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] This invention generally relates to oncolytic herpes simplex virus (oHSV) vectors that express molecules that stimulate the immune system. Background Technology

[0002] Oncolytic viruses (OVs) have become a treatment option that specifically destroys cancer cells through the oncolytic effect. Their killing mechanism is characterized by the lysis of cancer cells through viral replication.

[0003] This invention overcomes the shortcomings of current commercial oncolytic viruses, and has low toxicity, high expression level, and good stability. Summary of the Invention

[0004] In short, this disclosure relates to protection of herpes simplex virus vectors (HSV vectors) targeting one or more of IL12, IL15, and / or IL receptor 15α subunits. In one embodiment, the herpes simplex virus HSV vector comprises an expression cassette of IL12, IL15, and IL15 receptor α subunits, the expression cassette having a modified ICP47 promoter flanking it.

[0005] In one embodiment, the sequence of the modified ICP47 promoter contains 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, a nucleic acid sequence encoding a self-cleaving peptide 2A is contained within a frame between the coding sequences of IL12, IL15, and the IL15 receptor α subunit.

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

[0009] VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFNAHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNFSLLKLAGDVELNPGP, EGRGSLLTCGDVE ENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP, or CDAQRQKLLLSGDIEQNPGP.

[0010] In one implementation, one or more IRES sequences are located between the coding sequences of IL12, IL15 and the IL15 receptor α subunit.

[0011] In one implementation, IL15 and the IL15 receptor α subunit are expressed via a bidirectional promoter.

[0012] In one implementation, IL15 and the IL15 receptor α subunit are each followed by a nucleic acid sequence encoding Lys5 or Glu5.

[0013] In one embodiment, the hIL15 receptor α 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), and variant 4 (SEQ ID NO:6).

[0014] In one embodiment, an 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.

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

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

[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 linked to the 3'-end of the PD-L1 blocking peptide is a sequence encoding the IgG4 Fc region.

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

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

[0021] This article also discloses a formulation composed of the following components:

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

[0023] The concentration of glycerol in the formulation is 5%. + 3%.

[0024] The disclosure herein also relates to a pharmaceutical composition comprising the herpes simplex virus (HSV) vector of the present invention and a pharmaceutically acceptable vector.

[0025] The disclosures herein also relate to the use of the herpes simplex virus (HSV) vector, the formulations, or the pharmaceutical compositions described herein in the preparation of medicaments for treating cancer.

[0026] In one embodiment, the cancer is selected from liver cancer, stomach cancer, intestinal cancer, lung cancer, breast cancer, nasopharyngeal carcinoma, head and neck tumors, bladder cancer, colon cancer, rectal cancer, kidney 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 myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, piloblastic lymphoma, Burkitt's lymphoma, acute or chronic myeloid leukemia, melanoma, endometrial cancer, head and neck cancer, glioblastoma, osteosarcoma leukemia, lymphoma, myeloma, and sarcoma.

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

[0028] The Summary of the Invention has briefly introduced some concepts, which will be described in further detail in the Detailed Description section. Unless otherwise stated, the Summary of the Invention is neither intended to identify key or essential features of the subject matter claimed herein, nor is it intended to limit the scope of the subject matter claimed herein.

[0029] The effects of the invention

[0030] This invention overcomes the shortcomings of current commercial oncolytic viruses, and has low toxicity, high expression level, and good stability.

[0031] This invention modifies the structure of viruses, such as modifying the promoter regions of certain viral genes, so that the modified viruses can remain in infected cells for a longer period of time after infecting cells, thus evading immune cell attacks and enabling better replication and expression of foreign genes.

[0032] The present invention also improves the exogenous gene carried by the vector, for example, by constructing a fusion protein formed by an exogenous polypeptide and an appropriate terminal structure, so that the expressed protein has good stability and is suitable for practical applications.

[0033] The present invention also provides an optimized medium for preserving viruses. After the virus is expressed using the vector of the present invention, the optimized medium can maintain the virus activity for a longer period of time, which facilitates the industrial application of the vector and the virus.

[0034] One or more embodiments will be described in detail below. Features shown or described in conjunction with an exemplary embodiment can be combined with features of other embodiments. Therefore, any of the various embodiments described herein can be combined to provide further embodiments. When it is necessary to employ the concepts identified herein in the various patents, applications, or disclosures, aspects of these embodiments can be modified to provide further embodiments. Other features, objects, and advantages will be apparent from the specification, drawings, and claims. Attached Figure Description

[0035] The exemplary features, nature, and various advantages of the disclosure herein will be understood from the accompanying drawings and the following detailed description of various embodiments. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings, wherein, unless otherwise indicated, similar labels and numerals in the various views represent similar parts. The size and relative positions of the elements in the drawings are not necessarily drawn to scale. For example, the shapes of the elements have been chosen, enlarged, and placed to improve the readability of the drawings. Specific shapes of the elements as drawn have been selected for easy identification in the drawings. One or more embodiments are described below with reference to the accompanying drawings, wherein:

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

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

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

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

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

[0041] Figures 6A-6C The ELISA and Western blot analyses of IL-12 expression are shown after cells were infected with hVG001-1-2.

[0042] Figures 7A-7C The ELISA and Western blot analyses of IL-15 expression are shown after cells were infected with hVG001-1-2.

[0043] Figures 8A-8C The ELISA and Western blot analyses of IgG4 expression following cell infection with hVG001-1-2 are shown.

[0044] Figures 9A-9C For: (A) A schematic diagram of an exemplary construct in which the bi-CMV promoter drives the expression of the Sushi domain of IL-15Rα and IL-15, and (BC) DNA sequence and schematic diagram (SEQ ID No:557).

[0045] Figures 10A-10C For: (A) A schematic diagram of an exemplary construct in which the bi-CMV promoter drives the expression of IL-15 and IL-15Rα variant 4, and (BC) DNA sequence and schematic diagram (SEQ ID No: 558).

[0046] Figures 11A-11C For: (A) A schematic diagram of an exemplary construct in which the bi-CMV promoter drives the expression of IL-15-K5 and IL-15RαSushi domain-E5, and (BC) DNA sequence and schematic diagram (SEQ ID No:559).

[0047] Figures 12A-12DFor: (A) A schematic diagram of an exemplary construct in which the bi-CMV promoter drives the expression of IL-15-K5 and IL-15Rα variant 4-E5, and (BD) DNA sequence and schematic diagram (SEQ ID No: 560).

[0048] Figures 13A-13D For: (A) A schematic diagram of an exemplary construct in which the EF1α promoter controls the expression of the IL-15-IRES-IL-15RαSushi domain, and (BD) a DNA sequence and schematic diagram (SEQ ID No:561).

[0049] Figures 14A-14D For: (A) A schematic diagram of an exemplary construct in which the EF1α promoter controls the expression of IL-15-IRES-IL-15Rα variant 4, and (BD) a DNA sequence and schematic diagram (SEQ ID No: 562).

[0050] Figures 15A-15D For: (A) A schematic diagram of an exemplary construct in which the EF1α promoter controls the expression of the IL-15K5-IRES-IL-15RαSushi domain E5, and (BD) a DNA sequence and schematic diagram (SEQ ID No:563).

[0051] Figures 16A-16D For: (A) A schematic diagram of an exemplary construct in which the EF1α promoter controls the expression of the IL-15K5-IRES-IL-15Rα variant 4E5, and (BD) a DNA sequence and schematic diagram (SEQ ID No: 564).

[0052] Figures 17A-17E For: (A) A schematic diagram of an exemplary construct in which the CMV promoter controls the expression of the IL-12-p2A-IL-15-p2A-IL-15RαSushi domain, and (BE) a DNA sequence and schematic diagram (SEQ ID No:565).

[0053] Figures 18A-18E For: (A) A schematic diagram of an exemplary construct in which the CMV promoter controls the expression of IL-12-p2A-IL-15-p2A-IL-15Rα variant 1, and (BE) a DNA sequence and schematic diagram (SEQ ID No: 566).

[0054] Figures 19A-19DFor: (A) A schematic diagram of an exemplary construct in which the CMV promoter controls the expression of the IL-12-p2A-IL-15K5-p2A-IL-15RαSushi domain E5, and (BD) DNA sequence and schematic diagram (SEQ ID No:567).

[0055] Figure 20A , Figure 20B , Figure 20C , Figure 20D , Figure 20D continued For example: (A) a schematic diagram of an 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) are DNA sequences and schematic diagrams (SEQ ID No: 568).

[0056] Figure 21A , Figure 21B , Figure 21C A graph showing the percentage inhibition of PD-L1 by blocking peptide binding to PD-1 is presented.

