Modified oncolytic viruses, compositions and uses thereof

Abstract

The present application provides a modified oncolytic virus having a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immune activator. A pharmaceutical composition and a method for treating cancer are also provided, the pharmaceutical composition comprising the modified oncolytic virus, and the method for treating cancer comprises administering the modified oncolytic virus or the pharmaceutical composition to a subject.

Description

Technical Field

[0001] The present disclosure generally relates to modified oncolytic viruses, compositions comprising the modified oncolytic viruses, and uses thereof in tumor treatment. Background Art

[0002] Cancer is diagnosed in more than 14 million people worldwide each year. Despite numerous advances in medical research, cancer accounts for approximately 16% of all deaths.

[0003] Malignant tumors are usually resistant to conventional therapies and represent a major therapeutic challenge. For example, micrometastasis can occur very early in the development of primary tumors. Therefore, at the time of diagnosis, many tumor patients already have micrometastasis. Tumor-reactive T cells can find and destroy these micrometastasis without harming surrounding healthy tissues. However, the naturally occurring T cell response to malignant tumors is usually not enough to cause regression of primary or metastatic tumors.

[0004] Oncolytic viruses have shown potential as anti-tumor agents. Unlike conventional gene therapy, oncolytic viruses can spread in tumor tissues by virtue of viral replication and accompanying cell lysis. However, oncolytic viruses themselves are not sufficient to treat primary or metastatic tumors.

[0005] Therefore, there is a particularly urgent need to enhance the efficacy of oncolytic viruses and eliminate metastatic tumor cells. Summary of the invention

[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 immune activator.

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

[0008] In certain embodiments, the modified oncolytic virus is attenuated and can replicate in tumor cells. In certain embodiments, the viral genome includes at least one deletion or destruction that enables the virus to selectively replicate in tumor cells. In certain embodiments, the deletion or destruction is in an open reading frame (ORF) encoding at least a portion of an enzyme, which is both necessary for the replication of the virus and preferentially expressed in tumor cells compared to non-tumor cells. In certain embodiments, the enzyme is a kinase. In certain embodiments, the enzyme is a 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 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 comprising SEQ ID NO: 5 or a homologous sequence having at least 80% sequence identity thereto. In certain embodiments, the first heavy chain comprises an 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 an amino acid sequence of SEQ ID NO: 12, or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the first light chain comprises an amino acid sequence of SEQ ID NO: 13, or a homologous sequence thereof having at least 80% sequence identity.

[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 certain embodiments, the immune activator is a co-stimulatory activator. In certain embodiments, the immune activator is a second antibody or antigen-binding fragment thereof that binds a co-stimulatory molecule.

[0016] In certain embodiments, 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 certain embodiments, the second antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO:16.

[0018] In certain embodiments, the second antibody or antigen-binding fragment thereof comprises a second heavy chain comprising SEQ ID NOs: 17, 18, and 19. In certain embodiments, the second heavy chain comprises a variable region having an amino acid sequence of SEQ ID NO: 20, or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the second heavy chain comprises an amino acid sequence of SEQ ID NO: 21, or a homologous sequence thereof having at least 80% sequence identity.

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

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

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

[0022] In certain embodiments, 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 thereto.

[0023] In certain embodiments, the immune activator is an NK activator that stimulates NK cell activity. In certain embodiments, the NK activator is a second antibody or an antigen-binding fragment thereof that binds to a NK molecule. In certain embodiments, the NK molecule is selected from the group consisting of sialic acid-binding immunoglobulin-like lectin (Siglec), TIGIT, KIR, and NKG2A / D.

[0024] In certain embodiments, the immune activator is a macrophage activator that stimulates macrophage activity. In certain embodiments, the macrophage activator is a second antibody or an antigen-binding fragment thereof that binds to a macrophage molecule. In certain embodiments, the macrophage molecule is selected from the group consisting of CSF1R, CSF1 kinase, PS, and CD47.

[0025] In certain embodiments, the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and the immune activator is an antibody or an antigen-binding fragment thereof that specifically binds to CD137.

[0026] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are inserted at the location of the deletion.

[0027] In certain embodiments, the first heterologous polynucleotide is immediately upstream of the second heterologous polynucleotide or immediately downstream of the second heterologous polynucleotide.

[0028] In certain embodiments, the first heterologous polynucleotide encodes a first heavy chain and a first light chain of a first antibody. In certain embodiments, the first heterologous polynucleotide further comprises a first promoter capable of driving expression of the first heavy chain and a second promoter capable of driving expression of the first light chain, wherein the first promoter and the second promoter are in a head-to-head orientation.

[0029] In certain embodiments, the second heterologous polynucleotide encodes a second heavy chain and a second light chain of the second antibody. In certain embodiments, the second heterologous polynucleotide further comprises a third promoter capable of driving expression of the second heavy chain and a fourth promoter capable of driving expression of the second light chain, wherein the third promoter and the fourth promoter are in a head-to-head orientation.

[0030] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed in the same or different stages of the replication cycle of the modified oncolytic virus.

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

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

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

[0034] In certain embodiments, the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune activator expressed by the second heterologous polynucleotide are expressed as separate proteins.

[0035] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the modified oncolytic virus of the present disclosure and a pharmaceutically acceptable carrier.

[0036] In another aspect, the present disclosure relates to a method for treating a tumor, comprising administering an effective amount of the modified oncolytic virus of the present disclosure or the pharmaceutical composition of the present disclosure to a subject.

[0037] In certain embodiments, the subject is a human.

[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, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer, or hepatocellular carcinoma.

[0039] In certain embodiments, the route of administration is local administration. In certain embodiments, the route of administration is intratumoral injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Structure showing thymidine kinase (TK) deletion and anti-PD-1 and anti-4-1BB antibody insertion in WR-GS-600.

[0041] Figure 2Structure showing TK deletion and anti-PD-1 antibody insertion in WR-GS-620.

[0042] Figure 3 is a schematic diagram of the recombination steps of WR-GS-600.

[0043] Figure 4 is a schematic diagram of the recombination steps of WR-GS-620.

[0044] Figure 5 Primer positions relative to the GS-600 viral genome are shown.

[0045] Figure 6 Primer positions relative to the GS-610 viral genome are shown.

[0046] Figure 7 Primer positions relative to the GS-620 viral genome are shown.

[0047] Figure 8 Alignment of WR-GS-600 and WR wild-type virus strains is shown.

[0048] Fig. 9 Alignment of WR-GS-610 and WR wild-type virus strains is shown.

[0049] Fig.10 Alignment of WR-GS-620 and WR wild-type virus strains is shown.

[0050] Fig.11 Results showing immunofluorescence detection of human IgG expression. Fig.11 a shows a phase contrast image of U2OS cells. Fig.11 b shows background staining. Fig.11 c shows images of U2OS cells infected with WR-GS-600. Fig.11 d shows images of U2OS cells infected with WR-GS-620.

[0051] Fig.12 Shown are 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 detected two bands with molecular weights of approximately 50 kDa and 25 kDa, which correspond to the heavy and light chains of human antibodies, respectively.

[0052] Fig.13Shown are Western blotting results of human antibodies expressed by recombinant viruses (WR-GS-600, WR-GS-610, and WR-GS-620) using supernatants, where P600, P610, and P620 refer to WR-GS-600, WR-GS-610, and WR-GS-620, respectively. This Western blotting experiment detected two bands with molecular weights of approximately 50 kDa and 25 kDa, which correspond to human antibody heavy and light chains, respectively.

[0053] Fig.14 Bands generated by PCR amplification using WR-GS-600, WR-GS-610, and WR-GS-620 viral DNA are shown.

[0054] Fig.15 The amino acid sequence of the heavy chain of anti-huPD-1 and its coding sequence are shown.

[0055] Fig.16 The amino acid sequence of the light chain of anti-huPD-1 and its coding sequence are shown.

[0056] Fig.17 The amino acid sequence of the heavy chain of anti-hu4-1BB and its coding sequence are shown.

[0057] Fig.18 The amino acid sequence of the light chain of anti-hu4-1BB and its coding sequence are shown.

[0058] Fig.19 ELISA results of PD-1 binding analysis of WR-GS-620 infected supernatants are shown.

[0059] Fig. 20 ELISA results of 4-1BB binding analysis of WR-GS-600 infected supernatants and WR-GS-610 infected supernatants are shown.

[0060] Figure 21-23 Viability of HCT-116, HT-29, MC-38, and CT-26 cells infected with WR, WR-GS-600, WR-GS-610, and WR-GS-620 is shown.

[0061] Fig.24 Shown is the culture plate setup for viral titer determination.

[0062] Fig.25 Representative plate scans of WR-GS-610 virus titer assays are shown.

[0063] Fig.26 Representative plate scans showing WR virus titer assays.

[0064] Fig. 27 Representative plate scans of WR-GS-620 virus titer assays are shown.

[0065] Fig.28 Representative plate scans of WR-GS-600 virus titer assays are shown.

[0066] Fig.29 A representative plate scan of the control group treated with formulation buffer (FB) is shown.

[0067] Fig.30 In vivo virus distribution in tumors after intratumoral injection is shown.

[0068] Fig.31 In vivo virus distribution in the ovary after intratumoral injection is shown.

[0069] Fig.32 In vivo virus distribution in brain, spleen, liver and lung after intratumoral injection is shown. Integrated photon emission (1.928E10 vs. 1.554E10) is considered proportional to the number of tumor cells. Based on the data, GS-600 controls tumor growth.

[0070] Fig.33 Shown are changes in tumor volume in the syngeneic CT-26 murine tumor model following intratumoral injection (IT) of FB, WR, WR-GS-600, WR-GS-610, and WR-GS-620.

[0071] Fig.34 Model showing efficacy in isogenic mouse model treated with different viruses.

[0072] Fig.35 and 36 Shown are flow cytometry results of a humanized HT-29-Luc subcutaneous tumor model injected intravenously with human PBMCs.

[0073] Fig.37 and 38 Shown are the changes in tumor volume of the humanized HT-29 subcutaneous tumor model after intratumoral injection (IT) of FB, WR, WR-GS-600, WR-GS-610, and WR-GS-620.

[0074] Fig.39 Humanized HT-29-Luc subcutaneous tumor model showing tumor staining, where mice were not injected with hPBMCs.

[0075] Fig.40Tumors are shown stained for a humanized HT-29-Luc subcutaneous tumor model in which mice were injected with hPBMCs. 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 was infected with WR-GS-620, the third mouse was infected with WR-GS-610, the fourth mouse was infected with WR, and the fifth mouse was infected with FB. Mice in cage 7 were infected with WR-GS-620.

[0076] Fig.41 Humanized HT-29-Luc intraperitoneal tumor model showing tumor staining.

[0077] Fig.42 The percentage change of chemiluminescence intensity after treatment with different viruses within one week is shown. DETAILED DESCRIPTION

[0078] In one aspect, the present disclosure relates to a modified oncolytic virus comprising a viral genome inserted with a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immune activator.

[0079] Oncolytic viruses

[0080] As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in tumor cells in vitro or in vivo and slow tumor cell growth or induce tumor cell death, while having no effect or minimal effect on normal cells. In certain embodiments, an oncolytic virus contains a viral genome packaged into a virion (or viral particle) and is infectious (i.e., capable of infecting and entering a host cell or subject). In certain embodiments, an oncolytic virus may be a DNA virus or an RNA virus, and may be in any suitable form, such as a DNA viral vector, an RNA viral vector, or a virion.

[0081] As used herein, the term "selective replication" refers to that the replication rate of an oncolytic virus in tumor cells is significantly higher than that of non-tumor cells (e.g., healthy cells). In certain embodiments, an oncolytic virus shows a dissolution rate of at least 50%, 60%, 70%, 80%, 90%, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times, or 1000 times higher in tumor cells compared to non-tumor cells (e.g., healthy cells).

