, where P600, P610, and P620 refer to WR-GS-600, WR-GS-610, and WR-GS-620, respectively. This Western blotting experiment detects two bands with molecular weights of approximately 50 kDa and 25 kDa, corresponding to the heavy and light chains of the human antibodies, respectively. [Figure 14] Figure 14 shows the bands produced by PCR amplification using WR-GS-600, WR-GS-610, and WR-GS-620 viral DNA. [Figure 15] Figure 15 shows the amino acid sequence and coding sequence of the heavy chain of anti-huPD-1. [Figure 16] Figure 16 shows the amino acid sequence and coding sequence of the light chain of anti-huPD-1. [Figure 17] Figure 17 shows the amino acid sequence and coding sequence of the heavy chain of anti-hu4-1BB. [Figure 18] Figure 18 shows the amino acid sequence and coding sequence of the light chain of anti-hu4-1BB. [Figure 19] Figure 19 shows the ELISA results of a PD-1 binding assay on the supernatant infected with WR-GS-620. [Figure 20] Figure 20 shows the ELISA results of the 4-1BB binding assay for supernatants infected with WR-GS-600 and WR-GS-610. [Figure 21] Figure 21 shows the survival rates of HCT-116, HT-29, MC-38, and CT-26 cells infected with WR, WR-GS-600, WR-GS-610, and WR-GS-620, respectively. [Figure 22] Figure 22 shows the survival rates of HCT-116, HT-29, MC-38, and CT-26 cells infected with WR, WR-GS-600, WR-GS-610, and WR-GS-620, respectively. [Figure 23] Figure 23 shows the survival rates of HCT-116, HT-29, MC-38, and CT-26 cells infected with WR, WR-GS-600, WR-GS-610, and WR-GS-620, respectively. [Figure 24] Figure 24 shows the plate setup for identifying viral titers. [Figure 25] Figure 25 shows representative plate scan results for WR-GS-610 virus titer identification. [Figure 26] Figure 26 shows representative plate scan results for WR virus titer identification. [Figure 27] Figure 27 shows representative plate scan results for WR-GS-620 virus titer identification. [Figure 28] Figure 28 shows representative plate scan results for WR-GS-600 virus titer identification. [Figure 29] Figure 29 shows representative plate scan results for the control group treated with formulation buffer (FB). [Figure 30] Figure 30 shows the in vivo distribution of the virus in tumors after intratumoral injection. [Figure 31] Figure 31 shows the in vivo viral distribution in the ovary after intratumoral injection. [Figure 32] Figure 32 shows the in vivo viral distribution in the brain, spleen, liver, and lungs after intratumoral injection. The cumulative photon emission (1.928E10 vs. 1.554E10) is thought to be proportional to the number of tumor cells. Based on this data, GS-600 controls tumor growth. [Figure 33] Figure 33 shows the tumor volume changes in syngeneic CT-26 mouse tumor models after intratumor injection (IT) of FB, WR, WR-GS-600, WR-GS-610, and WR-GS-620. [Figure 34] Figure 34 shows the effect model of syngeneic mice treated with various viruses. [Figure 35] Figure 35 shows the flow cytometry results of a humanized HT-29-Luc subcutaneous tumor model in which human PBMCs were intravenously injected. [Figure 36] Figure 36 shows the flow cytometry results of a humanized HT-29-Luc subcutaneous tumor model in which human PBMCs were intravenously injected. [Figure 37] Figure 37 shows the tumor volume changes in humanized HT-29 subcutaneous tumor models after intratumor injection (IT) of FB, WR, WR-GS-600, WR-GS-610, and WR-GS-620. [Figure 38] Figure 38 shows the tumor volume changes in humanized HT-29 subcutaneous tumor models after intratumor injection (IT) of FB, WR, WR-GS-600, WR-GS-610, and WR-GS-620. [Figure 39] Figure 39 shows a humanized HT-29-Luc subcutaneous tumor model with stained tumors, in which the mice were not injected with hPBM. [Figure 40]Figure 40 shows a humanized HT-29-Luc subcutaneous tumor model with stained tumors, in which the mice were injected with hPBM. Mice in cage 2 were infected with WR-GS-600. Mice in cage 3 were infected with WR. Mice in cage 4 were infected with FB. Mice in cage 5 were infected with WR-GS-610. In cage 6, the first mouse was infected with WR-GS-600, the second mouse with WR-GS-620, the third mouse with WR-GS-610, the fourth mouse with WR, and the fifth mouse with FB. Mice in cage 7 were infected with WR-GS-620. [Figure 41] Figure 41 shows a humanized HT-29-Luc intraperitoneal tumor model with tumor staining. [Figure 42] Figure 42 shows the percentage change in chemiluminescence intensity after one week of treatment with various viruses. [Modes for carrying out the invention]

[0041] In one embodiment, the disclosure relates to a modified oncolytic virus comprising a viral genome in which a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immunoactivator are inserted.

[0042] Oncolytic viruses The term “oncolytic virus,” as used herein, means a virus that can selectively replicate in tumor cells, either in vitro or in vivo, without affecting or with minimal affecting normal cells, and can slow the growth of tumor cells or induce tumor cell death. In certain embodiments, the oncolytic virus comprises a viral genome packaged within a viral particle (or virion) and is infectious (i.e., can infect and enter a host cell or object). In certain embodiments, the oncolytic virus may be a DNA virus or an RNA virus and may be in any preferred form, such as a DNA viral vector, an RNA viral vector, or a viral particle.

[0043] The term “selectively replicates,” as used herein, means that the replication rate of an oncolytic virus is significantly higher in tumor cells than in non-tumor cells (e.g., healthy cells). In certain embodiments, an oncolytic virus exhibits a lysis rate at least 50%, 60%, 70%, 80%, 90%, 1x, 2x, 3x, 4x, 5x, 10x, 50x, 100x, or 1000x higher in tumor cells than in non-tumor cells (e.g., healthy cells).

[0044] In certain embodiments, the oncolytic viruses of the present disclosure can selectively replicate in liver tumor cells (e.g., Hepal-6 cells, Hep3B cells, 7402 cells, and 7721 cells), breast tumor cells (e.g., MCF-7 cells), tongue tumor cells (e.g., TCa8113 cells), adenoid cystic tumor cells (e.g., ACC-M cells), prostate tumor cells (e.g., LNCaP cells), human embryonic kidney cells (e.g., HEK293 cells), lung tumor cells (e.g., A549 cells), or cervical tumor cells (e.g., HeLa cells).

[0045] The oncolytic viruses described herein include poxviruses (e.g., vaccinia virus), adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, and AD5 / 3-D24-GMCSF), reoviruses (e.g., REOLYSIN), measles virus, herpes simplex virus (e.g., HSV, OncoVEX GMCSF), Newcastle disease virus (e.g., 73-T PV701 and HDV-HUJ strains), as described in the following literature: Phuangsab et al., 2001, Cancer Lett. 172(1): 27-36; Lorence et al., 2007, Curr. Cancer Drug Targets 7(2): 157-67; and Freeman et al., 2006, Mol. Ther. 13(1): It can be derived from retroviruses (e.g., influenza virus), myxoma virus, rhabdoviruses (e.g., vesicular stomatitis virus; as described in the following literature: Stojdl et al., 2000, Nat. Med. 6(7): 821-5 and Stojdl et al., 2003, Cancer Cell 4(4): 263-75), picornaviruses (e.g., Seneca Valley virus; SW-001 and NTX-010), coxsackievirus, or parvovirus.

[0046] In certain embodiments, the oncolytic viruses of this disclosure are derived from poxviruses. The term “poxvirus,” as used herein, means viruses belonging to the subfamily Poxvirinae. In certain embodiments, poxvirus is a virus belonging to the subfamily Chordopoxviridae. In certain embodiments, poxvirus is a virus belonging to the subfamily Orthopoxvirus. Genome sequences of various poxviruses, such as vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, and myxoma virus, are available in the Art and specialized databases, such as GenBank (accession numbers NC_006998, NC_003663, NC_005309, NC_004105, and NC_001132, respectively).

[0047] In certain embodiments, the oncolytic viruses of this disclosure are derived from vaccinia viruses. Vaccinia viruses are members of the Poxviridae family, characterized by a double-stranded DNA genome of approximately 190 kb that encodes numerous viral enzymes and factors that enable the virus to replicate independently of host cellular mechanisms. In certain embodiments, the vaccinia viruses of this disclosure are derived from the Elstree, Copenhagen, Western Reserve, or Wyeth strains. In certain embodiments, the vaccinia viruses of this disclosure are the Western Reserve strain. The Western Reserve strain has been well-characterized, and its complete sequence is available on the NCBI site (www.ncbi.nlm.nih.gov) under access number AY243312.

[0048] The term "modified oncolytic virus," as used herein, means an oncolytic virus modified by the introduction of heterologous nucleic acids or proteins or by alteration of native nucleic acids or proteins. In certain embodiments, the modified oncolytic viruses provided herein are genetically engineered by deletions and / or additions of nucleic acid sequences. In certain embodiments, the modified oncolytic viruses provided herein include deletions of thymidine kinase (TK) genes. In certain embodiments, the modified oncolytic viruses provided herein include additions of nucleic acid sequences encoding anti-human PD-1 and / or anti-human 4-1BB antibodies.

[0049] In certain embodiments, the modified oncolytic viruses of this disclosure are attenuated. In certain embodiments, the modified oncolytic viruses have reduced pathogenicity (e.g., less than 90%, 80%, 70%, 60%, or 50%) in normal cells (e.g., healthy cells) compared to their wild-type counterparts, or are undetectable.

[0050] The modified oncolytic viruses of this disclosure can be derived from any oncolytic virus known in the art to be oncolytic by their tendency to selectively replicate in tumor cells compared to non-tumor cells and to cause tumor cell death. Oncolytic viruses can be oncolytic in nature or can be made oncolytic by genetic engineering techniques, for example, by modifying one or more genes to increase tumor selectivity and / or preferential replication in tumor cells. Examples of such genes for modification include those involved in DNA replication, nucleic acid metabolism, host targeting, surface adhesion, pathogenicity, host cell lysis, and viral dispersal (see, e.g., Kirn et al., 2001, Nat. Med. 7: 781; Wong et al., 2010, Viruses 2: 78-106).

