CN116042723A - Oncolytic herpes simplex virus vectors expressing immune system-stimulating molecules - Google Patents

Abstract

The present invention provides an oncolytic herpes simplex virus vector expressing immune system-stimulating molecules comprising an expression cassette of IL12, IL15 and IL15 receptor alpha subunits flanked by modified ICP47 promoters. The herpes simplex virus vector of the invention can better replicate and express exogenous genes through structural modification, and the expressed protein has good stability and is suitable for practical application.

Description

Oncolytic herpes simplex virus vectors expressing immune system-stimulating molecules

Technical Field

The present invention relates generally to oncolytic herpes simplex virus (oHSV) vectors expressing molecules that stimulate the immune system.

Background

Oncolytic Viruses (OVs) have become a therapeutic approach to specifically destroy cancer cells by oncolytic effects, the killing mechanism of which is characterized by the lysis of cancer cells by the process of viral lytic replication.

The invention overcomes the defects of the current commercial oncolytic virus, and has low toxicity, high expression level and good stability.

Disclosure of Invention

Briefly, the present disclosure relates to herpes simplex virus vectors (HSV vectors) that protect against one or more of IL12, IL15, and/or IL receptor 15 alpha subunits. In one embodiment, the herpes simplex virus HSV vector comprises an expression cassette of IL12, IL15 and IL15 receptor alpha subunits flanked by modified ICP47 promoters.

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

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

In one embodiment, the nucleic acid sequence encoding self-cleaving peptide 2A is in-frame between the coding sequences of IL12, IL15 and IL15 receptor alpha subunits.

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

VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFNAHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNFSLLKLAGDVELNPGP, EGRGSLLTCGDVEENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP or CDAQRQKLLLSGDIEQNPGP.

In one embodiment, one or more IRES sequences are located between the coding sequences of IL12, IL15, and IL15 receptor alpha subunits.

In one embodiment, the IL15 and IL15 receptor alpha subunits are expressed by a bi-directional promoter.

In one embodiment, the IL15 and IL15 receptor alpha subunit are each followed by a nucleic acid sequence encoding Lys5 or Glu 5.

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

In one embodiment, an expression cassette comprising IL12, IL15 and IL15 receptor alpha subunits is inserted into an internal repeat region of HSV or a terminal repeat region of the HSV genome.

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

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

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.

In one embodiment, the sequence of the Fc region linked to the 3' -end of the PD-L1 blocking peptide is a sequence encoding an IgG4 Fc region.

In one embodiment, NFkB and OCT4/SOX2 enhancing elements are further included in the ICP4 or ICP27 regulatory region of the herpes simplex virus HSV vector.

In one embodiment, the ICP34.5 gene portion of the herpes simplex virus HSV vector is deleted or nonfunctional.

The disclosure also relates to a formulation consisting of the following components:

Suspension of virus expressed using the herpes simplex virus HSV vector according to any one of claims 1 to 15, glycerol, water;

wherein the concentration of glycerol in the preparation is 5+3％。

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

The disclosure also relates to the use of a herpes simplex virus HSV vector as described herein, or a formulation as described herein, or a pharmaceutical composition as described herein, in the manufacture of a medicament for treating cancer.

In one embodiment, the cancer is selected from liver cancer, stomach cancer, intestinal cancer, lung cancer, breast cancer, nasopharyngeal cancer, head and neck tumor, bladder cancer, colon cancer, rectal cancer, kidney cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma, burkitt's lymphoma, acute or chronic myelogenous leukemia, melanoma, endometrial cancer, head and neck cancer, glioblastoma, osteosarcoma leukemia, lymphoma, myeloma, and sarcoma.

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

The summary has briefly introduced some concepts, which will be described in further detail in the detailed description section. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter unless otherwise described.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention overcomes the defects of the current commercial oncolytic virus, and has low toxicity, high expression level and good stability.

The invention can make the modified virus exist in the infected cells for a longer time after infecting the cells to avoid immune cell attack by modifying the virus structure, such as modifying the promoter region of some genes of the virus, thereby being capable of better replicating and expressing the exogenous genes.

The invention also improves the exogenous gene carried by the vector, for example, constructs fusion protein formed by exogenous polypeptide and proper terminal structure, so that the expressed protein has good stability and is suitable for practical application.

The invention also provides an optimized medium for preserving viruses, and after the viruses are expressed by using the vector disclosed by the invention, the optimized medium can maintain the virus activity for a long time, so that the vector and the industrial application of the viruses are facilitated.