[0082] In certain embodiments, the oncolytic viruses of the present disclosure can selectively replicate in the following: 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).

[0083] The oncolytic viruses disclosed herein can be derived from 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, and those described in Phuangsab et al., 2001, Cancer Lett. 172(1):27-36; Lorence et al., 2007, Curr. Cancer Drug Target Research Recent Progress (Curr. Targets 7(2):157-67; and Freeman et al., 2006, Mol. Ther. 13(1):221-8), a retrovirus (e.g., influenza virus), a myxoma virus, a rhabdovirus (e.g., vesicular stomatitis virus; those described in Stojdl et al., 2000, Nat. Med. 6(7):821-5 and Stojdl et al., 2003, Cancer Cell 4(4):263-75), a picornavirus (e.g., Seneca Valley virus; SW-001 and NTX-010), a coxsackievirus, or a parvovirus.

[0084] In certain embodiments, the oncolytic virus of the present disclosure is derived from poxvirus. As used herein, the term "poxvirus" refers to a virus belonging to the Poxviridae subfamily. In certain embodiments, poxvirus is a virus belonging to the subfamily of vertebrate poxviruses (Chordopoxviridae). In certain embodiments, poxvirus is a virus belonging to the subfamily of orthopoxviruses (Orthopoxvirus). The genomes of various poxviruses, such as vaccinia virus, cowpox virus, canarypox virus, mousepox (Ectromelia) virus, myxoma virus genome sequences can be obtained in the art and in dedicated databases, such as gene banks (Genbank) (accession numbers are NC_006998, NC_003663, NC_005309, NC_004105, NC_001132, respectively).

[0085] In certain embodiments, the oncolytic virus of the present disclosure is derived from vaccinia virus. Vaccinia virus is a member of the poxvirus family, characterized by encoding a large number of viral enzymes and factors that enable the virus to replicate independently of the host cell mechanism. Approximately 190kb double-stranded DNA genome. In certain embodiments, the vaccinia virus of the present disclosure is derived from Elstree, Copenhagen, Western Reserve or Wyeth virus strains. In certain embodiments, the vaccinia virus of the present disclosure is a Western Reserve virus strain. Western Reserve virus strains have been well characterized, and their complete sequences are available on the NCBI website (www.ncbi.nlm.nih.gov) with accession number AY243312.

[0086] As used herein, the term "modified oncolytic virus" refers to an oncolytic virus modified by introducing heterologous nucleic acids or proteins or changing natural nucleic acids or proteins. In certain embodiments, the modified oncolytic virus provided herein is genetically altered by the deletion and / or addition of a nucleotide sequence. In certain embodiments, the modified oncolytic virus provided herein includes the deletion of a thymidine kinase (TK) gene. In certain embodiments, the modified oncolytic virus provided herein includes the addition of a nucleotide sequence encoding anti-human PD-1 and / or anti-human 4-1BB antibodies.

[0087] In certain embodiments, the modified oncolytic virus of the present disclosure is attenuated. In certain embodiments, in normal cells (e.g., healthy cells), the modified oncolytic virus has a reduced (e.g., low at least 90%, 80%, 70%, 60%, 50%) or undetectable toxicity compared to its wild-type counterpart.

[0088] The modified oncolytic virus disclosed herein may be derived from any oncolytic virus known in the art to be oncolytic, and the virus is known to be oncolytic because of its selective replication and the tendency to kill tumor cells compared to non-tumor cells. Oncolytic viruses can be naturally oncolytic, or can be genetically engineered, such as by modifying one or more genes to improve tumor selectivity and / or preferentially replicate in tumor cells to make it oncolytic. Examples of such genes for modification include those genes involved in DNA replication, nucleic acid metabolism, host tropism, surface connection, toxicity, host cell lysis and virus propagation (see, e.g., Kirn et al., 2001, Nature Medicine 7: 781; Wong et al., 2010, Viruses 2: 78-106).

[0089] In certain embodiments, the viral genome of the modified oncolytic virus disclosed herein comprises at least one deletion or destruction that enables the virus to selectively replicate in tumor cells. For example, the deletion or destruction can reduce the expression or function of an enzyme necessary for viral replication, so that the ability of the virus to replicate in the absence of such an enzyme becomes weaker. In some embodiments, viral replication depends on the presence and / or level of such an enzyme in the cell, and the higher the level of the enzyme, the higher the ability or rate of viral replication.

[0090] In certain embodiments, the deletion or disruption is in an open reading frame (ORF). As used herein, the term "open reading frame" or "ORF" or "coding sequence" refers to a DNA sequence that can be translated into an amino acid sequence. An ORF usually begins with a start codon (e.g., ATG), followed by amino acid coding codons, and ends with a stop codon (e.g., TGA, TAA, TAG).

[0091] In certain embodiments, the ORF encodes at least a portion of an enzyme that is necessary for viral replication and is preferentially expressed in tumor cells compared to non-tumor cells. As used herein, the term "expression" refers to the process by which a protein or peptide sequence is produced by its encoding DNA or RNA sequence. In certain embodiments, the enzyme is a kinase.

[0092] In certain embodiments, the deletion in an ORF is 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 certain embodiments, the deletion in an ORF accounts for 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 (optionally contiguous).

[0093] In certain embodiments, the ORF of thymidine kinase (TK) is deleted or destroyed. TK is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in normal cells because these cells generally have low concentrations of nucleotides, while TK is not essential in tumor cells containing high nucleotide concentrations. In poxviruses, the thymidine kinase encoding gene is located at locus J2R. In certain embodiments, TK is completely deleted.

[0094] In certain embodiments, the ORF of ribonucleotide reductase (RR) is deleted or disrupted. RR catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which is a key step in DNA biosynthesis. The viral enzyme consists of two heterologous subunits designated R1 and R2, which are 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, through GenBank Accession Nos. DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391, NC_003389, NC_003310, M-35027, AY 154, DQ011153, Y16780, X71982, AF438165, U60315, AF410153, AF380138, U86916, L22579, NC_006998, DQ121394 and NC_008291. In the context of 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 interfered with.

[0095] In certain embodiments, the viral genome of the modified oncolytic virus further comprises additional deletions or destructions, which further improve the tumor specificity of the virus. In certain embodiments, additional deletions or destructions are in the ORF of at least a portion of a tumor-specific protein preferentially or specifically expressed in a tumor cell. A representative example of a tumor-specific protein is VGF. VGF is a secretory protein expressed early in cells after viral infection, and its function seems to be important for the spread of viruses in normal cells. Another example is the A56R gene encoding hemagglutinin (Zhang et al., 2007, "Cancer Research (Cancer Res.)" 67: 10038-46). Another example is the F2L gene, which encodes a viral deoxyuridine triphosphatase (dUTPase) involved in maintaining DNA replication fidelity and providing precursors for the production of TMP by thymidylate synthase (Broyles et al., 1993, "Virol. (Virol.)" 195: 863-5). The sequence of the vaccinia virus F2L gene can be obtained in the gene bank by accession number M25392.

[0096] Immune checkpoint inhibitors

[0097] The modified oncolytic viruses provided herein comprise a viral genome having a first heterologous polynucleotide encoding an immune checkpoint inhibitor.

[0098] As used herein, the term "heterologous" means that the sequence is not endogenous to the wild-type virus.

[0099] As used herein, the terms "encode" or "encoding for" refer to being capable of being transcribed into mRNA and / or translated into a peptide or protein.

[0100] As used herein, the term "immune checkpoint protein" refers to a protein directly or indirectly involved in an immune pathway that is important for preventing uncontrolled immune responses, and therefore important for maintaining self-tolerance and / or tissue protection. As used herein, one or more immune checkpoint regulators can independently act at any step of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, transport to antigens and inflammatory sites, execution of direct effector functions, and signaling through cytokines and membrane ligands.

[0101] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that can negatively regulate the function of immune checkpoint proteins. Immune checkpoint inhibitors can be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microRNA, shRNA, peptides, antibodies, spherical nucleic acids, TALEN, zinc finger nucleases, and CRISPR / Cas9.

[0102] In certain embodiments, the immune checkpoint inhibitor is a natural or engineered antagonist of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM3 (e.g., galectin-9), ligands of the A2a receptor (e.g., adenosine, Regadenoson), ligands of LAG3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod, NLG919).

[0103] In certain embodiments, the immune checkpoint inhibitor is an antibody (e.g., antagonist antibody) selected from the group consisting of anti-PD-1 (e.g., Nivolumab, Pililizumab, 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, MSB0010718C, STI-A1010, MPDL3280A, Dapirolizumab CDP-7657, MEDI-4920, or those described in PCT / US2001 / 020964), anti-PD-L2, anti-(both PD-L1 and PD-L2) (e.g., AUR-012 and AMP-224), anti-CT LA-4 (e.g., Ipilimumab, Tremelimumab, or KAHR-102), anti-IDO (e.g., D1-methyl-tryptophan (Lunate)), anti-KIR (e.g., Lirilumab, IPH2101, or IPH4102), anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, or C9B7W), anti-TIM3 (e.g., F38-2E2, or ENUM005), anti-VISTA (e.g., Such as VA.F6) anti-BTLA (e.g. AF3354), anti-CD73 (e.g. OSU-HDAC42 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.

[0104] In certain embodiments, the immune checkpoint inhibitor is an antibody or antigen binding fragment thereof 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.

[0105] PD-1 inhibitors

[0106] In certain embodiments, the first heterologous polynucleotide of the present disclosure encodes a PD-1 inhibitor.

[0107] As used herein, the term "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and acts as a co-inhibitory receptor to negatively regulate 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. The representative amino acid sequence of human PD-1 is disclosed in Gene Bank Accession No.: NP_005009.2, and the representative nucleic acid sequence encoding human PD-1 is shown in Gene Bank Accession No.: NM_005018.2.

[0108] PD-1 negatively regulates T cell activation, and this inhibitory function is associated with the immunoreceptor tyrosine-based inhibitory motif (ITIM) of its cytoplasmic domain (Parry et al., 2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitory function of PD-1 can cause autoimmunity. The persistent negative signal generated by PD-1 is involved in many pathological situations, such as tumor immune escape and T cell dysfunction in chronic viral infection.

[0109] A PD-1 inhibitor can be any agent that inhibits PD-1 activity, such as those that reduce PD-1 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

[0110] The activity (e.g., the activity of PD-1) can be reduced due to, for example, inhibition of binding between a functional protein and its ligand (e.g., binding between PD-1 and PD-L1), inhibition of its biological activation (e.g., activation of PD-1), and / or reduced level (e.g., PD-1 level).

[0111] In certain embodiments, the PD-1 inhibitor is an antibody (eg, an antagonist antibody) that can specifically bind to PD-1.

[0112] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human and / or monkey PD-1 with a binding affinity (KD) ≤ 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 As used herein, KD refers to the ratio of the dissociation rate to the association rate (k off / k on ), which can be determined using surface plasmon resonance methods, for example using an instrument such as Biacore.

[0113] In certain embodiments, the PD-1 inhibitor is a full-length monoclonal antibody directed against PD-1.

[0114] In certain embodiments, the PD-1 antibody specifically binds to SEQ ID NO:1.

[0115] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises a first heavy chain comprising SEQ ID NOs: 2, 3, and 4.

[0116] As used herein with respect to an amino acid sequence (or nucleic acid sequence), the term "identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to obtain the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues are not considered identical residues. Alignment for the purpose of determining the percentage of amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), 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 website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997); MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007))) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool, or can customize the parameters suitable for the comparison, for example, by selecting a suitable algorithm.

[0117] In certain embodiments, the heavy chain comprises a variable region having SEQ ID NO: 5 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 6 or a homologous sequence having at least 80% sequence identity thereto.

[0118] In certain embodiments, the polynucleotide comprises a 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 a 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.