[0051] In certain embodiments, the modified viral genome of the oncolytic virus of this disclosure includes at least one deletion or disruption that allows the virus to selectively replicate within tumor cells. For example, the deletion or disruption may reduce the expression or function of an enzyme essential for viral replication, thereby reducing the virus's ability to replicate in the absence of such enzyme. In some embodiments, viral replication depends on the presence and / or level of such enzyme in the cell, with higher levels of the enzyme resulting in a higher viral replication capacity or rate.

[0052] In certain embodiments, the deletion or disruption is located in an open reading frame (ORF). The terms “open reading frame,” “ORF,” or “coding sequence,” as used herein, mean a DNA sequence that can be translated into an amino acid sequence. An ORF typically begins with a start codon (e.g., ATG), followed by an amino acid coding codon, and ends with a stop codon (e.g., TGA, TAA, TAG).

[0053] In certain embodiments, the ORF encodes at least a portion of an enzyme essential for viral replication that is preferentially expressed in tumor cells compared to non-tumor cells. The term “express” as used herein means the process by which a protein or peptide sequence is produced from its encoding DNA or RNA sequence. In certain embodiments, the enzyme is a kinase.

[0054] In a particular embodiment, the deletions in the ORF constitute 100%, greater than 99%, greater than 98%, greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 65%, greater than 60%, greater than 55%, greater than 50%, greater than 45%, greater than 40%, greater than 35%, greater than 30%, greater than 25%, greater than 20%, greater than 15%, or greater than 10% of the total length of the ORF. In a particular embodiment, the deletion in the ORF comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 500, 800, 1000, 1200, 1500, 1800, 2000, 2200, 2400, 2500, or more nucleotides (possibly consecutive nucleotides).

[0055] In certain embodiments, the ORF of thymidine kinase (TK) is deleted or destroyed. TK is involved in the synthesis of deoxyribonucleotides. Because normal cells generally have low concentrations of nucleotides, TK is required for viral replication in these normal cells, whereas in tumor cells, which have high nucleotide concentrations, TK is not necessarily required. In poxviruses, the thymidine kinase coding gene is located at the J2R locus. In certain embodiments, TK is completely deleted.

[0056] In certain embodiments, the ORF of ribonucleotide reductase (RR) is deleted or disrupted. RR catalyzes the reduction of ribonucleotides to deoxyribonucleotides, a crucial step in DNA biosynthesis. The viral enzyme consists of two heterologous subunits, named R1 and R2, encoded by the I4L and F4L loci, respectively. The sequences of the I4L and F4L genes and their locations in the genomes of various poxviruses are available in public databases, for example, GeneBank accessions DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391, NC_003389, NC_003310, M-3 These are available under 5027, AY243312, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, Y16780, X71982, AF438165, U60315, AF410153, AF380138, U86916, L22579, NC_006998, DQ121394, and NC_008291. In connection with the present invention, either or both of the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) may be deleted or disrupted.

[0057] In certain embodiments, the viral genome of the modified oncolytic virus further includes additional deletions or disruptions that further increase the tumor specificity of the virus. In certain embodiments, the additional deletions or disruptions are located in ORFs that encode at least some tumor-specific proteins that are preferentially or specifically expressed in tumor cells. A representative example of a tumor-specific protein is VGF, a secreted protein expressed early after viral infection of cells, whose function is thought to be important for viral spread in normal cells. Another example is the A56R gene encoding hemagglutinin (Zhang et al., 2007, Cancer Res. 67: 10038-46). A further example is the F2L gene encoding a viral dUTPase involved in both maintaining the accuracy of DNA replication and providing a precursor for the production of TMP by thymidylate synthase (Broyles et al., 1993, Virol. 195: 863-5). The sequence of the vaccinia virus F2L gene is available in GenBank under accession number M25392.

[0058] Immune checkpoint inhibitors The modified oncolytic viruses provided herein include a viral genome having a first heterologous polynucleotide encoding an immune checkpoint inhibitor.

[0059] As used herein, the term "heterogeneous" means that the sequence is not endogenous to the wild-type virus.

[0060] The terms “encode” or “to code,” as used herein, mean “to be transcribed into mRNA and / or to be translated into peptides or proteins.”

[0061] The term “immune checkpoint protein,” as used herein, means a protein that is directly or indirectly involved in immunological pathways that are important for preventing uncontrolled immune responses and thus for maintaining autoimmune tolerance and / or tissue protection. One or more immune checkpoint modulators as used herein may function independently in any step of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, transport to sites of antigen and inflammation, performance of direct effector functions, and signaling by cytokines and membrane ligands.

[0062] The term “immune checkpoint inhibitor,” as used herein, means a molecule that can negatively modulate the function of immune checkpoint proteins. Immune checkpoint inhibitors may be any of the molecular modalities known in the art, including, but not limited to, aptamers, mRNA, siRNA, microRNA, shRNA, peptides, antibodies, globular nucleic acids, TALENs, zinc finger nucleases, and CRISPR / Cas9.

[0063] In certain embodiments, the immune checkpoint inhibitor is a natural or genetically engineered antagonist of an inhibitory immune checkpoint molecule, such as a ligand for CTLA-4 (e.g., B7.1, B7.2), a ligand for TIM3 (e.g., galectin-9), a ligand for the A2a receptor (e.g., adenosine, regadenoson), a ligand for LAG3 (e.g., an MHC class I or MHC class II molecule), a ligand for BTLA (e.g., HVEM, B7-H4), a ligand for KIR (e.g., an MHC class I or MHC class II molecule), a ligand for PD-1 (e.g., PD-L1, PD-L2), or a ligand for IDO (e.g., NKTR-218, indoximod, NLG919).

[0064] In certain embodiments, immune checkpoint inhibitors include anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, BMS-936558, atezolizumab, lambrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042, or ANB011), anti-PD-L1 (e.g., KY-1003, MCLA-145, atezolizumab, MEDI-4736), (as cited in MSB0010718C, STI-A1010, MPDL3280A, dapirolizumab CDP-7657, MEDI-4920, or PCT / US2001 / 020964), anti-PD-L2, anti-(both PD-L1 and PD-L2) (e.g., AUR-012 and AMP-224), anti-CTLA-4 (e.g., ipilimumab, tremelimumab, or KAHR-102), anti-IDO (For example, Dl-methyltryptophan (Lunate), anti-KIR (for example, lirilumab, IPH2101, or IPH4102), anti-LAG3 (for example, BMS-986016, IMP701, IMP321, or C9B7W), anti-TIM3 (for example, F38-2E2 or ENUM005), anti-VISTA (for example, VA.F6), anti-BTLA (for example, AF3354), anti-CD73 (for example, OSU-HDA) The antibody is selected from the group consisting of anti-B7-H3 (e.g., C42 or MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, or 8H9), anti-A2aR, anti-B7-1, anti-B7-H3 (e.g., MGA271), anti-B7-H4, anti-(both B7-H3 and B7-H4), anti-CD52 (e.g., alemtuzumab), anti-IL-10, anti-IL-35, anti-MICA (e.g., IPH43), and anti-CD39 (e.g., an antagonist antibody).

[0065] In certain embodiments, the immune checkpoint inhibitor is an antibody or its antigen-binding fragment that can specifically bind to an immune checkpoint protein, selected from the group consisting of PD-1, PD-L1 / 2, CTLA-4, B7-H3 / 4, LAG3, TIM-3, VISTA, and CD160. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2. In certain embodiments, the immune checkpoint inhibitor is an anti-B7-H3 or anti-B7-H4 antibody, or an inhibitor of both B7-H3 and B7-H4.

[0066] PD-1 inhibitors In a particular embodiment, the first heterologous polynucleotide of this disclosure encodes a PD-1 inhibitor.

[0067] The term "PD-1," as used herein, means programmed cell death protein, which belongs to the immunoglobulin superfamily and functions as a co-inhibitory receptor that negatively modulates the immune system. PD-1 is a member of the CD28 / CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. A representative amino acid sequence of human PD-1 is disclosed in GenBank accession number: NP_005009.2, and a representative nucleic acid sequence encoding human PD-1 is shown in GenBank accession number: NM_005018.2.

[0068] PD-1 negatively modulates T cell activation, and this inhibitory function is related to an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (Parry et. al, 2005, Mol. Cell. Biol. 25:9543-53). Interference with this inhibitory function of PD-1 can lead to autoimmunity. Persistent negative signaling by PD-1 has been associated with T cell dysfunction in many pathological conditions, such as tumor immune evasion and chronic viral infections.

[0069] A PD-1 inhibitor can be any agent that inhibits the activity of PD-1, such as an agent that reduces the activity of PD-1 by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95%, or more.

[0070] Activity (e.g., of PD-1) can be reduced as a result of, for example, inhibition of the binding between a functional protein and its ligand (e.g., the binding between PD-1 and PD-L1), inhibition of its bioactivation (e.g., activation of PD-1), and / or reduction in level (e.g., level of PD-1).

[0071] In certain embodiments, the PD-1 inhibitor is an antibody (e.g., an antagonist antibody) that can specifically bind to PD-1.

[0072] As used herein, the term "specific binding" or "specifically binds" means a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment provided herein has a binding affinity (KD) of ≦ 10 -6 M (e.g., ≦ 5 × 10 -7 M, ≦ 2 × 10 -7 M, ≦ 10 -7 M, ≦ 5 × 10 -8 M, ≦ 2 × 10 -8 M, ≦ 10 -8 M, ≦ 5 × 10 -9 M, ≦ 2 × 10 -9 M, ≦ 10 -9 M, ≦ 10 -10 M) and specifically binds to human and / or monkey PD-1. KD, as used herein, means the ratio of the association rate to the dissociation rate (k off / k on ), which can be determined using surface plasmon resonance methods using an instrument such as a Biacore.

[0073] In certain embodiments, the PD-1 inhibitor is a full-length monoclonal antibody against PD-1.

[0074] In a particular embodiment, the PD-1 antibody specifically binds to SEQ ID NO: 1.

[0075] In a particular embodiment, the PD-1 antibody or its antigen-binding fragment comprises a primary heavy chain including SEQ ID NOs: 2, 3, and 4.