[0057] Figures 22A-22B The effects of PD-L1 inhibitory peptide on the cytotoxicity of target cells via anti-CD3 stimulated human peripheral blood mononuclear cells were demonstrated.

[0058] Figure 23A and Figure 23B The effects of IL-12 alone, IL-15 alone plus IL-15Rα, and IL-12 and IL-15 / IL-15Rα together on the production of IFNγ and TNFα in human peripheral blood mononuclear cells were shown.

[0059] Figure 24A and Figure 24B The effects of IL-12 and IL-15 / IL-15Rα on the cytotoxicity of U87 and MDA-MB-23 tumor cells via peripheral blood mononuclear cells were demonstrated.

[0060] Figures 25A-25C The expression of IL12, IL15 and PD-L1 blocking peptide is shown after tumor cells are infected with viruses (VG001-PLBh and VG001-15h) carrying PD-L1 blocking peptide or human IL15 / 15Ra. Figure 25D , Figure 25E The results show the effects of infecting tumor cells with viruses (VG001-PLBh and VG001-15h) carrying PD-L1 blocking peptides or human IL15 / 15Ra.

[0061] Figures 26A-26D The results of in vitro measurements for various constructs are shown. Figures 26A-26BThe results of cell transfection using the IL-TF-Fc plasmid expressing IL-12, IL-15 and PD-L1 blockers are shown. Figures 26C-26D The results of cell transfection with various mutant viruses, including hVG001-1-2, are shown.

[0062] Figures 27A-27E The results of cell viability assays for hVG001-1-2 and HSV-345 in human tumor cell lines and Vero cell lines are shown.

[0063] Figures 28A-28J The results of in vitro measurements for various constructs are shown. Figures 28A-28E The results of cell viability assays for mVG001-1-2 and HSV-345 in mouse tumor cell lines and Vero cell lines are shown. Figures 28F-28H The characterization of transgene expression following infection of CT26 mouse tumor cells with mVG001-1-2 is shown. Figures 28I-28J Characterization of the release of cytotoxic factors from peripheral monocytes after infection of CT26 cells with mVG001-1-2 is shown.

[0064] Figures 29A-29C Results of in vitro characterization of transgene expression following infection with hVG001-1-2 or VG001-1.7 in various cell lines are shown. Figures 29D-29E The study demonstrated that infected tumor cells stimulate peripheral monocytes to release cytokines and exhibit cytotoxic activity.

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

[0066] Figures 31A-31G The results of in vivo inhibition of tumor growth by the mVG001-1-2 and hVG001-1-2 constructs are shown.

[0067] Figures 32A-32C The 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 in mouse tumor cell lines and Vero cell lines are shown.

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

[0070] Figures 35A-35D The effects of viral modification are shown.

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

[0072] Figure 37A and Figure 37B The expression of exogenous genes carried by the virus within the tumor was provided in a mouse model after viral injection.

[0073] Figures 38A-38C The effects of VG001-1-2 on immune response are provided.

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

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

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

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

[0078] Figure 43 The modified terminal repeat (TR) region (SEQ ID NO: 576) in VG001-1-2 is shown, which carries an expression cassette encoding IL-12, IL-15 and the IL-15 receptor α subunit and has a US2 (ICP47) flanking.

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

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

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

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

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

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

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

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

[0087] Figure 52 It is the sequence of the short (S) type promoter A (SEQ ID NO:583).

[0088] Figure 53 The sequence is the (M) type promoter A sequence (SEQ ID NO:584).

[0089] Figure 54 It is the sequence of the long (S) type promoter A (SEQ ID NO:585).

[0090] Figure 55 The sequence of promoter B (survivin) is (SEQ ID NO:586).

[0091] Figures 56A-56B The results show the assay results of the cytotoxicity of VG001-1-2 virus on (A) human cancer cells and (B) mouse cancer cells.

[0092] Figures 57A-57C The images show that after cells were infected with VG001-1-2 virus, (A) they expressed IL-12, (B) they expressed IL-15, and (C) they expressed PD-L1 blockers.

[0093] Figure 58 The results of ELISA analysis of PD-L1 blockers generated after cell infection with VG001-1-2 are shown.

[0094] Figure 59 The results of a cell-based analysis of the PD-L1 blocker generated after cell infection with VG001-1-2 are shown.

[0095] Figures 60A-60DThe study demonstrated that IL-12, IL-15 / IL-15RA, and PD-L1 blockers synergistically promote immune cell function in vitro.

[0096] Figures 61A-61D The effect of intratumoral inoculation with VG001-1-2 virus is shown.

[0097] Figures 62A-62B The effects of treating tumor cells with mVG001-1-2 virus on gene expression and T cell activity are shown.

[0098] Figures 63A-63D The effect of mVG001-1-2 treatment on the intratumoral lymphocyte population is shown.

[0099] Figures 64A-64B The effects of minimum doses of human IL12 or human IL15 / IL15RA on immune cell function are shown.

[0100] Figure 65A , Figure 65B The biodistribution of the VG001-1-2 virus is shown.

[0101] Figure 66 The results show the quantitative detection of immune-related gene expression levels in tumor-bearing mice treated with VG001-1-2.

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

[0103] Figure 68 The results of titer analysis of viruses preserved in media with different formulations are shown.

[0104] Figure 69 The results of stability tests on fusion proteins with different structures expressed by the virus are shown.

[0105] Figure 70 The structure of the VG001-1-2 carrier is shown. Detailed Implementation

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

[0107] Disclosure Overview

[0108] The invention can be more readily understood by referring to the following detailed description of preferred embodiments and examples included herein. In short, the disclosure herein provides oncolytic herpes simplex virus type 1 or 2 vectors that express immunostimulatory molecules. Representative vectors comprise expression cassettes encoding one or more IL-12, IL-15, and IL-15Rα. Some vectors encode mouse or human IL-12, human IL-15, and human IL-15Rα. In some embodiments, the vector encodes mouse or human IL-12, hIL15, and the hIL15 receptor α subunit. In other embodiments, the vector encodes hIL-12, hIL15, and the hIL15 receptor α subunit. These three proteins can be expressed on one, two, or three transcripts. When expressed on the same transcript, subsequent post-transcriptional processes result in the expression of a single protein. In such cases, the coding region is separated by sequences encoding self-splicing peptide 2A or IRES. The coding region can also be expressed via a bidirectional promoter. The HSV vector optionally expresses one or more PD-L1 blocking peptides that it can secrete.

[0109] A. oHSV vector

[0110] Oncolytic viruses are viruses that preferably selectively lyse cancer cells. Viruses that selectively replicate in differentiated cells more than in undifferentiated cells are often oncolytic. Oncolytic viruses suitable for use in this article include herpes simplex viruses 1 and 2, and may also include non-human herpesviruses such as BHV or others.

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

[0112] Suitable oncolytic HSV can be derived from HSV-1 or HSV-2, including any laboratory strain or clinical isolate. In some embodiments, oHSV can be or may 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 or other non-laboratory strain JS-1. Other suitable HSV-1 viruses include HrrR3 (Goldsten and Weller, J. Virol. 62, 196-205, 1988); G2O7 (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 of Sciences, 2000; 97(5):2208-2213); NV1042 (Passer et al., Cancer Gene Therapy. 2013; 20(1):17-24); G2O7-IL2 (Carew et al., Molecular Therapy, 2001; 4(3):250-256); rQNestin34.5 (Kambara et al., Cancer Research, 2005; 65(7):2832-2839); G47Δ-mIL-18 (Fukuhara et al., Cancer Research, 2005; 65(23):10663-10668); and those vectors disclosed in PCT / US2017 / 030308 entitled "HSV Vectors with Enhanced Replication in Cancer Cells" and PCT / US2017 / 018539 entitled "Compositions and Methods of Using Stat1 / 3 Inhibitors with Oncolytic Herpes Virus", all of which are incorporated herein by reference.

[0113] The oHSV vector may have at least one modified, mutated, or deleted γ34.5 gene. The vector lacks the complete γ34.5 gene. In some embodiments, both genes are deleted, mutated, or modified. In other embodiments, one gene is deleted while the other is mutated or modified. Either endogenous 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 (e.g., nucleotide alterations, insertions, and deletions) render the gene unexpressable or inactivated. The γ34.5 gene can be modified in its 3'UTR using a miRNA target sequence. The target sequence binds to a miRNA expressed in tumor cells at a lower rate than in its normal control. In some embodiments, the modified or mutated γ34.5 gene is constructed in vitro and inserted into the oHSV vector as a substitute 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 other variations, such as having an exogenous promoter.