[0119] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof further comprises a light chain comprising SEQ ID NOs: 9, 10 and 11. In certain embodiments, the light chain comprises a variable region having an amino acid sequence of SEQ ID NO: 12 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the light chain comprises an amino acid sequence of SEQ ID NO: 13 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0120] In certain embodiments, the polynucleotide further comprises a 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 a 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.

[0121] Immune activators

[0122] The modified oncolytic viruses provided herein comprise a viral genome having a second heterologous polynucleotide encoding an immune activator.

[0123] As used herein, the term "immune activator" refers to any agent that is capable of enhancing the immune system.

[0124] As used herein, the term "enhancing the immune system" refers to the ability of an agent to stimulate the production of T cell activity, B cell activity, macrophage activity, and / or NK cell activity.

[0125] In certain embodiments, the immune activator is a co-stimulatory activator, a NK activator, or a macrophage activator.

[0126] Co-stimulatory molecule activator

[0127] In certain embodiments, the immune activator is a co-stimulatory molecule activator.

[0128] As used herein, the term "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or an Fc receptor, which provides a second signal required for the effective activation and function of T lymphocytes when binding an antigen. Examples of such co-stimulatory molecules include CD137 (i.e., 4-1BB), CD27, CD70, CD86, CD80, CD28, CD40, CD122, TNFRS25, OX40 (CD134), GITR, neuropilin, and ICOS (i.e., CD278).

[0129] In certain embodiments, the co-stimulatory activator can be a peptide, polypeptide (e.g., antibody) that can enhance the cellular immune system. In certain embodiments, the co-stimulatory activator is an antibody or antigen-binding fragment of such an antibody that binds to a co-stimulatory molecule and thereby stimulates the activity of a co-stimulatory molecule, such as a CD137 antibody (e.g., BMS-663513 or PF-05082566), a CD28 antibody (e.g., TGN-1412), a CD40 antibody (e.g., CP-870, 893, CDX1140, BI-655064, BMS-986090, APX005 or APX005M), OX40 (CD 134) antibodies (e.g., MEDI6383, MEDI6469, MEDI0562, or those described in U.S. Pat. No. 7,959,925), anti-GITR (e.g., TRX518, INBRX-110, or NOV-120301), CD70 antibodies, CD86 antibodies, CD80 antibodies, CD122 antibodies, TNFRS25 antibodies, neuropilin antibodies, and CD27 antibodies (e.g., CDX-1127, BION-1402, or hCD27.15).

[0130] CD137 Activators

[0131] In certain embodiments, the second heterologous polynucleotide of the present disclosure encodes a CD137 activator.

[0132] CD137, also known as 4-1BB, is a member of the tumor necrosis factor receptor (TNFR) gene family, which contains proteins involved in regulating cell proliferation, differentiation and programmed cell death (A. Ashkenazi, Nature, 2:420-430, (2002)). CD137 is mainly expressed in activated T cells (including CD4 + and CD8 +NK cells and NK T 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)).

[0133] The CD137 activator can 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.

[0134] In certain embodiments, the CD137 activator is an antibody that specifically binds to CD 137. In certain embodiments, the CD137 activator is a full-length antibody.

[0135] In certain embodiments, the CD137 antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO:16.

[0136] In certain embodiments, the CD137 antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NOs: 17, 18, and 19.

[0137] In certain embodiments, the heavy chain comprises a variable region having an 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 comprises an 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.

[0138] In certain embodiments, the polynucleotide comprises a 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 a 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.

[0139] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain comprising SEQ ID NO: 24, 25 and 26. In certain embodiments, the light chain comprises a variable region having an 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 a 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.

[0140] 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 thereto.

[0141] 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.

[0142] NK activators

[0143] In certain embodiments, the immune activator is a NK activator that stimulates NK cell activity. In certain embodiments, the NK activator is a second antibody or antigen-binding fragment thereof that binds to a NK molecule.

[0144] In certain embodiments, the NK activator is selected from the group consisting of a sialic acid-binding immunoglobulin-like lectin (Siglec) antibody, a TIGIT antibody, a KIR antibody, and a NKG2A / D antibody (eg, monalizumab).

[0145] Macrophage activators

[0146] In certain embodiments, the immune activator is a macrophage activator that stimulates macrophage activity. In certain embodiments, the macrophage activator is a second antibody or antigen-binding fragment thereof that binds to a macrophage molecule.

[0147] In certain embodiments, the macrophage activator is selected from the group consisting of a CSF1R antibody (eg, FPA008), a CSF1 kinase antibody, a PS antibody, and a CD47 antibody (eg, CC-90002, TTI-621, or VLST-007).

[0148] Antibody

[0149] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second and third constant region, 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. The antibody is in a "Y" shape, wherein the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises 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, wherein the first constant region of the heavy chain is connected to the second constant region by a hinge region. The variable regions of the light and heavy chains are responsible for the generation of antigen binding specificity. The variable regions in both chains generally contain three highly variable loops, called complementarity determining regions (CDRs) (the light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (for details, 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)). The three CDRs are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold that supports the structure of the variable region. The constant regions of the heavy and light chains are not associated with antigen binding specificity, but exhibit various effector functions. Antibodies are classified according to 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, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as 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).

[0150] As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs, but does not comprise a complete antibody structure. Examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single domain antibodies, and nanobodies. Antigen binding fragments are capable of binding to the same antigen that the parent antibody binds to.

[0151] As used herein, the term "Fab" refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) bound by disulfide bonds to the variable region and the first constant region of a single heavy chain.

[0152] As used herein, the term "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0153] As used herein, the term "F(ab')2" refers to a dimer of Fab'.

[0154] As used herein, the term "Fv" refers to an Fv fragment consisting of the variable region of a single light chain and the variable region of a single heavy chain.

[0155] As used herein, the term "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or through a peptide linker sequence (see, e.g., Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0156] As used herein, the term "scFv dimer" refers to a polymer formed by two scFvs.

[0157] The term "camelized single domain antibody", also called "heavy chain antibody" or "HCAb" (heavy chain antibody only) refers to an antibody containing two heavy chain variable regions but no light chain (see, e.g., 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. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries and llamas). Although devoid of light chains, camelized antibodies have a bona fide 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), each of which is incorporated herein by reference in its entirety).

[0158] As used herein, the term "Nanobody" refers to an antibody consisting of the heavy chain variable region from a heavy chain antibody and two constant regions, CH2 and CH3.

[0159] 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 antibodies or their antigen-binding fragments are fully human antibodies, which are optionally produced by transgenic rats, such as transgenic rats with inactivated endogenous rat immunoglobulin gene expression, and carrying recombinant human immunoglobulin loci with J locus deletion and C-κ mutation, and the antibodies may also be expressed by engineered cells (e.g., CHO cells).

[0160] As used herein, with respect to antibodies or antigen-binding fragments, the term "fully human" 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 derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody repertoire or other human antibody encoding sequences.

[0161] As used herein, with respect to antibodies or antigen-binding fragments, the term "humanized" refers to antibodies or antigen-binding fragments that contain CDRs derived from non-human animals, FR regions derived from humans, and, when applicable, constant regions derived from humans. In certain embodiments, humanized antibodies or antigen-binding fragments are suitable for use as human therapeutic agents 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 is substantially entirely composed of human sequences except that the CDR sequences are non-human sequences.

[0162] As used herein, with respect to antibodies or antigen-binding fragments, the term "chimeric" refers to antibodies or antigen-binding fragments having a portion of heavy and / or light chains derived from one species and the remainder of heavy and / or light chains derived from a different species. In certain embodiments, a chimeric antibody may comprise a constant region derived from a human and a variable region from a non-human species, such as from a mouse or rabbit.

[0163] As used herein, with respect to amino acid sequences, the term "conservative substitution" refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiological and chemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0164] Polynucleotide

[0165] In certain embodiments, the modified oncolytic virus of the present disclosure contains 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.

[0166] As used herein, the term "polynucleotide" or "nucleic acid" refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA) or a mixed ribonucleic acid-deoxyribonucleic acid, such as a DNA-RNA hybrid. The polynucleotide or nucleic acid can be single-stranded or double-stranded DNA or RNA or a DNA-RNA hybrid. The polynucleotide or nucleic acid can be linear or circular. In certain embodiments, when the virus is a DNA virus, the first and second heterologous polynucleotides are both DNA; or when the virus is an RNA virus, the first and second heterologous polynucleotides are both RNA. In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are both double-stranded DNA.

[0167] The first heterologous polynucleotide and the second heterologous polynucleotide can be introduced into the modified oncolytic virus using conventional methods known in the art, for example, by being synthesized by polymerase chain reaction (PCR) and connected to the viral genome with compatible restriction ends. For more 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.

[0168] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are introduced at the position of the deletion in the ORF. In certain embodiments, the first heterologous polynucleotide is immediately upstream of the second heterologous polynucleotide or immediately downstream of the second heterologous polynucleotide. As used herein, the term "immediately upstream or immediately downstream" means that the first heterologous polynucleotide and the second heterologous polynucleotide are positioned sufficiently close to each other on the viral genome so that they are spaced no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides apart. For example, if the 3' end of the upstream polynucleotide is spaced no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides apart from the 5' end of the downstream polynucleotide, the 3' end of the upstream polynucleotide is adjacent to the 5' end of the downstream polynucleotide. In certain embodiments, there is no ORF between the first heterologous polynucleotide and the second heterologous polynucleotide. In certain embodiments, there is a restriction site between the first heterologous polynucleotide and the second heterologous polynucleotide.

[0169] In certain embodiments, the first heterologous polynucleotide encodes a first heavy chain and a first light chain of a first antibody. In certain embodiments, the first heterologous polynucleotide further comprises a first promoter capable of driving expression of the first heavy chain and a second promoter capable of driving expression of the first light chain, wherein the first promoter and the second promoter are in a head-to-head orientation.

[0170] In certain embodiments, the first heterologous polynucleotide encodes the variable region of the first heavy chain of the first antibody, a 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 does not encode the first light chain of the first antibody.

[0171] As used herein, the term "head-to-head orientation" means that the two promoters are next to each other on the viral genome and they drive protein expression in opposite directions. An illustrative example is shown in Figure 2 middle.

[0172] In certain embodiments, the second heterologous polynucleotide encodes a second heavy chain and a second light chain of the second antibody. In certain embodiments, the second heterologous polynucleotide further comprises a third promoter capable of driving expression of the second heavy chain and a fourth promoter capable of driving expression of the second light chain, wherein the third promoter and the fourth promoter are in a head-to-head orientation.

[0173] As used herein, the term "promoter" refers to a polynucleotide sequence that can control the transcription of a coding sequence. The promoter sequence comprises a specific sequence sufficient for RNA polymerase recognition, binding and transcription initiation. In addition, the promoter sequence may comprise a sequence that regulates this recognition, binding and transcription initiation activity of RNA polymerase. The promoter may affect the transcription of a gene that is located on the same nucleic acid molecule as itself or a gene that is located on a different nucleic acid molecule as itself. Depending on the nature of regulation, the function of the promoter sequence may be constitutive or may be induced by stimulation. As used herein, a "constitutive" promoter refers to a promoter that is used to continuously activate gene expression in a host cell. As used herein, an "inducible" promoter refers to a promoter that activates gene expression in a host cell in the presence of a certain stimulus or some stimulation.