[0076] When the term "identity" is used herein in relation to amino acid sequences (or nucleic acid sequences), it means the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after the sequences have been aligned and, if necessary, identical amino acid (or nucleic acid) gaps have been introduced to achieve the maximum number. Conservative substitutions of amino acid residues are not considered identical residues. Alignment for the purpose of determining the percentage of identity of amino acid (or nucleic acid) sequences can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the National Center for Biotechnology Information (NCBI) website; see also Altschul SF et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Institute for Bioinformatics website; see also Higgins DG et al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or they may customize the parameters appropriately for alignment by, for example, selecting a suitable algorithm.

[0077] In certain embodiments, the heavy chain includes a variable region having a homologous sequence thereof with sequence number 5 or at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the heavy chain includes the amino acid sequence of sequence number 6 or a homologous sequence thereof with at least 80% sequence identity.

[0078] In certain embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0079] In certain embodiments, the PD-1 antibody or its antigen-binding fragment further comprises a light chain comprising SEQ ID NOs. 9, 10, and 11. In certain embodiments, the light chain comprises a variable region having the amino acid sequence of SEQ ID NO. 12 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the light chain comprises the amino acid sequence of SEQ ID NO. 13 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0080] In certain embodiments, the polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 14 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 15 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0081] Immunoactivator The modified oncolytic viruses provided herein include a viral genome having a second heterologous polynucleotide encoding an immunoactivator.

[0082] The term "immunoactivator," as used herein, means any drug that can enhance the immune system.

[0083] When used herein, the term "enhances the immune system" means the ability of a drug to stimulate the development of T cell activity, B cell activity, macrophage activity, and / or NK cell activity.

[0084] In a particular embodiment, the immunoactivator is a co-stimulation activator, an NK activator, or a macrophage activator.

[0085] Co-stimulatory molecule activator In a particular embodiment, the immunoactivator is a co-stimulatory molecule activator.

[0086] As used herein, the term “costimulatory molecule” refers to a cell surface molecule other than an antigen receptor or Fc receptor that provides a secondary signal necessary for the efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include CD137 (i.e., 4-1BB), CD27, CD70, CD86, CD80, CD28, CD40, CD122, TNFRS25, OX40 (CD134), GITR, neutrophilin, and ICOS (i.e., CD278).

[0087] In certain embodiments, the co-stimulatory activator may be a peptide, polypeptide (e.g., an antibody) that can enhance the cellular immune system. In certain embodiments, the co-stimulatory activator may be an antibody that binds to a costimulatory molecule and thereby stimulates the activity of the costimulatory molecule, or an antigen-binding fragment of such an antibody, e.g., CD137 antibody (e.g., BMS-663513 or PF-05082566), CD28 antibody (e.g., TGN-1412), CD40 antibody (e.g., CP-870, 893, CDX1140, BI-655064, BMS-986090, APX005, or APX005M), OX4 Examples include 0 (CD134) antibodies (e.g., those described in MEDI6383, MEDI6469, MEDI0562, or U.S. Patent No. 7,959,925), anti-GITR (e.g., TRX-518, INBRX-110, or NOV-120301), CD70 antibodies, CD86 antibodies, CD80 antibodies, CD122 antibodies, TNFRS25 antibodies, neutrophilin antibodies, and CD27 antibodies (e.g., CDX-1127, BION-1402, or hCD27.15).

[0088] CD137 Activator In a particular embodiment, the second heterologous polynucleotide of the present disclosure encodes a CD137 activator.

[0089] CD137, also known as 4-1BB, is a member of the tumor necrosis factor receptor (TNFR) gene family, which includes proteins involved in regulating cell proliferation, differentiation, and programmed cell death (A. Ashkenazi, Nature, 2: 420-430, (2002)). CD137 is CD4 + and CD8 + It is predominantly expressed in activated T cells, including both NK cells and NKT cells (see B. Kwon et al., Mol. Cell 10: 119-126, (2000); J. Hurtado et al, J. Immunol. 155: 3360-3365, (1995); and L. Melero et al., Cell. Immunol. 190: 167-172, (1998)).

[0090] A CD137 activator may be any agent that enhances PD-1 activity, such as those that enhance CD137 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95%, or more.

[0091] In certain embodiments, the CD137 activator is an antibody that specifically binds to CD137. In certain embodiments, the CD137 activator is a full-length antibody.

[0092] In a particular embodiment, the CD137 activator or its antigen-binding fragment specifically binds to SEQ ID NO: 16.

[0093] In a particular embodiment, the CD137 activator or its antigen-binding fragment comprises a heavy chain including SEQ ID NOs: 17, 18, and 19.

[0094] In certain embodiments, the heavy chain includes a variable region having the amino acid sequence of SEQ ID NO: 20 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 21 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0095] In certain embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 22 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 23 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0096] In certain embodiments, the antibody or its antigen-binding fragment further comprises a light chain comprising SEQ ID NOs: 24, 25, and 26. In certain embodiments, the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 27 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, the polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 28 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0097] In certain embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 29 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0098] In certain embodiments, the polynucleotide further comprises the nucleic acid sequence of SEQ ID NO: 30 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0099] NK Activator In certain embodiments, the immunoactivator is an NK activator that stimulates NK cell activity. In certain embodiments, the NK activator is a secondary antibody or its antigen-binding fragment that binds to an NK molecule.

[0100] In a particular embodiment, the NK activator is selected from the group consisting of Siglec antibody, TIGIT antibody, KIRs antibody, and NKG2A / D antibody (e.g., monalizumab).

[0101] Macrophage activator In certain embodiments, the immunoactivator is a macrophage activator that stimulates macrophage cell activity. In certain embodiments, the macrophage activator is a second antibody or its antigen-binding fragment that binds to macrophage molecules.

[0102] In a particular embodiment, the macrophage activator is selected from the group consisting of CSF1R antibodies (e.g., FPA008), CSF1 kinase antibodies, PS antibodies, and CD47 antibodies (e.g., CC-90002, TTI-621, or VLST-007).

[0103] antibody The term “antibody,” as used herein, encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a particular antigen. Naturally intact antibodies consist of two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies have a “Y” shape, where the stem of the Y consists of the second and third constant regions of two heavy chains joined together by a disulfide bond. Each arm of the Y contains a variable region and a first constant region of a single heavy chain bound to a variable region and a constant region of a single light chain, in which case the first constant region of the heavy chain is linked to the second constant region via a hinge region. The variable regions of the light and heavy chains are responsible for antigen-binding specificity. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs): the light (L) chain CDRs, which include LCDR1, LCDR2, and LCDR3; and the heavy (H) chain CDRs, which include HCDR1, HCDR2, and HCDR3. The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or specified according to the conventions of Kabat, Chothia, or Al-Lazikani (see Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991) for further details).The three CDRs are interposed between adjacent stretch regions known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold for supporting the structure of the variable regions. The constant regions of the heavy and light chains are independent of antigen-binding specificity but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc.

[0104] The term "antigen-binding fragment," as used herein, means an antibody fragment formed from a portion of an antibody containing one or more CDRs, but does not include an intact antibody structure. Examples of antigen-binding fragments, but not limited to, include Fab, Fab', F(ab')2, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single-domain antibodies, and nanobodies. Antigen-binding fragments can bind to the same antigen to which the parent antibody binds.

[0105] As used herein, the term "Fab" refers to a portion of an antibody consisting of a single light chain (both variable and constant regions) attached by disulfide bonds to the variable and primary constant regions of a single heavy chain.

[0106] The term "Fab" as used herein means a Fab fragment that includes a portion of the hinge region.

[0107] The term "F(ab')2," as used herein, refers to the dimer of Fab'.

[0108] As used herein, the term "Fv" refers to an Fv fragment consisting of a single light chain variable region and a single heavy chain variable region.

[0109] The terms "single-chain Fv antibody" or "scFv," as used herein, mean a genetically engineered antibody consisting of a light-chain variable region and a heavy-chain variable region linked to each other directly or via a peptide linker sequence (see, for example, Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0110] The term "scFv dimer," as used herein, refers to a polymer formed from two scFv molecules.

[0111] The term "camelized single-domain antibody" is also known as a "heavy-chain antibody" or "HCAb" (heavy-chain-only antibody) and refers to an antibody that contains two heavy-chain variable regions but no light chain (see, for example, Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; and U.S. Patent No. 6,005,079). Heavy-chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Camelized antibodies lack a light chain but possess a legitimate antigen-binding repertoire (see Hamers-Casterman C. et al., Nature. 363(6428):446-8 (1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. 54(1):39-47 (2002); and Nguyen VK. et al., Immunology. 109(1):93-101 (2003), which are incorporated herein by reference in their entirety).

[0112] As used herein, the term "nanobody" refers to an antibody consisting of a heavy chain variable region derived from a heavy chain antibody and two constant regions, CH2 and CH3.

[0113] In certain embodiments, the antibodies provided herein are fully human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, or rabbit antibodies. In certain embodiments, the antibodies provided herein are polyclonal antibodies, monoclonal antibodies, or recombinant antibodies. In certain embodiments, the antibodies provided herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies. In certain embodiments, the antibodies provided herein may be further labeled. In certain embodiments, the antibody or its antigen-binding fragment is a fully human antibody, which may optionally be produced by transgenic rats, for example, transgenic rats in which the expression of the endogenous rat immunoglobin gene has been inactivated, and which have a recombinant human immunoglobin locus with a J locus deletion and a C-κ mutation, and may also be expressed by genetically engineered cells (e.g., CHO cells).

[0114] When the term "fully human" is used herein in relation to an antibody or antigen-binding fragment, it means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or is derived from a non-human source, such as a transgenic non-human animal utilizing the human antibody repertoire, or is another human antibody-coding sequence.

[0115] When used herein in relation to antibodies or antigen-binding fragments, the term “humanized” means an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, an FR region derived from a human, and, where appropriate, a constant region derived from a human. Humanized antibodies or antigen-binding fragments are useful as therapeutic agents for humans in certain embodiments because they have reduced immunogenicity. In certain embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In certain embodiments, the humanized antibody or antigen-binding fragment consists substantially entirely of human sequences, except for the non-human CDR sequence.

[0116] When the term “chimera” is used herein in relation to an antibody or antigen-binding fragment, it means an antibody or antigen-binding fragment having a portion of the heavy chain and / or light chain from one species and the rest of the heavy chain and / or light chain from a different species. In certain embodiments, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human species, such as a mouse or rabbit.

