Oncolytic herpes simplex virus vector expressing an immune system-stimulating molecule

Abstract

The present application provides a kind of oncolytic herpes simplex virus vector of expression immune system-stimulating molecule, it includes the expression cassette of IL12, IL15 and IL15 receptor alpha subunit, expression cassette is flanked with modified ICP47 promoter.The herpes simplex virus vector of the present application is restructured, can better replicate and express foreign gene, the protein expressed has good stability, is suitable for practical application.

Description

TECHNICAL FIELD

[0001] The present application relates generally to oncolytic herpes simplex virus (oHSV) vectors expressing molecules that stimulate the immune system. BACKGROUND

[0002] Oncolytic viruses (OVs) have emerged as a therapeutic option for the specific destruction of cancer cells through an oncolytic effect, the killing mechanism of which is characterized by the lysis of cancer cells through a process of replication of the virus.

[0003] The present application overcomes the shortcomings of the current commercialized oncolytic viruses and has low toxicity, high expression level and good stability. SUMMARY

[0004] Briefly, the present disclosure relates to protecting a herpes simplex virus vector (HSV vector) against one or more of IL12, IL15 and / or IL15 receptor alpha subunit. In one embodiment, the herpes simplex virus HSV vector comprises an expression cassette for IL12, IL15 and IL15 receptor alpha subunit, flanked by a modified ICP47 promoter.

[0005] In one embodiment, the sequence of the modified ICP47 promoter comprises at least SEQ ID No. 584.

[0006] In one embodiment, the sequence of the modified ICP47 promoter is SEQ ID No. 584.

[0007] In one embodiment, there is an in-frame nucleic acid sequence encoding a self-cleaving peptide 2A between the coding sequences of IL12, IL15 and IL15 receptor alpha subunit.

[0008] In one embodiment, the amino acid sequence of the self-cleaving peptide 2A is:

[0009] VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNFSLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFNAHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNFSLLKLAGDVELNPGP, EGRGSLLTCGDVEENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP, or CDAQRQKLLLSGDIEQNPGP.

[0010] In one embodiment, one or more IRES sequences are located between the coding sequences for IL12, IL15, and IL15 receptor a subunit.

[0011] In one embodiment, IL15 and IL15 receptor a subunit are expressed by a bi-directional promoter.

[0012] In one embodiment, IL15 and IL15 receptor a subunit each follow a nucleic acid sequence encoding Lys5 or Glu5.

[0013] In one embodiment, the hIL15 receptor a subunit is selected from the group consisting of variant 1 (SEQ ID NO: 3), variant 2 (SEQ ID NO: 4), variant 3 (SEQ ID NO: 5), variant 4 (SEQ ID NO: 6).

[0014] In one embodiment, the expression cassette comprising IL12, IL15, and IL15 receptor a subunit is inserted into the internal repeat region of HSV or the terminal repeat region of the HSV genome.

[0015] In one embodiment, the herpes simplex virus (HSV) vector further comprises an expression cassette for one or more PD-L1 blocking peptides.

[0016] In one embodiment, the expression cassette for the PD-L1 blocking peptide is inserted between UL3 and UL4 of the HSV viral genes.

[0017] In one embodiment, the herpes simplex virus (HSV) vector further comprises a sequence encoding an Fc region linked to the 3’-end of the PD-L1 blocking peptide.

[0018] In one embodiment, the sequence of the Fc region connected to the 3'-end of the PD-L1 blocking peptide is the sequence encoding the IgG4 Fc region.

[0019] In one embodiment, further comprising NFkB and OCT4 / SOX2 enhancer elements in the ICP4 or ICP27 regulatory region of the herpes simplex virus HSV vector.

[0020] In one embodiment, the ICP34.5 gene of the herpes simplex virus HSV vector is partially deleted, or non-functional.

[0021] The present disclosure also relates to a preparation consisting of:

[0022] a suspension of viruses expressed by the herpes simplex virus HSV vector according to any one of claims 1 to 15, glycerol, water;

[0023] wherein the concentration of glycerol in the preparation is 5 + 3%.

[0024] The present disclosure also relates to a pharmaceutical composition comprising the herpes simplex virus HSV vector of the present application, and a pharmaceutically acceptable carrier.

[0025] The present disclosure also relates to the use of the herpes simplex virus HSV vector described herein, or the preparation described herein, or the pharmaceutical composition described herein, for the manufacture of a medicament for the treatment of cancer.

[0026] In one embodiment, the cancer is selected from the group consisting of hepatocarcinoma, gastric cancer, intestinal cancer, lung cancer, breast cancer, nasopharyngeal cancer, head and neck tumor, bladder cancer, colon cancer, rectal cancer, renal cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, acute or chronic myelogenous leukemia, melanoma, endometrial cancer, head and neck cancer, glioblastoma, osteosarcoma leukemia, lymphoma, myeloma and sarcoma.

[0027] In one embodiment, the treatment of cancer is administered by subcutaneous injection, intratumoral injection or intravenous injection

[0028] The Summary has been presented in order to provide a brief overview of certain concepts and in the Detailed Description their further description will be presented. Unless otherwise noted, nothing in the Summary is intended to limit the key or essential features of the subject matter claimed herein or limit the scope of the subject matter claimed herein.

[0029] Effects of the Invention

[0030] The present application overcomes the shortcomings of the current commercialized oncolytic viruses, and has low toxicity, high expression level and good stability.

[0031] The present application, by modifying the structure of the virus, such as modifying the promoter region of some genes of the virus, enables the modified virus to exist in the infected cells for a longer time to avoid the attack of immune cells after infecting the cells, so as to better replicate and express the foreign genes.

[0032] The present application also improves the foreign genes carried by the vector, such as constructing a fusion protein of the foreign polypeptide and a suitable terminal structure, so that the expressed protein has good stability and is suitable for practical application.

[0033] The present application also provides an optimized medium for preserving the virus, which can maintain the activity of the virus for a long time after the virus is expressed by the vector of the present application, facilitating the industrial application of the vector and the virus.

[0034] One or more embodiments will be described in detail below. Features shown or described with respect to one exemplary embodiment can be combined with features of other embodiments. Thus, any one of the various embodiments described herein can be combined with one another to provide further embodiments. Other aspects, features, and advantages of the present application will become apparent to those of ordinary skill in the art upon examination of the description, figures, and claims. It is intended that all such additional embodiments be included within this description, be within the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0035] Exemplary features of the disclosure, its nature and various advantages will be understood more readily according to the following detailed description of various embodiments with reference to the accompanying drawings. The implementation described is non-limiting and non-exhaustive. In the drawings, like reference numerals and characters refer to like parts throughout the various views. The size and relative positions of the elements in the drawings attached are not necessarily drawn to scale. For example, the shapes of the various elements have been chosen for the purpose of presentation and illustration and not with a desire to limit the scope of the disclosure. Certain specific shapes are chosen to illustrate the example nature of the disclosure. One or more embodiments are described below with reference to the drawings, wherein:

[0036] Figure 1A and Figure 1B is a schematic diagram of an exemplary oHSV vector.

[0037] Figure 2 A schematic diagram of the modified ICP34.5 region of the virus hVG001-1-2 (SEQ ID NO: 572) is shown.

[0038] Figure 3 A schematic of the modified UL54 promoter region of the virus hVG001-1-2 (SEQ ID NO: 573) is shown.

[0039] Figure 4 A schematic of the hVG001-1-2 viral genome with PD-L1 blocker insertion (SEQ ID NO: 574) is shown.

[0040] Figure 5 A schematic of the hVG001-1-2 modified TR region (SEQ ID NO: 575) is shown.

[0041] Figures 6A-6C ELISA and Western blot analysis of IL-12 expression following infection of cells with hVG001-1-2 is shown.

[0042] Figures 7A-7C ELISA and Western blot analysis of IL-15 expression following infection of cells with hVG001-1-2 is shown.

[0043] Figures 8A-8C ELISA and Western blot analysis of IgG4 expression following infection of cells with hVG001-1-2 is shown.

[0044] Figures 9A-9C (A) Schematic of exemplary construct where a bi-CMV promoter drives expression of IL-15Rα and Sushi domain of IL-15, and (B-C) DNA sequence and schematic (SEQ ID No: 557).

[0045] Figures 10A-10C (A) Schematic of exemplary construct where a bi-CMV promoter drives expression of IL-15 and IL-15Rα variant 4, and (B-C) DNA sequence and schematic (SEQ ID No: 558).

[0046] Figures 11A-11C (A) Schematic of exemplary construct where a bi-CMV promoter drives expression of IL-15-K5 and IL-15Rα Sushi domain-E5, and (B-C) DNA sequence and schematic (SEQ ID No: 559).

[0047] Figures 12A-12D(A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0048] Figures 13A-13D (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0049] Figures 14A-14D (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0050] Figures 15A-15D (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0051] Figures 16A-16D (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0052] Figures 17A-17E (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0053] Figures 18A-18E (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0054] Figures 19A-19D (A) a schematic of an exemplary construct in which the EF1 alpha promoter controls expression of IL-15-IRES-IL-15R alpha Sushi domain, and (B-D) DNA sequence and schematic (SEQ ID No: 561).

[0055] Figure 20A 、 Figure 20B 、 Figure 20C 、 Figure 20D 、 Figure 20D continued (A) Exemplary construct in which the CMV promoter controls the expression of IL-12-p2A-IL-15K5-p2A-IL-15Rα variant 1-E5, and (B), (C), (D), (D) cont. are DNA sequences and schematics (SEQ ID No: 568).

[0056] Figure 21A 、 Figure 21B 、 Figure 21C Graph showing the percent inhibition of PD-L1 by blocking peptide binding to PD-1.

[0057] Figures 22A-22B Graph showing the effect of PD-L1 inhibiting peptide on cytotoxicity of target cells by anti-CD3 stimulated human peripheral blood mononuclear cells.

[0058] Figure 23A and Figure 23B Graph showing the effect of IL-12 alone, IL15 plus IL-15Rα, and IL-12 and IL15 / IL-15Rα together on production of IFNy and TNFα in human peripheral blood mononuclear cells.

[0059] Figure 24A and Figure 24B Graph showing the effect of IL-12 and IL15 / IL-15Rα on cytotoxicity of U87 and MDA-MB-23 tumor cells by peripheral blood mononuclear cells.

[0060] Figures 25A-25C Graph showing expression of IL12, IL15, and PD-L1 blocking peptide after infection of tumor cells with viruses carrying PD-L1 blocking peptide or human IL15 / 15Ra (VG001-PLBh and VG001-15h). Figure 25D 、 Figure 25E Graph showing results after infection of tumor cells with viruses carrying PD-L1 blocking peptide or human IL15 / 15Ra (VG001-PLBh and VG001-15h).

[0061] Figures 26A-26D Graph showing results of in vitro assays of various constructs. Figures 26A-26B Graph showing results of cell transfection with IL-TF-Fc plasmid expressing IL-12, IL-15, and PD-L1 blocker. Figures 26C-26D Graph showing results of cell transfection with various mutant viruses including hVG001-1-2.

[0062] Figures 27A-27E Results of cell viability assays against hVG001-1-2 and HSV-345 performed on human tumor cell lines and Vero cell lines are shown.

[0063] Figures 28A-28J Results of in vitro assays of various constructs are shown. Figures 28A-28E Results of cell viability assays of mVG001-1-2 and HSV-345 on mouse tumor cell lines and Vero cell lines are shown; Figures 28F-28H Characterization of transgene expression following mVG001-1-2 infection of CT26 mouse tumor cells is shown, Figures 28I-28J Characterization of stimulation of peripheral mononuclear cells to release cytotoxic factors following infection of CT26 cells with mVG001-1-2 is shown.

[0064] Figures 29A-29C Results of in vitro characterization of transgene expression following infection of various cell lines with hVG001-1-2 or VG001-1.7 are shown. Figures 29D-29E Infected tumor cells stimulate peripheral mononuclear cells to release cytokines and cell-killing activity are shown.

[0065] Figures 30A-30G Results of assays to assess the ability of hVG001-1-2 to kill various human cancer cells in vitro are shown.

[0066] Figures 31A-31G Results of mVG001-1-2 and hVG001-1-2 constructs to inhibit tumor growth in vivo are shown.

[0067] Figures 32A-32C Growth curves of different viruses on three different human cell lines are shown.

[0068] Figures 33A-33D Growth curves of mVG001-1-2 and HSV-345 on mouse tumor cell lines and Vero cell lines are shown.

[0069] Figures 34A-34E Growth curves of hVG001-1-2 and HSV-345 on human tumor cell lines and Vero cell lines are shown.

[0070] Figures 35A-35D Effects of virus modification are shown.

[0071] Figures 36A-36D Effectiveness of exogenous genes expressed in vitro.

[0072] Figure 37A And Figure 37B Expression of exogenous genes carried by viruses intratumorally following injection of viruses in mouse models is provided.

[0073] Figures 38A-38C The impact of VG001-1-2 on the immune response is provided.

[0074] Figure 39 is a schematic of the modified exemplary oHSV viral vector (VG001-1-2).

[0075] Figure 40 The modified ICP34.5 region of the virus VG001-1-2 is shown (SEQ ID NO: 599).