One or more embodiments will be described in detail below. Features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Thus, any of the various embodiments described herein can be combined to provide further embodiments. Aspects of the embodiments can be modified, where necessary to employ concepts of the various patents, applications or publications identified herein, to provide yet further embodiments. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

Drawings

The exemplary features of the disclosure, its nature, and various advantages 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 following figures, wherein like reference numerals refer to like parts throughout the various views unless otherwise specified. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of the elements are selected, exaggerated, and placed so as to improve the readability of the drawings. The particular shapes of the elements as drawn, have been chosen for ease of recognition in the drawings. One or more embodiments are described below with reference to the drawings, in which:

Fig. 1A and 1B are schematic diagrams of exemplary oHSV vectors.

FIG. 2 shows a schematic representation of the modified ICP34.5 region (SEQ ID NO: 572) of virus hVG 001-1-2.

FIG. 3 shows a schematic representation of the modified UL54 promoter region of virus hVG001-1-2 (SEQ ID NO: 573).

FIG. 4 shows a schematic representation of the hVG001-1-2 viral genome with PD-L1 blocker insertion (SEQ ID NO: 574).

FIG. 5 shows a schematic representation of the hVG001-1-2 modified TR region (SEQ ID NO: 575).

FIGS. 6A-6C show ELISA and Western blot analysis of IL-12 expression following cell infection hVG 001-1-2.

FIGS. 7A-7C show ELISA and Western blot analysis of IL-15 expression following cell infection hVG 001-1-2.

FIGS. 8A-8C show ELISA and Western blot analysis of IgG4 expression following cell infection hVG 001-1-2.

Fig. 9A to 9C are: (A) Schematic representation of exemplary constructs in which the bi-CMV promoter drives expression of the Sushi domains of IL-15Rα and IL-15, as well as (B-C) DNA sequences and schematic representation (SEQ ID No: 557).

Fig. 10A to 10C are: (A) Schematic representation of exemplary constructs in which the bi-CMV promoter drives expression of IL-15 and IL-15Rα variant 4, as well as (B-C) DNA sequences and schematic representation (SEQ ID No: 558).

Fig. 11A to 11C are: (A) Schematic representation of exemplary constructs in which the bi-CMV promoter drives the expression of IL-15-K5 and IL-15Rα Sushi domain-E5, as well as (B-C) DNA sequences and schematic representation (SEQ ID No: 559).

Fig. 12A to 12D are: (A) Schematic representation of exemplary constructs in which the bi-CMV promoter drives the expression of IL-15-K5 and IL-15Rα variants 4-E5, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 560).

Fig. 13A to 13D are: (A) Schematic representation of exemplary constructs in which the EF 1. Alpha. Promoter controls the expression of the IL-15-IRES-IL-15R. Alpha. Sushi domain, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 561).

Fig. 14A to 14D are: (A) Schematic representation of exemplary constructs in which the EF 1. Alpha. Promoter controls the expression of IL-15-IRES-IL-15Rα variant 4, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 562).

Fig. 15A to 15D are: (A) Schematic representation of exemplary constructs in which the EF 1. Alpha. Promoter controls the expression of IL-15K5-IRES-IL-15Rα Sushi domain E5, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 563).

Fig. 16A to 16D are: (A) Schematic representation of exemplary constructs in which the EF 1. Alpha. Promoter controls the expression of IL-15K5-IRES-IL-15Rα variant 4E5, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 564).

Fig. 17A to 17E are: (A) Schematic representation of exemplary constructs in which the CMV promoter controls the expression of the IL-12-p2A-IL-15-p2A-IL-15Rα Sushi domain, as well as (B-E) DNA sequences and schematic representation (SEQ ID No: 565).

Fig. 18A to 18E are: (A) Schematic representation of exemplary constructs in which the CMV promoter controls expression of IL-12-p2A-IL-15-p2A-IL-15Rα variant 1, as well as (B-E) DNA sequences and schematic representation (SEQ ID No: 566).

Fig. 19A to 19D are: (A) Schematic representation of exemplary constructs in which the CMV promoter controls the expression of IL-12-p2A-IL-15K5-p2A-IL-15Rα Sushi domain E5, as well as (B-D) DNA sequences and schematic representation (SEQ ID No: 567).

Fig. 20A, 20B, 20C, 20D, and 20D are the following: schematic representation of (A) exemplary constructs in which the CMV promoter controls the expression of IL-12-p2A-IL-15K5-p2A-IL-15Rα variants 1-E5, and (B), (C), (D) follow the DNA sequence and schematic representation (SEQ ID No: 568).

Fig. 21A, 21B, 21C show graphs of percent inhibition of PD-L1 binding to PD-1 by blocking peptide.

FIGS. 22A-22B show the effect of PD-L1 inhibitory peptides on cytotoxicity of target cells by anti-CD 3 stimulated human peripheral blood mononuclear cells.

FIGS. 23A and 23B show the effect of IL-12 alone, IL-15 alone plus IL-15Rα, and IL-12 and IL-15/1L-15 Rα together on IFNγ and TNF α production in human peripheral blood mononuclear cells.