[0174] In certain embodiments, the promoter of the present disclosure is a eukaryotic promoter, such as a promoter from CMV (e.g., CMV immediate early promoter (CMV promoter)), Epstein barr virus (EBV) promoter, human immunodeficiency virus (HIV) promoter (e.g., HIV long terminal repeat (LTR) promoter), Moloney virus promoter, mouse mammary tumor virus (MMTV) promoter, Rous sarcoma virus (Rous sarcoma virus) promoter, etc. virus; RSV) promoter, SV40 early promoter, promoters from human genes (such as human myosin promoter, human heme 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 (TK), human elongation factor 1α promoter (EF1A), cauliflower mosaic virus (CaMV) 35S promoter, E2F-1 promoter (promoter of E2F1 transcription factor 1), α-fetoprotein promoter, etc. The promoters of the proteins are: promoters of cholecystokinin, promoters of carcinoembryonic antigen, promoters of C-erbB2 / neu oncogene, promoters of cyclooxygenase, promoters of CXC-chemokine receptor 4 (CXCR4), promoters of human epididymis protein 4 (HE4), promoters of type II hexokinase, promoters of L-ectin, promoters of mucin-like glycoprotein (MUC1), promoters of prostate-specific antigen (PSA), promoters of survivin, promoters of tyrosinase-related protein (TRP1), and promoters of tyrosinase.

[0175] In certain embodiments, the promoter of the present disclosure may be a tumor-specific promoter. As used herein, the term "tumor-specific promoter" refers to a promoter that is used to preferentially or exclusively activate gene expression in tumor cells and is not active or has reduced activity in non-tumor cells or non-tumor cells. Illustrative examples of tumor-specific promoters include, but are not limited to, E2F-1 promoter, promoter of α-fetoprotein, promoter of cholecystokinin, promoter of carcinoembryonic antigen, promoter of C-erbB2 / neu oncogene, promoter of cyclooxygenase, promoter of CXCR4, promoter of HE4, promoter of type II hexokinase, promoter of L-netrin, promoter of MUC1, promoter of PSA, promoter of survivin, promoter of TRP1, and promoter of tyrosinase.

[0176] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured so that they are expressed in the same or different stages of the replication cycle of the modified oncolytic virus. For example, both polynucleotides can be driven by an early promoter induced in the early stage of viral replication, or alternatively, both driven by a late promoter induced in the late stage of viral replication, or alternatively, one driven by an early promoter and the other driven by a late promoter.

[0177] In certain embodiments, the first promoter and the second promoter are the same or different. In certain embodiments, the first promoter and the second promoter are both late promoters. In certain embodiments, the late promoter is pSL.

[0178] In certain embodiments, the third promoter and the fourth promoter are the same or different. In certain embodiments, the third promoter and the fourth promoter are both early and late promoters. In certain embodiments, the early and late promoter is pSE / L.

[0179] In certain embodiments, the modified oncolytic virus comprises the following elements in frame in the 5′ to 3′ direction of the sense strand: a polynucleotide encoding the light chain of an antibody that binds to CD137 - a first early and late promoter - a second early and late promoter - 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 - a first late promoter - a second late promoter - a polynucleotide encoding the light chain of an antibody that binds to PD-1.

[0180] In certain embodiments, the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune activator expressed by the second heterologous polynucleotide are expressed as separate proteins. In other words, it is not expressed as a fusion protein, and is not connected to each other (whether covalently linked or through a joint). In certain embodiments, the immune checkpoint inhibitor expressed by the first heterologous polynucleotide is not fused with any other protein, and the immune activator expressed by the second heterologous polynucleotide is not fused with any other protein.

[0181] In certain embodiments, in addition to the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not include any other heterologous polynucleotides encoding immune checkpoint inhibitors or immune activators. In certain embodiments, in addition to the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not include any heterologous polynucleotides encoding other proteins.

[0182] Pharmaceutical composition

[0183] In another aspect, the present disclosure provides a pharmaceutical composition comprising the modified oncolytic virus described in the present disclosure and a pharmaceutically acceptable carrier.

[0184] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. In certain embodiments, pharmaceutically acceptable compounds, materials, compositions and / or dosage forms refer to those listed in the pharmacopoeia (such as the US Pharmacopoeia, the China Pharmacopoeia or the European Pharmacopoeia) approved by an administrative agency (such as the US Food and Drug Administration, the China Food and Drug Administration or the European Medicines Agency) or recognized as being available for animals and particularly humans.

[0185] The pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention may include, but is not limited to, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous vehicle (e.g., sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection or Ringer's dextrose and lactate injection), a non-aqueous vehicle (e.g., fixed oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), an antimicrobial agent, an isotonic agent (e.g., sodium chloride or dextrose), a buffer (e.g., phosphate or citrate buffer), an antioxidant (e.g., sodium bisulfate), an anesthetic (e.g., procaine hydrochloride), hydrochloride), suspending / dispersing agents (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinyl pyrrolidone), chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (such as polysorbate 80 (Tween-80)), diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants or emulsifiers.

[0186] In certain embodiments, the pharmaceutical composition is an oral formulation. Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, dragees (for a flavor base, typically sucrose and acacia or tragacanth), powders, granules, or aqueous or non-aqueous solutions or suspensions, or water-in-oil or oil-in-water emulsions, or elixirs or syrups, or candy lozenges (for an inert base, such as gelatin and glycerin or sucrose or acacia) and / or mouthwashes and the like.

[0187] In certain embodiments, the pharmaceutical composition can be an injectable formulation, comprising a sterile aqueous solution or dispersion, suspension or emulsion. In all cases, the injectable formulation should be sterile and should be liquid for injection. It should be stable under manufacturing and storage conditions, and should be resistant to microbial (such as bacteria and fungi) infection. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and its suitable mixture and / or vegetable oil. The injectable formulation should maintain appropriate fluidity, and the fluidity can be maintained in a variety of ways, such as using a coating (such as lecithin), using a surfactant, etc. Antimicrobial contamination can be achieved by adding various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

[0188] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles.All preparations for parenteral administration should be sterile and pyrogen-free, as is known and practiced in the art.

[0189] Treatment

[0190] In another aspect, the present disclosure provides a method for treating a tumor, comprising administering an effective amount of the modified oncolytic virus of the present disclosure or the pharmaceutical composition of the present disclosure to a subject.

[0191] As used herein, the term "subject" refers to humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Subject" can also be livestock animals (such as cattle, pigs, goats, chickens, rabbits or horses), or rodents (such as rats or mice), or primates (such as gorillas or monkeys) or domestic animals (such as dogs or cats). "Subject" can be male or female, and can also be at different ages. In certain embodiments, the subject is a human. Human "subject" can be Caucasian, African, Asian, Sumerian or other races, or mixed races of different races. Human "subject" can be an elderly, adult, teenager, child or infant.

[0192] As used herein, the term "tumor" refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation or metastasis, and includes both solid tumors and non-solid tumors such as leukemia. In the present disclosure, "tumor" can be used interchangeably with the terms "cancer", "malignant tumor", "hyperproliferation" and "neoplasm". Unless otherwise clearly indicated, the term "tumor cell" can be interchanged with the terms "cancer cell", "malignant cell", "hyperproliferative cell" and "neoplastic cell". In certain embodiments, the tumor is selected from the group consisting of head and neck tumors, breast tumors, colorectal tumors, liver tumors, pancreatic cancer, 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 carcinoma and melanoma. 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's lymphoma, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer or hepatocellular carcinoma.In certain embodiments, the tumor is difficult to treat with previous therapy (e.g., separate administration of oncolytic virus, immune checkpoint inhibitors and / or immune activators).

[0193] As used herein, the term "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, producing complete or partial regression of the condition, curing the condition, or some combination thereof. With respect to a tumor, "treating" may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell development, proliferation, or metastasis, or some combination thereof. With respect to a tumor, "treating" includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0194] The modified oncolytic virus and pharmaceutical composition can be administered by any suitable route known in the art, including but not limited to parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary and subcutaneous administration. In certain embodiments, the administration route is topical administration. In certain embodiments, the administration route is intratumoral injection.

[0195] In certain embodiments, the modified oncolytic virus and pharmaceutical composition are administered in a therapeutically effective dose. As used herein, the term "therapeutically effective dose" refers to the amount of a drug that can improve or eliminate a subject's disease or symptom, or preventively inhibit or prevent the occurrence of a disease or symptom. A therapeutically effective amount can be the amount of a drug that improves one or more diseases or symptoms of a subject to a certain extent; the amount of a drug that can partially or completely restore one or more physiological or biochemical parameters associated with the cause of the disease or symptom to normal; and / or the amount of a drug that can reduce the likelihood of a disease or symptom occurring.

[0196] The therapeutically effective dose of the modified oncolytic virus and pharmaceutical composition depends on various factors known in the art, such as the subject's weight, age, pre-existing medical condition, currently accepted therapy, health status, and the intensity of drug interactions, allergies, hypersensitivity and side effects, and the route of administration and the extent of disease development. A person skilled in the art (e.g., a physician or veterinarian) may reduce or increase the dosage according to these or other conditions or requirements.

[0197] In certain embodiments, the modified oncolytic virus and pharmaceutical composition can be about 10 4 PFU to about 10 14 A therapeutically effective dose of PFU (e.g., about 10 4 PFU, about 2*10 4 PFU, about 5*10 4 PFU, about 10 5 PFU, about 2*10 5 PFU, about 5*10 5 PFU, about 10 6 PFU, about 2*10 6 PFU, about 5*10 6 PFU, about 10 7 PFU, about 2*10 7 PFU, about 5*10 7 PFU, about 10 8 PFU, about 2*10 8 PFU, about 5*10 8 PFU, about 10 9 PFU, about 2*10 9 PFU, about 5*10 9 PFU, about 10 10 PFU, about 2*10 10 PFU, about 5*10 10 PFU, about 10 11 PFU, about 2*10 11 PFU, about 5*10 11 PFU, about 10 12 PFU, about 2*1012 PFU, about 5*10 12 PFU, about 10 13 PFU, about 2*10 13 PFU, about 5*10 13 PFU or about 10 14 In certain embodiments, the modified oncolytic virus and pharmaceutical composition are administered at about 10 11 PFU or less. In some of these embodiments, the dose is 5*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 less or 10 9 PFU or less. A specific dose can be divided and applied multiple times at time intervals, such as 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 longer. In certain embodiments, the applied dose can be changed during the course of treatment. For example, in certain embodiments, the initial applied dose may be higher than the subsequent applied dose. In certain embodiments, the applied dose is adjusted during the course of treatment, depending on the response of the subject being administered.

[0198] As used herein, the term "PFU" refers to plaque forming unit, which is a measure of the number of particles capable of forming plaques.

[0199] Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0200] combination

[0201] 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.

[0202] In certain embodiments, the other drug is an anti-tumor agent. Any agent known to have anti-tumor activity can be used as an anti-tumor agent. In certain embodiments, the anti-tumor agent is selected from the group consisting of a chemical agent, a polynucleotide, a peptide, a protein, or any combination thereof.

[0203] In certain embodiments, the anti-tumor agent is a chemical agent. Illustrative examples of anti-tumor chemical agents include, but are not limited to, Mitomycin C, Daunorubicin, Doxorubicin, Etoposide, Tamoxifen, Paclitaxel, Vincristine, and Rapamycin.

[0204] In certain embodiments, the anti-tumor agent is a polynucleotide. Illustrative examples of anti-tumor polynucleotides include, but are not limited to, antisense oligonucleotides, such as bcl-2 antisense oligonucleotides, clusterin antisense oligonucleotides, and c-myc antisense oligonucleotides; and RNAs capable of RNA interference (including small interfering RNAs (siRNAs), short 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.

[0205] In certain embodiments, the anti-tumor agent is a peptide or protein. Illustrative examples of anti-tumor peptides or proteins include, but are not limited to, antibodies such as Trastuzumab, Rituximab, Edrecolomab, Alemtuzumab, Daclizumab, Nimotuzumab, Gemtuzumab, Ibritumomab and Edrecolomab, protein therapeutics such as Endostatin, Angiostatin K1-3, Leuprolide, Sex Hormone Binding Globulin and Bikunin.

[0206] Medical Uses

[0207] In another aspect, the present disclosure provides use of the modified oncolytic virus of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a tumor.

[0208] In another aspect, the present disclosure provides a modified oncolytic virus of the present disclosure or a pharmaceutical composition of the present disclosure for use in treating a tumor.