[0076] Figure 41 The modified UL54 promoter region of the virus VG001-1-2 is shown (SEQ ID NO: 596).

[0077] Figure 42 The region in VG001-1-2 in which a PD-L1 blocker is inserted is shown (SEQ ID NO: 589).

[0078] Figure 43 The modified terminal repeats (TR) region of VG001-1-2 is shown (SEQ ID NO: 576), which carries an expression cassette encoding IL-12, IL-15 and IL-15 receptor alpha subunit, and is flanked by US2 (ICP47).

[0079] Figure 44 is a schematic of the short (S) type promoter A (SEQ ID NO: 583).

[0080] Figure 45 is a schematic of the medium (M) type promoter A (SEQ ID NO: 584).

[0081] Figure 46 is a schematic of the long (L) type promoter A (SEQ ID NO: 585).

[0082] Figure 47 is a schematic of the promoter B (survivin) (SEQ ID NO: 586).

[0083] Figures 48A-48F is the sequence of the IL12-IL15-IL15RA1 expression cassette carried in VG001-1-2 and its flanking sequences (SEQ ID NO: 576).

[0084] Figures 49A-49C is the sequence of the modified UL54 (ICP27) promoter-regulatory region in VG001-1-2 (SEQ ID NO. 596).

[0085] Figures 50A-50D Sequence for the region of the gene in VG001-1-2 inserted between UL3 and UL4 (SEQ ID No. 589) for the PD-L1 blocker.

[0086] Figure 51 Sequence for the modified ICP34.5 region contained in VG001-1-2 (SEQ ID NO. 599).

[0087] Figure 52 Sequence for short (S) promoter A (SEQ ID NO: 583).

[0088] Figure 53 Sequence for medium (M) promoter A (SEQ ID NO: 584).

[0089] Figure 54 Sequence for long (L) promoter A (SEQ ID NO: 585).

[0090] Figure 55 Sequence for promoter B (survivin) (SEQ ID NO: 586).

[0091] Figures 56A-56B Results of assays showing that the VG001-1-2 virus causes cytotoxicity to (A) human cancer cells, (B) mouse cancer cells.

[0092] Figures 57A-57C (A) IL-12, (B) IL-15, and (C) PD-L1 blocker expression following infection of cells with the VG001-1-2 virus.

[0093] Figure 58 Results of ELISA analysis of the PD-L1 blocker produced following infection of cells with VG001-1-2.

[0094] Figure 59 Results of cell-based analysis of the PD-L1 blocker produced following infection of cells with VG001-1-2.

[0095] Figures 60A-60D IL-12, IL-15 / IL-15RA, and PD-L1 blocker synergistically promote immune cell function in vitro.

[0096] Figures 61A-61D Results showing the effect of intratumoral inoculation with the VG001-1-2 virus.

[0097] Figures 62A-62BEffects of gene expression and T cell activity produced by treatment of tumor cells with mVG001-1-2 virus are shown.

[0098] Figures 63A-63D Effects of mVG001-1-2 treatment on intra-tumoral lymphocyte populations are shown.

[0099] Figures 64A-64B Effects of minimal dose human IL12 or human IL15 / IL15RA on immune cell function are shown.

[0100] Figure 65A 、 Figure 65B Biodistribution of VG001-1-2 virus is shown.

[0101] Figure 66 Results of quantification of immune-related gene expression levels in tumor-bearing mice treated with VG001-1-2 are shown.

[0102] Figure 67 Flow analysis results of cells infected with different viruses are shown.

[0103] Figure 68 Titer analysis results of viruses stored in different formulations of media are shown.

[0104] Figure 69 Stability test results of different configurations of fusion proteins expressed by the virus are shown.

[0105] Figure 70 Structure of VG001-1-2 vector is shown. DETAILED DESCRIPTION

[0106] The present disclosure can be more easily understood through the following detailed description of the preferred embodiments and examples of the application included herein.

[0107] Disclosure Overview

[0108] The present application can be more easily understood through a detailed description of the preferred embodiments and examples of the application included herein. Briefly, the present disclosure provides oncolytic herpes simplex virus type 1 or 2 vectors that express immunostimulatory molecules. Representative vectors comprise expression cassettes encoding one or more of IL-12, IL-15, and IL-15Rα. Certain vectors encode murine or human IL-12, human IL-15, human IL-15Rα. In certain embodiments, the vector encodes murine or human IL-12, hIL15, and hIL15 receptor alpha subunit. In other embodiments, the vector encodes hIL-12, HIL15, and HIL15 receptor alpha subunit. The three proteins can be expressed on one, two, or three transcripts. When expressed on the same transcript, post-transcriptional processes ensue that lead to expression of the individual proteins. In such cases, the coding regions are separated by a self-cleaving peptide 2A or an IRES sequence. The coding regions can also be expressed by a bi-directional promoter. The HSV vector optionally expresses one or more PD-L1 blocking peptides that it is capable of secreting.

[0109] A. oHSV vector

[0110] An oncolytic virus is a virus that preferentially lyses cancer cells (oncolysis), preferably in a selective manner. Viruses that replicate selectively in differentiated cells over non-differentiated cells are often oncolytic. Suitable oncolytic viruses for use herein include herpes simplex viruses 1 and 2, and can also include non-human herpes viruses, such as BHV or others.

[0111] Herpes simplex viruses (HSV) 1 and 2 are members of the Herpesviridae family that infect humans. The HSV genome contains two unique regions, referred to as the long unique (UL) region and the short unique (US) region. Each of these regions is flanked by a pair of inverted terminal repeat sequences. There are 75 known open reading frames. The viral genome has been engineered to develop oncolytic viruses for use in, for example, cancer therapy. Tumor-selective replication of HSV can be achieved by mutation of the HSV ICP34.5 (also referred to as γ34.5) gene. HSV contains two copies of ICP34.5, and mutants that inactivate one or both copies of the ICP34.5 gene lack neurovirulence, i.e., are non-pathogenic / non-neurovirulent and are oncolytic.

[0112] Suitable oncolytic HSVs can be derived from HSV-1 or HSV-2, including any laboratory strain or clinical isolate. In some embodiments, the oHSV can be or can be derived from one of the laboratory strains HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG52. In other embodiments, it can be any clinical isolate strain or other non-laboratory strain JS-1. Other suitable HSV-1 viruses include HrrR3 (Goldsten and Weller, J. Virol. 62, 196-205, 1988); G207 (Mineta et al., Nature Medicine. 1(9):938-943, 1995; Kooby et al., The FASEB Journal, 13(11): 1325-1334, 1999); G47Delta (Todo et al. Proceedings of the National Academy of Sciences. 2001; 98(11): 6396-6401); HSV 1716 (Mace et al. Head & Neck, 2008; 30(8): 1045-1051; Harrow et al., Gene Therapy. 2004; 11(22): 1648-1658); HF10 (Nakao et al., Cancer Gene Therapy. 2011; 18(3): 167-175); NV1020 (Fong et al., Molecular Therapy, 2009; 17(2): 389-394); T-VEC (Andtbacka et al., Journal of Clinical Oncology, 2015: 33(25): 2780-8); J100 (Gaston et al., PloS one, 2013; 8(11): e81768); M002 (Parker et al., Proceedings of the National Academy of Sciences, 2000; 97(5): 2208-2213); NV1042 (Passer et al., Cancer Gene Therapy. 2013; 20(1): 17-24); G207-IL2 (Carew et al., Molecular Therapy, 2001; 4(3): 250-256); rQNestin34.5 (Kambara et al., Cancer Research, 2005; 65(7): 2832-2839); G47A-mIL-18 (Fukuhara et al., Cancer Research, 2005; 65(23): 10663-10668); and those disclosed in PCT applications entitled "HSV Vectors with Enhanced Replication in Cancer Cells" PCT / US2017 / 030308 and "Compositions and Methods of Using Stat 1 / 3 Inhibitors with Oncolytic Herpes Virus" PCT / US2017 / 018539, among others, all of which are incorporated by reference and made a part hereof.

[0113] The oHSV vector can have at least one modification, mutation, or deletion of the γ34.5 gene. The vector lacks a complete γ34.5 gene. In some embodiments, both genes are deleted, mutated, or modified. In other embodiments, one gene is deleted and the other is mutated or modified. Either native γ34.5 gene can be deleted. In one embodiment, the terminal repeat region containing the γ34.5 gene and the ICP4 gene is deleted. Mutations, such as nucleotide changes, insertions, and deletions, render the gene non-expressible or the product inactive. The γ34.5 gene can be modified with a miRNA target sequence in its 3' UTR. The target sequence binds a miRNA that is expressed at a lower level in tumor cells than in their normal controls. In some embodiments, the modified or mutated γ34.5 gene is constructed in vitro and inserted into the oHSV vector as a replacement for the viral gene. When the modified or mutated γ34.5 gene replaces only one γ34.5 gene, the other γ34.5 gene is deleted. The γ34.5 gene can contain additional changes, such as having an exogenous promoter.

[0114] oHSV can have additional mutations, which can include disabling mutations (e.g., deletions, substitutions, insertions) that affect the virulence of the virus or its ability to replicate. For example, mutations can be made in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP 24, ICP56. Preferably, the mutation in one of these genes (optionally in both copies of the gene where appropriate) results in the inability of the HSV to express the corresponding functional polypeptide (or a reduction in that ability). In some embodiments, the promoter of a viral gene is replaced with a promoter that is selectively activated in the target cell, or inducible upon delivery of an inducer or upon a cellular event or in a particular environment. In particular embodiments, a tumor-specific promoter drives expression of a viral gene essential for HSV replication. In certain embodiments, expression of ICP4 or ICP27 or both is under the control of an exogenous promoter, e.g., a tumor-specific promoter. Exemplary tumor-specific promoters include survivin or telomerase; other suitable tumor-specific promoters can be specific to a single tumor type and are known in the art. Other elements can be present. In some cases, an enhancer (e.g., NF-kB / OCT4 / SOX2 enhancer) is present, e.g., in the regulatory region of ICP4 or ICP27 or both. Also, the 5' UTR can be exogenous, e.g., a 5' UTR from a growth factor gene (e.g., FGF).

[0115] oHSV can also have genes and nucleotide sequences of non-HSV origin. For example, a sequence encoding a prodrug, a sequence encoding a cytokine or other immune stimulatory factor, a tumor-specific promoter, an inducible promoter, an enhancer, a sequence homologous to a host T cell, and other sequences can be present in the oHSV genome. Exemplary sequences encode IL12, IL15, OX40L, a PD-L1 blocker, or a PD-1 blocker. For sequences encoding a product, they are operatively linked to the expression of a desired or desired promoter sequence and other regulatory sequences (e.g., enhancers, polyadenylation signal sequences).

[0116] The regulatory regions of the viral genes can be modified to include response elements that affect expression. Exemplary response elements include NF-κΒ, Oct-3 / 4-SOX2, enhancers, silencers, cAMP response elements, CAAT enhancer binding sequences, and insulator response elements. Other response elements can also be included. The viral promoters can be replaced with different promoters. The choice of the promoter depends on a number of factors, such as the HSV vector that is desired to be used, the treatment of the patient, the underlying state or condition, and the ease of application of an inducer (for inducible promoters). For cancer treatment, often when the promoter is replaced, it will have a cell-specific or tissue-specific or tumor-specific promoter. Tumor-specific, cell-specific, and tissue-specific promoters are known in the art. Other genetic elements can also be modified. For example, the 5' UTR of the viral genes can be replaced with an exogenous UTR.

[0117] B. Immune stimulating molecules

[0118] The oHSV vector includes nucleic acid sequences encoding one or more immunostimulatory molecules (e.g., IL-12, IL-15, and IL-15Rα). Exemplary amino acid sequences for IL-12, IL-15, and IL-15Rα are set forth in the Sequence Listing (SEQ ID NOs: 1-6). Any DNA sequence encoding the amino acid sequences is suitable, but codons are typically selected for preferred expression in the intended population of subjects receiving the oHSV.

[0119] 1. IL-12

[0120] Interleukin 12 (IL-12) is produced primarily by dendritic cells, macrophages, and monocytes in response to bacteria (e.g., lipopolysaccharides), pathogens, or activated T cells. IL-12 is able to induce IFNγ production, cell proliferation, and activation of natural killer cells and T cells. It is also critical for T cell differentiation into Thl cells. IL-12 is also able to inhibit tumor growth. Murine IL-12 is equally active on murine and human cells, and both are suitable for use in oHSV vectors.

[0121] Biologically active IL-12 is a heterodimeric molecule composed of 35 kDa (p35) and 40 kDa (p40) subunits covalently linked by disulfide bridges. Simultaneous expression of both subunits is necessary for production of the heterodimer. In an oHSV vector, IL-12 expression can be achieved in a number of ways. The two subunits can be expressed in separate constructs, each with a promoter, or in one construct from a bi-directional promoter, or from a construct with an element in the coding region, such as an IRES or a self-cleaving peptide. Alternatively, the subunits can be expressed as a single chain. For example, a functional single chain IL-12 fusion protein can be produced by linking the coding regions of p40 and p35 with a linker, which is typically composed of Ser or Gly or a combination of Ser and Gly, such as Ser5, (Gly4Ser)3, or Gly6Ser (e.g., Lieschke et al., Nature Biotechnology 15:35, 1997; Lode et al., PNAS 95:2475, 1998; alternative fusion constructs also see WO 2015 / 095249). The sequence and length of the linker is typically chosen in such a way that the structure has the most flexibility (Chen et al., Adv Drug Deliv Rev. 65:1357, 2013). Computer programs can be utilized to select linker sequences. One such program is called LINKER (Crasto and Feng, Protein Eng Design & Selection 13:309). One exemplary single chain IL-12 has the amino acid sequence of SEQ ID NO: 1. Amino acid substitutions, insertions, and deletions can be made, as long as the IL-12 remains functional.