FIGS. 24A and 24B show the effect of IL-12 and IL-15/IL-15 Rα on cytotoxicity of U87 and MDA-MB-23 tumor cells by peripheral blood mononuclear cells.

FIGS. 25A-25C show the expression of IL12, IL15 and PD-L1 blocking peptides after infection of tumor cells with viruses carrying PD-L1 blocking peptides or human IL15/15Ra (VG 001-PLBh and VG001-15 h). FIGS. 25D and 25E show the results after infection of tumor cells with viruses carrying PD-L1 blocking peptide or human IL15/15Ra (VG 001-PLBh and VG001-15 h).

Fig. 26A-26D show the results of in vitro assays of the various constructs. FIGS. 26A-26B show the results of cell transfection with IL-TF-Fc plasmids expressing IL-12, IL-15 and PD-L1 blockers. FIGS. 26C-26D show the results of cell transfection of various mutant viruses, including hVG 001-1-2.

FIGS. 27A-27E show the results of cell viability assays for hVG001-1-2 and HSV-345 performed on human tumor cell lines and Vero cell lines.

FIGS. 28A-28J show the results of in vitro assays of the various constructs. FIGS. 28A-28E show the results of cell viability assays of mVG001-1-2 and HSV-345 on mouse tumor cell lines and Vero cell lines; FIGS. 28F-28H show characterization of transgene expression following infection of CT26 mouse tumor cells with mVG001-1-2, and FIGS. 28I-28J show characterization of cytotoxic factor release by peripheral mononuclear cells stimulated following infection of CT26 cells with mVG 001-1-2.

FIGS. 29A-29C show the results of in vitro characterization of transgene expression following hVG001-1-2 or VG001-1.7 infection of various cell lines. FIGS. 29D-29E show that infected tumor cells stimulated peripheral monocytes to release cytokines and cell killing activity.

FIGS. 30A-30G show the results of an assay to evaluate hVG 001-1-2's ability to kill various human cancer cells in vitro.

FIGS. 31A-31G show the results of inhibition of tumor growth in vivo for the mVG001-1-2 and hVG001-1-2 constructs.

FIGS. 32A-32C show growth curves of different viruses on three different human cell lines.

FIGS. 33A-33D show growth curves of mVG001-1-2 and HSV-345 on mouse tumor cell lines and Vero cell lines.

FIGS. 34A-34E show growth curves of hVG001-1-2 and HSV-345 on human tumor cell lines and Vero cell lines.

FIGS. 35A-35D show the effect of virus modification.

FIGS. 36A to 36D are graphs showing the effectiveness of foreign genes expressed in vitro.

FIGS. 37A and 37B provide the expression of foreign genes carried by the virus in the tumor following virus injection in a mouse model.

FIGS. 38A-38C provide the effect of VG001-1-2 on immune response.

FIG. 39 is a schematic diagram of a modified exemplary oHSV viral vector (VG 001-1-2).

FIG. 40 shows the modified ICP34.5 region of virus VG001-1-2 (SEQ ID NO: 599).

FIG. 41 shows the modified UL54 promoter region of virus VG001-1-2 (SEQ ID NO: 596).

FIG. 42 shows the region of VG001-1-2 into which the PD-L1 blocker is inserted (SEQ ID NO: 589).

FIG. 43 shows the modified Terminal Repeats (TR) region (SEQ ID NO: 576) of VG001-1-2 carrying the expression cassette encoding IL-12, IL-15 and IL-15 receptor alpha subunits and flanked by US2 (ICP 47).

FIG. 44 is a schematic diagram of a short (S) type promoter A (SEQ ID NO: 583).

FIG. 45 is a schematic diagram of a middle (M) type promoter A (SEQ ID NO: 584).

FIG. 46 is a schematic diagram of a long (L) type promoter A (SEQ ID NO: 585).

FIG. 47 is a schematic representation of promoter B (survivin) (SEQ ID NO: 586).

FIGS. 48A-48F show the sequences flanking the IL12-IL15-IL15RA1 expression cassette (SEQ ID NO: 576) carried in VG 001-1-2.

FIGS. 49A-49C are sequences of modified UL54 (ICP 27) promoter-regulatory regions in VG001-1-2 (SEQ ID NO. 596).

FIGS. 50A-50D are sequences of VG001-1-2 (SEQ ID No. 589) with the PD-L1 blocker inserted in the gene region between UL3 and UL 4.

FIG. 51 is the sequence of the modified ICP34.5 region (SEQ ID NO. 599) contained in VG 001-1-2.

FIG. 52 shows the sequence of short (S) type promoter A (SEQ ID NO: 583).

FIG. 53 shows the sequence of the middle (M) type promoter A (SEQ ID NO: 584).