[0209] Examples

[0210] The following examples are set forth to aid the understanding of the present disclosure and should not be construed in any way to limit the scope of the present invention as defined in the claims that follow.

[0211] Example 1: Virus Construction

[0212] The starting WR strain of vaccinia virus was obtained from ATCC (www.atcc.org: VR-1354). Since multiple genes were involved, a stepwise engineering approach was used to establish WR-GS-600. Briefly, in the first step, WR DNA was recombined with a modified pSEM-1 vector (Rintoul et al., 2011) to insert the marker / selection gene into the TK locus. This allows for easy differentiation from the wild-type parent for further engineering. Afterwards, a transgenic line with flanking sequences of J1R and J3R and encoding anti-human PD-1 (the amino acid sequence of anti-human PD-1 and the nucleic acid sequence encoding anti-human PD-1 are shown in Fig.15 and 16 The amino acid sequence of anti-human 4-1BB and the nucleic acid sequence encoding anti-human 4-1BB are shown in Fig.17 and 18 The recombinant plasmid (in the middle) was transfected into U2OS cells infected with WR to completely delete TK and insert the antibody sequence. Figure 1 Structure showing thymidine kinase (TK) deletion, anti-PD-1 and anti-4-1BB antibody insertion in WR-GS-600, and Figure 3 Schematic diagram showing the recombination steps to generate WR-GS-600.

[0213] The recombination reactions were performed using U2OS cells from the characterization master working cell bank. Three rounds of plaque purification were performed using U2OS cells and one round of plaque purification was performed using HeLa cells. Afterwards, a filtration step using a 0.65 μm filter was incorporated to ensure that the final plaques picked were clonal. The details are described in Table 1 below.

[0214] Table 1: WR-GS-600 plaque generation and purification procedures

[0215]

[0216]

[0217] After further plaque purification, antibody expression was monitored by immunofluorescence and flow cytometry.U2OS and HeLa cells were mock infected (ie, infected with a control solution without virus), infected with a control virus with antibody expression, or infected with a purified clone of WR-GS-600, respectively.

[0218] Finally, in two roller bottles (1700cm2 ) was amplified in a unique clone with a verified DNA sequence and high-level antibody expression. The cells were precipitated and then resuspended in 1mM Tris pH 9.0. After a round of freeze-thaw (-80 / 37 degrees), the mixture was precipitated again. The supernatant was aliquoted into 12 cryogenic test tubes, 1ml per test tube (pre-made master virus library). The precipitated cells were resuspended in 3ml 1mM Tris pH 9.0 and subjected to another round of freeze / thaw. The supernatant was collected after precipitation and then subjected to overnight benzonase treatment and sucrose purification. The titer of pre-made MVB and purified benzonase was determined using U2OS cells. For a total of 5mL stock solution, the titer was found to be between 1.0-2.1*10 9 pfu / mL range, which is similar to the titer of the parental WR virus.

[0219] WR-GS-610 (with the gene encoding anti-human 4-1BB inserted) and WR-GS-620 (with the gene encoding anti-human PD-1 inserted) were manufactured by the same protocol as WR-GS-600, except that WR-GS-600 was inserted with both the gene encoding anti-human PD-1 and the gene encoding anti-human 4-1BB. Figure 2 The structure showing the deletion of TK and the insertion of anti-4-1BB antibody in WR-GS-620, and Figure 4 Schematic diagram showing the recombination steps of WR-GS-620.

[0220] Example 2: Characterization of WR-GS-600, WR-GS-610, and WR-GS-620

[0221] During the engineering process of these new viruses, their genome integrity and protein expression were closely monitored.

[0222] PCR, sequencing, and restriction digestion

[0223] Viral genomic DNA was isolated from a sucrose cushion purified by treating the viral preparation with the universal nuclease endonuclease, by sucrose precipitation, followed by proteinase K and detergent treatment, and the DNA was subsequently extracted and recovered using phenol / chloroform / isoamyl alcohol extraction and ethanol precipitation.

[0224] To ensure that the viral genome has the expected sequence carrying the designed antibody sequence, a series of primers have been designed, including primers within the recombination region and primers outside the engineered segment. The identity of the virus (WR-GS-600, WR-GS-610 and WR-GS-620) was confirmed by qPCR (TaqMan). The primers used in PCR are shown in Table 2. The positions of the primers in the viral genome are shown in Figures 5 to 7 The predicted sizes of the PCR bands are shown in Figures 5 to 7 The results of PCR amplification of genomic DNA of WR-GS-600, WR-GS-610 and WR-GS-620 are shown in Fig.14 middle.

[0225] The TK deletion was also verified by Sanger sequencing. Figure 8 , 9 Figures 1 and 2 show the genetic changes of WR after insertion of antibody encoding genes. Sanger sequencing alignment of the WR-GS-600 viral genome relative to the designed DNA sequences for expression of anti-hu4-1BB and anti-huPD-1 in WR-GS-600 was performed. The alignment showed that the viral genome of WR-GS-600 was identical to the designed DNA sequences.

[0226] Table 2: Primers used for PCR and sequencing

[0227]

[0228] The restriction enzyme HindIII cuts near the TK region of WR and produces a band of 5004bp. When TK is missing in WR-GS-600 and anti-huPD-1 and / or anti-hu4-1BB antibodies are inserted, two additional HindIII restriction sites are introduced, which produce three bands of 1638, 2548 and 4666bp respectively. For WR-GS-620, the 5004bp band of wild-type WR is replaced by two bands of 1922 and 4666bp. These differences in restriction digestion patterns can be used to quickly identify these viruses. The results are shown in Table 3.

[0229] Table 3: HindIII digestion of viral genome

[0230]

[0231] Immunofluorescence

[0232] Transgenic expression of human antibodies was verified by immunofluorescence against human IgG (see Fig.11 FITC-conjugated goat anti-human IgG (H+L) (Invitrogen, catalog number 62-8411) was used to stain virus-infected U2OS cells.

[0233] Flow cytometric analysis

[0234] Flow cytometric analysis of mock-infected, control WR virus (WR-mCherry)-infected, WR-GS-600-infected, and WR-GS-620-infected HeLa cells confirmed the specific expression of human antibodies when infected with WR-GS-600 and WR-GS-620. Human IgG from infected supernatants detected in Western blots provided further evidence of human antibody expression.

[0235] Western blotting

[0236] Western blots detecting human IgG from infected supernatants provided further evidence of antibody expression. Fig.12 and 13 Anti-PD-1 and anti-41-BB antibody expression by recombinant viruses (WR-GS-600, WR-GS-610, and WR-GS-620) using cell lysates and supernatants, respectively, is shown by Western blotting.

[0237] Functional characterization of expressed anti-PD-1 antibodies using PD-1 binding ELISA

[0238] Recombinant human PD-1 Fc chimera (R&D Systems, Minneapolis, MN) was resuspended to 0.2 mg / ml with Dulbecco's Phosphate Buffered Saline (DPBS) containing 0.1% bovine serum albumin (BSA) and diluted to a final concentration of 0.03 μg / ml with DPBS. Nunc-Immuno Maxisorp 96-well culture plates were coated with 0.1 ml of recombinant PD-1 Fc chimera per well, leaving empty wells for nonspecific binding controls, and incubated overnight at 4°C. The coating solution was removed and the culture plates were washed with wash buffer (DPBS containing 0.05% Tween-20, 200 μL per well each time). Blocking buffer (5% skim milk powder, 0.05% Tween-20 in DPBS, 200 μL per well at a time) was added to all wells and incubated for 1 hour at 4°C with mixing. Blocking buffer was removed and the culture plate was washed with wash buffer. Serial dilutions of WR-GS-620 and WR-GS-600 supernatants were prepared in DPBS, and the diluted supernatants (100 μL per well) were added to the culture plate. The culture plate was incubated for 1.5 hours at room temperature. The antibody-containing supernatant solution was removed, and the culture plate was washed with wash buffer. Horseradish peroxidase-labeled goat anti-human IgG, F(ab')2 specific F(ab')2 antibody (Jackson Immunoresearch, West Grove, PA) was diluted with DPBS, and 100 μL per well was added to the culture plate. The culture plate was incubated for 1 hour at room temperature and washed with wash buffer. 100 μL SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Labs Gaithersburg, MD) was added per well and incubated at room temperature for 20 minutes. The reaction was stopped by adding an equal volume of 2M H2SO4 and the absorbance was read at 450 nm on a Molecular Devices Spectra Max 340 (Molecular Devices, Sunnyvale, CA).

[0239] Supernatants from U2OS infected at MOI 0.05 for 48 h were used for analysis and the results are shown in Fig.19 These results indicate that the anti-PD-1 antibody expressed by GS-620 can specifically bind to PD-1 and the binding is concentration-dependent.

[0240] Functional characterization of expressed anti-4-1BB antibodies using 4-1BB binding ELISA

[0241] Human 4-1BB IgG1Fc chimera (Andy Bio, Minneapolis, Minnesota) was resuspended to 0.2 mg / ml with Dulbecco's phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA), and diluted to a final concentration of 0.03 μg / ml with DPBS. Nunc-Immuno Maxisorp 96-well culture plates were coated with 0.1 ml of recombinant 4-1BB chimera per well, leaving empty wells for nonspecific binding controls, and incubated overnight at 4 ° C. The 4-1BB solution was removed and the culture plates were washed with wash buffer (DPBS containing 0.05% Tween-20). Blocking buffer (DPBS containing 5% skim milk powder, 0.05% Tween-20) was added to all wells and incubated for 1 hour at 4 ° C with mixing. The blocking buffer was removed and the culture plates were washed with wash buffer. Serial dilutions of WR-GS-610 and WR-GS-600 supernatants were prepared in DPBS, and the diluted supernatants were added to the culture plates. The culture plates were incubated at room temperature for 1.5 hours. The supernatant solution containing the antibodies was removed, and the culture plates were washed with wash buffer. Horseradish peroxidase-labeled goat anti-human IgG, F(ab')2 specific F(ab')2 antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania) were diluted with DPBS and added to the culture plates. The culture plates were incubated at room temperature for 1 hour and washed with wash buffer. SureBlue TMB microporous peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was added, and incubated at room temperature for 20 minutes. The reaction was stopped by adding the same volume of 2M H2SO4, and the absorbance was read at 450nm on a Molecular Devices Spectra Max 340 (Molecular Instruments, Sunnyvale, California).

[0242] Supernatants from U2OS infected at MOI 0.05 for 48 h were used for analysis and the results are shown in Fig. 20 These results indicate that the anti-4-1BB antibodies expressed by GS-600 and GS-610 can specifically bind to 4-1BB and the binding is concentration-dependent.

[0243] The above tests showed that WR-GS-600, WR-GS-610 and WR-GS-620 were well constructed and could express functional corresponding antibodies (anti-PD1 antibody for WR-GS-600 and WR-GS-620, and anti-4-1BB antibody for WR-GS-600 and WR-GS-610).

[0244] Example 3: In vivo studies of WR-GS-600, WR-GS-610 and WR-GS-620 recombinant viruses

[0245] The following studies were performed to determine whether WR-GS-600, WR-GS-610, and WR-GS-620 recombinant viruses are safe for mice and whether the recombinant viruses can target and penetrate tumors in mice. The delivery route can be intravenous (IV) or intraperitoneal (IP). All animal experiments were performed in accordance with the guidance of the local animal care committee.

[0246] Cells of WR-GS-600, WR-GS-610 and WR-GS-620 in CT26, MC38, HT-29 and HCT-116 cell lines Measurement of toxicity (cell killing data)

[0247] Colorectal cancer cell lines CT26-LacZ (murine), MC38-Luc (murine), HT-29-Luc (human) and HCT-116-Luc (human) were used for in vitro cytotoxicity testing. WR-GS-600, WR-GS-610 and WR-GS-620 were prepared at three different MOIs, namely 0.01 MOI (3E2 PFU), 0.1 MOI (3E3 PFU) and 1.0 MOI (3E4 PFU). Measurements were performed at three different time points, namely 24 hours, 48 ​​hours and 72 hours.