[0122] 2. IL-15

[0123] IL-15 is a cytokine that regulates natural killer cell as well as T cell activation and proliferation, and can have other biological activities. There are two isoforms that differ in signal peptide sequence and have identical mature protein sequences. The sequence of the isoform with the longer signal peptide (sometimes referred to as LSP-IL15) is GenBank (NCBI) accession number NP 000576, while the sequence of the isoform with the shorter signal peptide (sometimes referred to as SSP-IL15) is accession number NP 751915. Either isoform is suitable for use in an oHSV vector. There can be amino acid insertions, deletions, and substitutions, such as found in polymorphisms, as long as the protein binds IL-15.

[0124] In some embodiments, both IL-15 and IL-15Ra have C-terminal peptides of a coiled coil with selective dimerization. A number of suitable peptides are taught in the literature (see, e.g., Tripet et al., Protein Engineering 9: 1029, 1996; Aronsson et al., Sci Rep 5: 14063, 2015). Typically, the amino acid sequence of the coiled coil has heptad repeats of hydrophobic (h) and polar (p) residues in the form hpphppp. Two exemplary coiled coils are K-coil (KVSALKE, SEQ ID No. 7) and E-coil (EVSALEK, SEQ ID NO. 8). Typically, 3-6 tandem copies are used. In some embodiments herein, 5 tandem copies are used. K5 (KVSALKEKVSALKEKVSALKEKVSALKEKVSALKE, SEQ ID NO. 9) and E5 (EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK, SEQ ID NO. 10). The K-coil and E-coil are designed with opposite charges, so IL-15 is fused to one coiled coil, while IL-15Ra is fused to a coiled coil with opposite charges. An exemplary Sushi domain fused to E5 is shown in SEQ ID NO: 12, an exemplary IL-15Ra variant 4 fused to E5 is shown in SEQ ID NO: 13, an exemplary IL-15Ra variant 1 fused to E5 is shown in SEQ ID NO: 14, and an exemplary IL-15 fused to K5 is shown in SEQ ID NO: 15.

[0125] 3. IL-15Ra subunit

[0126] The interleukin-15 receptor alpha subunit (IL-15Rα) is one of the three subunits of the complex that binds IL-15. This alpha subunit binds IL-15 with high affinity and is able to bind to IL-15 independently of the other subunits. There are at least four variants (isoforms), referred to herein as variant 1 (NP 002180.1) (SEQ ID NO:3); variant 2 (NP 751950.2) (SEQ ID NO:4); variant 3 (NP 001230468.1) (SEQ ID NO:5); and variant 4 (NP_001243694) (SEQ ID NO:6). The alpha subunit contains a Sushi domain (aka complement control protein (CCP), short consensus repeats (SCRs), or SUSHI repeat), which is the shortest region that retains IL-15 binding activity. A typical Sushi domain is about 60-70 amino acids, which contains four cysteines that form two disulfide bonds and is a common motif in protein-protein interactions. The Sushi domain of IL-15Rα comprises residues 31 to about 95 (corresponding to variant 1) (SEQ ID NO: 11). The location of the Sushi domain in the other variants is known. Amino acid substitution of the cysteines in sIL-15Rα abrogates its ability to suppress acute inflammation and T cells respond to alloantigen in vivo (Wei et al., J Immunol. 167:277, 2001).

[0127] The oHSV vector comprises a nucleic acid sequence encoding IL-15Rα, a variant of IL-15Rα, or a Sushi domain. Typically, the protein is expressed with a leader peptide, and in some embodiments, the leader peptide is from IL-15Rα. Other leader peptides are known in the art. Amino acid substitutions can be present, so long as the protein binds IL-15. Natural substitutions, polymorphisms are known.

[0128] 4. PD-L1 blocking peptide

[0129] Programmed death ligand 1 (PD-L1) plays a role in suppressing the immune system, possibly by binding to the PD-1 receptor. Blocking the protein-protein interaction has been shown to be able to improve cancer treatment.

[0130] An oHSV vector can express a PD-L1 blocking peptide. Suitable peptides include TAHPSPSPRSAGQF (SEQ ID NO: 16), EYRMSPSNQT (SEQ ID NO: 17), YYRMSPSNQT (SEQ ID NO: 18), TRYPSPSPKPEGRF (SEQ ID NO: 19), and WNRLSPSNQT (SEQ ID NO: 20). Other suitable peptides include those listed in Table 4 (SEQ ID NOs: 21-500). Typically, the blocking peptide is expressed from a leader sequence. Leader sequences are well known in the art. They include the immunoglobulin kappa chain leader sequence (METDTLLLWVLLLWVPGSTG; SEQ ID NO: 501) and the IL-2 leader sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 502). When there is more than one blocking peptide, the peptides are typically separated by a flexible linker peptide. The linker is typically Gly or Ser or Gly / Ser rich. Examples of suitable linkers are shown in (SEQ ID NOs: 503-519) (see also, Chichili et al., Protein Science 22:153, 2013). There can be one copy of the peptide or two copies or three copies or more copies. The multiple copies are typically random and can have linkers between the copies. The blocking peptide construct can also include an Fc sequence at the C-terminus of the peptide, either with or without the hinge region of an immunoglobulin Fc sequence. Although any Fc region can be used, typically, the Fc will be from one of the IgG subclasses, e.g., human IgGl, human IgG2, human IgG3, and human IgG4 or their murine counterparts.

[0131] C. Component organization

[0132] The molecules IL-12, IL-15, and IL-15Ra can have various different configurations in an oHSV vector. For example, each molecule can be expressed individually from separate promoters / regulatory regions or co-expressed from one or two separate promoters / regulatory regions.

[0133] In certain embodiments, two or three of the molecules are expressed from one promoter in a single transcript and their coding sequences are separated by an IRES (internal ribosome entry site) sequence. IRES regions attract the eukaryotic ribosomal translation initiation complex and thus enable translation initiation to occur in the middle of the mRNA and independently of the usual 5'-end cap structure. Suitable IRES sequences are well known and many suitable IRES sequences can be found in the IRESite's Expression Base of experimentally confirmed IRES sequences (see, e.g., http: / / iresite.org / IRESite_web.php?page=browse_plasmids; accessed May 26, 2016).

[0134] In various embodiments, the three genes are present in any order and separated by one or more IRES sequences. The IRES sequences can be the same or different. Additional sequences can be present at the gene / IRES junction or the IRES / IRES junction.

[0135] In certain embodiments, two or three of the molecules are expressed from one promoter in a single transcript and their coding sequences are separated by one or more self-cleaving peptide 2A. These peptides are short peptides (about 18-22 amino acids) and are inserted in frame between the coding sequences. During translation, the ribosome skips the synthesis of the glycolyl-prolyl peptide bond at the C-terminus of the 2A peptide, resulting in cleavage between the 2A peptide and its adjacent nearest downstream protein. Thus, they produce equimolar levels of multiple gene products from the same mRNA. The "cleavage" occurs between the gly-pro residues at the C-terminus, which means that the upstream cistron will have an additional residue added to its C-terminus, while the downstream cistron begins with a proline. Exemplary p2A peptide sequences are shown in SEQ ID NOs: 520-535.

[0136] Other ways to affect co-expression of the molecules is to use a bi-directional promoter. Bi-directional promoters are a common feature of the human genome (Trinklein et al., Genome Res 14:62, 2004). Bi-directional promoters initiate transcription in both directions and often contain shared elements that regulate both genes. In addition to natural bi-directional promoters, bi-directional promoters have been synthesized. One such promoter is bi-CMV. pBI-CMVl is a mammalian bidirectional expression vector that enables constitutive expression of two proteins of interest. Protein expression is driven by two constitutively active minimal human cytomegalovirus promoters (PminCMVl and PminCMV2 in opposite orientation). An exemplary DNA sequence for the bi-directional CMV promoter is SEQ ID NO. 536.

[0137] Bi-directional promoters (e.g., bi-CMV promoters) are primarily used to achieve co-expression of hIL15 and IL-15Ra (or Sushi domain). When two molecules are co-expressed using an IRES or p2A sequence, it is typically hIL15 and IL-15Ra (or Sushi domain). In these cases, IL-12 and PD-L1 blocking peptide can be co-expressed using a bi-directional promoter or as a polycistronic transcript with an IRES or p2A sequence, or they can be expressed separately from their own promoter / regulatory region.

[0138] Other promoters can be used. Cellular promoters, viral promoters, and the like are suitable. The promoter can be constitutive or inducible or cell / tissue specific. Many promoters are well known. One particular promoter that can be used is the constitutive EF-1 alpha promoter.

[0139] The sequences are assembled in one or more expression cassettes. Examples provide specific versions of some exemplary expression cassettes. The expression cassettes can be inserted into the HSV genome at any location that does not disrupt critical functions (e.g., replication). In certain embodiments, the expression cassettes are inserted in the internal or terminal repeat regions after the first deletion of the repeat region. Other suitable insertion regions include between viral genes, for example, between the UL3 and UL4 viral genes, between the UL50 and UL51 genes, and between US1 and US2.

[0140] In certain embodiments, expression cassettes expressing PD-L1 blocking peptides are inserted between viral genes (e.g., UL3 and UL4, UL50 and UL51, and / or US1 and US2). In other embodiments, expression cassettes expressing IL-12, IL-15, and IL-15Ra are inserted in place of the terminal repeat regions and expression cassettes expressing PD-L1 peptides are inserted between the UL3 and UL4 genes.

[0141] D. Therapeutic Composition

[0142] Therapeutic compositions that can be used to prevent, treat, or lessen the effects of a disease, e.g., cancer, are provided. More specifically, therapeutic compositions comprising at least one oncolytic virus as described herein are provided. Representative examples include oHSV with one or more expression cassettes for IL12, IL15, and / or IL receptor 15a subunits. In one embodiment, the expression cassettes express all of IL12, IL15, and IL receptor 15a subunits. In preferred embodiments, the expression cassettes comprise murine or human IL12, hIL15, and hIL15 receptor a subunits.

[0143] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable carrier" means any and all solvents, diluents, or vehicles, which are compatible with the oncolytic virus and not injurious to the subject to whom the composition is administered (generally see Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21stedition (May 1, 2005) and in The United States Pharmacopeia: The National Formulary (USP 40-NF 35 and Supplement).

[0144] In the context of the oncolytic viruses described herein, non-limiting examples of suitable pharmaceutical vehicles include phosphate buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various forms of wetting agents, sterile solutions, and the like. Additional pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantable elements containing the oncolytic virus, or any other vehicle, delivery agent, or dispersion device or material. Such vehicles can be configured by conventional methods and can be administered to a subject in an effective dose. Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycols, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like) and the salts of organic acids (such as acetates, propionates, malonates, benzoates, and the like). Such pharmaceutically acceptable (pharmaceutical grade) vehicles, diluents, and excipients for delivery of oHSV to target cancer cells preferably do not induce an immune response in the individual (subject) receiving the composition (and are preferably administered in a manner that is not unduly toxic).

[0145] The compositions provided herein can be provided in a variety of different concentrations. For example, the dose of oncolytic virus provided can range from about 10 6 pfu to about 10 9 pfu. In further embodiments, the dosage form of the treatment can range from about 10 6 to about 10 8 pfu / ml, with up to 4 ml injected into patients with large lesions (e.g., >5 cm) and smaller amounts (e.g., up to 0.1 ml) in patients with small lesions (e.g., <0.5 cm) every 2-5 weeks.

[0146] In certain embodiments of the present application, substandard doses can be used. Thus, in certain embodiments, less than about 10 6 pfu / ml can be administered to a patient (up to 4 ml injected into a patient every 2-3 weeks).

[0147] The compositions can be stored at temperatures that are beneficial for stable shelf life, including room temperature (about 20°C), 4°C, -20°C, -80°C, in liquid nitrogen. Compositions intended for in vivo use are typically stored at low temperatures, as they generally do not have preservatives. Compositions can be stored in dry form (e.g., lyophilized) or in liquid form.

[0148] E. Dosage

[0149] In addition to the compositions described herein, various methods are provided for treating or ameliorating cancer using such compositions, including the step of administering to a subject an effective dose or amount of an HSV vector as described herein.

[0150] The terms "effective dose" and "effective amount" refer to the amount of oncolytic virus that is sufficient to affect the targeted cancer treatment, e.g., an amount effective to reduce the size or load of the targeted tumor, or to impede the growth rate of the targeted tumor cells. More specifically, such terms refer to the amount of oncolytic virus that, when administered at the necessary dose and for the duration of treatment, is effective to achieve the desired result. For example, in the context of treating cancer, an effective amount of the compositions described herein is an amount that causes regression, reduces tumor load, and / or prevents tumor spread or cancer growth. The effective amount can vary depending on various factors, such as the disease state, age, sex, and weight of the subject, as well as the drug formulation, route of administration, and the like, but can be routinely determined by one of skill in the art.