[0114] oHSV can have additional mutations, which may include disabling mutations (e.g., deletions, substitutions, insertions) that affect viral virulence or its ability to replicate. For example, mutations can be made in any one or more of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24, and ICP56. Preferably, a mutation in one of these genes (optionally in two copies of the suitable gene) results in HSV's inability to express the corresponding functional polypeptide (or a reduced ability to do so). In some embodiments, the promoter of the viral gene is replaced with a promoter that is selectively activated in target cells, or that is inducible after elicitor delivery, or that is inducible after a cellular event or in a specific environment. In certain embodiments, tumor-specific promoters drive the expression of viral genes essential for HSV replication. In some embodiments, the expression of ICP4 or ICP27, or both, is controlled by an exogenous promoter, such as a tumor-specific promoter. Exemplary tumor-specific promoters include survivin or telomerase; other suitable tumor-specific promoters may be specific to a single tumor type and are known in the art. Other elements may be present. In some cases, enhancers (e.g., NF-kB / OCT4 / SOX2 enhancers) are present, for example in the regulatory regions of ICP4 or ICP27 or both. Moreover, the 5'UTR can be exogenous, such as the 5'UTR of a growth factor gene (e.g., FGF).

[0115] oHSV can also contain gene and nucleotide sequences not derived from HSV. For example, the oHSV genome may contain sequences encoding prodrugs, sequences encoding cytokines or other immunostimulatory factors, tumor-specific promoters, inducible promoters, enhancers, sequences homologous to host T cells, and other sequences. Exemplary sequences encode IL12, IL15, OX40L, PD-L1 blockers, or PD-1 blockers. For sequences encoding products, they are operatively linked to promoter sequences and other regulatory sequences (e.g., enhancers, polyadenylation signaling sequences) for expression.

[0116] The regulatory region of a viral gene can be modified to include response elements that influence expression. Exemplary response elements include NF-κB, Oct-3 / 4-SOX2, enhancers, silencers, cAMP response elements, CAAT enhancer binding sequences, and isolators. Other response elements may also be included. The viral promoter can be replaced with different promoters. The choice of promoter depends on a number of factors, such as the desired HSV vector, the patient's treatment, baseline condition or disease status, and the ease of applying the inducible promoter (for inducible promoters). For cancer treatment, when the promoter is replaced, it is typically a cell-specific, tissue-specific, or tumor-specific promoter. Tumor-specific, cell-specific, and tissue-specific promoters are known in the art. Other gene elements can also be modified. For example, the 5' UTR of a viral gene can be replaced with a foreign UTR.

[0117] B. Immune stimulating molecules

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

[0119] 1.IL-12

[0120] Interleukin-12 (IL-12) is primarily produced by dendritic cells, macrophages, and monocytes in response to bacteria (e.g., lipopolysaccharides), pathogens, or activated T cells. IL-12 can induce IFNγ production, cell proliferation, and activate natural killer cells and T cells. It is also crucial for T cell differentiation into Th1 cells. IL-12 can also inhibit tumor growth. Mouse IL-12 exhibits equal activity in both mouse and human cells and is 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 the generation of the heterodimer. IL-12 expression in the oHSV vector can be achieved in several ways. Both subunits can be expressed in a single construct, each with a promoter, or in a construct starting from a bidirectional promoter, or from a construct containing an element such as IRES or a self-cleaving peptide in the coding region. Alternatively, the subunit can be expressed as a single strand. For example, a viable single-chain IL-12 fusion protein can be generated by linking the coding regions of p40 and p35 to 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 linker sequence and length are typically selected in a manner that maximizes the flexibility of the structure (Chen et al., Adv Drug Deliv Rev. 65:1357, 2013). The linker sequence can be selected using computer programs. One such program is called LINKER (Crasto and Feng, Protein EngDesign & Selection 13:309). An exemplary single-chain IL-12 has the amino acid sequence of SEQ ID NO:1. Amino acid substitutions, insertions, and deletions can be performed, as long as the IL-12 retains its function.

[0122] 2.IL-15

[0123] IL-15 is a cytokine that regulates the activation and proliferation of natural killer cells and T cells, and also possesses other biological activities. Two isoforms exist, with different signal peptide sequences but identical mature protein sequences. The GenBank (NCBI) accession number for the isoform with the longer signal peptide (sometimes referred to as LSP-IL15) is NP000576, while the accession number for the isoform with the shorter signal peptide (sometimes referred to as SSP-IL15) is NP 751915. Either isoform is suitable for use in oHSV vectors. Amino acid insertions, deletions, and substitutions are permissible, as observed in polymorphisms, as long as the protein binds to IL-15.

[0124] In some embodiments, both IL-15 and IL-15Rα are C-terminal peptides of a selectively dimerized coiled helix. A large number of suitable peptides are taught in the literature (see, for example, Tripet et al., Protein Engineering 9:1029, 1996; Aronsson et al., Sci Rep 5:14063, 2015). Typically, the amino acid sequence of the coiled helix has heptapeptide repeats of hydrophobic (h) and polar (p) residues in the form of hpphppp. Two exemplary coiled helices are the K-helix (KVSALKE, SEQ ID No. 7) and the E-helix (EVSALEK, SEQ ID No. 8). Typically, 3–6 tandem copies are used. In some embodiments described herein, 5 tandem copies are used: K5 (KVSALKEKVSALKEKVSALKEKVSALKE, SEQ ID No. 9) and E5 (EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK, SEQ ID No. 10). The K-helix and E-helix are designed to carry opposite charges, so IL-15 is fused with a coiled helix, while IL-15Rα is fused to a coiled helix with the opposite charge. An exemplary Sushi domain fused to E5 is shown in SEQ ID NO:12, an exemplary IL-15Rα variant 4 fused to E5 is shown in SEQ ID NO:13, an exemplary IL-15Rα 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-15Rα subunit

[0126] The interleukin-15 receptor α subunit (IL-15Rα) is one of the three subunits of the IL-15-binding complex. This α subunit binds to IL-15 with high affinity and can bind independently of the other subunits. At least four variants (isotypes) exist, 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 α subunit contains a Sushi domain (aka complement control protein (CCP), short consensus repeats (SCRs), or SUSHI repeats), which is the shortest region retaining IL-15 binding activity. A typical Sushi domain consists of approximately 60-70 amino acids, containing four cysteine ​​residues that form two disulfide bonds and serve as a shared motif in protein-protein interactions. The Sushi domain of IL-15Rα contains residues 31 to approximately 95 (corresponding to variant 1) (SEQ ID NO: 11). The position of the Sushi domain in other variants is known. Amino acid substitutions of cysteine ​​residues in sIL-15Rα eliminate its ability to suppress acute inflammation and cause T cells to respond in vivo to alloantigens (Wei et al., J Immunol. 167:277, 2001).

[0127] The oHSV vector contains a nucleic acid sequence encoding IL-15Rα, a variant of IL-15Rα, or a Sushi domain. Typically, the protein is expressed by a lead peptide, and in some embodiments, the lead peptide is derived from IL-15Rα. Other lead peptides are known in the art. Amino acid substitutions may be present, provided the protein binds to IL-15. Natural substitutions and 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 through binding to the PD-1 receptor. Blocking protein-protein interactions has been shown to improve cancer treatment.

[0130] The oHSV vector can express PD-L1 blocking peptides. 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 by a leader sequence. Leader sequences are well known in the art. They include the immunoglobulin κ chain leader sequence (METDTLLLWVLLLWVPGSTG; SEQ ID NO:501) and the IL-2 leader sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO:502). When more than one blocking peptide is present, the peptide is usually separated by a flexible linker. This linker is typically Gly or Ser or rich in Gly / Ser. Examples of suitable adapters are shown in (SEQ ID NOs: 503-519) (see also Chichili et al., Protein Science 22:153, 2013). There may be one, two, three, or more copies of the peptide. Multiple copies are generally randomized and adapters may be present between copies. The blocking peptide construct may also include an Fc sequence at the C-terminus of the peptide, or an immunoglobulin Fc sequence with or without a hinge region. Although any Fc region is used, typically the Fc will be from one of the IgG subclasses, such as human IgG1, human IgG2, human IgG3, and human IgG4 or their mouse counterparts.

[0131] C. Component organization

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

[0133] In some implementations, two or three of the molecules are expressed in a single transcript from a single promoter, and their coding sequences are encoded by IRES (Internal Ribosome Entry Site) sequences. The IRES region attracts the eukaryotic ribosomal translation initiation complex and thus allows translation initiation to occur in the middle of the mRNA, independent of the commonly used 5'-cap structure. Suitable IRES sequences are well known, and many suitable IRES sequences can be found in the IRESite's expression bases of experimentally confirmed IRES sequences (see, for example, http: / / iresite.org / IRESite_web.php?page=browse_plasmids; received May 26, 2016).