[0248] Cell preparation: Each cell line was first seeded in two 15 cm tissue culture dishes and incubated until near confluence was between 75-90%. The cells were washed and counted using conventional methods known to those of ordinary skill in the art. Each well of a 96-well flat bottom culture plate was seeded with approximately 3E4 cells. Nine culture plates were required for each cell type for nine different experimental conditions.

[0249] Preparation of virus dilutions: The virus was thawed on ice and then thawed in a 37°C water bath to ensure complete thawing. The thawed virus was vortexed twice at maximum speed, and each time lasted for 20 seconds. WR-GS-600, WR-GS-610 and WR-GS-620 viruses were prepared at three different concentrations, namely MOI 1.0, MOI0.1 and MOI 0.01. 50 μL of virus was added to the corresponding wells, and then the 96-well flat-bottom culture plate was gently shaken in 4 quadrants to mix. The culture plate was incubated at 37°C supplemented with 5% CO2. MOI 1.0 corresponds to 3E4 PFU / 50μL or 600PFU / μL or 6E5PFU / mL. MOI 0.1 corresponds to 3E3PFU / 50μL or 60PFU / μL or 6E4 PFU / mL. MOI 0.01 corresponds to 3E2 PFU / 50 μL or 6 PFU / μL or 6E3 PFU / mL.

[0250] Alamar Blue was used to detect the cytotoxicity of the virus in the four cell lines mentioned above using conventional methods known to those of ordinary skill in the art. For each condition, cell viability was calculated with 6 replicates. Figure 21-23 It was shown that at three different concentrations and three different time points, there was no significant difference in the cell viability of the four cell types mentioned above treated with WR, WR-GS-600, WR-GS-610 and WR-GS-620. This indicates that incorporating the polynucleotide sequence of the checkpoint inhibitor antibody into the vaccinia virus (WR) genome does not change the cytotoxic properties of the virus. Figure 21-23 It was further shown that after viral treatment, the cell viability of HT-29 and HCT-116 cell lines decreased more significantly than that of CT-26 and MC-38, indicating that human cancer cells are more sensitive to viral infection and killing.

[0251] Measurement of biodistribution of viral vectors

[0252] Tissue Homogenization:

[0253] 25 Balb / C mice (Jackson Lab) in 5 different groups were killed one at a time. After disinfection spray, mice were cut open and 50 to 100 mg tumors, lungs, spleen, liver, brain or ovaries were excised therefrom. The remaining tissues were frozen with OCT. The excised tissue was weighed and placed in a 2.0 mL Eppendorf tube. Tissue samples were frozen overnight at -80 ° C. Tissue samples were homogenized the next day in a manner known to those of ordinary skill in the art. In brief, two autoclaved 5 mm TissueLyser beads were distributed in each tube. A total of 48 tubes were loaded into TissueLyser. Homogenization was performed at 28 Hz for 1 minute. Then, the insert of the adapter was turned 180 °, and homogenization was run for another 1 minute to achieve uniform homogenization. Thereafter, 500 μL DMEM was added to each sample. The tube was centrifuged at 3500 g for 2 minutes. The supernatant was transferred to a 1.5 mL eppendorf tube and stored at -80°C prior to titer determination.

[0254] 24-well format for titration:

[0255] U2OS cells were used for virus titer determination, and 10E2 PFU / mL JX594 stock solution (a) and 31.0 PFU / mL JX594 stock solution (b) were prepared and used as positive controls.

[0256] For five tissues, namely brain (B), liver (V), lung (L), ovary (O) and spleen (S), three concentrations of each virus were prepared for WR-GS-600, WR-GS-610 and WR-GS-620: (1) pure 150 μL for infection; (2) 98 μL from (1) in 212 μL DMEM, mixed, and 150 μL was used for infection; and (3) 98 μL from (2) in 212 μL DMEM, mixed, and 150 μL was used for infection.

[0257] For controls (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.

[0258] For tumor (T), six concentrations of each virus were prepared for WR-GS-600, WR-GS-610, and WR-GS-620: (1) 150 μL pure for infection; (2) 98 μL from (1) in 212 μL DMEM, mixed, and 150 μL was used for infection; (3) 98 μL from (2) in 212 μL DMEM, mixed, and 150 μL was used for infection; (4) 98 μL from (3) in 212 μL DMEM, mixed, and 150 μL was used for infection; (5) 98 μL from (4) in 212 μL DMEM, mixed, and 150 μL was used for infection; and (6) 98 μL from (5) in 212 μL DMEM, mixed, and 150 μL was used for infection.

[0259] like Fig.24 24-well plates were set up as shown. Each plate was seeded with tumor, lung, spleen, liver, brain, and ovarian cells prepared from one mouse using the methods described in the tissue homogenization section.

[0260] As mentioned above, 25 mice were divided into 5 groups, each group had 5 mice, i.e. 5 culture plates. The tumor, lung, spleen, liver, brain and ovarian cells in group 1 were infected with WR-GS-610, the cells in group 2 were infected with WR, the cells in group 3 were infected with WR-GS-620, the cells in group 4 were infected with WR-GS-600, and all cells in group 5 (except the cells in the positive control wells) were treated with formulation buffer (FB) as a negative control, wherein the formulation buffer contained 30 mM Tris, 10% sucrose and 150 mM NaCl, pH 7. Fig.25 WR-GS-610 virus plaques were shown to be present only in tumor cell wells. Fig.26WR virus plaques were shown to be present in both tumor cell wells and ovarian cell wells. Fig. 27 WR-GS-620 virus plaques were shown to be present in both tumor cell wells and ovarian cell wells. Fig.28 WR-GS-600 virus plaques were shown to be present only in tumor cell wells. Fig.29 Virus plaques were not shown to be present in the 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.

[0261] In vivo virus distribution in subcutaneous tumors and other tissues:

[0262] Objective: Safety and biodistribution of viral vectors

[0263] Study plan

[0264] i. Order 35 Balb / C mice (Charles River). Mice were assigned to 5 treatment groups, PBS control (FB) and WR, WR-GS-600, WR-GS-610 and WR-GS-620 groups.

[0265] ii. Treatment started when tumors in the tumor group reached 5 mm in size.

[0266] iii. Three virus injections were performed via tail vein injection (scheduled on days 1, 4, and 7).

[0267] iv. Monitor the weight and health of mice.

[0268] v. On day 9, mice were sacrificed and tissues from brain, lung, liver, ovary, spleen were collected. Vaccinia titers in different tissues were determined by plaque assay on U2OS cells.

[0269] Tables 3-5 below summarize the treatment groups, treatment schedule and anesthesia, endpoints and euthanasia.

[0270] Table 3. Treatment groups:

[0271] Group No. Group size First injection dose Scheduling 1 5 Control - Preparation Buffer n / a Day 1, Day 4, Day 7 2 5 P600 1E7 PFU Day 1, Day 4, Day 7 3 5 P610 1E7 PFU Day 1, Day 4, Day 7 4 5 P620 1E7 PFU Day 1, Day 4, Day 7 7 5 WR 1E7 PFU Day 1, Day 4, Day 7

[0272] Table 4. Processing schedule:

[0273] date Day program July 26, 2019 -11 Weighing and ear cropping July 26, 2019 SC injection of CT26 cells into the right flank (#) August 6, 2019 1 Inject IT 50μL (1E7 PFU) virus August 9, 2019 4 Inject IT 50μL (1E7 PFU) virus August 12, 2019 7 Inject ITμL (1E7 PFU) of virus August 14, 2019 9 Heart bleeding, cervical dislocation. Collection of spleen, liver, ovary, lung and brain

[0274] Table 5. Anesthesia, Endpoints, and Euthanasia:

[0275]

[0276]

[0277] Data are presented by averaging 5 mice per group. Figure 30-32 WR-GS-600 and WR-GS-610 were preferentially present in tumors, and very little WR-GS-600 and WR-GS-610 virus was observed in the ovaries, brain, spleen, liver, and lungs. In contrast, after intratumoral injection, a large amount of WR-GS-620 virus was observed in tumors, ovaries, brain, spleen, liver, and lungs. These data confirm that WR-GS-600 and WR-GS-610 have higher tumor targeting specificity than WR-GS-620. In addition, Figure 30-32 The second and third bars in the bar graph of show that although the PFU per gram of tissue for WR-GS-600 and WR-GS-610 are similar in ovary (WR-GS-610 is slightly higher than WR-GS-600), brain, spleen, liver, and lung, the PFU per gram of tumor tissue for WR-GS-600 is about three times higher than that for WR-GS-610. This suggests that while other tissues are similarly infected by WR-GS-600 and WR-GS-610, tumors can be more severely infected by WR-GS-600 than by WR-GS-610.

[0278] Previous studies have shown that the vaccinia virus strain can infect normal mouse organs, especially the ovaries (Zhao Y. et al., Viral Immunology, 2011, 24, 387). Fig.31 The results presented in are consistent.

[0279] Collectively, these data demonstrate that incorporating a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding an immune activator into an oncolytic virus, such as WR, results in a synergistic effect of tumor targeting and infection that cannot otherwise be achieved by a wild-type oncolytic virus or a modified oncolytic virus having only the first heterologous polynucleotide or only the second heterologous polynucleotide.

[0280] Measurement of tumor size changes in the CT-26 murine tumor model after infection with different viruses

[0281] Twenty-five Balb / C mice (Jackson Laboratories) were implanted with CT26 tumors (CT-26LacZ 5E6 cells SG right flank). The mice were further divided into 5 treatment groups: formulation buffer (FB), WR, WR-GS-600, WR-GS-610 and WR-GS-620. Treatment was started when the tumors of the tumor groups reached 5 mm in size. 1E7 pfu of different viruses were injected intratumorally on days 1, 4 and 7, and the weight and health of the mice were monitored. Tumor growth was followed by measuring tumor size with a caliper.

[0282] Changes in tumor size were recorded and the results are summarized in Fig.33 where the % change in tumor volume on day x was calculated by comparing the tumor volume on day x with the tumor volume on day 1. Fig.33 The results showed that the increase in tumor volume size when treated with WR, WR-GS-600, WR-GS-610 and WR-GS-620 was much smaller than that when treated with formulation buffer (FB) on days 1, 4 and 7 after virus injection, indicating the tumor inhibitory effect of the above-mentioned viruses in vivo.

[0283] Efficacy measurements in syngeneic mouse models

[0284] A subcutaneous CT-26LacZ tumor model was established in Balb / C mice. Different viruses of 1E7 were injected via tail vein injection on days 1, 3, and 7, and the weight and health of the mice were monitored. The endpoint was set at tumors >1,700 mm 3 , and the study was terminated on day 31. The mouse survival results are shown in Fig.34 middle.

[0285] Measurement of tumor size changes in the humanized HT-29-Luc subcutaneous tumor model after infection with different viruses

[0286] The experiment is briefly described as follows:

[0287] Day 1: Order 30 Rag2 - / - IL2Rg-deficient mice (Jackson Laboratories) were implanted with HT-29 tumors (HT-29Luc 5E6 cells SQ right flank).

[0288] Day 7: Tumor growth was examined by IVIS.

[0289] Day 8: 5.8E6 human PBMCs were administered IP via intravenous injection.

[0290] Day 14: IVIS assignment to group.

[0291] Day 15: Processed 1E7 IT.

[0292] Day 18: IVIS and processing of 1E7 IT.

[0293] Day 24: IVIS.

[0294] Confirmation of human peripheral blood mononuclear cell transplantation

[0295] Treated mice were bled submandibularly and 100 μL of blood was obtained and added to sodium heparin. Red blood cells were lysed and stained for hCD45, CD3, CD8 and CD4. Fluorescence results were read on the LSR Fortessa and are summarized in Fig.35 and 36, which demonstrated successful transplantation of human peripheral blood mononuclear cells into immunodeficient mice.