[0151] The therapeutic compositions are administered to a subject diagnosed with or suspected of having cancer. The subject can be a human or a non-human animal.

[0152] The compositions are used to treat cancer. As used herein, the terms "treatment" or "treatment" mean a process for obtaining a beneficial or desired result, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions of a disease detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, prevention of disease progression, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of the disease state, and remission, whether partial or total. The terms "treatment" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0153] Representative forms of cancer include carcinomas, leukemias, lymphomas, myelomas, and sarcomas. Further examples include, but are not limited to, cholangiocarcinoma, brain cancer (e.g., glioblastoma), breast cancer, cervical cancer, colorectal cancer, CNS cancer (e.g., acoustic neuroma, astrocytoma, craniopharyngioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, retinocytoma, neuroblastoma, oligodendroglioma, pinealoma, and retinoblastoma), endometrial lining cancer, hematopoietic cell cancer (e.g., leukemia and lymphoma), kidney cancer, laryngeal cancer, lung cancer, liver cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (e.g., melanoma and squamous cell carcinoma), and thyroid cancer. Cancer can comprise a solid tumor (e.g., a sarcoma such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteosarcoma), a diffuse cancer (e.g., a leukemia), or some combination of these (e.g., a metastatic cancer having both a solid tumor and diffuse or disseminated cancer cells). Cancer can be resistant to conventional therapy (e.g., conventional chemotherapy and / or radiotherapy).

[0154] Other disorders of benign tumors and unwanted cell proliferation can also be treated.

[0155] The oHSV described herein can be administered by routes such as oral, topical, parenteral, systemic, intraarterial, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, or transdermal administration. In certain embodiments, the oncolytic virus can be delivered through a cannula, through a catheter, or by direct injection. The site of administration can be intratumoral or at a location remote from the tumor. The route of administration often depends on the type of cancer to be treated.

[0156] The attending physician can readily determine optimum or appropriate dosages of the oncolytic virus, depending on the expression of the patient, the observation of the patient, and various clinical factors, including, for example, the size, surface area, age, sex, and specific oncolytic virus administered, time and route of administration, the type of cancer being treated, the general health of the patient, and the other medications the patient can be taking. According to certain embodiments, the subject is treated with the oncolytic virus described herein can be used in conjunction with other types of therapy, such as chemotherapy, for example, with a chemotherapeutic agent such as etoposide, ifosfamide, doxorubicin, vincristine, doxycycline, and the like.

[0157] The oHSV can be formulated into pharmaceutical and pharmaceutical compositions for clinical use and can be combined with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The formulation depends at least in part on the route of administration. Suitable formulations can include the virus and inhibitor in a sterile culture medium. The formulation can be in fluid, gel, patch, or solid form. The formulation can be provided to the subject or to a medical professional.

[0158] A therapeutically effective amount is preferably administered. This is an amount sufficient to show benefit to the subject. The actual amount administered and timing of administration will depend, at least in part, on the nature of the cancer, the condition of the subject, the delivery site, and other factors.

[0159] In further embodiments of the application, the oncolytic virus can be administered intratumorally, or before or after surgical resection of the tumor.

[0160] The following examples are provided by way of illustration, and not by way of limitation.

[0161] Example

[0162] All constructs were generated using standard recombinant techniques, including chemical synthesis.

[0163] Example 1

[0164] Schematic of an exemplary OHSV vector

[0165] Figure 1A and Figure 1B An exemplary schematic of a representative oHSV vector is provided.

[0166] Example 2

[0167] Exemplary constructs

[0168] In this example, various constructs and their sequences are presented.

[0169] hVG001-1-2 comprises a modified ICP34.5 region Figure 2 ; SEQ ID NO. 572), a modified UL54 promoter-regulatory region Figure 3 ; SEQ ID NO. 573), an insertion of a PD-L1 blocker Figure 4 ; SEQ ID No. 574) in the gene region between UL3 and UL4, and a modified terminal repeat (TR) region Figure 5 ; SEQ ID NO. 575) carrying an expression cassette encoding IL-12, IL-15, and IL-15 receptor a subunit. The virus also has a modified and partially deleted ICP 34.5 region.

[0170] mVG001-1-2 is functionally identical to the human version, hVG001-1-2, which carries human IL-12 and a human PD-L1 blocker, except that mVG001-1-2 carries a mouse version of IL-12 and a mouse PD-L1 blocker in the same locations on the viral genome as hVG001-1-2.

[0171] Example 3

[0172] Abbreviations used in subsequent examples

[0173] TF-Fc: PD-L1 blocking peptide (TF) fused to Fc and used to construct VG001-1-2.

[0174] IL-TF-Fc: Plasmid carrying IL-12, IL-15, and PD-L1 blocker.

[0175] HSV-345: ICP34.5-deleted virus.

[0176] OS-ICP27 2-11: ICP34.5-deleted virus with Oct4 / Sox2 binding site and surviving promoter (OS) inserted in the promoter-regulatory region of ICP27 (OS-ICP27) not used to construct VG001-1-2.

[0177] OS-ICP27 5-7: ICP34.5-deleted virus with OS-ICP27 mutation not used to construct VG001-1-2.

[0178] NO-ICP27 1-4-4 (also referred to as NO-ICP27-145): ICP34.5-deleted virus with NF-kB response element and Oct4 / Sox2 binding site (NO) inserted in the promoter-regulatory region of ICP27 (NO-ICP27) upstream of the transcription start site at 145 bp not used to construct VG001-1-2.

[0179] NO-ICP27 5-2-2 (also referred to as NO-ICP27-99): ICP34.5-deleted virus with NF-kB response element and Oct4 / Sox2 binding site (NO) inserted in the promoter-regulatory region of ICP27 (NO-ICP27) upstream of the transcription start site at 99 bp not used to construct VG001-1-2.

[0180] VG001-1.7 (also referred to as HSV 1-VG 001-1.7): Backbone virus used to construct VG001-1-2 (NO-ICP27 1-4-4 mutant carrying exogenous promoter and poly(A) flanked in the deleted terminal repeat region of the viral genome with empty MCS, which was subsequently used for insertion of the IL-12 / IL-15 expression cassette).

[0181] VG001-15h (also referred to as VG001-1-2-15h): VG001 carrying human IL-15.

[0182] VG001-1215h (also referred to as VG001-1-2-1215h): VG001 carrying human IL-12 and human IL-15.

[0183] VG001-PLBh (also referred to as VG001-1-2-PLBh): VG001 carrying a human PD-L1 blocker inserted into the intergenic region between UL3 and UL4.

[0184] 8-8-15RA1-PDL1b: VG001 carrying human IL-15 and human PD-L1 blocker.

[0185] VG001-1-2-1215PLBm (also referred to as mVG001-1-2): VG001 carrying mouse IL-12, human IL-15, and mouse PD-L1 blocker.

[0186] VG001-1-2-1215PLBh (also referred to as hVG001-1-2 or VG001-1-2): VG001 carrying human IL-12, human IL-15, and human PD-L1 blocker.

[0187] Example 4

[0188] Expression of IL-12 after infection of cells with HVG001-1-2

[0189] In this example, Western blot analysis and ELISA expression of IL-12 are shown.

[0190] Figure 6A Western blot analysis results of VG001-1-2-1215PLBh virus infection are shown. H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 24 hours. Cell lysates were prepared, loaded onto a 12% SDS-PAGE gel, and transferred to a PVDF membrane. The membrane was blotted with an anti-human IL-12 antibody, followed by analysis with an HRP-conjugated anti-mouse IgG secondary antibody, and images were detected and analyzed with the Bio-Rad ImageLab system.

[0191] Figures 6B-6CUpregulation of human IL-12 production following VG001-1-2-1215PLBh virus infection is shown. LS174T or H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 48 hours. Infected cell supernatants were harvested and bound to 96-well Immuno Maxisorp flat bottom plates coated with anti-human IL-12 capture antibody. Binding was detected via biotinylated anti-human IL-12 antibody, avidin-horseradish peroxidase (HRP), and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450 nm using a plate reader. Concentrations of human IL-12 in the incubated supernatants were calculated based on a human IL-12 standard curve.

[0192] Example 5

[0193] Expression of IL-15 following infection of cells with HVG001-1-2

[0194] In this example, Western blot analysis and ELISA expression of IL-15 expression are shown.

[0195] Figure 7A Western blot analysis results following VG001-1-2-1215PLBh virus infection are shown. H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 24 hours. Cell lysates were prepared, loaded onto a 12% SDS-PAGE gel, and transferred to a PVDF membrane. The membrane was probed with an anti-human IL-15 antibody, followed by analysis with an HRP-conjugated anti-mouse IgG secondary antibody, and images were detected and analyzed using the Bio-Rad ImageLab system.

[0196] Figures 7B-7C Upregulation of human IL-15 production following VG001-1-2-1215PLBh virus infection is shown. LS174T or H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 48 hours. Infected cell supernatants were harvested and bound to 96-well Immuno Maxisorp flat bottom plates coated with anti-human IL-15 capture antibody. Binding was detected via biotinylated anti-human IL-15 antibody, avidin-horseradish peroxidase (HRP), and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450 nm using a plate reader. Concentrations of human IL-15 in the incubated supernatants were calculated based on a human IL-15 standard curve.

[0197] Example 6

[0198] Expression of IL-4 after infection of cells with HVG001-1-2

[0199] In this example, Western blot analysis and ELISA expression of IgG4 expressed are shown.

[0200] Figure 8A Results of Western blot analysis after infection of VG001-1-2-1215PLBh virus are shown. H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 24 hours. Cell lysates were prepared, loaded onto 12% SDS-PAGE gels, and transferred to PVDF membranes. Membranes were blotted with HRP-conjugated anti-human IgG antibodies, and images were detected and analyzed using the Bio-Rad ImageLab system.

[0201] Figures 8B-8C Upregulation of human PD-L1 blocker (fused to human Fc region) production after infection of VG001-1-2-1215PLBh virus is shown. LS174T or H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI = 1) for 48 hours. Infected cell supernatants were harvested and bound to 96-well ImmunoMaxisorp flat bottom plates coated with anti-human IgG4 capture antibody. Binding was detected via biotinylated anti-human IgG4 antibody, avidin-horseradish peroxidase (HRP), and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450 nm using a plate reader. Concentrations of human IL-4 in the incubated supernatants were calculated based on a human IL-4 standard curve.

[0202] Example 7

[0203] Constructs comprising PD-L1 blocking peptides

[0204] PD-L1 blocking peptides were generated with Ig kappa chain leader sequence (SEQ ID NO: 501). When two or more blocking peptides were in the same construct, they were linked with a Gly-Ser rich sequence (Gly4Ser)3(SEQ ID NO: 503). The following constructs were made.

[0205] TF only: METDTLLLWVLLLWVPGSTGTAHPSPSPRSAGQF (SEQ ID NO: 537);

[0206] ET + TF:

[0207] METDTLLLWVLLLWVPGSTGEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF (SEQ ID NO: 538);

[0208] TF + WT:

[0209] METDTLLLWVLLLWVPGSTGYYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF (SEQ ID NO: 539);

[0210] Mouse TF: METDTLLLWVLLLWVPGSTGTRYPSPSPKPEGRF (SEQ ID NO: 540);

[0211] Mouse WT + TF:

[0212] METDTLLLWVLLLWVPGSTGWNRLSPSNQTGGGGSGGGGSGGGGSTRYPSPSPKPEGRF (SEQ ID NO: 541).

[0213] Triple TF + ET:

[0214] METDTLLLWVLLLWVPGSTGTAHPSPSPRSAGQFTAHPSPSPRSAGQFTAHPSPSPRSAGQFGGGGSGGGGSGGGGSEYRMSPSNQTEYRMSPSNQTEYRMSPSNQT (SEQ ID NO: 542)

[0215] METDTLLLWVLLLWVPGSTGEYRMSPSNQTEYRMSPSNQTEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFTAHPSPSPRSAGQFTAHPSPSPRSAGQF (SEQ ID NO: 543).

[0216] Additional constructs were made using the IL-2 signal sequence (MYRMQLLSCIALSLALVTNS (SEQ ID NO: 502), and either a human IgG4 Fc region (with hinge region) (SEQ ID NO: 544) or a murine IgGl Fc region (with hinge region) (SEQ ID NO: 545). The constructs were:

[0217] TF only:

[0218] MYRMQLLSCIALSLALVTNSTAHPSPSPRSAGQFISAMVRSPPCPSCPAPEFLGG

[0219] PSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK

[0220] PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR

[0221] EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:546)

[0222] ET+TF:

[0223] MYRMQLLSCIALSLALVTNSEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFISAMVRSPPCPSCPPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:547)

[0224] YT+TF:

[0225] MYRMQLLSCIALSLALVTNSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 549)

[0226] Mouse TF:

[0227] MYRMQLLSCIALSLALVTNSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 549)

[0228] Mouse WT + TF:

[0229] MYRMQLLSCIALSLALVTNSWNRLSPSNQTGGGGSGGGGSGGGGSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 550).