[0134] In various implementations, these three genes exist in any order and are separated by one or more IRES sequences. These IRES sequences may be the same or different. Additional sequences may exist at gene / IRES junctions or IRES / IRES junctions.

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

[0136] Another way to influence the co-expression of molecules is through the use of bidirectional promoters. Bidirectional promoters are a common feature of the human genome (Trinklein et al., Genome Res 14:62, 2004). Bidirectional promoters initiate transcription in two directions and typically contain shared elements regulating both genes. In addition to natural bidirectional promoters, bidirectional promoters have been synthesized. One such promoter is bi-CMV. pBI-CMV1 is a mammalian bidirectional expression vector that enables constitutive expression of two proteins of interest. Protein expression is driven by two constitutively activated minimal human cytomegalovirus promoters (PminCMV1 and PminCMV2 with opposite orientations). An exemplary DNA sequence of the bidirectional CMV promoter is shown in SEQ ID NO. 536.

[0137] Bidirectional promoters (e.g., bi-CMV promoters) are primarily used to achieve the co-expression of hIL15 and IL-15Rα (or the Sushi domain). When two molecules are co-expressed using an IRES or p2A sequence, it is typically hIL15 and IL-15Rα (or the Sushi domain). In these cases, IL-12 and PD-L1 blocking peptides can be co-expressed using a bidirectional promoter or as a polyci-reverse transcript with an IRES or p2A sequence, or they can be expressed individually from their own promoter / regulatory regions.

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

[0139] The sequence is assembled in one or more expression cassettes. Examples provide specific versions of some exemplary expression cassettes. The expression cassette can be inserted into the HSV genome at any location without disrupting critical functions (e.g., replication). In some implementations, the expression cassette is inserted into an internal or terminal repeat region after the initial deletion of the repeat region. Other suitable insertion regions include between viral genes, such as between the UL3 and UL4 viral genes, between the UL50 and UL51 genes, and between US1 and US2.

[0140] In some embodiments, an expression cassette expressing a PD-L1 blocking peptide is inserted between viral genes (e.g., UL3 and UL4, UL50 and UL51 and / or US1 and US2). In other embodiments, an expression cassette expressing IL-12, IL-15, and IL-15Rα is inserted instead of the terminal repeat region, and an expression cassette expressing a PD-L1 peptide is inserted between the UL3 and UL4 genes.

[0141] D. Therapeutic Composition

[0142] Therapeutic compositions are provided that can be used to prevent, treat, or mitigate the effects of diseases, such as, for example, cancer. More specifically, therapeutic compositions comprising at least one oncolytic virus as described herein are provided. Representative examples include oHSV having an expression cassette having one or more IL12, IL15, and / or IL receptor 15α subunits. In one embodiment, the expression cassette expresses all IL12, IL15, and IL receptor 15α subunits. In a preferred embodiment, the expression cassette comprises mouse or human IL12, hIL15, and hIL15 receptor α subunits.

[0143] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier” means any solvent, diluent, or excipient that does not interfere with the potency of the oncolytic virus’s biological activity and is non-toxic to the subject receiving the drug (see Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005) and in The United States Pharmacop E1A: The National Formulary (USP 40–NF 35 and Supplements)).

[0144] In the case of oncolytic viruses described herein, non-limiting examples of suitable pharmaceutically acceptable solvents include phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various forms of wetting agents, sterile solutions, and the like. Other pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantable elements containing oncolytic viruses, or any other solvent, delivery agent, or dispersion device or material. Such solvents can be prepared by conventional methods and can be administered to subjects at effective doses. Other pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts may also be included, such as inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.) and salts of organic acids (e.g., acetate, propionate, malonate, benzoate, etc.). Such pharmaceutically acceptable (pharmaceutical grade) solvents, diluents, and excipients that can be used to deliver oHSV to target cancer cells preferably do not induce an immune response in the individual receiving the composition (subject) (and are preferably administered in a manner free from inappropriate toxicity).

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

[0146] In some embodiments of the invention, a lower-than-standard dose may be used. Therefore, in some embodiments, less than about 10 [units of something] may be administered to the patient. 6 pfu / ml (4ml injected into the patient every 2-3 weeks).

[0147] This composition can be stored at temperatures beneficial to its stable shelf life, including room temperature (approximately 20°C), 4°C, -20°C, and -80°C, in liquid nitrogen. Since the composition is generally not intended for in vivo use, it is typically stored at low temperatures. The composition can be stored in a dry form (e.g., lyophilized) or in a liquid form.

[0148] E. Dosage

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

[0150] The terms "effective dose" and "effective amount" refer to an amount of oncolytic virus sufficient to affect the treatment of a target cancer, such as an amount that effectively reduces the size or load of a target tumor or inhibits the growth rate of target tumor cells. More specifically, such terms refer to an amount of oncolytic virus, administered at the necessary dose and during the treatment period, that effectively achieves the desired outcome. For example, in the case of treating cancer, the effective amount of the composition described herein is an amount that causes a reaction, reduces the tumor burden, and / or prevents tumor spread or cancer growth. The effective amount can vary depending on various factors, such as the subject's disease state, age, sex, and weight, as well as the drug formulation, route of administration, etc., but can be routinely determined by those skilled in the art.

[0151] The treatment composition is administered to subjects diagnosed with or suspected of having cancer. Subjects may be humans or non-human animals.

[0152] This composition is used to treat cancer. As used herein, the terms “treatment” or “management” mean a process for achieving a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions of a detectable or undetectable disease, reduction of disease severity, stable (i.e., non-aggravating) state of disease, prevention of disease spread, delay or slowing of disease progression, relief or reduction of disease state, reduction of disease recurrence, and control (partial or complete). The terms “treatment” and “management” may also mean prolonged survival compared to expected survival without treatment.

[0153] Representative forms of cancer include carcinomas, leukemia, lymphoma, myeloma, and sarcoma. Further examples include, but are not limited to, cholangiocarcinoma, brain cancer (e.g., glioblastoma), breast cancer, cervical cancer, colorectal cancer, CNS cancers (e.g., acoustic neuroma, astrocytoma, craniopharyngioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, reticulum cystoma, neuroblastoma, oligodendroglioma, pineal tumor, and retinoblastoma), endometrial lining cancer, hematopoietic carcinomas (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 include solid tumors (e.g., sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteosarcoma), diffuse cancers (e.g., leukemia), or combinations of these (e.g., metastatic cancer with both solid tumors and diffuse or diffuse cancer cells). Cancer can be resistant to conventional treatments (e.g., conventional chemotherapy and / or radiation therapy).

[0154] It can also treat benign tumors and other conditions involving unwanted cell proliferation.

[0155] The oHSV described herein can be administered via routes such as oral, local, parenteral, systemic, intravenous, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, or percutaneous. In some embodiments, the oncolytic virus can be delivered via cannula, catheter, or direct injection. The site of administration can be intratumoral or remote from the tumor. The route of administration often depends on the type of cancer being treated.

[0156] Attending physicians in this field can readily determine the optimal or appropriate dosage regimen of oncolytic viruses based on patient presentations, patient observations, and various clinical factors, including, for example, subject size, body surface area, age, sex, and the specific oncolytic virus administered, the timing and route of administration, the type of cancer to be treated, the patient's overall health status, and other drug treatments the patient has received. According to some embodiments, the oncolytic viruses described herein can be used in combination with other types of treatment, such as chemotherapy, for example, using chemotherapeutic agents such as etoposide, ifosfamide, doxorubicin, vincristine, doxycycline, etc.

[0157] oHSV can be formulated into pharmaceuticals and pharmaceutical compositions for clinical use and can be combined with pharmaceutically acceptable carriers, diluents, excipients, or excipients. The formulation depends at least in part on the route of administration. Suitable formulations may contain the virus and inhibitor in a sterile culture medium. The formulation may be in fluid, gel, patch, or solid form. The formulation may be provided to subjects or healthcare professionals.

[0158] Preferably, a therapeutically effective dose is administered. This is a dose sufficient to demonstrate benefit to the subject. The actual dose and administration time will depend at least in part on the nature of the cancer, the subject's condition, the delivery site, and other factors.

[0159] In another embodiment of the invention, the oncolytic virus can be administered intratumorally or before or after surgical removal of the tumor.

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

[0161] Example

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

[0163] Example 1

[0164] A schematic diagram of an exemplary OHSV carrier

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

[0166] Example 2

[0167] Exemplary builder

[0168] In this embodiment, various constructs and their sequences are proposed.