[0117] When the term “conservative substitution” is used herein in relation to amino acid sequences, it means the substitution of an amino acid residue with a different amino acid residue having a side chain of similar physiological and chemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues having acidic side chains (e.g., Asp, Glu), between amino acids having basic side chains (e.g., His, Lys, and Arg), or between residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of a protein and thus can maintain the biological activity of the protein.

[0118] Polynucleotides In a particular embodiment, the modified oncolytic virus of the present disclosure comprises a first heterologous polynucleotide encoding an inhibitory antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and a second heterologous polynucleotide encoding an activating antibody or an antigen-binding fragment thereof that specifically binds to CD137.

[0119] The terms “polynucleotide” or “nucleic acid,” as used herein, mean ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or mixed ribonucleic acid-deoxyribonucleic acid, e.g., DNA-RNA hybrids. Polynucleotides or nucleic acids may be single-stranded or double-stranded DNA or RNA or DNA-RNA hybrids. Polynucleotides or nucleic acids may be linear or cyclic. In certain embodiments, both the first and second heterologous polynucleotides are DNA if the virus is a DNA virus, or both are RNA if the virus is an RNA virus. In certain embodiments, both the first and second heterologous polynucleotides are double-stranded DNA.

[0120] The first and second heterologous polynucleotides can be introduced into modified oncolytic viruses by using conventional methods known in the art, such as synthesis by polymerase chain reaction (PCR) and ligation with a viral genome having interchangeable control ends. For further details, see, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, NY (1989)), which is incorporated herein by reference in its entirety.

[0121] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are introduced at the site of a deletion in the ORF. In certain embodiments, the first heterologous polynucleotide is located immediately upstream or immediately downstream of the second heterologous polynucleotide. The term “immediately upstream or immediately downstream,” as used herein, means that the first heterologous polynucleotide and the second heterologous polynucleotide are located very close together on the viral genome, separated from each other by nucleotides 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less. For example, if the 3' end of the upstream polynucleotide is separated from the 5' end of the downstream polynucleotide by nucleotides 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less, then the 3' end of the upstream polynucleotide is immediately adjacent to the 5' end of the downstream polynucleotide. In certain embodiments, there is no ORF between the first and second heterologous polynucleotides. In certain embodiments, there is a restriction site between the first and second heterologous polynucleotides.

[0122] In a particular embodiment, the first heterologous polynucleotide encodes the first heavy chain and the first light chain of the first antibody. In a particular embodiment, the first heterologous polynucleotide further comprises a first promoter capable of driving the expression of the first heavy chain and a second promoter capable of driving the expression of the first light chain, wherein the first and second promoters are head-to-head oriented.

[0123] In certain embodiments, the first heterologous polynucleotide encodes the variable region of the first heavy chain, the linker, and the variable region of the first light chain of the first antibody. In certain embodiments, the first heterologous polynucleotide encodes the first heavy chain of the first antibody but not the first light chain.

[0124] When used herein, the term "head-to-head orientation" means that two promoters are located very close to each other on the viral genome and drive protein expression in opposite directions. A specific example is shown in Figure 2.

[0125] In a particular embodiment, the second heterologous polynucleotide encodes the second duplex and second light chain of the second antibody. In a particular embodiment, the second heterologous polynucleotide further comprises a third promoter capable of driving the expression of the duplex and a fourth promoter capable of driving the expression of the second light chain, wherein the third and fourth promoters are head-to-head oriented.

[0126] The term “promoter,” as used herein, means a polynucleotide sequence capable of controlling the transcription of a coding sequence. A promoter sequence includes a specific sequence that is sufficient for RNA polymerase recognition, binding, and transcription initiation. Furthermore, a promoter sequence may include sequences that modulate this recognition, binding, and transcription initiation activity of RNA polymerase. A promoter can influence the transcription of genes located on the same nucleic acid molecule as itself or on a different nucleic acid molecule. The function of a promoter sequence may be constitutive or stimulus-induced, depending on the nature of its regulation. A “constitutive” promoter, as used herein, means a promoter that functions to continuously activate gene expression in a host cell. A “stimulative” promoter, as used herein, means a promoter that activates gene expression in a host cell in the presence of a particular stimulus.

[0127] In certain embodiments, the promoters of this disclosure include eukaryotic cell promoters, e.g., CMV-derived promoters (e.g., CMV very early promoter (CMV promoter)), Epstein-Barr virus (EBV) promoter, human immunodeficiency virus (HIV) promoters (e.g., HIV long-terminal repeat (LTR) promoter, Moloney virus promoter, mouse mammary cancer virus (MMTV) promoter, Roussarcoma virus (RSV) promoter, SV40 early promoter), human gene-derived promoters, e.g., human myosin promoter, human hemoglobin promoter, human muscle creatine promoter, human metallothionein β-actin promoter, human ubiquitin C promoter (UBC), mouse phosphoglycerate kinase 1 promoter (PGK), human thymidine kinase promoter These include the promoter of protein 1 (TK), human prolongation factor 1 alpha promoter (EF1A), cauliflower mosaic virus (CaMV) 35S promoter, E2F-1 promoter (promoter of E2F1 transcription factor 1), α-fetoprotein promoter, cholecystokinin promoter, carcinoembryonic antigen promoter, C-erbB2 / neu tumorigenicity promoter, cyclooxygenase promoter, CXC-chemokine receptor 4 (CXCR4) promoter, human epididymal protein 4 (HE4) promoter, hexokinase type II promoter, L-plastin promoter, mucin-like glycoprotein (MUC1) promoter, prostate-specific antigen (PSA) promoter, survivorin promoter, tyrosinase-related protein (TRP1) promoter, and tyrosinase promoter.

[0128] In certain embodiments, the promoters of this disclosure may be tumor-specific promoters. The term “tumor-specific promoter,” as used herein, means a promoter that functions to preferentially or exclusively activate gene expression in tumor cells and is inactive or has reduced activity in non-tumor cells or non-tumor cells. Specific examples of tumor-specific promoters, but not limited to, include the E2F-1 promoter, the α-fetoprotein promoter, the cholecystokinin promoter, the carcinoembryonic antigen promoter, the C-erbB2 / neu tumorigenicity promoter, the cyclooxygenase promoter, the CXCR4 promoter, the HE4 promoter, the hexokinase type II promoter, the L-plastin promoter, the MUC1 promoter, the PSA promoter, the survivin promoter, the TRP1 promoter, and the tyrosinase promoter.

[0129] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured to be expressed at the same or different stages in the replication cycle of the modified oncolytic virus. For example, the two polynucleotides may both be driven by an early promoter induced in the early stages of viral replication, or both may be driven by a late promoter induced in the later stages of viral replication, or one may be driven by an early promoter and the other by a late promoter.

[0130] In certain embodiments, the first promoter and the second promoter are the same or different. In certain embodiments, both the first promoter and the second promoter are late promoters. In certain embodiments, the late promoter is a pSL (progressive single promoter).

[0131] In certain embodiments, the third and fourth promoters are the same or different. In certain embodiments, both the third and fourth are early and late promoters. In certain embodiments, the early and late promoters are pSE / L.

[0132] In a particular embodiment, the modified oncolytic virus includes, in the 5' to 3' direction of the sense strand, the following elements in frame: a polynucleotide encoding the light chain of an antibody that binds to CD137 — first early and late promoters — second early and late promoters — a polynucleotide encoding the heavy chain of an antibody that binds to CD137 — a polynucleotide encoding the heavy chain of an antibody that binds to PD-1 — first late promoter — second late promoter — a polynucleotide encoding the light chain of an antibody that binds to PD-1.

[0133] In certain embodiments, the immune checkpoint inhibitor expressed from the first heterologous polynucleotide and the immunoactivator expressed from the second heterologous polynucleotide are expressed as separate proteins. In other words, they are not expressed as a fusion protein and are not linked to each other (neither covalently nor by linkers). In certain embodiments, the immune checkpoint inhibitor expressed from the first heterologous polynucleotide is not fused with any other protein, and the immunoactivator expressed from the second heterologous polynucleotide is not fused with any other protein.

[0134] In certain embodiments, the modified oncolytic virus contains no other heterologous polynucleotides encoding immune checkpoint inhibitors or immunoactivators, except for the first and second heterologous polynucleotides. In certain embodiments, the modified oncolytic virus contains no other proteins encoding heterologous polynucleotides, except for the first and second heterologous polynucleotides.

[0135] Pharmaceutical composition In another embodiment, the Disclosure provides a pharmaceutical composition comprising a modified oncolytic virus as described herein and a pharmaceutically acceptable carrier.

[0136] The term “pharmaceutically acceptable,” as used herein, means a compound, material, composition, and / or dosage form that, within the bounds of full medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that meets a reasonable benefit-to-risk ratio. In certain embodiments, a pharmaceutically acceptable compound, material, composition, and / or dosage form means one that is approved by a regulatory body (e.g., the U.S. Food and Drug Administration, the China Food and Drug Administration, or the European Medicines Agency) or listed in a generally accepted pharmacopoeia (e.g., the United States Pharmacopoeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia) for use in animals, and more specifically in humans.

[0137] The pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the present invention are not limited to these, but include, for example, pharmaceutically acceptable liquid, gel, or solid carriers; aqueous vehicles (e.g., sodium chloride injection, Ringer's solution, isotonic glucose injection, sterile water injection, or Ringer's solution with glucose and lactate); non-aqueous vehicles (e.g., non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil); antimicrobial agents; isotonic agents (e.g., sodium chloride or glucose); and buffers (e.g., phosphate buffer or citrate buffer). These may include, for example, antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / distributing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, auxiliaries, excipients, or non-toxic auxiliaries, other components known in the art, or various combinations thereof. Preferred components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, or emulsifiers.

[0138] In certain embodiments, the pharmaceutical composition is an oral preparation. Oral preparations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (usually sucrose and acacia or tragacanth as flavoring agents), powders, granules, or aqueous or non-aqueous solutions or suspensions, or water-in-oil emulsions or oil-in-water emulsions, or elixirs or syrups, or confectionery tablets (such as gelatin and glycerin, or sucrose or acacia as inert agents), and / or mouthwashes and similar products.