[0230] Example 8

[0231] Constructs comprising IL-15 and IL-15Ra under the control of a bi-directional CMV promoter

[0232] In this example, various constructs were generated to co-express IL-15 and IL-15Ra under the control of a bi-directional CMV promoter.

[0233] In construct 1, the bi-CMV promoter drives expression of IL-15Ra and the Sushi domain of IL-15 (Figure 9, SEQ ID No. 557).

[0234] In construct 2, the bi-CMV promoter drives expression of IL-15 and IL-15Ra variant 4 (Figure 10, SEQ ID No. 558).

[0235] In construct 3, the bi-CMV promoter drives expression of IL-15-K5 and IL-15Ra Sushi domain-E5 (Figure 11, SEQ ID No. 559).

[0236] In construct 4, the bi-CMV promoter drives expression of IL-15-K5 and IL-15Ra variant 4-E5 (Figure 12, SEQ ID No. 560).

[0237] Example 9

[0238] Constructs comprising IL-15 and IL-15Ra genes under the control of an EF1a promoter

[0239] In this example, various constructs were generated to express IL-15 and IL-15Rα in a multi-cistronic transcript under the control of the EFla promoter (SEQ ID NO: 551). IL-15 and IL-15Rα were linked by an exemplary IRES sequence (SEQ ID NO: 552).

[0240] In Construct 1, the EFla promoter controls expression of IL-15-IRES-IL-15Rα Sushi domain (Figure 13, SEQ ID No. 561).

[0241] In Construct 2, the EFla promoter controls expression of IL-15-IRES-IL-15Rα variant 4 (Figure 14, SEQ ID No. 562).

[0242] In Construct 3, the EFla promoter controls expression of IL-15K5-IRES-IL-15Rα Sushi domain E5 (Figure 15, SEQ ID No. 563).

[0243] In Construct 4, the EFla promoter controls expression of IL-15K5-IRES-IL-15Rα variant 4 E5 (Figure 16, SEQ ID No. 564).

[0244] Example 10

[0245] Constructs containing IL-12, IL-15, and IL-15Rα genes under the control of the CMV promoter

[0246] In this example, various constructs were generated to express IL-12, IL-15, and IL-15Rα in a multi-cistronic transcript under the control of the CMV promoter. IL-12, IL-15, and IL-15Rα were linked by an exemplary p2A sequence (SEQ ID NO: 554).

[0247] In Construct 1, the CMV promoter (SEQ ID NO: 553) controls

[0248] expression of IL-12-p2A-IL-15-p2A-IL-15Rα Sushi domain (Figure 17, SEQ ID Nos. 565, 569).

[0249] In Construct 2, the CMV promoter controls expression of IL-12-p2A-IL-15-p2A-IL-15Rα variant 1 (Figure 18, SEQ ID Nos. 566, 570).

[0250] In Construct 3, the CMV promoter controls expression of IL-12-p2A-IL-15K5-p2A-IL-15Ra Sushi domain E5 (Figure 19, SEQ ID Nos. 567, 571).

[0251] In Construct 4, the CMV promoter controls expression of IL-12-p2A-IL-15K5-IRES-IL-15Ra variant 1 E5 (Figure 20, SEQ ID Nos. 568, 572).

[0252] Example 11

[0253] Constructs inserted between UL3 and UL4 containing PD-L1 blockers

[0254] In this example, constructs were generated to express a PD-L1 blocking peptide in the intergenic region between bases 829 and 830. In SEQ ID NO: 556, bases 1-675: UL3 coding sequence; bases 676-829: region between UL3 and UL4 and upstream of the PD-L1 blocker cassette; bases 830-833: region between UL3 and UL4 downstream of the PD-L1 blocker cassette; bases 834-1433: UL4 coding sequence.

[0255] Example 12

[0256] Inhibition of human PD-L1 binding to PD-1 by blocking peptides

[0257] Recombinant human PD-L1 Fc protein was coated to the bottom of a 96-well flat bottom plate at 4°C overnight. After the coated plate was incubated overnight, different PD-L1 blockers were added to each well of the plate and incubated at room temperature for 2 hours before adding recombinant human PD-1 Fc protein. Biotinylated anti-human IgG antibody and streptavidin-HRP were then added to each well, and binding of human PD-1 to PD-L1 was detected by adding TMB substrate. The color development was measured by a microplate reader at 450 nm wavelength. The percentage of inhibition was calculated by comparison to the non-synthetic peptide control. Figure 21 shows the percentage inhibition by peptides ET, ET+TF, YT, YT+TF, TW, TW+TF, WT, WT+TF, and TF at two different concentrations (3 μΜ and 10 μΜ). At 10 μΜ, the range of inhibition was about 22% to about 48%.

[0258] Example 13

[0259] Blocking PD-L1 enhances cytotoxicity against tumor cells by blocking peptide binding

[0260] Human peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3 antibody + human IL-2 for 24 hours and then incubated with different synthetic PD-L1 blockers and calcein-AM labeled target cells for 4 hours. After 4 hours of incubation, cells from the incubated supernatant were harvested and the released calcein-AM fluorescence was measured by a microplate reader. The percentage of cytotoxicity was calculated based on the following formula: [(sample reading - minimal release) / (maximal release - minimal release)] x 100.

[0261] Figures 22A-22B Results are shown for four different tumor cells: H460, U87, LS147T and MDA-MB-231 cells. On some tumor cells, the cytotoxicity statistically significantly increased for all peptides except TF.

[0262] Example 14

[0263] Synergistic effect of IL-12 and IL-15 on cytokine production

[0264] Human PBMC were incubated with medium control, IL-12 only, IL-15RA only, or IL-12, IL-15 and IL-15Ra1 + neutralizing anti-IL-12 or anti-IL-15 antibodies for 48 hours. Cultured cell supernatants were harvested for determination of human IFNy and TNFa production by ELISA.

[0265] Figure 23A and Figure 23B Results are shown for cytokine production. The combination of IL-12 and IL-15Ra1 resulted in statistically significant increase of cytokines human IFNy and TNFa. Anti-IL-12 antibodies inhibited their production.

[0266] Example 15

[0267] Synergistic effect of IL-12 and IL-15 on cytotoxicity against tumor cells

[0268] Human PBMC were incubated with tumor target cells and medium control, IL-12 only, IL-15RA only, or IL-12, IL-15 and IL-15Ra1 + neutralizing anti-IL-12 or anti-IL-15 antibodies for 24 hours. Cultured cell supernatants were harvested for determination of cytotoxicity by LDH. The percentage of cytotoxicity was calculated based on the following formula: [(sample reading - minimal release) / (maximal release - minimal release)] x 100.

[0269] Figure 24A and Figure 24BIL-12 and IL-15 together were shown to increase cytotoxicity in a statistically significant manner. In the MDA-MB-231 cell line, the addition of anti-IL-12 or IL-15 antibodies significantly reduced this effect.

[0270] Example 16

[0271] In vitro potency of viruses VG001-1-2-PLBH and VG001-1-2-15H

[0272] In this example, 3x10 4 H460 or LS174T tumor cells were seeded in each well of a 96-well plate and incubated overnight at 37°C. The next day, seeded cells were infected with VG001-1.7 backbone, VG001-1-2-PLBh or VG001-1-2-15h viruses (MOI=1) for 24 hours and production of human IL-12, human IL-15 and human IgG4 was assessed by ELISA Figure 25A ) and subsequently 3x10 5 human PBMC were added to the culture and co-cultured for 24 hours to assess cytotoxicity by LDH assay Figure 25D ) or for 48 hours to assess human IFNg production by ELISA Figure 25E ) For the cytotoxicity assay, the percentage of cytotoxicity was calculated based on the following formula: [(actual reading - minimal release) / (maximal release - minimal release)]x100%. Supernatants harvested from tumor cells incubated with media only were used as minimal release, while supernatants harvested from tumor cells incubated with lysis buffer were used as maximal release.

[0273] Example 17

[0274] In vitro potency of various constructs

[0275] Figures 26A-26D Results of in vitro assays of various constructs are shown.

[0276] Figures 26A-26B Results of transfection of cells with IL-TF-Fc plasmids carrying IL-12, IL-15 and PD-L1 blockers are shown. In Figures 26A-26B different tumor cell lines were transfected with IL-TF-Fc plasmid DNA for 24 hours and subsequently human PBMC were added to the culture. Cell supernatants were harvested after 24 hours for quantification of cytotoxicity by LDH assay Figure 26A ) and after 48 hours for detection of human IFNg production by ELISA assay Figure 26B

[0277] Figures 26C-26D ​Results of infection of cells with various mutant viruses including hVG001-1-2 are shown. Virus-encoded IL12, IL15, and PD-L1 blocker synergistically increase IFNg production and cytotoxicity. H460 tumor cells were seeded into each well of a 96-well plate and incubated at 37°C overnight. The next day, seeded cells were infected with the indicated viruses at MOI = 1 for 24 hours. Human PBMCs were then added to the cultures and co-incubated for 24 hours to assess cytotoxicity by LDH assay Figure 26C ), or co-incubated for 48 hours to assess human IFNg production by ELISA Figure 26D ). For the cytotoxicity assay, the percent cytotoxicity was calculated based on the following formula: [(actual reading - minimal release) / (maximal release - minimal release)] x 100%. Supernatant from tumor cells incubated with media only was used as the minimal release, while supernatant from tumor cells incubated with lysis buffer was used as the maximal release.

[0278] In Figures 27A-27E , a panel of 9 different human tumor cell lines (+ Vero cells) were infected with VG001-1-2-1212PLBh (VG001-1-2h) and HSV-345 viruses at MOI 0, 0.04, 0.2, 1, and 5. Cell viability was quantitatively analyzed at 48 hours post-infection using an MTT assay.

[0279] Figures 28A-28J Results of in vitro assays of various constructs are shown. Figures 28A-28E Results of cell viability assays of mVG001-1-2 and HSV-345 performed on mouse tumor cell lines and Vero cell line are shown; Figures 28F-28J Characterization of transgene expression following infection of CT26 mouse tumor cells with mVG001-1-2 or VG001-1.7 is shown.

[0280] In Figures 28A-28E , a panel of 6 different mouse tumor cell lines (+ Vero cells) were infected with VG001-1-2m and HSV-345 viruses at MOI 0, 0.04, 0.2, 1, and 5. Cell viability was quantitatively analyzed at 48 hours post-infection using an MTT assay.

[0281] In Figures 28F-28J , 3 x 10 4CT26 tumor cells were seeded into each well of a 96-well plate and incubated overnight at 37°C. The next day, the seeded cells were infected with VG001-1.7 backbone or VG001-1-2-1215PLBm virus (MOI=1) for 24 hours, and the production of mouse IL-12, human IL-15, and mouse IgG was assessed. Subsequently, 3 × 10⁶ cells from Balb / c mice were... 5 Spleen cells were added to the culture and co-cultured for 24 hours to assess cytotoxicity by LDH assay or for 48 hours to assess mouse IFNg production by ELISA. For cytotoxicity assays, cytotoxicity was calculated based on the following formula: [(actual reading – minimum release) / (maximum release – minimum release)] × 100%. The supernatant harvested from tumor cells incubated only with the culture medium was used for minimum release, while the supernatant harvested from tumor cells incubated with lysis buffer was used for maximum release.

[0282] exist Figures 29A-29E In the middle, 3×10 4 H460, LS174T, or UMUC3 tumor cells were seeded into each well of a 96-well plate and incubated overnight at 37°C. The next day, the seeded cells were infected with VG001-1.7 backbone and VG001-1-2-1215h virus (MOI=1) for 24 hours, and the production of human IL-12, human IL-15, and human IgG4 (18R) was measured. Subsequently, 3 × 10⁻⁶ cells were cultured... 5 Personal BMCs were added to the culture and co-cultured for 24 hours to assess cytotoxicity (18S) by LDH assay or for 48 hours to assess human IFNγ production (18T) by ELISA. For cytotoxicity assays, the percentage of cytotoxicity was calculated based on the following formula: [(actual reading – minimum release) / (maximum release – minimum release)] × 100%. The supernatant harvested from tumor cells incubated only with the culture medium was used for minimum release, while the supernatant harvested from tumor cells incubated with lysis buffer was used for maximum release.

[0283] exist Figures 30A-30G In this study, the antitumor effect of VG001-1-2-1215PLBh (hVG001-1-2) virus was evaluated in various human cancer cells (including U87, MCF7, H460, LNCaP, LS174T, MDA, and PC3) at 72 hours post-infection and MOIs ranging from 0 to 5. Cell viability percentage was quantitatively analyzed by MTT assay. VG001-1-2-1215PLBh virus exhibited potent T-cell killing ability in all tested human tumor cell lines.

[0284] Example 18

[0285] In vivo efficacy of VG001-1-2 virus constructs

[0286] In vivo efficacy of VG001-1-2 virus constructs Figures 31A-31B In vivo efficacy of VG001-1-2 virus constructs 7 PFU / mouse of VG001-1-2-1215PLBm (mVG001-1-2) virus or VG001-1.7 backbone virus or injected with PBS (vehicle control). Tumor size measurements were taken after the indicated number of injections. Mice treated with VG001-1-2-1215PLBm exhibited a significant (P<0.05) reduction in tumor volume compared to mice treated with PBS.