[0296] Changes in tumor volume after viral infection are summarized in Fig.37 and 38 middle. Fig.37 It was shown that mice treated with WR-GS-600 exhibited minimal increase in tumor volume compared to mice treated with WR-GS-610, WR-GS-620 or WR compared to the control group. More interestingly, after infection of mice with WR-GS-600 on day 32 after HT-29-Luc injection, tumor size did not increase significantly and even decreased from day 8 of WR-GS-600 treatment. Fig.38 WR and WR-GS-620 were shown to have earlier endpoints than WR-GS-600 and WR-GS-610, which was due to the higher toxicity of WR and WR-GS-620 than WR-GS-600 and WR-GS-610. Fig.38 It was further shown that WR-GS-600 and WR-GS-610 can control tumor growth when compared to formulation buffer. Together, these data indicate that WR-GS-600 and WR-GS-610 are less toxic than WR and WR-GS-620, and that both WR-GS-600 and WR-GS-610 can control tumor growth.

[0297] Fig.39 and 40 Shown is the growth of human tumor HT-29 in NCG mice with or without human PBMCs measured by in vivo imaging IVIS (IVIS Spectrum, PerkinElmer).

[0298] Fig.41 and 42 It was shown that for the humanized HT-29-Luc intraperitoneal mouse model injected intraperitoneally with virus, WR-GS-600 and WR-GS-620 infection can significantly reduce the chemiluminescence intensity of the tumor, indicating the tumor inhibitory efficacy 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 the tumor, indicating the tumor growth control efficacy of WR and WR-GS-610.

[0299] The aforementioned in vitro and in vivo results indicate that WR, WR-GS-600, WR-GS-610 and WR-GS-620 can kill cancer cells and control tumor growth. However, WR and WR-GS-620 exhibit higher toxicity, which leads to early termination of drug testing. WR-GS-600 and WR-GS-610 are more effective in tumor growth control, wherein the tumor targeting specificity of WR-GS-600 is higher than that of WR-GS-610. More importantly, in the humanized HT-29 intraperitoneal tumor mouse model, intraperitoneal injection of WR-GS-600 reduces tumor size, while intraperitoneal injection of WR-GS-610 does not stop the increase in tumor size, but the percentage of tumor size increase is much smaller than when treated with WR. These data show that the incorporation of heterologous polynucleotides encoding immune checkpoint inhibitors and heterologous polynucleotides encoding immune activators can reduce toxicity, provide tumor targeting specificity, and improve tumor control efficacy.

[0300] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application.

[0301] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0302] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Sequence Listing <110> Shanghai Jinsi Biotechnology Co., Ltd. <120> Modified oncolytic viruses, compositions and uses thereof <130> 068615-8001WO02 <150> PCT / CN2018 / 104830 <151> 2018-09-10 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 268 <212> PRT <213> Human <400> 1 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55 60 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 115 120 125 Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 130 135 140 Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly Leu Leu Gly Ser 145 150 155 160 Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala 165 170 175 Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp 180 185 190 Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe 195 200 205 Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu 210 215 220 Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser 225 230 235 240 Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro 245 250 255 Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 260 265 <210> 2 <211> 5 <212> PRT <213> Synthetic <400> 2 Asn Ser Gly Met His 1 5 <210> 3 <211> 17 <212> PRT <213> Synthetic <400> 3 Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 4 <211> 4 <212> PRT <213> synthetic <400> 4 Asn Asp Asp Tyr 1 <210> 5 <211> 113 <212> PRT <213> synthetic <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 6 <211> 440 <212> PRT <213> Synthetic <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 7 <211> 339 <212> DNA <213> Synthetic <400> 7 cgaacttacg gtcaccaatg tgccctgtcc ccagtaatcg tcgtttgtgg cgcagtagta 60 tacggctgtg tcctcagccc gcagtgaatt catctgtagg aacaatgtat ttttagagtt 120 gtccctacta atggtgaacc gtcccttcac agagtcggcg taataacgct tggagccgtc 180 ataccagata actgccaccc actccaagcc cttgcctggc gcttgccgta cccagtgcat 240 cccgctgtta gaaaaggtga tgccggaagc cttacagtcc aatcttagtg atctgcctgg 300 ctggacaact ccgccgcctg actcaaccag ctgtacttg 339 <210> 8 <211> 890 <212> DNA <213> Synthetic <400> 8 ctgacgaccc gataggttga attgaattgt tcctcgcggg gtttcgtctt tgcgttatgc 60 acttcaacgc cgtctacgta ccagttgaat tgcacttcgg gatcctcttg agagacatca 120 accacgacac aagtcacttc cggggtccgg ctaatcatta gggtgtcctt aggttttggt 180 gggaacagaa acacggacgg tccacccaag aactcggggg caggacacgg agggcagggg 240 ggtccatact tggattcgac ccgcttatct actttagtat tagagggctt gtggtcaacg 300 ttacaggtat aagttttcgt gcctagacta gatgaaggaa cagtgaccac ggaagatagt 360 gaatagaggc ccgagctctg cagcacggca gggaatgtgt gcactccaga ggtcagggcg 420 ccgctgttcc aggacacagt caccggttct ggaaagtagt ccttcacgag acatcctagc 480 gcagctgtgc tttcgcttgt gctcctactg cagggggcga gcgggaacac tgagggtccc 540 ttggtgcttg ccgaacttac ggtcaccaat gtgccctgtc cccagtaatc gtcgtttgtg 600 gcgcagtagt atacggctgt gtcctcagcc cgcagtgaat tcatctgtag gaacaatgta 660 tttttagagt tgtccctact aatggtgaac cgtcccttca cagagtcggc gtaataacgc 720 ttggagccgt cataccagat aactgccacc cactccaagc ccttgcctgg cgcttgccgt 780 acccagtgca tcccgctgtt agaaaaggtg atgccggaag ccttacagtc caatcttagt 840 gatctgcctg gctggacaac tccgccgcct gactcaacca gctgtacttg 890 <210> 9 <211> 11 <212> PRT <213> Synthetic <400> 9 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 10 <211> 7 <212> PRT <213> synthetic <400> 10 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 11 <211> 9 <212> PRT <213> synthetic <400> 11 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 12 <211> 107 <212> PRT <213> synthetic <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 214 <212> PRT <213> Synthetic <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 320 <212> DNA <213> Synthetic <400> 14 gaaatcgtac tcacgcagtc ccctgctact ctgagtctct caccaggaga acgcgctacc 60 ctttcttgcc gtgcgtcaca gtcagtatcg tcctatctgg cttggtatca gcaaaaacca 120 ggtcaggccc cccgattatt gatttatgat gcatctaacc gggctacagg gattcctgcc 180 agatttagcg gtagcgggag tggaactgac ttcactctaa cattagctcc cttgagccag 240 aggatttcgc cgtctactac tgtcagcagt cttccaactg gcctcgtact ttcggacagg 300 gaacaaaggt ggaaatcaaa 320 <210> 15 <211> 642 <212> DNA <213> Synthetic <400> 15 gaaatcgtac tcacgcagtc ccctgctact ctgagtctct caccaggaga acgcgctacc 60 ctttcttgcc gtgcgtcaca gtcagtatcg tcctatctgg cttggtatca gcaaaaacca 120 ggtcaggccc cccgattatt gatttatgat gcatctaacc gggctacagg gattcctgcc 180 agatttagcg gtagcgggag tggaactgac ttcactctaa ccattagctc ccttgagcca 240 gaggatttcg ccgtctacta ctgtcagcag tcttccaact ggcctcgtac tttcggacag 300 ggaacaaagg tggaaatcaa acgtaccgtg gctgcaccca gcgtgttcat ttttccacca 360 agcgacgagc agctcaagag cggaaccgca tccgtagtat gtctcctcaa taacttctac 420 ccacgagaag ccaaagtgca gtggaaggtg gataatgcct tgcaatccgg aaacagccaa 480 gaaagcgtga ccgaacagga ttcaaaagac agcacctatt ctctgtccag cacattgaca 540 ctgagtaaag ctgattatga gaagcacaag gtctacgcgt gtgaggttac acatcaagga 600 ttgtcttcac cagtcaccaa gagtttcaat agaggagagt gc 642 <210> 16 <211> 238 <212> PRT <213> Human <400> 16 Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala 1 5 10 15 Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro 20 25 30 Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys 35 40 45 Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr 50 55 60 Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala 65 70 75 80 Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr 85 90 95 Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys 100 105 110 Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser 115 120 125 Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser 130 135 140 Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro 145 150 155 160 Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu Ala 165 170 175 Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg 180 185 190 Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 195 200 205 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 210 215 220 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 225 230 235 <210> 17 <211> 5 <212> PRT <213> Synthetic <400> 17 Gly Tyr Tyr Trp Ser 1 5 <210> 18 <211> 16 <212> PRT <213> Synthetic <400> 18 Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser 1 5 10 15 <210> 19 <211> 13 <212> PRT <213> Synthetic <400> 19 Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu 1 5 10 <210> 20 <211> 121 <212> PRT <213> Synthetic <400> 20 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 21 <211> 448 <212> PRT <213> Synthetic <400> 21 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 22 <211> 363 <212> DNA <213> Synthetic <400> 22 caggttcagc tacagcagtg gggagcaggt ctgctgaaac caagcgaaac tttgtccttg 60 acgtgtgcag tctatggagg gtcctttagc ggttattatt ggtcctggat tagacagtcc 120 cctgagaagg ggctcgagtg gataggcgaa atcaatcatg gggggtatgt gacttacaat 180 ccctccctcg agtcccgggt aactatcagc gtggacactt cgaagaatca attttcacta 240 aagcttagtt ctgtcactgc tgctgataca gctgtctact attgtgcgcg tgattacgga 300 ccaggtaatt acgattggta tttcgacttg tgggggaggg gtaccttggt cacagtatca 360 tcc 363 <210> 23 <211> 1344 <212> DNA <213> Synthetic <400> 23 caggttcagc tacagcagtg gggagcaggt ctgctgaaac caagcgaaac tttgtccttg 60 acgtgtgcag tctatggagg gtcctttagc ggttattatt ggtcctggat tagacagtcc 120 cctgagaagg ggctcgagtg gataggcgaa atcaatcatg gggggtatgt gacttacaat 180 ccctccctcg agtcccgggt aactatcagc gtggacactt cgaagaatca attttcacta 240 aagcttagtt ctgtcactgc tgctgataca gctgtctact attgtgcgcg tgattacgga 300 ccaggtaatt acgattggta tttcgacttg tgggggaggg gtaccttggt cacagtatca 360 tccgcaagta cgaagggccc ttccgtgttt ccactcgctc cctgcagtcg aagcacctca 420 gaatcaaccg ccgctctggg gtgtctcgtg aaggactact tcccggaacc tgtgaccgtc 480 agctggaact ccggggccct gacgagcgga gtgcacacct tccccgccgt gctccagagt 540 agtggacttt actccttatc ttccgtcgtc acagtgccta gttcatctct ggggaccaag 600 acatacactt gcaacgtgga ccataaacct tcaaacacga aagtcgataa acgcgtcgag 660 tctaaatacg gtcctccatg tccgccttgc cctgcccccg agtttctagg aggaccatca 720 gtctttcttt tcccaccaaa accgaaggac acgctcatga tttcacggac ccccgaagtg 780 acctgcgtgg tggtggacgt atcccaggag gatccagagg tgcagtttaa ttggtatgtg 840 gacggggtag aagttcataa cgctaaaacg aagcctcgcg aggaacaatt caatagtacc 900 tatagagtgg tgtcagtgct cactgtactg caccaggatt ggctcaacgg caaggagtac 960 aaatgtaagg tgtccaataa ggggctgccc agttctattg agaagacaat cagcaaggcg 1020 aagggccagc caagggagcc acaggtctat acactaccac caagccagga agagatgaca 1080 aagaaccagg tgtcactgac ttgtctggtc aagggctttt atccatctga tattgccgtg 1140 gagtgggagt ccaacggaca gccagagaac aactacaaga ccaccccccc cgtcctggac 1200 tctgacggct cattctttct gtatagcaga ctgaccgtgg ataagtctcg gtggcaggaa 1260 gggaacgtct tctcgtgcag cgtcatgcac gaggccctgc acaaccacta cacgcagaag 1320 tctctctcgc tttccctagg gaag 1344 <210> 24 <211> 11 <212> PRT <213> Synthetic <400> 24 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 25 <211> 7 <212> PRT <213> synthetic <400> 25 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 26 <211> 11 <212> PRT <213> synthetic <400> 26 Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr 1 5 10 <210> 27 <211> 109 <212> PRT <213> synthetic <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 327 <212> PRT <213> Synthetic <400> 28 Thr Thr Thr Ala Ala Thr Thr Thr Cys Cys Ala Cys Cys Thr Thr Gly 1 5 10 15 Gly Thr Ala Cys Cys Ala Cys Cys Ala Cys Cys Gly Ala Ala Cys Gly 20 25 30 Thr Cys Ala Gly Ala Gly Cys Gly Gly Gly Ala Gly Gly Cys Cys Ala 35 40 45 Ala Thr Thr Ala Gly Ala Cys Cys Thr Cys Thr Gly Thr Thr Gly Ala 50 55 60 Cys Ala Ala Thr Ala Gly Thr Ala Ala Ala Cys Thr Gly Cys Gly Ala 65 70 75 80 Ala Ala Thr Cys Cys Thr Cys Gly Gly Gly Thr Thr Cys Cys Ala Ala 85 90 95 Cys Gly Ala Ala Cys Thr Ala Ala Thr Gly Gly Thr Cys Ala Gly Thr 100 105 110 Gly Thr Gly Ala Ala Gly Thr Cys Gly Gly Thr Gly Cys Cys Gly Cys 115 120 125 Thr Gly Cys Cys Gly Gly Ala Thr Cys Cys Gly Gly Ala Ala Ala Ala 130 135 140 Cys Cys Thr Gly Gly Cys Thr Gly Gly Gly Ala Thr Gly Cys Cys Gly 145 150 155 160 Gly Thr Gly Gly Cys Cys Cys Gly Ala Thr Thr Gly Gly Ala Cys Gly 165 170 175 Cys Gly Thr Cys Ala Thr Ala Gly Ala Thr Cys Ala Gly Thr Ala Gly 180 185 190 Thr Cys Thr Gly Gly Gly Ala Gly Cys Cys Thr Gly Ala Cys Cys Cys 195 200 205 Gly Gly Thr Thr Thr Cys Thr Gly Cys Thr Gly Ala Thr Ala Cys Cys 210 215 220 Ala Ala Gly Cys Cys Ala Gly Ala Thr Ala Thr Gly Ala Thr Gly Ala 225 230 235 240 Ala Ala Cys Gly Cys Thr Cys Thr Gly Gly Gly Ala Thr Gly Cys Cys 245 250 255 Cys Thr Gly Cys Ala Gly Gly Ala Thr Ala Ala Gly Gly Thr Thr Gly 260 265 270 Cys Ala Cys Gly Cys Thr Cys Cys Cys Cys Thr Gly Gly Ala Gly Ala 275 280 285 Cys Ala Gly Ala Cys Thr Cys Ala Gly Gly Gly Thr Thr Gly Cys Thr 290 295 300 Gly Gly Gly Gly Ala Cys Thr Gly Cys Gly Thr Cys Ala Gly Thr Ala 305 310 315 320 Cys Thr Ala Thr Cys Thr Cys 325 <210> 29 <211> 216 <212> PRT <213> Synthetic <400> 29 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 30 <211> 648 <212> DNA <213> Synthetic <400> 30 gcactcgcct cgattgaagc tcttagtaac ggggctggat aagccctggt gtgtcacttc 60 gcaggcatac accttatgtt tctcgtagtc ggccttactc agggtcaggg ttgaagaaag 120 tgaatacgta gaatctttgg agtcttgctc ggtcacgctc tcttgactat tcccgctctg 180 cagggcattg tccaccttcc attggacttt tgcctccctg gggtaaaagt tgttgaggag 240 acagacgacg ctcgccgtgc cggacttgag ctgttcgtca gatggaggga agatgaaaac 300 gctgggagcc gcgacggttc ttttaatttc caccttggta ccaccaccga acgtcagagc 360 gggaggccaa ttagacctct gttgacaata gtaaactgcg aaatcctcgg gttccaacga 420 actaatggtc agtgtgaagt cggtgccgct gccggatccg gaaaacctgg ctgggatgcc 480 ggtggcccga ttggacgcgt catagatcag tagtctggga gcctgacccg gtttctgctg 540 ataccaagcc agatatgatg aaacgctctg ggatgccctg caggataagg ttgcacgctc 600 ccctggagac agactcaggg ttgctgggga ctgcgtcagt actatctc 648 <210> 31 <211> 30 <212> DNA <213> Synthetic <400> 31 atggatcaca accagtatct cttaacgatg 30 <210> 32 <211> 30 <212> DNA <213> Synthetic <400> 32 gaaatataga ttgttgtaga aatagtacct 30 <210> 33 <211> twenty four <212> DNA <213> Synthetic <400> 33 atatcgcatt ttctaacgtg atgg 24 <210> 34 <211> twenty four <212> DNA <213> Synthetic <400> 34 ggtttatcta acgacacaac atcc 24 <210> 35 <211> twenty four <212> DNA <213> Synthetic <400> 35 gatgcgattc aaaaaagaat cctc 24 <210> 36 <211> twenty four <212> DNA <213> Synthetic <400> 36 ggataaggtt gcacgctccc ctgg 24 <210> 37 <211> twenty four <212> DNA <213> Synthetic <400> 37 ctttactcct tatcttccgt cgtc 24 <210> 38 <211> twenty four <212> DNA <213> Synthetic <400> 38 gcaacgcttc gtgcatcacg gagc 24 <210> 39 <211> twenty four <212> DNA <213> Synthetic <400> 39 gtagtccttc acgagacatc ctag 24 <210> 40 <211> twenty four <212> DNA <213> Synthetic <400> 40 gccgtctact actgtcagca gtct 24 <210> 41 <211> 25 <212> DNA <213> Synthetic <400> 41 tgtgtaccgg gagcagatcc tatat 25 <210> 42 <211> twenty four <212> DNA <213> Synthetic <400> 42 cggcgcagtg agtaatcaag gtca 24 <210> 43 <211> twenty four <212> DNA <213> Synthetic <400> 43 attagccgga ccccggaagt gact 24 <210> 44 <211> twenty four <212> DNA <213> Synthetic <400> 44 ggcttggtgg tagtgtatag acct 24 <210> 45 <211> twenty four <212> DNA <213> Synthetic <400> 45 accccccatg attgatttcg ccta 24 <210> 46 <211> twenty four <212> DNA <213> Synthetic <400> 46 ctccaaagat tctacgtatt cact 24