FIG. 54 shows the sequence of a long (S) type promoter A (SEQ ID NO: 585).

FIG. 55 shows the sequence of promoter B (survivin) (SEQ ID NO: 586).

FIGS. 56A-56B show the results of cytotoxicity of VG001-1-2 virus on (A) human cancer cells and (B) mouse cancer cells.

FIGS. 57A-57C show (A) expression of IL-12, (B) expression of IL-15, and (C) expression of PD-L1 blocker after infection of cells with VG001-1-2 virus.

FIG. 58 shows the results of ELISA analysis of PD-L1 blockers produced after VG001-1-2 infection of cells.

FIG. 59 shows the results of a cell-based assay for PD-L1 blockers produced after VG001-1-2 infection of cells.

FIGS. 60A-60D show that IL-12, IL-15/IL-15RA and PD-L1 blockers synergistically promote immune cell function in vitro.

FIGS. 61A-61D show the effect of intratumoral vaccination with VG001-1-2 virus.

FIGS. 62A-62B show the effect of gene expression and T cell activity resulting from treatment of tumor cells with mVG001-1-2 virus.

FIGS. 63A-63D show the effect of mVG001-1-2 treatment on intratumoral lymphocyte populations.

FIGS. 64A-64B show the effect of minimum doses of human IL12 or human IL15/IL15RA on immune cell function.

FIGS. 65A and 65B show the biodistribution of VG001-1-2 virus.

FIG. 66 shows the results of quantitative determination of immune-related gene expression levels in tumor-bearing mice treated with VG 001-1-2.

FIG. 67 shows the results of flow analysis of cells after infection with different viruses.

FIG. 68 shows the results of titer analysis of viruses stored in different formulations of media.

FIG. 69 shows the results of stability tests of fusion proteins of different structures expressed by viruses.

FIG. 70 shows the structure of VG001-1-2 vector.

Detailed Description

The present disclosure may be understood more readily by reference to the following detailed description of preferred embodiments and examples of the invention included herein.

Summary of the disclosure

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments and examples of the invention included herein. Briefly, the present disclosure provides oncolytic herpes simplex virus type 1 or type 2 vectors that express an immunostimulatory molecule. Representative vectors include expression cassettes encoding one or more of IL-12, IL-15, and IL-15Rα. Certain vectors encode murine or human IL-12, human IL-15Rα. In certain embodiments, the vector encodes murine or human IL-12, hIL15, and hIL15 receptor alpha subunits. In other embodiments, the vector encodes hIL-12, HIL15 and the HIL15 receptor alpha subunit. These three proteins may be expressed on one, two or three transcripts. When expressed on the same transcript, the ensuing post-transcriptional process results in the expression of a single protein. In such cases, the coding regions are separated by a sequence encoding a self-cleaving peptide 2A or IRES. The coding region may also be expressed by a bi-directional promoter. The HSV vector optionally expresses one or more PD-L1 blocking peptides that it is capable of secreting.

A.oHSV vector

Oncolytic virus is a virus (oncosis) that lyses cancer cells, preferably in a selective manner. Viruses that selectively replicate in differentiated cells over non-differentiated cells are often oncolytic. Oncolytic viruses suitable for use herein include herpes simplex viruses 1 and 2, but may also include non-human herpesviruses such as BHV or others.

Herpes Simplex Virus (HSV) 1 and 2 are members of the herpesviridae family of infected humans. The HSV genome contains two distinct regions, referred to as a long distinct (UL) region and a short distinct (US) region. Each of these regions is flanked by a pair of inverted terminal repeats. There are 75 known open reading frames. The viral genome has been engineered to develop oncolytic viruses for use in, for example, cancer treatment. Tumor-selective replication of HSV can be achieved by mutation of the HSV ICP34.5 (also known as γ34.5) gene. HSV contains two copies of ICP34.5, and mutants that inactivate one or both copies of the ICP34.5 gene are known to be deficient in neurovirulence, i.e., non-pathogenic/non-neurotoxic and oncolytic.

Suitable oncolytic HSV may be derived from HSV-1 or HSV-2, including any laboratory strain or clinical isolate. In some embodiments, the oHSV may be or may be derived from one of the laboratory strains HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG 52. In other embodiments, it may be any clinically isolated strain 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, geneTherapy.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): e 81768); m002 (Parker et al, proceedings of the National Academy of Sciences,2000;97 (5): 2208-2213); NV1042 (Passer et al, cancer Gene therapy.2013;20 (1): 17-24); G2O7-IL2 (Carew et al Molecular Therapy,2001;4 (3): 250-256); rQNestin34.5 (Kambara et al, cancer Research,2005;65 (7): 2832-2839); G47.DELTA. -mIL-18 (Fukuhara et al, cancer Research,2005;65 (23): 10663-10668); and those disclosed in PCT application PCT/US2017/030308 entitled "HSV Vectors with Enhanced Replication in Cancer Cells (HSV vector enhancing replication in cancer cells)" and PCT/US2017/018539 entitled "Compositions and Methods of Using Stat1/3Inhibitors with Oncolytic Herpes Virus (compositions and methods utilizing Stat1/3inhibitor and oncolytic herpes virus)", the entire contents of which are incorporated herein by reference.