[0139] In certain embodiments, the pharmaceutical composition may be an injectable formulation, such as a sterile aqueous solution, dispersion, suspension, or emulsion. In all cases, the injectable formulation must be sterile and liquid to facilitate injection. It must be stable under manufacturing and storage conditions and resistant to microbial infection (e.g., bacteria and fungi). The carrier may be a solvent or dispersion medium, such as water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof and / or vegetable oils. The injectable formulation must maintain adequate fluidity, which can be maintained in various ways, such as the use of coatings such as lecithin or the use of surfactants. Antimicrobial contamination can be achieved by the addition of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

[0140] In certain embodiments, a unit dose of a parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration must be sterile and not pyrogenic.

[0141] Treatment method In another embodiment, the Disclosure provides a method for treating a tumor, comprising administering an effective amount of the modified oncolytic virus or the pharmaceutical composition of the Disclosure to the target.

[0142] The term “subject” as used herein means human or non-human animal. Non-human animals include all vertebrates, e.g., mammals and non-mammals. “Subject” may also be a domestic animal (e.g., a cattle, pig, goat, chicken, rabbit, or horse), or a rodent (e.g., a rat or mouse), or a primate (e.g., a gorilla or monkey), or a pet (e.g., a dog or cat). “Subject” may be male or female and may also be of different ages. In certain embodiments, subject is human. Human “subject” may be Caucasian, African, Asian, Sumerian, or of other races, or a hybrid of different races. Human “subject” may be elderly, adult, teenager, child, or infant.

[0143] When used herein, the term “tumor” means any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and encompasses both solid and non-solid tumors, such as leukemia. In this disclosure, “tumor” is used interchangeably with the terms “cancer,” “malignant tumor,” “overgrowth,” and “neoplasm.” The term “tumor cell” is interchangeable with the terms “cancer cell,” “malignant cell,” “overgrowth cell,” and “neoplasm,” unless otherwise specified. In certain embodiments, tumor is selected from the group consisting of head and neck tumors, breast tumors, colorectal tumors, liver tumors, pancreatic adenocarcinomas, gallbladder and bile duct tumors, ovarian tumors, cervical tumors, small cell lung tumors, non-small cell lung tumors, renal cell carcinoma, bladder tumors, prostate tumors, bone tumors, mesothelioma, brain tumors, soft tissue sarcomas, uterine tumors, thyroid tumors, nasopharyngeal carcinomas, and melanomas. In certain embodiments, tumor is a solid tumor. In certain embodiments, the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, squamous cell carcinoma of the head and neck, bladder cancer, colorectal cancer, or hepatocellular carcinoma. In certain embodiments, the tumor was refractory to previous treatments (e.g., separate administrations of oncolytic viruses, immune checkpoint inhibitors, and / or immunoactivators).

[0144] The terms “treating” or “treatment” of a condition, as used herein, include preventing or mitigating a condition, slowing the onset or progression of a condition, reducing the risk of the condition progressing, preventing or delaying the progression of symptoms associated with the condition, reducing or ending symptoms associated with the condition, producing complete or partial improvement of the condition, curing the condition, or any combination thereof. In relation to tumors, “treating” or “treatment” may mean inhibiting or slowing the growth, proliferation or metastasis of neogeneic or malignant cells, preventing or delaying the progression of the growth, proliferation or metastasis of neogeneic or malignant cells, or any combination thereof. In relation to tumors, “treating” or “treatment” may include eradicating all or part of a tumor, inhibiting or slowing the growth and metastasis of a tumor, preventing or delaying the progression of a tumor, or any combination thereof.

[0145] Modified oncolytic viruses and pharmaceutical compositions may be administered via any preferred route known in the art, for example, but not limited to, parenteral, oral, enteral, buccal, nasal, topical, transrectal, transvaginal, transmucosal, epithelial, transdermal, dermal, ophthalmic, transpulmonary, and subcutaneous administration routes. In certain embodiments, the route of administration is topical. In certain embodiments, the route of administration is intratumoral injection.

[0146] In certain embodiments, modified oncolytic viruses and pharmaceutical compositions are administered in a therapeutically effective dose. The term “therapeutic dose,” as used herein, means the amount of a drug that can improve or eliminate a disease or symptom of interest, or the amount of a drug that can preventively inhibit or prevent the onset of a disease or symptom. A therapeutically effective dose may be the amount of a drug that improves one or more diseases or symptoms in a subject to a certain degree; the amount of a drug that can partially or completely restore one or more physiological or biochemical parameters related to the cause of the disease or symptom to a normal state; and / or the amount of a drug that can reduce the likelihood of the disease or symptom occurring.

[0147] The therapeutically effective dose of modified oncolytic viruses and pharmaceutical compositions depends on various factors known in the art, such as body weight, age, existing medical status, current treatments, the health status of the subject, as well as the intensity of drug interactions, allergies, hyperallergenicity and side effects, as well as the route of administration and the degree of disease progression. A person skilled in the art (e.g., a physician or veterinarian) may reduce or increase the dose in accordance with these and other conditions or requirements.

[0148] In a particular embodiment, the modified oncolytic virus and pharmaceutical composition are about 10 4 From PFU 14 PFU (for example, about 10 4 PFU, approx. 2×10 4 PFU, approx. 5×10 4 PFU, approx. 10 5 PFU, approx. 2×10 5 PFU, approx. 5×10 5 PFU, approx. 10 6 PFU, approx. 2×10 6 PFU, approx. 5×10 6 PFU, approx. 10 7 PFU, approx. 2×10 7 PFU, approx. 5×10 7 PFU, approx. 10 8 PFU, approx. 2×10 8 PFU, approx. 5×10 8PFU, approx. 10 9 PFU, approx. 2×10 9 PFU, approx. 5×10 9 PFU, approx. 10 10 PFU, approx. 2×10 10 PFU, approx. 5×10 10 PFU, approx. 10 11 PFU, approx. 2×10 11 PFU, approx. 5×10 11 PFU, approx. 10 12 PFU, approx. 2×10 12 PFU, approx. 5×10 12 PFU, approx. 10 13 PFU, approx. 2×10 13 PFU, approx. 5×10 13 PFU, or about 10 14 It may be administered in a therapeutically effective dose of PFU. In certain embodiments of these, the modified oncolytic virus and pharmaceutical composition are about 10 11 It is administered in doses of 5 × 10⁻¹⁰ or less. In certain embodiments of these, the dose is 5 × 10⁻¹⁰. 10 PFU or less, 2×10 10 PFU or less, 5×10 9 PFU or less, 4×10 9 PFU or less, 3×10 9 PFU or less, 2×10 9 PFU or lower, or 10 9 The dose is less than or equal to PFU. A specific dose can be divided and administered in multiple doses at intervals, for example, once a day, twice a day or more, twice a month or more, once a week, once every two weeks, once every three weeks, once a month, or once every two months or more. In a particular embodiment, the dose administered may change during the course of treatment. For example, in a particular embodiment, the initial dose administered may be greater than subsequent doses. In a particular embodiment, the dose administered is adjusted during the course of treatment in accordance with the response of the subject.

[0149] The term "PFU," as used herein, means plaque-forming unit, which is an indicator of the number of particles capable of forming plaque.

[0150] The administration plan may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, or divided doses may be administered over time.

[0151] combination In certain embodiments, the pharmaceutical composition may be used in combination with one or more other drugs. In certain embodiments, the composition comprises at least one other drug.

[0152] In certain embodiments, the other drug is an antitumor agent. Any agent known to be active against tumors may be used as an antitumor agent. In certain embodiments, the antitumor agent is selected from the group consisting of chemical agents, polynucleotides, peptides, proteins, or any combination thereof.

[0153] In certain embodiments, the antitumor agent is a chemical agent. Specific examples of antitumor chemical agents include, but are not limited to, mitomycin C, daunorubicin, doxorubicin, etoposide, tamoxifen, paclitaxel, vincristine, and rapamycin.

[0154] In certain embodiments, the antitumor agent is a polynucleotide. Specific examples of antitumor polynucleotides include, but are not limited to, antisense oligonucleotides, such as bcl-2 antisense oligonucleotides, clatherin antisense oligonucleotides, and c-myc antisense oligonucleotides, as well as RNA capable of RNA interference (such as small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), and microinterfering RNAs (miRNAs)), such as anti-VEGF siRNAs, shRNAs, or miRNAs, anti-bcl-2 siRNAs, shRNAs, or miRNAs, and anti-claudin-3 siRNAs, shRNAs, or miRNAs.

[0155] In certain embodiments, the antitumor agent is a peptide or protein. Specific examples of antitumor peptides or proteins include, but are not limited to, antibodies such as trastuzumab, rituximab, edrecolomab, alemtuzumab, daclizumab, nimotuzumab, gemtuzumab, ibritumomab, and edrecolomab, and protein-based therapeutic agents such as endostatin, angiostatin K1-3, leuprolide, sex hormone-binding globulin, and bicinin.

[0156] medical use In another embodiment, the Disclosure provides the use of a modified oncolytic virus or a pharmaceutical composition of the Disclosure in the manufacture of a pharmaceutical for treating a tumor.

[0157] In another embodiment, the Disclosure provides a modified oncolytic virus or a pharmaceutical composition of the Disclosure for use in the treatment of tumors. [Examples]

[0158] The embodiments described below are described in detail to aid in understanding this disclosure and should not be construed in any way as limiting the scope of the invention as defined in the subsequent claims.

[0159] [Example 1] Virus Construction The starting material, a WR strain of vaccinia virus, was obtained from ATCC (www.atcc.org: VR-1354). WR-GS-600 was constructed using a stepwise recombination approach based on the involved synonymous genes. In short, the first step involved inserting a marker / selection gene into the TK locus by recombining WR DNA with a modified pSEM-1 vector (Rintoul et al., 2011). This allows for easy differentiation from wild-type parents for further genetic manipulation. Subsequently, recombinant plasmids containing the adjacent J1R and J3R sequences, encoding anti-human PD-1 (amino acid sequence of anti-human PD-1 and nucleic acid sequence encoding anti-human PD-1 shown in Figures 15 and 16, respectively) and anti-human 4-1BB (amino acid sequence of anti-human 4-1BB and nucleic acid sequence encoding anti-human 4-1BB shown in Figures 17 and 18, respectively), were transfected into WR-infected U2OS cells. Figure 1 shows the structure of WR-GS-600 with thymidine kinase (TK) deletion, anti-PD-1 antibody, and anti-4-1BB antibody insertion, while Figure 3 shows a schematic diagram of the recombination steps that produce WR-GS-600.