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

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

[0171] Example 3

[0172] Abbreviations used in subsequent embodiments

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

[0174] IL-TF-Fc: A plasmid carrying IL-12, IL-15 and PD-L1 blockers.

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

[0176] OS-ICP27 2-11: A virus with an Oct4 / Sox2 binding site and an ICP34.5-deleted 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: A virus with an ICP34.5 deletion that is not used to construct VG001-1-2 due to the OS-ICP27 mutation.

[0178] NO-ICP27 1-4-4 (also known as NO-ICP27-145): A virus with an NF-kB response element and an ICP34.5 deletion inserted 145 bp upstream of the transcription start site of ICP27 into the promoter-regulatory region of ICP27 (NO-ICP27) not used to construct VG001-1-2.

[0179] NO-ICP27 5-2-2 (also known as NO-ICP27-99): A virus with an NF-kB response element and an ICP34.5 deletion inserted 99 bp upstream of the transcription start site of ICP27 into the promoter-regulatory region of ICP27 (NO-ICP27) not used to construct VG001-1-2.

[0180] VG001-1.7 (also known as HSV 1-VG 001-1.7): The backbone virus used to construct VG001-1-2 (a NO-ICP27 1-4-4 mutant carrying an exogenous promoter and having an empty MCS in a deleted terminal repeat region of the viral genome, which was subsequently used to insert an IL-12 / IL-15 expression cassette).

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

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

[0183] VG001-PLBh (also known 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 blockers.

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

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

[0187] Example 4

[0188] IL-12 expression in cells infected with HVG001-1-2

[0189] In this embodiment, Western blot analysis and ELISA expression of IL-12 expression 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 anti-human IL-12 antibody, followed by analysis with HRP-conjugated anti-mouse IgG secondary antibody. Images were detected and analyzed using a Bio-Rad ImageLab system.

[0191] Figures 6B-6CThe upregulation of human IL-12 production following infection with VG001-1-2-1215PLBh virus is illustrated. 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 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. The concentration of human IL-12 in the cultured supernatant was calculated based on a human IL-12 standard curve.

[0192] Example 5

[0193] IL-15 expression in cells infected with HVG001-1-2

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

[0195] Figure 7A Western blot analysis results after infection with 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 a 12% SDS-PAGE gel, and transferred to a PVDF membrane. The membrane was blotted with anti-human IL-15 antibody, followed by analysis with HRP-conjugated anti-mouse IgG secondary antibody. Images were detected and analyzed using a Bio-Rad ImageLab system.

[0196] Figures 7B-7C The upregulation of human IL-15 production following infection with VG001-1-2-1215PLBh virus was demonstrated. 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 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. The concentration of human IL-15 in the cultured supernatant was calculated based on a human IL-15 standard curve.

[0197] Example 6

[0198] IL-4 expression in cells infected with HVG001-1-2

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

[0200] Figure 8A Western blot analysis results after infection with 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 a 12% SDS-PAGE gel, and transferred to a PVDF membrane. The membrane was blotted with HRP-conjugated anti-human IgG antibody, and the images were detected and analyzed using a Bio-Rad ImageLab system.

[0201] Figures 8B-8C The upregulation of human PD-L1 blocker (fused to the human Fc region) production following infection with VG001-1-2-1215PLBh virus was demonstrated. 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 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. The concentration of human IL-4 in the cultured supernatant was calculated based on a human IL-4 standard curve.

[0202] Example 7

[0203] Construct containing PD-L1 blocking peptide

[0204] The PD-L1 blocking peptide was generated using the Igκ chain leader sequence (SEQ ID NO:501). When two or more blocking peptides were present in the same construct, they were linked to a Gly-Ser-rich sequence (Gly4Ser)3 (SEQ ID NO:503). The following constructs were prepared.

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

[0206] ET+TF:

[0207] METDTLLLWVLLLWVPGSTGEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF(SEQ IDNO:538);

[0208] YT+TF:

[0209] METDTLLLWVLLLWVPGSTGYYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF(SEQ IDNO:539);

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

[0211] Mouse WT+TF:

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

[0213] Triple TF+ET:

[0214] METDTLLLWVLLLWVPGSTGTAHPSPSPRSAGQFTAHPSPSPRSAGQFTAHSPSPRSAGQFGGGGSGGGGSGGGGSEYRMSPSNQTEYRMSPSNQTEYRMSPSNQT(SEQ ID NO:542)

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

[0216] Other constructs were prepared using the IL-2 signal sequence (MYRMQLLSCIALSLALVTNS (SEQ ID NO:502) and the human IgG4 Fc region (with a hinge region) (SEQ ID NO:544) or the mouse IgG1 Fc region (with a hinge region) (SEQ ID NO:545). The constructs are as follows:

[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] MYRMQLLSCIALSLALVTNSYYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFISAMVRSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:548)

[0226] Mouse TF:

[0227] MYRMQLLSCIALSLALVTNSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:549)

[0228] Mouse WT + TF:

[0229] MYRMQLLSCIALSLALVTNSWNRLSPSNQTGGGGSGGGGSGGGGSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDI SKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFP EDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 550).

[0230] Example 8

[0231] Constructs containing IL-15 and IL-15Rα under the control of bidirectional CMV promoters

[0232] In this embodiment, various constructs were generated to co-express IL-15 and IL-15Rα under the control of a bidirectional CMV promoter.

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

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

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

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

[0237] Example 9

[0238] Constructs containing the IL-15 and IL-15Rα genes under the control of the EF1α promoter

[0239] In this embodiment, various constructs were generated to express IL-15 and IL-15Rα in a polycistronic transcript under the control of the EF1α promoter (SEQ ID NO:551). IL-15 and IL-15Rα are linked by an exemplary IRES sequence (SEQ ID NO:552).

[0240] In construct 1, the EF1α promoter controls the expression of the IL-15-IRES-IL-15RαSushi domain (Fig. 13, SEQ ID No. 561).

[0241] In construct 2, the EF1α promoter controls the expression of IL-15-IRES-IL-15Rα variant 4 (Fig. 14, SEQ ID No. 562).

[0242] In construct 3, the EF1α promoter controls the expression of the IL-15K5-IRES-IL-15RαSushi domain E5 (Fig. 15, SEQ ID No. 563).

[0243] In construct 4, the EF1α promoter controls the expression of IL-15K5-IRES-IL-15Rα variant 4E5 (Fig. 16, SEQ ID No. 564).

[0244] Example 10

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

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

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

[0248] Expression of the IL-12-p2A-IL-15-p2A-IL-15RαSushi domain (Figure 17, SEQ ID Nos. 565, 569).

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

[0250] In construct 3, CMV initiation controls the expression of the E5 domain of the IL-12-p2A-IL-15K5-p2A-IL-15RαSushi structure (Fig. 19, SEQ ID Nos. 567, 571).

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

[0252] Example 11

[0253] A construct containing a PD-L1 blocker inserted between UL3 and UL4

[0254] In this embodiment, a construct was generated to express the 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: the region between UL3 and UL4 and upstream of the PD-L1 blocking sub-box; bases 830-833: the region between UL3 and UL4 and downstream of the PD-L1 blocking sub-box; bases 834-1433: UL4 coding sequence.

[0255] Example 12

[0256] By blocking peptides to inhibit human PD-L1 binding to PD-1

[0257] Recombinant human PD-L1 Fc protein was coated onto the bottom of a 96-well plate overnight at 4°C. After overnight coating, different PD-L1 blockers were added to each well and incubated at room temperature for 2 hours, followed by the addition of 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. Colorimetric measurements were taken at 450 nm using a microplate reader. The percentage of inhibition was calculated by comparison with a non-synthetic peptide control. Figure 21 shows the percentage of inhibition produced by peptides ET, ET+TF, YT, YT+TF, TW, TW+TF, WT, WT+TF, and TF at two different concentrations (3 μM and 10 μM). At 10 μM, the inhibition ranged from approximately 22% to approximately 48%.

[0258] Example 13

[0259] Enhancing cytotoxicity against tumor cells by blocking PD-L1 binding to peptides.

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

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

[0262] Example 14

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

[0264] Human PBMCs were incubated for 48 hours with a culture medium control, IL-12 only, IL-15RA only, or a combination of IL-12, IL-15, and IL-15Rα1+ neutralized with anti-IL-12 or anti-IL-15 antibodies. The cultured cell supernatant was harvested for the determination of human IFNγ and TNFα production by ELISA.