[0287] In vivo efficacy of VG001-1-2 virus constructs Figures 31C-31D In vivo efficacy of VG001-1-2 virus constructs 6 PFU / mouse of VG001-1-2-1215PLBm (mVG001-1-2) virus or VG001-1.7 backbone virus or injected with PBS (vehicle control). Tumor size measurements were taken after the indicated number of injections. Mice treated with VG001-1-2-1215PLBm exhibited a significant (P<0.05) reduction in tumor volume compared to mice treated with PBS.

[0288] In vivo efficacy of VG001-1-2 virus constructs Figures 31E-31G In vivo efficacy of VG001-1-2 virus constructs 7 PFU / mouse of VG001-1-2-1215PLBm (mVG001-1-2) virus or VG001-1.7 backbone virus or injected with PBS (vehicle control). Tumor size measurements were taken after the indicated number of injections. Mice treated with VG001-1-2-1215PLBm exhibited a significant (P<0.05) reduction in tumor volume compared to mice treated with PBS.

[0289] In vivo efficacy of VG001-1-2 virus constructs Figure 31E ) compared to tumor volume at the time of virus or PBS. Tumor size measurements using calipers are expressed as the fold change in tumor volume at a given time point compared to tumor volume at the time of virus or PBS. (hVG001-1-2) virus, while the remaining 6 animals served as a vehicle control and were injected twice with an equal volume of PBS. Tumor size was measured using two different methods. Caliper measurements are expressed as the fold change in tumor volume at a given time point compared to tumor volume at the time of virus or PBS. Figure 31FCompared to the PBS-treated control, quantitative imaging of tumor growth using IVIS showed an even more significant reduction in tumor size in animals treated with oHSV, with tumor size decreasing to undetectable levels using fluorescence sedimentation 50 days post-transplantation. Figure 31G (Left side: two vector controls; Right side: two oHSV-treated mice).

[0290] Example 19

[0291] Replication of HVG001-1-2 in cell lines

[0292] Figures 32A-32C , Figures 33A-33D and Figures 34A-34E The growth curves and cytotoxicity expression data show hVG001-1-2 viral replication as well as parental HSV-345 virus. These expressions also show that the virus does not grow in mouse tumor cell lines compared to human cell lines, although HSV-1 is known to grow poorly in mouse cells.

[0293] Example 20

[0294] Assessment of viral modification

[0295] Human PBMCs were stimulated for 48 hours with culture medium alone, recombinant IL-12 alone, recombinant IL-15 alone, or different forms of IL-15 / IL-15RA1 complexes with or without anti-IL-12 (6 mg / ml) or anti-IL-15 (0.5 mg / ml) neutralizing antibodies. The cultured supernatant was then harvested against human IFNg and... Figure 35A and Figure 35B The figure shows the use of ELISA to measure human TNFα production.

[0296] To assess cytotoxicity against tumor cells, calcein-AM-labeled tumor cells were co-incubated with stimulated human PBMCs for 24 hours. The supernatant was harvested for measuring fluorescence release. The supernatant harvested from calcein-AM-labeled tumor cells was incubated with medium used only for minimum release, and the supernatant harvested from calcein-AM-labeled tumor cells was incubated with lysis buffer used for maximum release. The percentage of cytotoxicity was calculated based on the following formula: [(actual reading – minimum release) / (maximum release – minimum release)] × 100%. Figure 35C The results show the cytotoxicity of U87 tumor cells. Figure 35D The results of cytotoxicity of MDA-MB-231 tumor cells are shown in the figure.

[0297] Example 21

[0298] Effective expression in vitro

[0299] Human peripheral blood mononuclear cells (PBMCs) were stimulated for 48 hours with culture medium only, recombinant IL-12 only, recombinant IL-15 only, or an IL-1+IL-15 / IL-15RA1 complex with or without anti-IL-12 (6 mg / ml) or anti-IL-15 (0.5 mg / ml) neutralizing antibodies. The cultured supernatant was then harvested for the production of human IFNg and human TNFα, such as... Figure 36A and Figure 36B The results are shown using ELISA.

[0300] To evaluate cytotoxicity against tumor cells, 1×10 4 One calcein-AM-labeled tumor cell and 1×10 5 Human PBMCs were co-incubated for 24 hours. The supernatant was harvested for measuring the released fluorescence. The supernatant harvested from calcein-labeled tumor cells was incubated with medium used for minimum release, and the supernatant harvested from calcein-labeled tumor cells was incubated with lysis buffer used for maximum release. The percentage of cytotoxicity was calculated based on the following formula: [(actual reading – minimum release) / (maximum release – minimum release)] × 100%. Figure 36C The cytotoxicity results of U87 tumor cells were shown. Figure 36D The results for MDA-MB-231 tumor cells are shown.

[0301] Example 22

[0302] Tumor cells infected with VG001-1-2h produce human IL-12, human IL-15 / IL15Ra and human IgG4.

[0303] In short, LNCaP cells were transplanted into nude mice and injected with solvent, ICP27-, or VG001-1-2h virus. Serum and tumor samples were harvested 120 hours after injection, and the production of human IL-12, human IL-15 / IL-15Ra, and human IgG4 was evaluated by ELISA. Figure 37A The results are shown in the figure.

[0304] Fadu cells were transplanted into nude mice and injected with solvent, HSV 1-VG 001-1.7, or VG001-1-2h virus. Tumor samples were harvested 24 hours after injection, and the production of human IL-12, human IL-15 / IL-15Ra, and human IgG4 was evaluated by ELISA. Figure 37B The results are shown in the figure.

[0305] Example 23

[0306] The effect of VG001-1-2m on immune response

[0307] CT26 colon cancer cells were implanted into balb / c mice and received PBS, HSV 1-VG 001-1.7 or VG001-1-2m virus injection. Tumor cells were harvested at 24 hours post injection and the percentage of CD8+ T cells, CD4+ T cells, or NK cells were measured by flow cytometry. Figures 38A-38C Results are shown in Table 2.

[0308] Example 24

[0309] Another exemplary construct

[0310] In this example, the construct was further engineered, particularly the US12 (ICP47) promoter region flanking the IL12-IL15-IL15RA1 expression cassette was engineered, thus another construct and its sequence were presented.

[0311] VG001-1-2 contains a modified ICP34.5 region Figure 40 ; SEQ ID NO. 599), a modified UL54 promoter-regulatory region Figure 41 ; SEQ ID NO. 596), an insertion of a PD-L1 blocker Figure 42 ; SEQ ID No. 589) in the gene region between UL3 and UL4, and a modified terminal repeat (TR) region Figure 43 ; SEQ ID NO. 576) carrying an expression cassette encoding IL-12, IL-15 and IL-15 receptor alpha subunit (IL12-IL15-IL15RA1). The protein sequence expressed by the IL12-IL15-IL15RA1 expression cassette is SEQ ID No. 577. SEQ ID Nos. 578-582 are the sequences of IL12, IL15, IL15RA1, upstream of self-cleaving linker P2A, and self-cleaving linker P2A, respectively.

[0312] VG001-1-2 carries human IL-12 and human PD-L1 blocker. mVG001-1-2 is the corresponding mouse version, which is functionally identical to VG001-1-2 except that in the same locations on the viral genome it carries is the mouse version of IL-12 and the mouse version of PD-L1 blocker.

[0313] The US12 (ICP47) promoter region flanking the IL12-IL15-IL15RA1 expression cassette carried by VG001-1-2 was engineered to construct multiple versions of the flanking US12 (ICP47) promoter: short (S; SEQ ID No. 583), medium (M; SEQ ID No. 584), long (L; SEQ ID No. 585), survivin (SEQ ID No. 586), etc. Depending on the version of the flanking ICP47 promoter, the corresponding VG001-1-2 can be specifically designated as VG001-1-2 (S), VG001-1-2 (M), VG001-1-2 (L), VG001-1-2 (surviving), etc. In the subsequent examples, unless otherwise specified, VG001-1-2 refers to VG001-1-2 (M), i.e., the vector carries the IL12-IL15-IL15RA1 expression cassette flanked by the medium (M) version of the ICP47 promoter. The structure of the VG001-1-2 vector is shown in Figure 70 .

[0314] Example 25

[0315] Cytotoxicity of VG001-1-2

[0316] As shown in Figure 56A , U87, H460, MCF-7, LS174T, and MDA-MB-231 human cancer cell monolayers were infected with VG001-1-2 virus at MOIs of 0, 0.04, 0.2, 1, and 5, and cell survival percentage was quantified by MTT assay 72 hours post-infection to evaluate the cytotoxicity of VG001-1-2. As shown in Figure 56B , four mouse tumor cell lines, B16-F10, 4T1, CT26, and A20, were infected with mVG001-1-2 virus at MOIs of 0, 0.04, 0.2, 1, and 5, and cell viability was quantified by MTT assay 72 hours post-infection.

[0317] Example 26

[0318] In vitro characterization of IL12, IL15, and PD-L1 blocker expressed by VG001-1-2

[0319] As shown in Figure 57, H460 human lung cancer cells and LS174T colon cancer cells were infected with VG001-1-2 or its backbone virus HSV 1-VG 001-1.7 (MOI = 1) for 24 hours. Transgene expression was quantified by immunoblotting (left column) and ELISA (right column). Figure 57A Expression of human IL-2 is shown, Figure 57BExpression of human IL-5 is shown, Figure 57C Expression of PD-L1 blocker is shown.

[0320] Example 27

[0321] In vitro characterization of PD-L1 blocker expressed by VG001-1-2

[0322] As Figure 58 shown, TF+Fc peptide containing supernatant harvested from VG001-1-2 infected 293FT cells was mixed with recombinant human PD-1 Fc and bound to human PD-L1 Fc coated 96 well Immuno Maxi Sorp flat bottom plates. Binding was detected by biotinylated anti-PD-1 antibody, streptavidin-horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450 nm with a plate reader. The percent (%) increase in human PD-1 / PD-L1 inhibition was compared to the no peptide sample.

[0323] Example 28

[0324] In vitro characterization of IL12 expressed by VG001-1-2

[0325] Figure 59 Results of cell based assay with TF+Fc peptide treatment are shown. 5 x 10 4 Jurkat T cells were activated with 1 pg / ml of PHA and 50 ng / ml of PMA and mixed with 1 x 10 5 PD-L1 expressing tumor cells with supernatant containing PD-L1 blocking peptide for 48 hours at 37°C. After 48 hours, cell culture supernatants were harvested and IL-2 produced by Jurkat T cells was determined by IL-2 ELISA.

[0326] Example 29

[0327] IL-12, IL-15 / IL-15RA and PD-L1 blocker synergistically enhance immune cell function

[0328] U87 human glioma cells and MDA-MB-231 human breast cancer cells overexpressing IL12, IL15 / IL15RA or co-expressing IL12 and IL15 / IL15RA were treated with human PBMCs.

[0329] Figure 60A Production of cytokines IFN-γ and TNF-α was determined by ELISA.

[0330] Figure 60B Immune cell induced cytotoxicity was determined by LDH.

[0331] Figure 60C This study demonstrates the synergistic activation of PBMCs via virus-encoded IL12, IL15, and PD-L1 inhibitors: H460 tumor cells were seeded into each well of a 96-well plate and cultured overnight at 37°C. The cells were then infected with VG001-1-2 virus at MOI=1 for 24 hours and co-incubated with human PBMCs for 48 hours. IFN-γ production was quantified by ELISA. The viruses tested included HSV1-VG 001-1.7 (without immunomodulator), HSV 1-VG001-1.7-PDL1b (PD-L1 blocker), HSV 1-VG 001-1.7-15RA1 (IL15 / IL15RA), HSV 1-VG 001-1.7-RA1-PDL1b (IL15 / IL15RA+PD-L1 blocker), HSV 1-VG001-1.7-h1215 (IL12)+IL15 / IL15RA), and VG001-1-2 (IL12+IL15 / IL15RA+PD-L1 blocker).

[0332] Figure 60D The experiment showed that PHA-activated human PBMCs (n=4) were co-incubated with recombinant human PD-L1 protein and supernatant from VG001-1-2 infected cells for 48 hours. Antibody-mediated neutralization of IL-1 and / or IL-15 occurred concurrently with the depletion of the PD-L1 inhibitor. Co-incubation with supernatant from uninfected cells served as a negative control. Human IFN-γ production was assessed by ELISA.

[0333] Example 30

[0334] In vivo efficacy of VG001-1-2 intratumoral inoculation

[0335] Figure 61A The efficacy of VG001-1-2 in the U87 human glioblastoma model was demonstrated: U87 cells were implanted into the lower abdomen of 7 nude mice, followed by intratumoral injection of 1×10⁻⁶ cells twice at 2-day intervals. 7 PFU / mouse VG001-1-2 or vector control.

[0336] Figure 61B Demonstrates immune-mediated, non-injection-based distal tumor clearance: A20 cells were implanted into the flanks of immunocompetent mice. mVG001-1-2 or HSV 1-VG 001-1.7 backbone virus was administered at 5 × 10⁻⁶. 6PFU / mouse / day was injected into tumors on only one side for 5 consecutive days. Sixteen mice were treated with mVG001-1-2, and six mice were treated with HSV1-VG 001-1.7. Mice treated with 6 / 16 of the HSV1-VG 001-1.7 mice experienced complete tumor regression on both sides compared with mice treated with 0 / 6 of the HSV1-VG 001-1.7 mice.