[0160] Recombination was performed using U2OS cells from a characterized master working cell bank. Three rounds of plaque purification were performed using U2OS cells, followed by one round using HeLa cells. A filtration step using a 0.65 μm filter was then incorporated to ensure that the selected final plaques were clonal. Detailed information is provided in Table 1 below.

[0161] [Table 1]

[0162] After further plaque purification, antibody expression was monitored by immunofluorescence and flow cytometry. U2OS and HeLa cells were separately infected either with a simulated infection (i.e., with a control solution without the virus), with a control virus exhibiting antibody expression, or with purified clones of WR-GS-600.

[0163] Finally, the only clone with a verified DNA sequence and high levels of antibody expression was collected in two roller bottles (1700cm³). 2 Cells were grown in ) and pelletized, then resuspended in 1 mMTris at pH 9.0. After one round of freeze-thaw cycles (-80 / 37°C), the mixture was pelletized again. The supernatant was divided into 1 ml portions into 12 cryogenic tubes (pre-Master Virus Bank). The pelleted cells were resuspended in 3 ml of 1 mMTris at pH 9.0 and subjected to further freeze / thaw cycles. The supernatant was collected after pelletizing, then treated with benzoase overnight and purified with sucrose. The titers of pre-MVB and purified benzoase were identified using U2OS cells. The titers were 1.0–2.1 × 10⁶ for a total of 5 mL of stock. 9 It was found to be in the range of PFU / mL, which is similar to that of the parent WR virus.

[0164] WR-GS-610 (with insertion of the gene encoding anti-human 4-1BB) and WR-GS-620 (with insertion of the gene encoding anti-human PD-1) were manufactured using the same protocol as WR-GS-600, except that both the gene encoding anti-human PD-1 and the gene encoding anti-human 4-1BB were inserted into WR-GS-600. Figure 2 shows the structure of WR-GS-620 with TK deletion and anti-4-1BB antibody insertion, and Figure 4 shows a schematic diagram of the recombination steps for WR-GS-620.

[0165] [Example 2] Characterization of WR-GS-600, WR-GS-610, and WR-GS-620 During the genetic engineering process of these novel viruses, the integrity of their genomes and protein expression were monitored in detail.

[0166] PCR, sequencing, and restriction digestion The viral preparation was treated with benzonase endonuclease, pelletized with sucrose, then treated with proteinase K and a surfactant, and subsequently the DNA was extracted. This DNA was recovered using phenol / chloroform / isoamyl alcohol extraction, and the purified sucrose cushion was precipitated with ethanol to isolate the viral genomic DNA.

[0167] To ensure that the viral genome contained the expected sequence, including the designed antibody sequence, a set of primers was designed, including those within the recombinant region and those outside the genetically modified section. The identity of the viruses (WR-GS-600, WR-GS-610, and WR-GS-620) was confirmed by qPCR (TaqMan). The primers used for PCR are shown in Table 2. The positions of the primers in the viral genome are shown in Figures 5 to 7, where the expected band sizes of the PCR are also shown. The results of PCR amplification of the genomic DNA of WR-GS-600, WR-GS-610, and WR-GS-620 are shown in Figure 14.

[0168] TK deletion was also verified by Sanger sequencing. Figures 8, 9, and 10 show the genetic changes from WR after insertion of the antibody-coding gene. Sanger sequencing alignment of the WR-GS-600 viral genome was performed against the DNA sequence designed to express anti-hu4-1BB and anti-huPD-1 in WR-GS-600. The alignment showed that the WR-GS-600 viral genome was identical to the designed DNA sequence.

[0169] [Table 2]

[0170] The restriction enzyme HindIII cleaved around the TK region of WR, producing a 5004 bp band. When the TK was deleted and anti-huPD-1 and / or anti-hu4-1BB antibodies were inserted into WR-GS-600, two extra HindIII restriction enzyme recognition sites were introduced, producing three bands of 1638 bp, 2548 bp, and 4666 bp, respectively. In WR-GS-620, the 5004 bp band of wild-type WR was replaced by two bands of 1922 bp and 4666 bp. These differences in restriction enzyme digestion patterns can be used for rapid identification of these viruses. The results are shown in Table 3.

[0171] [Table 3(1)]

[0172] Immunofluorescence Transgenic expression of human antibodies was verified by immunofluorescence against human IgG (see Figure 11). FITC-conjugated goat anti-human IgG (H+L) (Invitrogen, Cat #62-8411) was used to stain virus-infected U2OS cells.

[0173] Flow cytometry analysis Flow cytometry analysis of HeLa cells infected with simulated infection, control WR virus (WR-mCherry), WR-GS-600, and WR-GS-620 was performed separately to confirm the specific expression of human antibodies in cells infected with WR-GS-600 and WR-GS-620. Human IgG detected in the infected supernatant by Western blotting provided further evidence of human antibody expression.

[0174] Western blot method Western blotting to detect human IgG from infected supernatant provided further evidence of antibody expression. Figures 12 and 13 show the expression of anti-PD-1 and anti--41-BB antibodies by recombinant viruses (WR-GS-600, WR-GS-610, and WR-GS-620) using cell lysates and supernatants, as measured by Western blotting.

[0175] Functional characterization of expressed anti-PD-1 antibodies using PD-1-conjugated ELISA Recombinant human PD-1Fc chimera (R&D Systems, Minneapolis, MN) is resuspended in Dulbecco's phosphate-buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA) to 0.2 mg / ml, and then diluted with DPBS to a final concentration of 0.03 μg / ml. Nunc-Immuno Maxisorp 96-well plates are coated with the recombinant PD-L1-Fc chimera at a rate of 0.1 mL per well, with empty wells for nonspecific binding controls, and incubated overnight at 4°C. The coating solution is removed, and the plates are washed with wash buffer (0.05% Tween-20 in DPBS, 200 μL per well in each batch). Blocking buffer (5% skim milk powder, 0.05% Tween-20 in DPBS, 200 μL per well in each batch) is added to all wells, and incubated at 4°C for 1 hour with mixing. Remove the blocking buffer and wash the plate with wash buffer. Serially dilute the WR-GS-620 supernatant and WR-GS-600 supernatant with DPBS and add the diluted supernatant (100 μL per well) to the plate. Incubate the plate at room temperature for 1.5 hours. Remove the supernatant solution containing the antibody and wash the plate with wash buffer. Dilute horseradish peroxidase-labeled goat anti-human IgG, F(ab')2-specific F(ab')2 antibody (Jackson Immunoresearch, Westglobe, PA) with DPBS and add 100 μL per well to the plate. Incubate the plate at room temperature for 1 hour and wash with wash buffer. Add 100 μL per well of SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Labs, Gaizersberg, MD) and incubate at room temperature for 20 minutes. The reaction was stopped by adding the same amount of 2M H2SO4, and the absorbance at 450nm was read using a Molecular Devices Spectra Max 340 (Molecular Devices, Sunnybell, CA).

[0176] For analysis, supernatant from U2OS infected with MOI 0.05 for 48 hours was used, and the results are shown in Figure 19. These results suggest that the anti-PD-1 antibody expressed in GS-620 can specifically bind to PD-1, and that this binding is concentration-dependent.

[0177] Functional characterization of expressed anti-4-1BB antibodies using 4-1BB conjugated ELISA Human 4-1BBIgG1Fc chimera (R&D Systems, Minneapolis, MN) is resuspended in Dulbecco's phosphate-buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA) to 0.2 mg / ml, and then diluted with DPBS to a final concentration of 0.03 μg / ml. Nunc-Immuno Maxisorp 96-well plates are coated with recombinant 4-1BB chimera at a rate of 0.1 mL per well, with empty wells for nonspecific binding controls, and incubated overnight at 4°C. The 4-1BB solution is removed, and the plates are washed with wash buffer (0.05% Tween-20 in DPBS). Blocking buffer (5% skim milk powder, 0.05% Tween-20 in DPBS) is added to all wells, and incubated at 4°C for 1 hour with mixing. The blocking buffer is removed, and the plates are washed with wash buffer. Serial dilutions of WR-GS-610 and WR-GS-600 supernatants are prepared in DPBS, and the diluted supernatants are added to the plate. The plate is incubated at room temperature for 1.5 hours. The supernatant solution containing the antibody is removed, and the plate is washed with wash buffer. Horseradish peroxidase-labeled goat anti-human IgG, F(ab')2-specific F(ab')2 antibody (Jackson Immunoresearch, Westglobe, PA) is diluted in DPBS and added to the plate. The plate is incubated at room temperature for 1 hour and washed with wash buffer. SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Labs, Gaizersberg, MD) is added, and the plate is incubated at room temperature for 20 minutes. The reaction is stopped by adding an equal volume of 2M H2SO4, and the absorbance at 450 nm is read using a Molecular Devices Spectra Max 340 (Molecular Devices, Sunnybell, CA).

[0178] For analysis, supernatant from U2OS infected with MOI 0.05 for 48 hours was used, and the results are shown in Figure 20. These results suggest that anti-4-1BB antibodies expressed in GS-600 and GS-610 can specifically bind to 4-1BB, and that this binding is concentration-dependent.

[0179] The above tests demonstrate that WR-GS-600, WR-GS-610, and WR-GS-620 are well-constituted and capable of expressing functionally corresponding antibodies (anti-PD1 antibodies against WR-GS-600 and WR-GS-620, and anti-4-1BB antibodies against WR-GS-600 and WR-GS-610).

[0180] [Example 3] In vivo study of recombinant WR-GS-610 and WR-GS-620 viruses The following studies will be conducted to determine whether the recombinant WR-GS-600, WR-GS-610, and WR-GS-620 viruses are safe for mice and whether the recombinant viruses can target and enter tumors in mice. The delivery route will be intravenous (IV) or intraperitoneal (IP). All animal studies will be conducted in accordance with the guidelines of the local animal experimentation committee.

[0181] Measurement of cytotoxicity (cell lethality data) of WR-GS-600, WR-GS-610, and WR-GS-620 in CT26, MC38, HT-29, and HCT-116 cell lines. For in vitro cytotoxicity studies, colorectal cancer cell lines CT26-LacZ (mouse), MC38-Luc (mouse), HT-29-Luc (human), and HCT-116-Luc (human) were used. WR-GS-600, WR-GS-610, and WR-GS-620 were prepared to three different MOIs, namely 0.01 MOI (3E2 PFU), 0.1 MOI (3E3 PFU), and 1.0 MOI (3E4 PFU), respectively. Measurements were performed at three different time points: 24 hours, 48 ​​hours, and 72 hours.