[0265] Figure 23A and Figure 23B The results of cytokine production are shown. The combination of IL-12 and IL-15Rα1 resulted in a statistically significant increase in the cytokines human IFNγ and TNFα. Anti-IL-12 antibody inhibited their production.

[0266] Example 15

[0267] The synergistic effect of IL-12 and IL-15 on tumor cell cytotoxicity

[0268] Human PBMCs were incubated for 24 hours with tumor target cells, culture medium controls, IL-12 alone, IL-15RA alone, or a combination of IL-12, IL-15, and IL-15RA1+ neutralized with anti-IL-12 or anti-IL-15 antibodies. The cultured cell supernatant was harvested for cytotoxicity measurement via LDH. The percentage of cytotoxicity was calculated based on the following formula: [(sample reading – minimum release) / (maximum release – minimum release)] × 100.

[0269] Figure 24A and Figure 24BThe study showed that IL-12 and IL-15 together increased cytotoxicity in a statistically significant manner. In the MDA-MB-231 cell line, the addition of anti-IL-12 or anti-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 embodiment, 3×10 4 H460 or LS174T 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, VG001-1-2-PLBh, or VG001-1-2-15h virus (MOI=1) for 24 hours, and the production of human IL-12, human IL-15, and human IgG4 was assessed. Figure 25A -C). Then 3×10 5 Human PBMCs were added to the culture and co-cultured for 24 hours to assess cytotoxicity via LDH assay. Figure 25D Alternatively, co-culture for 48 hours to assess human IFNg production via ELISA. Figure 25E For cytotoxicity assays, the percentage of cytotoxicity is 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 is used as the minimum release, while the supernatant harvested from tumor cells incubated with lysis buffer is used as the maximum release.

[0273] Example 17

[0274] In vitro efficacy of various constructs

[0275] Figures 26A-26D The results of in vitro measurements for various constructs are shown.

[0276] Figures 26A-26B The results of transfecting cells with the IL-TF-Fc plasmid carrying IL-12, IL-15, and PD-L1 blockers are shown. Figures 26A-26B In this study, different tumor cell lines were transfected with IL-TF-Fc plasmid DNA for 24 hours, and human PBMCs were subsequently added to the cultures. Cell supernatant was harvested 24 hours later for quantitative analysis of cytotoxicity using LDH assay. Figure 26A ), and was used 48 hours later to detect human IFNg production by ELISA assay. Figure 26B ).

[0277] Figures 26C-26DResults of cell infection with various mutant viruses, including hVG001-1-2, are shown. Virus-encoded IL12, IL15, and PD-L1 blockers synergistically increased IFNg production and cytotoxicity. H460 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 the specified virus at MOI=1 for 24 hours. Human PBMCs were then added to the culture and co-incubated for 24 hours to assess cytotoxicity via LDH assay. Figure 26C ), or co-incubate for 48 hours to assess human IFNg production via ELISA ( Figure 26D For cytotoxicity assays, the percentage of cytotoxicity is 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 is used as the minimum release, while the supernatant harvested from tumor cells incubated with lysis buffer is used as the maximum release.

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

[0279] Figures 28A-28J The results of in vitro measurements for various constructs are shown. Figures 28A-28E The results of cell viability assays for mVG001-1-2 and HSV-345 in mouse tumor cell lines and Vero cell lines 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] exist Figures 28A-28E In this study, six different mouse tumor cell lines (+Vero cells) were infected with VG001-1-2m and HSV-345 virus at MOIs of 0, 0.04, 0.2, 1, and 5. Cell viability was quantitatively analyzed using MTT assay at 48 hours post-infection.

[0281] exist Figures 28F-28J In the middle, 3×10 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 the VG001-1-2 viral construct

[0286] exist Figures 31A-31B In this study, BALB / c mice bearing B-cell lymphoma tumors (A20 mice) underwent five intratumoral injections totaling 1×10⁻⁶ mg / L. 7 PFU / mouse VG001-1-2-1215PLBm (mVG001-1-2) virus or VG001-1.7 backbone virus or PBS (vector control) injection. Tumor size was measured after a specified number of injections. Mice treated with VG001-1-2-1215PLBm showed a significant reduction in tumor volume (P<0.05) compared to mice treated with PBS.

[0287] exist Figures 31C-31D In this study, CT26 mice bearing colon cancer BALB / c tumors underwent five intratumoral injections totaling 5 × 10⁶ cells / mL. 6 PFU / mouse VG001-1-2-1215PLBm (mVG001-1-2) virus or VG001-1.7 backbone virus or PBS (vector control) injection. Tumor size was measured after a specified number of injections. Mice treated with VG001-1-2-1215PLBm showed a significant reduction in tumor volume (P<0.05) compared to mice treated with PBS.

[0288] exist Figures 31E-31G In this study, oHSV therapy with xenografted human prostate tumors in mice was evaluated. LNCaP human prostate tumor cells were transplanted into the right lower abdomen of twelve mice. Thirty-five days post-transplantation, six animals in a randomized group received two intratumoral injections totaling 5 × 10⁻⁶ cells. 7 PFU / mouse VG001-1-2-1215PLBh

[0289] (hVG001-1-2) virus was used, while the remaining 6 animals served as vector controls and were injected twice with equal volumes 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 the tumor volume with virus or PBS. Figure 31E During the study, tumor-bearing mice treated with VG001-1-2-1215PLBh virus showed significant tumor shrinkage, with tumor size decreasing by more than 50% by the end of 15 days, while mice treated with the vector showed approximately a 3-fold increase in tumor volume during the same time period. Tumor growth was also monitored using a whole-animal bioluminescence imaging system (IVISImaging System; Xenogen, Mountain View, CA). Quantitative analysis of signal intensity was performed as the sum of all photons detected per second (…). 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 injected with PBS, HSV 1-VG 001-1.7, or VG001-1-2m virus. Tumor cells were harvested 24 hours after injection, and the percentage of CD8+ T cells, CD4+ T cells, or NK cells was measured by flow cytometry. Figures 38A-38C The results are shown in the figure.

[0308] Example 24

[0309] Another exemplary building block

[0310] In this embodiment, the construct is further modified, particularly the US12 (ICP47) promoter region located on the flank of the IL12-IL15-IL15RA1 expression box, thereby proposing another construct and its sequence.

[0311] VG001-1-2 contains a modified ICP34.5 region ( Figure 40 ; SEQ ID NO.599), modified UL54 promoter-control region ( Figure 41 (SEQ ID NO. 596), inserting a PD-L1 blocker into the gene region between UL3 and UL4 ( Figure 42 ; SEQ ID No. 589), and a modified terminal repeat (TR) region carrying an expression cassette encoding IL-12, IL-15 and the IL-15 receptor α subunit (IL12-IL15-IL15RA1). Figure 43 (SEQ ID NO. 576). 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 the self-cleaving linker peptide P2A, and the self-cleaving linker peptide P2A, respectively.

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

[0313] The US12 (ICP47) promoter region on the flank of the IL12-IL15-IL15RA1 expression cassette carried by VG001-1-2 was modified to construct various versions of the flank US12 (ICP47) promoter: short (S; SEQ ID No. 583), medium (M; SEQ ID No. 584), long (L; SEQ ID No. 585), and survivin (SEQ ID No. 586). According to the different versions of the flank ICP47 promoter, VG001-1-2 can be specifically divided into VG001-1-2(S), VG001-1-2(M), VG001-1-2(L), and VG001-1-2(surviving). In the following embodiments, unless otherwise stated, VG001-1-2 refers to VG001-1-2(M), that is, the IL12-IL15-IL15RA1 expression cassette carried by the vector has a medium (M) version of the ICP47 promoter on its flanking side. The structure of the VG001-1-2 vector is shown in Figure 70 .

[0314] Example 25

[0315] Cytotoxicity of VG001-1-2

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

[0317] Example 26

[0318] In vitro characterization of IL12, IL15 and PD-L1 blockers 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. Transgenic expression was quantified by Western blotting (left column) and ELISA (right column). Figure 57A This shows the expression of human IL-2. Figure 57BThis shows the expression of human IL-5. Figure 57C The expression of the PD-L1 blocker is shown.

[0320] Example 27

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

[0322] like Figure 58 As shown, supernatant containing TF+Fc peptide harvested from VG001-1-2 infected 293FT cells was mixed with recombinant human PD-1 Fc and bound to a 96-well Immuno Maxisorp plate coated with human PD-L1 Fc. 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 using a plate reader. The percentage increase (%) in human PD-1 / PD-L1 inhibition was compared with that of peptide-free samples.