The oHSV vector may have a modification, mutation, or deletion of at least one 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 and the other gene is mutated or modified. Any one of the endogenous (native) γ34.5 genes can be deleted. In one embodiment, the terminal repeat region comprising the γ34.5 gene and the ICP4 gene is deleted. Mutations (e.g., nucleotide changes, insertions, and deletions) render the gene unexpressible or product-inactivating. The γ34.5 gene can be modified in its 3' utr with miRNA target sequences. The target sequence binds to a miRNA that is expressed in tumor cells less than that expressed in its normal control. In some embodiments, the modified or mutated γ34.5 gene is constructed in vitro and inserted into an ohv vector as a surrogate 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 comprise further changes, for example with an exogenous promoter.

The oHSV may have additional mutations, which may include disabling mutations (e.g., deletions, substitutions, insertions), which affect the virulence of the virus or its ability to replicate. For example, mutations may be made in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP 24, ICP 56. Preferably, a mutation in one of these genes (optionally in both copies of the gene where appropriate) results in HSV being unable to express the corresponding functional polypeptide (or the ability to be reduced). In some embodiments, the promoter of the viral gene is replaced with a promoter that is selectively activated in the target cell, or that is inducible after delivery of the inducer, or that is inducible after a cellular event or in a specific environment. In particular embodiments, the tumor-specific promoter drives expression of viral genes that are critical for HSV replication. In certain embodiments, the expression of ICP4 or ICP27, or both, is under the control of an exogenous promoter, e.g., a tumor specific promoter. Exemplary tumor-specific promoters include survivin (survivin) or telomerase (telomerase); other suitable tumor-specific promoters may be specific for 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, such as in the regulatory region of ICP4 or ICP27, or both. Furthermore, the 5'UTR may be exogenous, such as a 5' UTR from a growth factor gene (e.g., FGF).

oHSV may also have genes and nucleotide sequences that are not of HSV origin. For example, the ohv genome may have a sequence encoding a prodrug, a sequence encoding a cytokine or other immunostimulatory factor, a tumor-specific promoter, an inducible promoter, an enhancer, a sequence homologous to a host T cell, and other sequences therein. Exemplary sequences encode IL12, IL15, OX40L, PD-L1 blocker or PD-1 blocker. For sequences encoding the product, it is operably linked to promoter sequences and other regulatory sequences (e.g., enhancers, polyadenylation signal sequences) necessary or desirable for expression.

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

B.Immunostimulatory molecules

The oHSV vector comprises nucleic acid sequences encoding one or more immunostimulatory molecules (e.g., IL-12, IL-15, and IL-15 ra). The amino acid sequences of exemplary IL-12, IL-15 and IL-15Rα are set forth in the sequence listing (SEQ ID NOs: 1-6). Any DNA sequence encoding this amino acid sequence is suitable, but typically codons will be selected for preferred expression in a population of subjects designated to receive oHSV.

1.IL-12

Interleukin 12 (IL-12) is produced mainly by dendritic cells, macrophages and monocytes corresponding to bacteria (e.g., lipopolysaccharides), pathogens or activated T cells. IL-12 is capable of inducing IFNγ production, cell proliferation, and activating natural killer cells and T cells. It is also critical for T cell differentiation into Th1 cells. IL-12 also inhibits tumor growth. Murine IL-12 is equally active on murine and human cells and is suitable for use in oHSV vectors.

Biologically active IL-12 is a heterodimeric molecule composed of 35kDa (p 35) and 40kDa (p 40) subunits covalently linked by disulfide bridges. Simultaneous expression of both subunits is necessary for the production of heterodimers. In the oHSV vector, IL-12 expression can be achieved in a variety of ways. Two subunits can be expressed in separate constructs, each with a promoter, or in one construct starting from a bi-directional promoter, or from a construct with elements such as IRES or self-cleaving peptides in the coding region. Alternatively, the subunit can be expressed as a single strand. For example, a functional single chain IL-12 fusion protein can be produced by ligating the coding regions of p40 and p35 with a linker, typically consisting of Ser or Gly or a combination of Ser and Gly, such as Ser5, (Gly 4 Ser) 3 or Gly6Ser (e.g., lieschke et al Nature Biotechnology 15:35,1997; lode et al, PNAS 95:2475,1998; alternative fusion constructs also see WO 2015/095249). The sequence and length of the linker is typically chosen in such a way that the structure and the greatest flexibility are achieved (Chen et al Adv Drug Deliv rev.65:1357,2013). The linker sequence can be selected using a computer program. One such program is known as LINKER (Crasto and Feng, protein Eng Design & 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 may be made as long as the IL-12 retains function.