[0182] Cell preparation: First, each cell line was seeded into two 15 cm tissue culture dishes and incubated until subconfluent between 75 and 90%. The cells were washed and counted using conventional methods known to those skilled in the art. Approximately 3E4 cells were seeded into each well of a 96-well flat-bottom plate. Each cell type requires nine plates for nine different experimental conditions.

[0183] Virus Dilution Preparation: The viruses were thawed on ice, and then thawed in a 37°C water bath to ensure complete thawing. The thawed viruses were vortexed twice at maximum speed for 20 seconds each time. WR-GS-600, WR-GS-610, and WR-GS-620 viruses were prepared to three different concentrations: MOI 1.0, MOI 0.1, and MOI 0.01. 50 μL of virus was added to the corresponding wells, and then the 96-well flat-bottom plate was gently shaken in four quadrants to mix. The plate was incubated at 37°C with 5% CO2 supplementation. MOI 1.0 corresponds to 3E4 PFU / 50 μL, or 600 PFU / μL, or 6E5 PFU / mL. MOI 0.1 corresponds to 3E3 PFU / 50 μL, or 60 PFU / μL, or 6E4 PFU / mL. MOI 0.01 corresponds to 3E2PFU / 50μL, or 6PFU / μL, or 6E3PFU / mL.

[0184] Using conventional methods known to those skilled in the art, cell viability was calculated by six replicate tests for each condition using Alamar Blue to detect the cytotoxicity of the virus in the four cell lines mentioned above. Figures 21–23 show that there were no significant differences in cell viability of the four cell types mentioned above after treatment with WR, WR-GS-600, WR-GS-610, and WR-GS-620 at three different concentrations and three different time points. This suggests that the incorporation of polynucleotide sequences for checkpoint inhibitor antibodies into the vaccinia virus (WR) genome does not alter the cytotoxicity of the virus. Figures 21–23 further show that, in response to viral treatment, the cell viability of HT-29 and HCT-116 cell lines was significantly reduced compared to CT-26 and MC-38, indicating that human cancer cells are more sensitive to viral infection and viral cytotoxicity.

[0185] Measurement of the in vivo distribution of viral vectors Organizational homogenization: One 25Balb / C mouse (Jackson Lab) from five different groups was sacrificed at a time. After disinfection spraying, the mice were dissected, and 50-100 mg of either the tumor, lung, spleen, liver, brain, or ovary was excised. The remaining tissue was flash-frozen by OCT. The excised tissue was weighed and placed in 2.0 mL Eppendorf tubes. The tissue samples were frozen overnight at -80°C. The following day, the tissue samples were homogenized in a manner known to those skilled in the art. Briefly, two autoclaved 5 mm TissueLyser beads were dispensed into each tube. A total of 48 tubes were placed in the TissueLyser. Homogenization was performed for 1 minute at 28 Hz. After this, the adapter was reversed 180° and homogenization was performed for another minute to achieve uniform homogenization. After this, 500 μL of DMEM was added to each sample. The tubes were centrifuged at 3500g for 2 minutes. The supernatant was transferred to a 1.5 mL Eppendorf tube and stored at -80°C until titration.

[0186] 24-well format for titer measurement: U2OS cells were used for viral titer measurement, and 10E2 PFU / mL JX594 stocks (a) and 31.0 PFU / mL JX594 stocks (b) were prepared and used as positive controls.

[0187] Three concentrations of each virus, WR-GS-600, WR-GS-610, and WR-GS-620, were prepared for five tissues: brain (B), liver (V), lung (L), ovary (O), and spleen (S): (1) 150 μL of pure virus for infection; (2) 98 μL from (1) in 212 μL of DMEM, mixed, and 150 μL taken for infection; and (3) 98 μL from (2) in 212 μL of DMEM, mixed, and 150 μL taken for infection.

[0188] Compared to the control (C), U2OS cells were treated with (1) 150 μL of 10E2 PFU / mL JX594 stock (a); (2) 150 μL of 31.0 PFU / mL JX594 stock (b); or (3) 150 μL of DMEM.

[0189] Six concentrations were prepared for each of the WR-GS-600, WR-GS-610, and WR-GS-620 viruses against tumors (T): (1) 150 μL of pure virus for infection; (2) 98 μL from (1) in 212 μL of DMEM, mixed, and 150 μL taken for infection; (3) 98 μL from (2) in 212 μL of DMEM, mixed, and 150 μL taken for infection; (4) 98 μL from (3) in 212 μL of DMEM, mixed, and 150 μL taken for infection; (5) 98 μL from (4) in 212 μL of DMEM, mixed, and 150 μL taken for infection; and (6) 98 μL from (5) in 212 μL of DMEM, mixed, and 150 μL taken for infection.

[0190] A 24-well plate was set up as shown in Figure 24. Tumor, lung, spleen, liver, brain, and ovarian cells prepared from one mouse were seeded into each plate using the method described in the section on tissue homogenization above.

[0191] As mentioned above, 25 mice were divided into 5 groups, each group containing 5 mice, i.e., 5 plates. In Group 1, tumor, lung, spleen, liver, brain, and ovarian cells were infected with WR-GS-610; in Group 2, WR was used; in Group 3, WR-GS-620; in Group 4, WR-GS-600; and all cells in Group 5 (excluding the positive control well cells) were treated with formulation buffer (FB) as a negative control. This formulation buffer contained 30 mM Tris, 10% sucrose, and 150 mM NaCl at pH 7. Figure 25 shows that WR-GS-610 viral plaques were present only in the tumor cell wells. Figure 26 shows that WR viral plaques were present in both the tumor cell wells and the ovarian cell wells. Figure 27 shows that WR-GS-620 viral plaques were present in both the tumor cell wells and the ovarian cell wells. Figure 28 shows that WR-GS-600 viral plaques are present only in tumor cell wells. Figure 29 shows that viral plaques are absent in tumor cell wells in group 5. These data suggest that WR-GS-600 and WR-GS-610 can target tumors more specifically than WR-GS-620 and WR.

[0192] In vivo virus distribution in injected subcutaneous tumors and other tissues Objective: Safety and in vivo distribution of viral vectors Research Protocol Order i.35Balb / C mice (Charles River). The mice will be divided into five treatment groups: PBS control (FB), and WR, WR-GS-600, WR-GS-610, and WR-GS-620. ii. When the tumors in the tumor group reach a size of 5 mm, treatment should be initiated. iii. Three doses of the virus administered via tail vein infusion (planned for days 1, 4, and 7) iv. Monitor the weight and health status of the mice. v. On day 9, mice are sacrificed and tissues are collected from the brain, lungs, liver, ovaries, and spleen. Plaque assays in U2OS cells are used to identify vaccinia titers in different tissues.

[0193] Tables 3-5 below summarize the treatment groups, treatment schedules, anesthesia, endpoints, and euthanasia.

[0194] [Table 3(2)]

[0195] [Table 4]

[0196] [Table 5]

[0197] Data were presented by taking the average of 5 mice per group. Figures 30–32 show that WR-GS-600 and WR-GS-610 were rather present in tumors, with only small amounts of WR-GS-600 and WR-GS-610 viruses detected in the ovaries, brain, spleen, liver, and lungs. In contrast, large amounts of WR-GS-620 virus were observed in tumors, ovaries, brain, spleen, liver, and lungs after intratumoral injection. These data support the idea that WR-GS-600 and WR-GS-610 have significantly higher tumor targeting specificity than WR-GS-620. Furthermore, the second and third bars in the bar graphs of Figures 30-32 show that the PFU values ​​per gram of tissue for WR-GS-600 and WR-GS-610 were similar in the ovaries (WR-GS-610 was slightly higher than WR-GS-600), brain, spleen, liver, and lungs, and that the PFU values ​​per gram of tumor tissue for WR-GS-600 were approximately three times higher than those for WR-GS-610. This suggests that, when other cells were similarly infected with WR-GS-600 and WR-GS-610, WR-GS-600 could infect tumors more effectively than WR-GS-610.

[0198] Previous studies have shown that the vaccinia Western Reserve strain can infect organs in normal mice, particularly the ovaries (Zhao Y. et al, Viral Immunology, 2011, 24, 387), which is consistent with the results shown in Figure 31.

[0199] In summary, these data suggest that the incorporation of a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immunoactivator into oncolytic viruses, such as WR, results in a synergistic effect of tumor targeting and infection, otherwise such a synergy cannot be achieved by wild-type oncolytic viruses or modified oncolytic viruses containing only the first or only the second heterologous polynucleotide.

[0200] Measurement of tumor size changes in CT-26 mouse tumor models after different viral infections. CT26 tumors were transplanted into 25Balb / C mice (Jackson Lab) (CT-26 LacZ 5E6 cells, SG, right flank). The mice were further divided into five treatment groups: formulation buffer (FB), WR, WR-GS-600, WR-GS-610, and WR-GS-620. Treatment was initiated when the tumors in each group reached a size of 5 mm. Different amounts of 1E7 PFU of virus were injected into the tumors on days 1, 4, and 7, and the mice's body weight and health status were monitored. After tumor growth, tumor size was measured using calipers.

[0201] The tumor size changes were recorded, and the results are summarized in Figure 33, where the percentage change in tumor volume on day x is calculated by comparing the tumor volume on day 1 with the tumor volume on day x. Figure 33 shows that the increase in tumor volume size on days 1, 4, and 7 after virus infusion, when treated with WR, WR-GS-600, WR-GS-610, and WR-GS-620, was significantly smaller than when treated with formulation buffer (FB), suggesting the in vivo tumor inhibitory effect of the virus mentioned above.

[0202] Measurement of effectiveness in a syngeneic mouse model A subcutaneous CT-26LacZ tumor model was prepared in Balb / C mice. Different 1E7 viruses were injected via tail vein infusion on days 1, 3, and 7, and the mice's body weight and health status were monitored. The endpoint was tumor >1,700 mm. 3 The settings were adjusted, and the study was concluded on day 31. The results of mouse survival are shown in Figure 34.