[0323] Example 28

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

[0325] Figure 59 Cell-based assays of TF+Fc peptide treatment are shown. The cells were activated with 1 μg / ml PHA and 50 ng / ml PMA for 5 × 10⁻⁶ cells / day. 4 Jurkat T cells, and 1×10 5 Tumor cells expressing PD-L1 were mixed with a supernatant containing a PD-L1 blocking peptide and incubated at 37°C for 48 hours. After 48 hours, the cell culture supernatant was harvested, and the IL-2 produced by Jurkat T cells was measured by IL-2 ELISA.

[0326] Example 29

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

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

[0329] Figure 60A The results show the production of cytokines IFN-γ and TNF-α as determined by ELISA.

[0330] Figure 60B The results show the determination of immune cell-induced cytotoxicity using 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 66As shown, tumors treated with mVG001-1-2 exhibited differential expression of innate and adaptive immune-related genes compared to tumors treated with HSV 1-VG 001-1.7 and PBS. Overexpression of the specified targets was validated by RT-qPCR.

[0354] Example 36

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

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

[0357] Specifically: 293T cells were seeded in 6-well plates (1×10⁶ cells / well) and cultured in DMEM medium supplemented with 10% serum. After the cells grew into a monolayer, 1×10⁶ pfu (MOI=1) of the following virus or blank culture medium was added to each well:

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

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

[0360] c. Non-infected control with only culture medium added (2 wells)

[0361] After viral infection, cells were cultured overnight, then digested with 750 μL trypsin, 1 mL DMEM was added, and cells were transferred to flow cytometry tubes. Cells were centrifuged (1500 RPM, 5 min), the supernatant was removed, and cells were washed with 2 mL PBS. This washing was repeated once, and the PBS was removed. One well in each of the three groups (ac) was inoculated with an antibody against MHC (histocompatibility complex) protein (Anti-Hu HLA-ABC): cells were resuspended in 100 μL PBS + 2% FBS, and 2 μL of antibody was added. Another well in each group was inoculated with 100 μL PBS + 2% FBS, without antibody. Cells were incubated in the dark for 1 hour, the antibody was removed, and cells were washed with 3 mL PBS + 2% FBS. Cells were centrifuged (1500 RPM, 5 min), and most of the washings were discarded before flow cytometry analysis. Results are shown below. Figure 67 .

[0362] Depend on Figure 67It is evident that infection with wild-type HSV-1 virus reduces the expression level of MHC proteins on the cell surface. Surprisingly, cells infected with VG001-1-2 virus exhibit even lower MHC expression levels than those infected with wild-type HSV-1 virus. This suggests that, compared to wild-type virus, by modifying the ICP47 promoter, VG001-1-2 virus can persist in infected cells for a longer period after infection, evading immune cell attack and thus better replicating and expressing exogenous genes.

[0363] Example 37: Exploratory Experiment of Oncolytic Virus Stabilizer

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

[0365] Table 1. Media with different formulations for preserving viruses

[0366]

[0367] Prepare samples according to Table 1, place them in a 37℃ incubator, and after 48 hours, transfer them to a -80℃ freezer until virus titration is performed. Sample G8 was removed from the -80℃ freezer before titration, diluted, and then titrated simultaneously with the other seven groups of samples. The result of G8 serves as a reference standard for determining the stability of the other groups of virus samples.

[0368] Arrange Vero cells at 8 × 10⁸ cells per well 5 Cells were seeded into 6-well plates at a rate of 3 ml per well and incubated at 37°C in a CO2 incubator for 24 h. Virus samples were serially diluted according to the protocol in Table 2.

[0369] Table 2 Virus titration and dilution process

[0370]

[0371] Remove the six-well plates, which have been replaced with FBS-free medium, from the 37°C incubator. Aspirate the medium and add 1 ml of diluent to each well. Perform four replicates per sample (two six-well plates per sample). After adding 1 ml of virus diluent to each well, incubate at 37°C for 60 minutes to allow virus adsorption. Aspirate the virus liquid, then add 2 ml / well of MEM (FBS-free) medium and 1.5% methylcellulose. Incubate at 37°C for 96 hours.

[0372] After 96 hours, discard the covering material, add 1 ml of 4% glutaraldehyde solution to each well, incubate at room temperature for 30 minutes, then discard the solution. Next, add 2% crystal violet staining solution (1 ml / well) and stain at room temperature for 15 minutes. Gently rinse with softened water and air dry. After the six-well plate has dried, the dead cells caused by the virus do not stain, forming white spots. Count the empty spots under a medical viewing lamp.

[0373] Results calculation: Wells with 20–150 empty plaques were used for titer counting. The average number of plaques was then multiplied by the viral dilution. For example, if the average number of empty plaques in tube 6 was 20, the viral titer would be 20 × 10⁻⁶. 6 This corresponds to 2.0 * 10⁷ PFU / ml. Each sample was tested in triplicate. The viral titer was calculated from the plaque results, and the results of G1-G7 were compared with G8 to determine the most stable protectant against the virus.

[0374] The results of the viral titers measured in each group are shown in the figure. Figure 68 Test results showed that the virus stored at 37℃ using formulation G5 maintained good activity, with virtually no difference in activity compared to the virus stored at -80℃. Although it is generally believed that increasing sucrose can improve the stability of the viral solution, surprisingly, the HSV-1 oncolytic virus, in the presence of sucrose, was not conducive to maintaining viral activity, regardless of the presence of glycerol.

[0375] Example 38: Exploratory study on the stability of the protein in mouse serum

[0376] VG001-1-2 carries a human PD-L1 blocking peptide (TF). The C-terminus of the PD-L1 blocking peptide may contain an Fc sequence. The Fc can originate from one of the IgG subclasses. To understand the stability of various fusion proteins formed by fusing Fc sequences from different sources to the TF terminus, the following experiments were conducted.

[0377] Cell culture

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

[0379] Plasmid extraction: For VG001-1-2, sequences encoding IgG1 Fc or IgG4 Fc fragments were inserted into the end of its PD-L1 coding region, resulting in 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 using the heat shock method and cultured overnight at 37°C on LB+AMP bacterial culture plates. Single colonies were picked and cultured overnight at 37°C on a shaker (150 rpm / min) in 2-3 ml of LB+AMP liquid medium.

[0380] Plasmids were extracted from the above-mentioned culture and turbid culture medium, labeled Fc-1 and Fc-4, and the extraction date was recorded.

[0381] The quality of the extracted plasmid was determined by agarose gel electrophoresis, and the concentration of plasmid DNA was detected by a DNA concentration analyzer.

[0382] 2.3 Plasmid transfection

[0383] 293 cells were seeded in 6-well plates and cultured overnight in a cell culture incubator to ensure adherence.

[0384] The two plasmids were mixed with the plasmid transfection reagent and serum-free medium according to the plasmid concentration and the instructions for plasmid transfection. The mixture was added to 293 cells and incubated for 4 hours. Then, the medium was replaced with 6% FBS serum and cultured for another 48 hours.

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

[0386] All sample supernatants were frozen at -80°C.

[0387] 2.4 Mixing of protein and serum

[0388] Take the supernatant (2 mL) after transfection and mouse serum from -80℃, thaw them on ice, and prepare samples according to the formula in Table 3.

[0389] Table 3: Preparation of Mixed Samples

[0390]

[0391]

[0392] After sample preparation, sample groups G01 / G02 were transferred to a -20°C freezer for storage, while the remaining groups were placed in a 37°C incubator. Sample groups G1 / G2 were transferred to a -20°C freezer on day 14, and sample groups G3 / G4 were transferred to a -20°C freezer on day 28. All samples were collected and ready for ELISA testing.

[0393] ELISA detection of samples

[0394] Thaw samples on ice. Take 300 μL of each sample and perform ELISA assay, 100 μL per well, with three replicates per sample. Calculate the protein content of each sample according to the kit instructions.

[0395] 1) Coat the ELISA plate with the antibodies from the kit and incubate overnight at 4°C;

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

[0397] 3) Add blocking solution and seal at room temperature for 2 hours;

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

[0399] 5) Use a sealing film to prevent liquid evaporation from the wells and incubate at room temperature for 2 hours;

[0400] 6) After incubation, wash four times with washing solution, and dry the liquid in the hole with absorbent paper each time;

[0401] 7) Add 100 μL of detection antibody per well according to the kit instructions, seal the plate, and incubate at room temperature for 1 hour;

[0402] 8) After incubation, wash four times with washing solution, and dry the liquid in the hole with absorbent paper each time;

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

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

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

[0406] 12) Analyze the data according to the kit requirements.

[0407] Based on the ELISA results, the ability of TF-Fc in each sample to bind to IgG4 or IgG1 antibodies in the ELISA kit was determined, and the stability of the IgG1 Fc / IgG4 Fc fusion protein in mouse serum was calculated. Figure 69 ).