[0337] Figure 61C Mice treated with mVG001-1-2 were protected from CT26 tumor re-attack: CT26 cells were implanted into the lower flank of 16 immune-active BALB / c mice, with 8 animals randomly assigned to the mVG001-1-2 treatment group and the other 8 to the vector control group. At 21 days post-implantation, all 8 vector control animals died from tumor burden. No animals in the mVG001-1-2 treatment group died from tumor, but 4 animals in this group died due to non-tumor-related conditions. The 4 surviving mice in the mVG001-1-2 treatment group underwent re-implantation of CT26 cells at the same site 90 days post-injection.

[0338] Figure 61D The tumor size 7 days after CT26 re-challenge is shown in age-matched control mice not treated with mVG001-1-2.

[0339] Example 31

[0340] Gene expression and T cell activity in tumors treated with MVG001-1-2

[0341] As shown in Figure 62, CT26 tumor cells were implanted into BALB / c mice, and then injected at a dose of 5 × 10⁻⁶ cells per day for 5 consecutive days. 6 Multiple injections of mVG001-1-2, HSV 1-VG 001-1.7, or PBS control were administered at a PFU / mouse / day dose. Splenocytes collected on days 5, 7, and 9 post-injection were used to measure mouse IFN-γ ELISpot. Quantitative results are plotted in the right figure, with two mice in each group.

[0342] Example 32

[0343] Effects of MVG001-1-2 treatment on tumor intratumoral lymphocyte populations

[0344] like Figure 63A As shown, BALB / c mice received CT26 cell engraftment and, 8 days later, were injected five times with PBS, HSV1-VG 001-1.7 backbone, or mVG001-1-2 virus. Tumors were harvested 24 or 120 hours after the last injection. Different T cell subsets within the tumor mass were analyzed by flow cytometry. Figure 63B) and immunosuppressive cells ( Figure 63C The percentage of ). Figure 63D Immunohistochemical analysis of excised CT26 tumor sections treated with PBS control (vector) or mVG001-1-2 is shown using monoclonal antibodies against CD3 and perforin and polyclonal antibodies against HSV-1.

[0345] Example 33

[0346] Effects of minimum doses of human IL12 or human IL15 / IL15RA on immune cell function

[0347] As shown in Figure 64, human IL-12 harvested from the supernatant of transfected 293FT cells was co-cultured with PHA-stimulated human PBMCs for 48 hours. Human IFN-γ production was assessed by ELISA. Figure 64B As shown, 293FT cells were transfected with plasmids expressing human IL12 or human IL15 / IL15RA for 48 hours. The supernatant was harvested and co-cultured with PHA-stimulated human PBMCs for 48 hours. Cell proliferation was assessed by MTT assay.

[0348] Example 34

[0349] Biodistribution of viruses

[0350] like Figure 65A , Figure 65B As shown, nude mice carrying LS174T tumors were treated with 5 × 10⁻⁶... 7 VG001-1-2 was administered intratumorally at a dose of PFU / mouse / day. Mice were euthanized at different time points, and genomic DNA was isolated from these organs for qPCR. Viral copy number was quantified using the IL15RA1 gene.

[0351] Example 35

[0352] qPCR measurement

[0353] CT26 tumor cells were implanted into mice, and the mice were injected daily with mVG001-1-2 and control virus HSV1-VG001-1.7 (total 5 × 10⁻⁶). 7 Five treatments were performed using either PFU or PBS. Tumors were harvested 24 and 48 hours after the last viral injection, RNA was isolated and purified, and then gene expression profiling was performed using a Qiagen Mouse Innate & Adaptive Immune Response RT2 Profiler™ PCR Array. Figure 66Differential expression of innate and adaptive immunity related genes in tumors treated with mVG001-1-2 compared to tumors treated with HSV 1-VG 001-1.7 and PBS. Overexpression of the indicated targets was verified by RT-qPCR.

[0354] Example 36

[0355] Changes in surface MHC of cells infected with viruses of different structures

[0356] To understand the effect of the promoter engineered VG001-1-2 virus on cells, cells were infected with VG001-1-2 and wild type (VG001-1-2-1215PLBh, ICP47 promoter not engineered) viruses under the same conditions.

[0357] Specifically: 293T cells were plated in 6-well plates (1 x 106cells / well) in DMEM medium with 10% serum. After the cells grew to a monolayer, 1 x 106pfu (MOI = 1) of the following viruses or blank medium were added to each well:

[0358] a. VG001-1-2 virus (2 wells)

[0359] b. Wild type virus (2 wells)

[0360] c. Non-infection control with medium only (2 wells)

[0361] After viral infection, the cells were incubated overnight, then the cells were trypsinized with 750 ul trypsin, 1 ml DMEM was added, and the cells were transferred to a flow cytometry tube. The cells were centrifuged (1500 RPM, 5 min), after the supernatant was removed, the cells were washed with 2 ml PBS, and the washing was repeated once, and the PBS was removed. 1 well of each of the above a-c groups was added with an antibody against MHC (histocompatibility complex) protein (Anti-Hu HLA-ABC): the cells were suspended with 100 ul PBS + 2% FBS, and 2 ul of the antibody was added. In addition, 1 well of each was added with 100 ul PBS + 2% FBS without the antibody. Incubate for 1 hour in the dark, remove the antibody, wash the cells with 3 ml PBS + 2% FBS, centrifuge the cells (1500 RPM, 5 min), and discard most of the cell washing solution for flow cytometry analysis. The results are shown in Figure 67 .

[0362] From Figure 67It can be seen that the wild type HSV-1 virus infection causes the expression amount of MHC protein on the cell surface to decrease. Unexpectedly, the MHC expression level of the cell infected with the VG001-1-2 virus is even further lower than that of the cell infected with the wild type HSV-1 virus. It can be seen that, compared with the wild type virus, by modifying the ICP47 promoter, the VG001-1-2 virus can exist in the infected cell for a longer time to avoid the attack of immune cells after infecting the cell, so as to better replicate and express the foreign gene.

[0363] Exploratory test of oncolytic virus stabilizer

[0364] In order to optimize the medium for preserving the oncolytic virus, different formulations of the medium were tested. A batch of VG001-1-2 virus samples were prepared, and the virus was preserved according to the formulations and conditions in Table 1.

[0365] Table 1 Medium of different formulations for preserving the virus

[0366]

[0367] The samples were prepared according to Table 1 and placed in a 37°C incubator. After 48 hours, the above samples were transferred to a -80°C refrigerator until the virus titer test was performed. The original virus of sample G8 was taken out from the -80°C refrigerator before titration, diluted, and then the virus titration was performed simultaneously with the other seven groups of samples. The results of G8 were used as a reference standard for determining the stability of the virus samples of the other groups.

[0368] Vero cells were inoculated into 6-well plates at a cell amount of 8x10 5 cells per well, 3ml per well, and cultured in a 37°C carbon dioxide incubator for 24h. The virus samples were gradient diluted according to the scheme in Table 2.

[0369] Table 2 Virus titration dilution process

[0370]

[0371] The six-well plates with FBS-free medium were taken out from the 37°C incubator, the medium was aspirated, 1ml of diluent was added to each well, and four replicate wells were prepared for each sample (2 six-well plates per sample). After adding 1ml of virus diluent per well, the virus was adsorbed for 60 minutes in a 37°C incubator, the virus liquid was aspirated, and 2ml / well of MEM (without FBS) culture solution and methyl cellulose (1.5%) were added. The plates were placed in a 37°C incubator for 96 hours.

[0372] 96 hours later, the covers were removed, 1 ml of 4% glutaraldehyde solution was added to each well, and the plate was left at room temperature for 30 minutes. The solution was then discarded, and the wells were stained with 2% crystal violet staining solution, 1 ml per well, for 15 minutes at room temperature. The wells were then gently rinsed with softened water and allowed to dry. After the six-well plate was dried, the dead cells caused by the virus did not stain, forming white spots. The number of plaques was counted under a medical film viewer.

[0373] Result calculation: The number of plaques in the wells was counted, and the average number of plaques was multiplied by the dilution of the virus. For example, if the average number of plaques in the wells of tube 6 was 20, the titer of the virus was 20 x 10 6 , i.e. 2.0 x 107PFU / ml. Each of the above samples was repeated three times. The titer of the virus was calculated from the number of plaques, and the results of G1-G7 were compared with those of G8 to determine the most stable protective agent for the virus.

[0374] The results of measuring the titer of the virus in each group are shown in Figure 68 . The test results show that the activity of the virus stored at 37°C with formula G5 remains good and is essentially indistinguishable from the activity of the virus stored at -80°C. Although it is generally believed that increasing the amount of sucrose can improve the stability of the virus solution, it was unexpected that HSV-1 oncolytic virus, whether or not glycerol is present, is not conducive to maintaining the activity of the virus in the presence of sucrose.

[0375] Example 38 Stability of protein in mouse serum

[0376] VG001-1-2 carries a human PD-L1 blocking peptide (TF), and the C-terminus of the PD-L1 blocking peptide can comprise an Fc sequence. The Fc can be from one of the IgG subclasses. In order to understand the stability of various fusion proteins formed by fusing the C-terminus of TF with Fc sequences from different sources, the following experiments were performed.

[0377] Cell culture

[0378] 293 cells were cultured in RPMI-1640 medium containing double antibodies and 10% fetal bovine serum in a 37°C incubator containing 5% CO2. When the cell density reached about 80%, the cells were digested with trypsin and subcultured at a ratio of 1:3-4.

[0379] Plasmid extraction: for VG001-1-2, the sequence encoding IgG1 Fc or IgG4 Fc fragment was inserted at the end of its PD-L1 coding region, respectively, to obtain plasmids VG001-1-2-Fc1 and VG001-1-2-Fc4, which can express PD-L1 fused with IgG1 Fc and PD-L1 fused with IgG4 Fc, respectively. The two plasmids were transformed into E. coli BL21 by heat shock method, and cultured overnight at 37°C on LB+AMP bacterial plates. Single colonies were picked and cultured overnight at 37°C in LB+AMP liquid medium (2-3 ml) in a shaker (150 rpm / min).

[0380] The plasmids were extracted from the above culture and turbid medium, labeled Fc-1 and Fc-4, and the extraction date.

[0381] The quality of the extracted plasmids was identified on agarose electrophoresis, and the concentration of plasmid DNA was detected using a DNA concentration detector.

[0382] 2.3 Plasmid transfection

[0383] 293 cells were plated in a 6-well plate and incubated overnight in a cell incubator.

[0384] The two plasmids were mixed with plasmid transfection reagents and serum-free medium according to the plasmid transfection instructions based on the plasmid concentration. After adding 293 cells and incubating for 4 hours, the medium was replaced with 6% FBS serum medium and incubated for 48 hours.

[0385] After plasmid transfection, the supernatant (2 ml) was collected at 48 hours.

[0386] All supernatant samples were frozen in a -80°C refrigerator.

[0387] 2.4 Protein and serum mixing

[0388] The above transfected supernatant (2 mL) and mouse serum were taken out from -80°C, thawed on ice, and then the samples were prepared according to the formula in Table 3.

[0389] Table 3: Configuration of mixed samples

[0390]

[0391]

[0392] After the samples were prepared, sample groups G01 / G02 were transferred to a -20°C refrigerator for storage, and the remaining groups were placed in a 37°C incubator. Sample groups G1 / G2 were transferred to a -20°C refrigerator for storage on the 14th day, and sample groups G3 / G4 were transferred to a -20°C refrigerator for storage on the 28th day. After all samples were collected, they were subjected to ELISA detection.

[0393] ELISA assay of samples

[0394] Samples were thawed on ice. 300ul of each sample was taken out for ELISA assay, 100ul per well, three replicates for each sample. The protein content of each sample was calculated according to the instruction of the kit.

[0395] 1) Coat ELISA plate with antibody in the kit, 4°C overnight;

[0396] 2) Wash the coated ELISA plate twice with washing buffer in the kit, and dry the liquid in the well with absorbent paper after each wash;

[0397] 3) Add blocking buffer, block at room temperature for 2 hours;

[0398] 4) After washing twice with washing buffer, add the prepared standard curve and the above samples and Assay buffer, two replicates for each sample;

[0399] 5) Prevent evaporation of liquid in the well with sealing film, incubate at room temperature for 2 hours;

[0400] 6) After incubation, wash four times with washing buffer, and dry the liquid in the well with absorbent paper after each wash;

[0401] 7) According to the requirements of the kit, add 100ul / well of detection antibody, seal the plate, and incubate at room temperature for 1 hour;

[0402] 8) After incubation, wash four times with washing buffer, and dry the liquid in the well with absorbent paper after each wash;

[0403] 9) Add 100ul of substrate to each well, incubate at room temperature in the dark for 15 minutes;

[0404] 10) Add 100ul of stop solution to each well, incubate at room temperature for 10-20 minutes;

[0405] 11) Adjust the ELISA enzyme reader, read the plate at wavelengths of 450nm and 570nm;

[0406] 12) According to the requirements of the kit, analyze the data.