[0203] Measurement of tumor size changes in humanized HT-29-Luc subcutaneous tumor models after different viral infections. The experiment in question can be briefly described as follows.

[0204] Day 1: Order 30 Rag2 - / -IL2Rg null mice (Jackson Lab) and transplant an HT-29 tumor (HT-29 Luc 5E6 cells, SQ, right flank).

[0205] Day 7: Check for tumor growth using IVIS.

[0206] Day 8: Administer 5.8E6 human PBMC IP by intravenous infusion.

[0207] Day 14: Group IVIS assignment.

[0208] Day 15: 1E7 IT processing.

[0209] Day 18: IVIS and 1E7 IT processing.

[0210] Day 24: IVIS.

[0211] Confirmation of human peripheral blood mononuclear cell transplantation The treated mice were subjected to submandibular hemorrhage, and 100 μL of blood was collected and added to sodium heparin. The red blood cells were lysed, and the fluorescence results stained for hCD45, CD3, CD8, and CD4 were read using LSR Fortessa, and the results are summarized in Figures 35 and 36, which confirm that human peripheral blood mononuclear cells were successfully transplanted into immunodeficient mice.

[0212] The changes in tumor volume after viral infection are summarized in Figures 37 and 38. Figure 37 shows that, compared to the control group, mice treated with WR-GS-600 showed the smallest increase in tumor volume compared to mice treated with WR-GS-610, WR-GS-620, or WR. More interestingly, mice infected with WR-GS-600 showed little increase in tumor size at 32 days after HT-29-Luc injection, and even decreased from 8 days after WR-GS-600 treatment. Figure 38 shows that WR and WR-GS-620 have earlier endpoints than WR-GS-600 and WR-GS-610, due to the higher toxicity of WR and WR-GS-620 compared to WR-GS-600 and WR-GS-610. Figure 38 further shows that WR-GS-600 and WR-GS-610 can control tumor growth when compared to formulation buffers. In summary, these data suggest that WR-GS-600 and WR-GS-610 have lower toxicity than WR and WR-GS-620, and that both WR-GS-600 and WR-GS-610 can control tumor growth.

[0213] Figures 39 and 40 show that human tumor HT-29 grows in NCG mice with and without human PBMCs, as measured by in vivo imaging IVIS (The IVIS spectrum, PerkinElmer).

[0214] Figures 41 and 42 show that in a humanized HT-29-Luc intraperitoneal mouse model in which the virus is injected into the peritoneal cavity, WR-GS-600 and WR-GS-620 infection can significantly reduce the chemiluminescence intensity of tumors, suggesting the tumor inhibitory efficiency of WR-GS-600 and WR-GS-620. Compared with the formulation buffer, WR and WR-GS-610 showed a smaller increase in the chemiluminescence intensity of tumors, suggesting the tumor growth control efficacy of WR and WR-GS-610.

[0215] The aforementioned in vitro and in vivo results indicate that WR, WR-GS-600, WR-GS-610, and WR-GS-620 can induce cancer cell death and control tumor growth. However, WR and WR-GS-620 exhibit higher toxicity, which has led to early termination of drug trials. WR-GS-600 and WR-GS-610 are more effective in controlling tumor growth, with WR-GS-600 exhibiting higher tumor targeting specificity than WR-GS-610. More importantly, in a humanized HT-29 intraperitoneal tumor mouse model, intraperitoneal injection of WR-GS-600 reduced tumor size, while intraperitoneal injection of WR-GS-610 did not halt tumor size increase, although the percentage of tumor size increase was significantly smaller than when treated with WR. These data suggest that the incorporation of both heterologous polynucleotides encoding immune checkpoint inhibitors and heterologous polynucleotides encoding immunoactivators can reduce toxicity, increase tumor targeting specificity, and improve tumor control efficacy.

[0216] With regard to the use of substantially any plural and / or singular terminology, a person skilled in the art can translate from plural to singular and / or singular to plural as appropriate for the context and / or application.

[0217] Furthermore, if the features and aspects of this disclosure are described in terms of the Markush group, a person skilled in the art will understand that the disclosure can also be described in terms of any individual member or subgroup of a member of the Markush group.

[0218] While various aspects and embodiments are disclosed herein, other aspects and embodiments will also be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A modified oncolytic virus comprising a viral genome having a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immunoactivator, The immune checkpoint inhibitor is an antibody or its antigen-binding fragment that specifically binds to PD-1, and the immunoactivator is an antibody or its antigen-binding fragment that specifically binds to CD137. The antibody that specifically binds to PD-1 comprises a primary heavy chain containing HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 3, and HCDR3 shown in SEQ ID NO: 4, and a primary light chain containing LCDR1 shown in SEQ ID NO: 9, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO:

11. The antibody that specifically binds to CD137 comprises a double chain containing HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, and HCDR3 shown in SEQ ID NO: 19, and a second light chain containing LCDR1 shown in SEQ ID NO: 24, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO:

26. The viral genome includes at least one deletion or disruption that enables the virus to selectively replicate within tumor cells. The deletion or disruption is located in an open reading frame (ORF) that encodes at least a portion of an enzyme essential for the replication of the virus and which is preferentially expressed in tumor cells rather than non-tumor cells. The enzyme is thymidine kinase. Modified oncolytic virus.

2. A modified oncolytic virus according to claim 1, selected from the group consisting of vaccinia, adenovirus, reovirus, measles, herpes simplex virus, Semryqui forest fever virus, Venezuelan encephalitis virus, parvovirus, avian anemia virus, measles virus, coxsackievirus, varicella stomatitis virus, Seneca Valley virus, Maraba virus, and Newcastle disease virus.

3. A modified oncolytic virus according to claim 1, which is attenuated and can replicate in tumor cells.

4. A modified oncolytic virus according to claim 2, derived from the Western Reserve strain.

5. The modified oncolytic virus according to claim 1, wherein the primary heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 5 or a homologous sequence having at least 90% sequence identity thereto.

6. The modified oncolytic virus according to claim 5, wherein the primary heavy chain comprises the amino acid sequence of SEQ ID NO: 6 or a homologous sequence having at least 80% sequence identity.

7. The modified oncolytic virus according to any one of claims 1 to 6, wherein the first light chain includes a variable region having the amino acid sequence of SEQ ID NO: 12 or a homologous sequence having at least 90% sequence identity thereto.

8. The modified oncolytic virus according to claim 7, wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 13 or a homologous sequence having at least 80% sequence identity.

9. The modified oncolytic virus according to any one of claims 1 to 8, wherein the double helix comprises a variable region having the amino acid sequence of SEQ ID NO: 20 or a homologous sequence having at least 90% sequence identity thereto.

10. The modified oncolytic virus according to claim 9, wherein the double helix comprises the amino acid sequence of SEQ ID NO: 21 or a homologous sequence having at least 80% sequence identity.

11. The modified oncolytic virus according to any one of claims 1 to 10, wherein the second light chain includes a variable region having the amino acid sequence of SEQ ID NO: 27 or a homologous sequence having at least 90% sequence identity thereto.

12. The modified oncolytic virus according to claim 11, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 29 or a homologous sequence having at least 80% sequence identity.

13. The modified oncolytic virus according to claim 1, wherein the first heterologous polynucleotide and the second heterologous polynucleotide are inserted at the site of the deletion.

14. The modified oncolytic virus according to claim 13, wherein the first heterologous polynucleotide is located immediately upstream or immediately downstream of the second heterologous polynucleotide.

15. The modified oncolytic virus according to claim 1, wherein the first heterologous polynucleotide encodes the first heavy chain and the first light chain of the first antibody.

16. The modified oncolytic virus according to claim 15, wherein the first heterologous polynucleotide further comprises a first promoter capable of driving the expression of the first heavy chain and a second promoter capable of driving the expression of the first light chain, and the first promoter and the second promoter are oriented head-to-head.

17. The modified oncolytic virus according to claim 1, wherein the second heterologous polynucleotide encodes the double hemisphere and the second light chain of the second antibody.

18. The modified oncolytic virus according to claim 17, wherein the second heterologous polynucleotide further comprises a third promoter capable of driving the expression of the double helix and a fourth promoter capable of driving the expression of the second light chain, and the third promoter and the fourth promoter are oriented head-to-head.

19. The modified oncolytic virus according to claim 1, wherein the first heterologous polynucleotide and the second heterologous polynucleotide are configured to be expressed at the same or different stages in the replication cycle of the modified oncolytic virus.

20. The modified oncolytic virus according to claim 16, wherein the first promoter and the second promoter are the same or different.

21. The modified oncolytic virus according to claim 16, wherein both the first promoter and the second promoter are late promoters.

22. The modified oncolytic virus according to claim 21, wherein the late promoter is pSL.

23. The modified oncolytic virus according to claim 18, wherein the third promoter and the fourth promoter are the same or different.

24. The modified oncolytic virus according to claim 18, wherein both the third promoter and the fourth promoter are early and late promoters.

25. The modified oncolytic virus according to claim 24, wherein the initial and late promoters are pSE / L.

26. A modified oncolytic virus according to claim 1, comprising, in the 5' to 3' direction of the sense strand, the following elements in frame: a polynucleotide encoding the light chain of an antibody that binds to CD137 - first early and late promoters - second early and late promoters - a polynucleotide encoding the heavy chain of an antibody that binds to CD137 - a polynucleotide encoding the heavy chain of an antibody that binds to PD-1 - first late promoter - second late promoter - a polynucleotide encoding the light chain of an antibody that binds to PD-1.

27. The modified oncolytic virus according to claim 1, wherein the immune checkpoint inhibitor expressed from the first heterologous polynucleotide and the immunoactivator expressed from the second heterologous polynucleotide are expressed as separate proteins.

28. A pharmaceutical composition comprising a modified oncolytic virus according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition according to claim 28 for use in a method of treating a tumor, comprising administering to a subject an effective amount of a modified oncolytic virus according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 28.

30. The pharmaceutical composition according to claim 29, wherein the subject is a human.

31. The pharmaceutical composition according to claim 29, wherein the tumor is a solid tumor.

32. The pharmaceutical composition according to claim 29, wherein the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, squamous cell carcinoma of the head and neck, bladder cancer, colorectal cancer, or hepatocellular carcinoma.

33. The pharmaceutical composition according to claim 29, wherein the route of administration is local.

34. The pharmaceutical composition according to claim 33, wherein the administration route is intratumor injection.

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