[0408] The test results show that, contrary to conventional wisdom, the TF-IgG1 Fc fusion structure is less stable in serum than the TF-IgG4 Fc fusion structure. After 28 days, the TF-IgG4Fc fusion protein exhibited a significantly higher binding capacity to PD-L1 compared to the TF-IgG1Fc fusion protein, with a statistically significant difference (p = 0.01).

[0409] The following are other exemplary implementations of the content disclosed herein:

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

[0411] 2) The HSV vector of Embodiment 1, wherein the nucleic acid sequence encoding the self-cleaving peptide 2A is located within a frame between the coding sequences of IL12, IL15, and the IL15 receptor α subunit. In a preferred embodiment, IL12 is a mouse or human sequence, IL15 is a human sequence, and the IL15 receptor α subunit is a human sequence.

[0412] 3) The HSV vector of Implementation Method 2, wherein the nucleic acid sequence encodes a sequence selected from...

[0413] VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFN-AHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNF-SLLKLAGDVELNPGP, EGRGSLLTCGDVEENP Self-cleaving peptide 2A of GP, 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 located between the coding sequences of IL12, IL15 and the IL15 receptor α subunit. In a preferred embodiment, IL12 is a mouse or human sequence, IL15 is a human sequence, and the IL15 receptor α subunit is a human sequence.

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

[0416] 6) An HSV vector according to any one of embodiments 1 to 5, wherein the IL15 and the IL15 receptor α subunit are expressed via a bidirectional promoter. In a preferred embodiment, IL12 is a mouse or human sequence, IL15 is a human sequence, and the IL15 receptor α subunit is a human sequence.

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

[0418] 8) An HSV vector of any one of embodiments 1 to 7, wherein each of IL15 and the IL15 receptor α subunit follows a nucleic acid sequence encoding Lys5 or Glu5. In a preferred embodiment, IL12 is a mouse or human sequence, IL15 is a human sequence, and the IL15 receptor α subunit is a human sequence.

[0419] 9) An HSV vector of any one of embodiments 1 to 8, wherein the hIL15 receptor α 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 comprises an expression cassette of one or more PD-L1 blocking peptides, or 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 comprises a sequence encoding a peptide linker between a plurality of PD-L1 blocking peptides.

[0422] 12) The HSV vector of any one of embodiments 1 to 11 further includes 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 comprises a sequence encoding an Fc region linked to the 3'-end of the PD-L1 blocking peptide.

[0424] 14) An 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 a sequence encoding a 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 carrier of any one of embodiments 1 to 15 further includes NFkB and OCT4 / SOX2 enhancement elements in the ICP4 or ICP27 control region.

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

[0428] 18) An HSV vector of any one of embodiments 1 to 17, wherein the expression cassette contains at least one bidirectional CMV promoter.

[0429] 19) An HSV vector of any one of embodiments 1 to 18, wherein the expression cassette contains at least one cell promoter.

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

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

[0432] 22) An HSV vector of any one of embodiments 1 to 21, wherein the ICP34.5 gene is regulated by means of a 3'UTR containing a target sequence of a miRNA that is poorly expressed in tumor cells.

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

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

[0435] 25) According to the method of embodiment 24, the cancer is selected from the group consisting of carcinomas, leukemia, lymphoma, myeloma and sarcoma.

[0436] All patents, publications, scientific articles, websites, and other documents and materials cited or referenced herein represent the skill level of a person skilled in the art to which this invention pertains, and each cited document and material is incorporated herein by reference as if it were individually or wholly incorporated in its entirety. The applicant reserves the right to incorporate any and all materials and information from any such patents, publications, scientific articles, websites, electronic information, and other references or documents into this specification.

[0437] The written description portion of this patent includes all claims. Furthermore, all claims, including all original claims and all claims in any and all priority documents, are incorporated in their entirety by reference into the written description portion of this specification, and the applicant reserves the right to incorporate them actually into any other part of the written description or this application, or any and all such claims. Therefore, for example, in no case should a patent be construed as providing a written description of the claims that does not precisely specify the wording of the claims in the original written document of the patent's written description portion.

[0438] The claims shall be interpreted in accordance with the law. However, despite claims or consideration that any claim or part thereof may be easily or difficult to interpret, in no event shall any adjustment or modification to any claim or any part thereof be construed as having forfeited any equivalent rights and not constituting part of the prior art during the term of the patent application.

[0439] All features disclosed in this specification can be combined arbitrarily. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a series of equivalent or similar features.

[0440] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Therefore, from the foregoing, although specific non-limiting embodiments of the invention have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are all included within the scope of the appended claims, and the invention is not limited to the appended claims.

[0441] The specific methods and compositions described herein represent preferred, non-limiting embodiments and are exemplary and not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon reading this specification and are included within the spirit of the invention as defined by the claims. It will be readily understood by those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention suitably described illustratively herein may be practiced without any elements or limitations that are not specifically disclosed herein. Therefore, for example, in each instance herein, in non-limiting embodiments or examples of the invention, the terms “comprising,” “including,” “containing,” etc., will be interpreted expansively and without limitation. The methods and approaches suitably set forth herein may be practiced in different sequences of steps and are not necessarily limited to the sequence of steps shown herein or in the claims.

[0442] The terms and expressions used are descriptive rather than restrictive, and their use is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed through various non-limiting embodiments and / or preferred non-limiting embodiments and optional features, any and all modifications and variations that can be made by those skilled in the art based on the disclosed concept are considered to be within the scope defined by the appended claims.

[0443] This document provides a broad and general description of the invention. Each narrower species and subspecies falling within the scope of this disclosure also constitutes a part of this invention. This includes a general description of the invention, but with attached conditions or negative limitations, namely, the removal of any subject matter from the appendices, regardless of whether the removed material is specifically described herein.

[0444] It should also be understood that, as used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “an,” “a,” and “the” include the plural forms, the terms “X and / or Y” mean “X” or “Y” or both “X” and “Y,” and the letter “s” following a noun indicates both the plural and the singular form of that noun. Furthermore, features or aspects of this invention are described in the form of the Markush group, and those skilled in the art will recognize that the invention encompasses and therefore is also described in the form of any individual member of the Markush group and any subgroup of any member, and the applicant reserves the right to amend the application or claims, particularly for any individual member of the Markush group or any subgroup of any member.

[0445] Other non-limiting embodiments are included in the appended claims. This patent shall not be construed as being limited to the specific or non-limiting embodiments or methods specifically and / or expressly disclosed herein. In no event shall this patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is expressly adopted by the applicant in a written response and is neither limiting nor reserved.

Claims

1. A herpes simplex virus (HSV) vector comprising an expression cassette encoding IL12, IL15, and an IL15 receptor α subunit, wherein, The expression box has modified ICP47 promoters on its side wings; The IL15 receptor α subunit is variant 1 as shown in SEQ ID NO.3; 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 sequence of which is SEQ ID NO.

599.

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

584.

3. The herpes simplex virus (HSV) vector 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 herpes simplex virus (HSV) vector according to claim 3, wherein, The amino acid sequence of the self-cleaving peptide 2A is: VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFNAHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNFSLLKLAGDVELNPGP, EGRGS LLTCGDVEENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP or CDAQRQKLLLSGDIEQNPGP.

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

7. The herpes simplex virus (HSV) vector 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 herpes simplex virus (HSV) vector 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 herpes simplex virus (HSV) vector 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 herpes simplex virus (HSV) vector 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 herpes simplex virus (HSV) vector 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. A pharmaceutical composition comprising the herpes simplex virus (HSV) vector as described in any one of claims 1 to 11, and a pharmaceutically acceptable vector.

13. The use of the herpes simplex virus (HSV) vector according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12, in the preparation of a medicament for treating cancer, wherein, The cancers mentioned are selected from liver cancer, stomach cancer, intestinal cancer, lung cancer, breast cancer, nasopharyngeal carcinoma, head and neck tumors, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, acute lymphoblastic leukemia, acute or chronic myeloid leukemia, melanoma, endometrial cancer, head and neck cancer, glioblastoma, osteosarcoma, lymphoma, myeloma, and sarcoma.

14. The application according to claim 13, wherein, The cancers mentioned are selected from colon cancer, rectal cancer, small cell lung cancer, non-small cell lung cancer, chronic myeloid leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, pilocellular lymphoma, and Burkitt's lymphoma.

15. The application according to claim 13 or 14, wherein the treatment of cancer is administered by subcutaneous injection, intratumoral injection or intravenous injection.

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