2.IL-15

IL-15 is a cytokine that regulates natural killer cell and T cell activation and proliferation, and can have other biological activities. There are two isoforms of different signal peptide sequences and having identical mature protein sequences. GenBank (NCBI) accession number NP 000576 for sequences with isoforms of longer signal peptides (sometimes referred to as LSP-IL 15) and NP 751915 for sequences with isoforms of shorter signal peptides (sometimes referred to as SSP-IL 15). Either isoform is suitable for use in oHSV vectors. Amino acid insertions, deletions and substitutions may be present, as found in polymorphisms, provided that the protein binds IL-15.

In some embodiments, IL-15 and IL-15R alpha both have a selectively dimerized coiled coil C-terminal peptide. Numerous suitable peptides are taught in the literature (see, e.g., tripet et al, protein Engineering9:1029,1996; aronsson et al, sci Rep 5:14063, 2015). Typically, the amino acid sequence of the coiled coil has heptapeptide repeats of hydrophobic (h) and polar (p) residues in the form of hppphppp. Two exemplary coiled-coil coils are the K-coil (KVSALKE, SEQ ID No. 7) and the E-coil (EVSALEK, SEQ ID No. 8). Typically, 3-6 tandem copies are used. In some embodiments herein, 5 tandem copies are used. K5 (KVSALKEKVSALKEKVSALKEKVSALKEKVSALKE, SEQ ID No. 9) and E5 (EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK, SEQ ID No. 10). The K and E helices are designed to be oppositely charged, so IL-15 fuses with one coiled coil, while IL-15Rα fuses to the oppositely charged coiled coil. 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.

IL-15Rα subunit

Interleukin-15 receptor alpha subunit (IL-15 ra) is one of three subunits of the complex that bind IL-15. The alpha subunit binds IL-15 with high affinity and is capable of binding to IL-15 independently of other subunits. There are at least four variants (isoforms), referred to herein as variant 1 (NP 002180.1) (SEQ ID NO: 3); variant 2 (NP 751950.2) (SEQ ID NO: 4); variant 3 (NP 001230468.1) (SEQ ID NO: 5); and variant 4 (NP-001243694) (SEQ ID NO: 6). The alpha subunit contains the Sushi domain (aka complement regulatory protein (complement control protein, CCP), short homologous repeats (short consensus repeats, SCRs) or Sushi repeats), which is the shortest region that retains IL-15 binding activity. Typical Sushi domains are about 60-70 amino acids, which contain four cysteines that form two disulfide bonds and are common motifs in protein-protein interactions. The Sushi domain of IL-15Rα comprises residues 31 to about 95 (corresponding to variant 1) (SEQ ID NO: 11). The position of the Sushi domain in other variants is known. Amino acid substitutions of cysteines in sIL-15Rα abrogate its ability to inhibit acute inflammation and T cells respond to alloantigens in vivo (Wei et al, J Immunol.167:277,2001).

The oHSV vector comprises a nucleic acid sequence encoding IL-15 ra, a variant of IL-15 ra, or a Sushi domain. Typically, the protein is expressed by a leader peptide, and in some embodiments, the leader peptide is from IL-15 ra. Other lead peptides are known in the art. Amino acid substitutions may be present as long as the protein binds IL-15. Natural substitution, polymorphism are known.

PD-L1 blocking peptides

Programmed death ligand 1 (PD-L1) plays a role in suppressing the immune system, probably due to the binding of the PD-1 receptor. Blocking protein-protein interactions has been shown to improve cancer treatment.

The oHSV vector can express a PD-L1 blocking peptide. Suitable peptides include TAHPSPSPRSAGQF (SEQ ID NO: 16), EYRMSPSNQT (SEQ ID NO: 17), YYRMSPSNQT (SEQ ID NO: 18), TRYPSPSPKPEGRF (SEQ ID NO: 19) and WNRLSPSNQT (SEQ ID NO: 20). Other suitable peptides include those listed in Table 4 (SEQ ID NOs: 21-500). Typically, the blocking peptide is expressed from a leader sequence. The leader sequences are well known in the art. It includes an immunoglobulin kappa chain leader sequence (METDTLLLWVLLLWVPGSTG; SEQ ID NO: 501) and an IL-2 leader sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 502). When more than one blocking peptide is present, the peptides are typically separated by a linking peptide that imparts flexibility. The linker is typically Gly or Ser or Gly/Ser rich. Examples of suitable linkers are shown in (SEQ ID NOs: 503-519) (see also Chichili et al, protein Science 22:153, 2013). There may be one peptide copy or two copies or three copies or more. Multiple copies are typically random and may have a splice between the copies. The blocking peptide construct may also comprise 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 useful, typically, fc will be from one of the IgG subclasses, e.g., human IgG1, human IgG2, human IgG3, and human IgG4 or its murine counterparts.