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 immune activator; The oncolytic virus is vaccinia virus; The first heterologous polynucleotide encodes a PD-1 inhibitor, which is a full-length monoclonal antibody against PD-1; wherein the full-length monoclonal antibody against PD-1 comprises a first heavy chain, which comprises SEQ ID NOs: 2, 3, and 4; and the full-length monoclonal antibody against PD-1 further comprises a first light chain, which comprises SEQ ID NOs: 9, 10, and 11; The second heterologous polynucleotide encodes a CD137 activator, which is a full-length antibody that specifically binds to CD137; wherein the full-length antibody that specifically binds to CD137 comprises a second heavy chain, which comprises SEQ ID NOs: 17, 18, and 19; and the full-length antibody that specifically binds to CD137 further comprises a second light chain, which comprises SEQ ID NOs: 24, 25, and 26; The first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed at different stages of the replication cycle of the modified oncolytic virus; wherein the modified oncolytic virus comprises the following elements in frame in the 5′ to 3′ direction of the sense strand: a polynucleotide encoding the light chain of an antibody that binds to CD137-a first early and late promoter-a second early and late promoter-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-a first late promoter-a second late promoter-a polynucleotide encoding the light chain of an antibody that binds to PD-1; wherein the first heterologous polynucleotide is immediately downstream of the second heterologous polynucleotide; The first early and late promoters are in a head-to-head orientation, and the first late promoter and the second late promoter are in a head-to-head orientation, which means that the two promoters are adjacent to each other on the viral genome and drive protein expression in opposite directions; wherein the viral genome comprises at least one deletion or disruption that enables the virus to replicate selectively in tumor cells; wherein the deletion or the disruption is in an open reading frame encoding at least a portion of an enzyme that is both essential for replication of the virus and preferentially expressed in tumor cells compared to non-tumor cells; wherein the enzyme is thymidine kinase; The first heterologous polynucleotide and the second heterologous polynucleotide are inserted at the location of the deletion or the disruption.

2. The modified oncolytic virus according to claim 1, wherein the modified oncolytic virus is attenuated and can replicate in tumor cells.

3. The modified oncolytic virus according to claim 1, wherein the oncolytic virus is derived from the WR strain.

4. The modified oncolytic virus according to claim 1, wherein the full-length monoclonal antibody against PD-1 specifically binds to SEQ ID NO:

1.

5. The modified oncolytic virus according to claim 1, wherein the first heavy chain comprises a variable region having SEQ ID NO:

5.

6. The modified oncolytic virus according to claim 1, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO:

6.

7. The modified oncolytic virus according to claim 1, wherein the first light chain comprises a variable region having an amino acid sequence of SEQ ID NO:

12.

8. The modified oncolytic virus according to claim 1, wherein the first light chain comprises the amino acid sequence of SEQ ID NO:

13.

9. The modified oncolytic virus according to claim 1, wherein the first heterologous polynucleotide comprises a nucleic acid sequence of SEQ IDNO:

15.

10. The modified oncolytic virus according to claim 1, wherein the full-length antibody that specifically binds to CD137 specifically binds to SEQ ID NO:

16.

11. The modified oncolytic virus according to claim 1, wherein the second heavy chain comprises a variable region having an amino acid sequence of SEQ ID NO:

20.

12. The modified oncolytic virus according to claim 1, wherein the second heterologous polynucleotide comprises a nucleic acid sequence of SEQ ID NO:

22.

13. The modified oncolytic virus according to claim 1, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO:

21.

14. The modified oncolytic virus according to claim 1, wherein the second heterologous polynucleotide comprises a nucleic acid sequence of SEQ ID NO:

23.

15. The modified oncolytic virus according to claim 1, wherein the second light chain comprises a variable region having an amino acid sequence of SEQ ID NO:

27.

16. A modified oncolytic virus according to claim 12, wherein the second light chain comprises the amino acid sequence of SEQ ID NO:

29.

17. The modified oncolytic virus of claim 1, wherein the late promoter is pSL.

18. The modified oncolytic virus of claim 1, wherein the early and late promoters are pSE / L.

19. The modified oncolytic virus according to any one of claims 1-18, wherein the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune activator expressed by the second heterologous polynucleotide are expressed as separate proteins.

20. A pharmaceutical composition comprising the modified oncolytic virus according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. Use of the modified oncolytic virus according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating a tumor.

22. The use according to claim 21, wherein the subject of the medicament is a human.

23. The use according to claim 21, wherein the tumor is a solid tumor.

24. The use according to claim 21, wherein the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer or hepatocellular carcinoma.

25. The use according to claim 21, wherein the route of administration of the drug is topical administration.

26. The use according to claim 25, wherein the administration route is intratumoral injection.

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