[0407] According to the ELISA results, determine the ability of TF-Fc in each sample to bind to IgG4 or IgG1 antibody in the ELISA kit, and obtain the stability of IgG1 Fc and IgG4 Fc fusion protein in mouse serum. Figure 69

[0408] ​As can be seen from the test results, contrary to the conventional belief, the structure of TF fused with IgG1 Fc is not as stable in serum as the structure of TF fused with IgG4 Fc. After 28 days, the ability of the TF-IgG4 Fc fusion protein to bind to PD-L1 is significantly higher than that of the TF-IgG1 Fc fusion protein, with a statistically significant difference (p=0.01).

[0409] The following are other exemplary embodiments of the disclosure:

[0410] 1) An HSV vector expressing one or more of IL12, IL15 and / or the IL receptor 15 alpha subunit. In one embodiment, the HSV vector comprises an expression cassette expressing IL12, IL15 and the IL receptor 15 alpha subunit. In various embodiments, the IL12, IL15 and IL15 receptor alpha subunit sequences expressed are of mammalian origin (e.g., murine or human origin). In preferred embodiments, the expression cassette expresses murine or human IL12, murine or human IL15 and murine or human IL15 receptor alpha subunit. In further embodiments, the expression cassette expresses murine or human IL12, hIL15, and murine and h15 receptor alpha subunit.

[0411] 2) The HSV vector of embodiment 1, wherein the nucleic acid sequence encoding the self-cleaving peptide 2A is in frame between the coding sequences for IL12, IL15 and IL15 receptor alpha subunit. In preferred embodiments, IL12 is of murine or human sequence, IL15 is of human sequence and IL15 receptor alpha subunit is of human sequence.

[0412] 3) The HSV vector of embodiment 2, wherein the nucleic acid sequence encodes a self-cleaving peptide 2A selected from the group consisting of

[0413] VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFN-AHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNF-SLLKLAGDVELNPGP, EGRGSLLTCGDVEENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP, and CDAQRQKLLLSGDIEQNPGP.

[0414] 4) The HSV vector of embodiments 1 to 3, wherein one or more IRES sequences are positioned between the coding sequences for IL12, IL15 and IL15 receptor alpha subunit. In preferred embodiments, IL12 is murine or human sequence, IL15 is human sequence, and IL15 receptor alpha subunit is human sequence.

[0415] 5) The HSV vector of embodiments 1 to 4, wherein the IL15 and IL15 receptor alpha subunit are co-expressed using an IRES sequence. In preferred embodiments, IL12 is murine or human sequence, IL15 is human sequence, and IL15 receptor alpha subunit is human sequence.

[0416] 6) The HSV vector of any one of embodiments 1 to 5, wherein the IL15 and IL15 receptor alpha subunit are expressed by a bi-directional promoter. In preferred embodiments, IL12 is murine or human sequence, IL15 is human sequence, and IL15 receptor alpha subunit is human sequence.

[0417] 7) The HSV vector of embodiment 6, wherein the bi-directional promoter is bi-CMV.

[0418] 8) The HSV vector of any one of embodiments 1 to 7, wherein each of IL15 and IL15 receptor alpha subunit is preceded by a nucleic acid sequence encoding Lys5 or Glu5. In preferred embodiments, IL12 is murine or human sequence, IL15 is human sequence, and IL15 receptor alpha subunit is human sequence.

[0419] 9) The HSV vector of any one of embodiments 1 to 8, wherein the hIL15 receptor alpha subunit is selected from the group consisting of variant 1, variant 2, variant 3, and variant 4.

[0420] 10) The HSV vector of any one of embodiments 1 to 9, further comprising an expression cassette for one or more PD-L1 blocking peptides, or wherein the expression cassette comprises one or more PD-L1 blocking peptides.

[0421] 11) The HSV vector of any one of embodiments 1 to 10, further comprising a sequence encoding a peptide linker between the plurality of PD-L1 blocking peptides.

[0422] 12) The HSV vector of any one of embodiments 1 to 11, further comprising one or more IRES sequences between the plurality of PD-L1 blocking peptides.

[0423] 13) The HSV vector of any one of embodiments 1 to 12, further comprising a sequence encoding an Fc region linked to the 3 '-end of the PD-L1 blocking peptide.

[0424] 14) The HSV vector of any one of embodiments 1 to 13, wherein the expression cassette is inserted into an internal repeat region or a terminal repeat region of the HSV genome.

[0425] 15) The HSV vector of embodiment 10, wherein the sequence encoding the PD-L1 blocking peptide is inserted between viral genes, for example, between the UL3 and UL4 viral genes, between the UL50 and UL51 genes, and / or between US1 and US2.

[0426] 16) The HSV vector of any one of embodiments 1 to 15, further comprising NFkB and OCT4 / SOX2 enhancer elements in the ICP4 or ICP27 regulatory regions.

[0427] 17) The HSV vector of any one of embodiments 1 to 16, wherein the ICP34.5 gene is deleted.

[0428] 18) The HSV vector of any one of embodiments 1 to 17, wherein the expression cassette comprises at least one bi-directional CMV promoter.

[0429] 19) The HSV vector of any one of embodiments 1 to 18, wherein the expression cassette comprises at least one cellular promoter.

[0430] 20) The HSV vector of any one of embodiments 1 to 19, wherein the expression cassette for IL12 / IL15 / IL15 receptor alpha subunit is inserted into an internal repeat region or a terminal repeat region, wherein the original viral sequence is replaced by the expression cassette.

[0431] 21) The HSV vector of any one of embodiments 1-20, wherein the HSV is HSV-1 or HSV-2.

[0432] 22) The HSV vector of any one of embodiments 1 to 21, wherein the ICP34.5 gene is regulated by a 3’UTR comprising a target sequence for a miRNA that is lowly expressed in tumor cells.

[0433] 23) A pharmaceutical composition comprising the HSV vector of any one of embodiments 1 to 22, and a pharmaceutically acceptable carrier.

[0434] 24) A method of treating cancer, comprising administering to a patient the HSV vector of any one of embodiments 1 to 22, or the pharmaceutical composition of embodiment 23.

[0435] 25) The method according to embodiment 24, wherein the cancer is selected from the group consisting of carcinomas, leukemias, lymphomas, myelomas, and sarcomas.

[0436] All patents, publications, scientific articles, webpages, and other documents and materials referenced or mentioned in this document are indicative of the level of skill of those skilled in the art to which the application pertains, and each such document and material is hereby incorporated by reference in its entirety to the same extent as if it had been incorporated individually or collectively in its entirety by reference herein. Applicant reserves the right to physically incorporate into the specification any and all material and information in any such patents, publications, scientific articles, webpages, electronic information, and other references or documents.

[0437] The written description portion of the patent contains all the claims. In addition, all claims, including all original claims and all claims in any and all priority documents, are hereby incorporated into the written description of the specification, in their entirety, by this reference, Applicant reserves the right to physically incorporate into the specification any and all material and information in any such patents, publications, scientific articles, webpages, electronic information, and other references or documents. Thus, for example, in no event will the patent be construed as claiming any subject matter that is not recited in the written description portion of the specification as originally filed.

[0438] Claims are construed according to law. However, notwithstanding any claim of or belief in ease or difficulty of construing any claim or portion thereof, in no event will any adjustment or modification of any claim or portion thereof during the pendency of this patent application be construed as having lost any equivalent rights, which do not constitute prior art.

[0439] All features disclosed in this specification may be combined in any combination. Where certain features of the application could be combined with, substituted, or modified for other features according to the application, each combination and sub-combination is also contemplated and disclosed by this application. Accordingly, unless otherwise indicated herein, no feature disclosed herein is essential to the application.

[0440] It is to be understood that, even though matters have been described which appear certain embodiment of the application, the foregoing description is intended to illustrate and not to limit the scope of the application as defined by the metes and bounds of the following claims. Accordingly, from the above description, it is clear that the application is susceptible of broad and various embodiments. Numerous specific non-limiting embodiments have been described for purposes of illustration only in accordance with the application and its practice. Other variations and modifications of the application can be adopted without departing from its spirit and scope, and it is intended to cover all such variations and modifications as come within the scope of the following claims. Other aspects, advantages, and modifications are within the scope of the following claims and it is intended to cover all such aspects, advantages, and modifications of the application as falling within the scope of the claims.

[0441] The specific methods and compositions described herein represent preferred non-limiting embodiments, and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the application disclosed herein without departing from the scope and spirit of the application. The application illustratively described herein suitably can be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein. Thus, for example, in each instance herein, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. The methods and processes illustratively described herein suitably can be practiced in different sequences, and he steps need not be performed in the order described herein or in the claims.

[0442] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by various non-limiting embodiments and / or optional features, other modifications and / or alterations, and any and all equivalents thereof, are within the scope of this invention as defined by the appended claims.

[0443] The present invention is described herein in broad and general terms. Each of the narrower species and subgeneric groups of the application fall within the scope of the disclosure. This includes the general description as well as specific examples that are not intended to be limiting, but rather to be illustrative of the general principles of the application.

[0444] It should also be understood that, as used herein and in the appended claims, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise. The term "X and / or Y" means "X", or "Y", or both "X" and "Y". The term "s" following a noun means both the plural and the singular form of that noun. Further, features or aspects of the present invention are described in terms of Markush groups and subgroups, and those skilled in the art will recognize that the present invention encompasses by way of example and without limitation any and all subgroups of any individual member of a Markush group as well as any individual member of a sub-group. Applicants reserve the right to amend the application or claims to expressly recite any individual member of a Markush group or sub-group.

[0445] Other non-limiting implementations are included in the appended claims. This patent is not to be construed as limited to the specific embodiments or examples disclosed or to the examples presented in the attached claims. In any event, the patent is not to be construed as limited to only those embodiments or examples that exist currently, described or presented herein, but also includes any and all embodiments or examples that are developed from a combination of the features and / or functions disclosed or suggested in the patent and / or the claims.

Claims

1. The use of a herpes simplex virus (HSV) vector formulation in the preparation of a medicament for treating cancer, wherein the formulation comprises the following components: A suspension of the virus expressed by the herpes simplex virus (HSV) vector. glycerin, water, The concentration of glycerol in the formulation is 5%. The herpes simplex virus (HSV) vector contains an expression cassette encoding IL12, IL15, and the IL15 receptor α subunit, wherein the expression cassette has a modified ICP47 promoter on its flanking side. The IL15 receptor α subunit is selected from the group consisting of variant 1 as shown in SEQ ID NO: 3, variant 2 as shown in SEQ ID NO: 4, variant 3 as shown in SEQ ID NO: 5, and variant 4 as shown in SEQ ID NO: 6; The herpes simplex virus HSV vector further comprises an expression cassette encoding one or more PD-L1 blocking peptides and a sequence encoding an IgG4 Fc region, wherein once expressed, the IgG4 Fc region is linked to the 3' end of the PD-L1 blocking peptide. The herpes simplex virus (HSV) vector further includes a modified ICP34.5 region; The cancers mentioned are selected from liver cancer, stomach cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, thyroid cancer, skin cancer, endometrial cancer, head and neck cancer, osteosarcoma, leukemia, lymphoma, myeloma, and sarcoma.

2. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The sequence of the modified ICP47 promoter contains SEQ ID No.

584.

3. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The box between the coding sequences of IL12, IL15 and the IL15 receptor α subunit contains a nucleic acid sequence encoding the self-cleaving peptide 2A.

4. The application of the herpes simplex virus (HSV) vector preparation according to claim 3, wherein, The amino acid sequence of the self-cleaving peptide 2A is: ATNF-SLLKQAGDVEENPGP.

5. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, One or more IRES sequences are located between the coding sequences of IL12, IL15 and the IL15 receptor α subunit.

6. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The IL15 and IL15 receptor α subunit are expressed via a bidirectional promoter.

7. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The IL15 and IL15 receptor α subunit are each followed by a nucleic acid sequence encoding Lys5 or Glu5.

8. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The expression cassette containing IL12, IL15, and the IL15 receptor α subunit is inserted into the internal repeat region of HSV or the terminal repeat region of the HSV genome.

9. The application of the herpes simplex virus (HSV) vector preparation according to claim 1, wherein, The expression cassette of the PD-L1 blocking peptide is inserted between UL3 and UL4 of the HSV viral gene.

10. The use of the herpes simplex virus (HSV) vector formulation according to any one of claims 1-9, wherein, The ICP4 or ICP27 regulatory region of the herpes simplex virus HSV vector further includes NFkB and OCT4 / SOX2 enhancement elements.

11. The use of the herpes simplex virus (HSV) vector formulation according to any one of claims 1-9, wherein, The ICP34.5 gene of the herpes simplex virus HSV vector is partially deleted or non-functional.

12. The use of the carrier formulation according to any one of claims 1-9, wherein the treatment of cancer is administered by subcutaneous injection, intratumoral injection or intravenous injection.

13. The application of the carrier formulation according to claim 10, wherein the treatment of cancer is administered by subcutaneous injection, intratumoral injection or intravenous injection.

14. The application of the carrier formulation according to claim 11, wherein the treatment of cancer is administered by subcutaneous injection, intratumoral injection or intravenous injection.

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