C.Organization of elements

The molecules IL-12, IL-15 and IL-15Rα can have a variety of different configurations in the oHSV vector. For example, each molecule may be expressed separately from a separate promoter/regulatory region or co-expressed from one or two separate promoter/regulatory regions.

In certain embodiments, two or three of the molecules are expressed from one promoter in a single transcript and their coding sequences are sequenced by an IRES (internal ribosome entry site). The IRES region attracts eukaryotic ribosomal translation initiation complexes and thus enables translation initiation in the middle of the mRNA and independent of the commonly used 5' -end cap structure. Suitable IRES sequences are well known and many suitable IRES sequences can be found in IRESITE's expression bases of experimentally confirmed IRES sequences (see, e.g., http:// IRESite. Org/IRESITE_web. Phppage=browse_plasmas; 2016, 5, 26, manuscript).

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

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

Other ways to affect the co-expression of molecules are to use bi-directional promoters. The bi-directional promoter is a common feature of the human Genome (Trinklein et al Genome Res 14:62, 2004). Bi-directional promoters initiate transcription in two directions and typically contain common elements that regulate both genes. In addition to natural bi-directional promoters, bi-directional promoters have been synthesized. One such promoter is bi-CMV. pBI-CMV1 is a mammalian bi-directional expression vector that enables constitutive expression of two proteins of interest. Protein expression was driven by two constitutively activated minimal human cytomegalovirus promoters (pmincv 1 and pmincv 2 in opposite orientations). An exemplary DNA sequence for a bi-directional CMV promoter is SEQ ID NO.536.

Bi-directional promoters (e.g., bi-CMV promoters) are mainly used to achieve co-expression of hIL15 and IL-15 ra (or Sushi domain). When two molecules are co-expressed using IRES or p2A sequences, they are typically hIL15 and IL-15Rα (or Sushi domain). In these cases, the IL-12 and PD-L1 blocking peptides may be co-expressed using a bi-directional promoter or as a polycistronic transcript with IRES or p2A sequences, or they may be expressed separately from their own promoter/regulatory regions.

Other promoters may be used. Cellular promoters, viral promoters, and the like are suitable. The promoter may be constitutive or inducible or cell/tissue specific. Many promoters are well known. One specific promoter that may be used is the constitutive EF-1. Alpha. Promoter.

The sequences are assembled in one or more expression cassettes. Examples provide specific versions of some of the exemplary expression cassettes. The expression cassette may be inserted into the HSV genome at any location that does not disrupt critical functions (e.g., replication). In certain embodiments, the expression cassette is inserted in the internal or terminal repeat after the first deletion of the repeat. Other suitable insertion regions include between viral genes, e.g., between UL3 and UL4 viral genes, between UL50 and UL51 genes, and between US1 and US 2.

In certain embodiments, an expression cassette that expresses a PD-L1 blocking peptide is inserted between viral genes (e.g., UL3 and UL4, UL50 and UL51, and/or US1 and US 2). In other embodiments, the terminal repeat region is replaced by an expression cassette that expresses IL-12, IL-15, and IL-15Rα, and an expression cassette that expresses a PD-L1 peptide is inserted between the UL3 and UL4 genes.

D.Therapeutic compositions

Therapeutic compositions are provided that can be used to prevent, treat, or mitigate the effects of a disease, 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 expression cassettes for one or more IL12, IL15, and/or IL receptor 15 alpha subunits. In one embodiment, the expression cassette expresses all of the IL12, IL15 and IL receptor 15 alpha subunits. In a preferred embodiment, the expression cassette comprises murine or human IL12, hll 15 and hll 15 receptor alpha subunits.

In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable carrier" is intended to encompass any vehicle, diluent or excipient that does not interfere with the efficacy of the oncolytic viral biological activity and that is non-toxic to the subject receiving the administration (see generally Remington: the Science and Practice of Pharmacy, lippincott Williams & Wilkins; 21 st edition (month 5 of 2005, day and in The United States PharmacopE A: the National Formulary (USP 40-NF 35 and journal).

In the case of the oncolytic viruses described herein, non-limiting examples of suitable pharmaceutically acceptable vehicles include phosphate buffered saline solutions, water, emulsions (e.g., oil/water emulsions), various forms of wetting agents, sterile solutions, and the like. Additional pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, oncolytic virus containing implant components, or any other vehicles, delivery agents, or dispersing devices or materials. Such vehicles can be formulated by conventional methods and can be administered to a subject in an effective dose. Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts may also be included, for example, 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) vehicles, diluents and excipients that may be used to deliver oHSV to target cancer cells preferably do not induce an immune response in the subject receiving the composition (and are preferably administered in a manner that is devoid of undue toxicity).

The compositions provided herein can be provided in a variety of different concentrations. For example, the oncolytic virus may be provided in a dosage range of about 10 ⁶ pfu to about 10 ⁹ pfu. In a further embodiment, the therapeutic agentThe range of quantitative forms can be about 10 ⁶ To about 10 ⁸ pfu/ml, with large lesions every 2-5 weeks (e.g.,>5 cm) of the patient, up to 4ml of the injection, and in cases with small lesions (e.g.,<0.5 cm) to hold a small amount (e.g., up to 0.1 ml).

In certain embodiments of the invention, sub-standard dosages may be used. Thus, in certain embodiments, less than about 10 may be administered to a patient ⁶ pfu/ml (up to 4ml per 2-3 weeks of patient injection).

The composition may be stored at temperatures conducive to stable shelf life, including room temperature (about 20 ℃), 4 ℃, -20 ℃, -80 ℃, in liquid nitrogen. Since compositions intended for in vivo use are generally free of preservatives, they are typically stored at low temperatures. The composition may be stored in dry form (e.g., lyophilized) or in liquid form.

E.Administration of drugs

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

The terms "effective dose" and "effective amount" refer to an amount of oncolytic virus sufficient to affect a treatment of a target cancer, e.g., an amount effective to reduce the size or load of a target tumor, or to interfere with the growth rate of target tumor cells. More specifically, such terms refer to the amount of oncolytic virus administered at the necessary dose and during the treatment period effective to achieve the desired result. For example, in the case of treating cancer, an effective amount of a composition described herein is an amount that causes a link, reduces tumor burden, and/or prevents tumor spread or cancer growth. The effective amount may vary depending on various factors, such as the disease state, age, sex and weight of the subject, as well as the pharmaceutical formulation, route of administration, etc., but can be routinely determined by one skilled in the art.

The therapeutic composition is administered to a subject diagnosed with or suspected of having cancer. The subject may be a human or a non-human animal.

The composition is used for treating cancer. As used herein, the term "treatment" or "treatment" means a process for obtaining a beneficial or desired result, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions of a detectable or undetectable disease, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delaying or slowing of disease progression, diminishment or palliation of disease state, diminishment or control of disease recurrence (in part or in whole). The terms "treatment" and "treatment" may also mean an increase in survival compared to the expected survival without treatment.

Representative forms of cancer include carcinoma (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 cancer (e.g., acoustic neuroma, astrocytoma, craniopharyngioma, ependymoma, glioblastoma, angioblastoma, medulloblastoma, reticuloblastoma, neuroblastoma, oligodendroglioma, pineal tumor, and retinoblastoma), endometrial (endometrial lining) carcinoma, hematopoietic cancer (e.g., leukemia and lymphoma), renal cancer, laryngeal carcinoma, lung cancer, liver cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (e.g., melanoma and squamous cell carcinoma), and thyroid cancer. The cancer may comprise a solid tumor (e.g., sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteosarcoma), a diffuse cancer (e.g., leukemia), or some combination of these (e.g., metastatic cancer with both solid tumors and diffuse or diffuse cancer cells). The cancer can be resistant to conventional treatment (e.g., conventional chemotherapy and/or radiation therapy).

Benign tumors and other conditions of unwanted cell proliferation may also be treated.

The oHSV described herein can be administered, for example, orally, topically, parenterally, systemically, intravenously, intramuscularly, intraocularly, intrathecally, intratumorally, subcutaneously, or transdermally. In certain embodiments, the oncolytic virus can be delivered through a cannula, through a catheter, or by direct injection. The site of administration may be intratumoral or remote from the location of the tumor. The route of administration often depends on the type of cancer to be treated.

The physician in the art can readily determine the optimal or appropriate dosage regimen for an oncolytic virus based on the patient's expression, patient observations and various clinical factors including, for example, subject size, body surface area, age, sex and particular oncolytic virus administered, time and route of administration, type of cancer to be treated, general health of the patient, and other drug treatment the patient is undergoing. According to certain embodiments, the subject is administered an oncolytic virus as described herein in combination with other types of treatment, such as chemotherapy, e.g., with a chemotherapeutic agent such as etoposide, ifosfamide, doxorubicin, vincristine, doxycycline, and the like.

The oHSV may be formulated into medicaments and pharmaceutical compositions for clinical use and may be combined with pharmaceutically acceptable carriers, diluents, excipients or adjuvants. The formulation depends at least in part on the route of administration. Suitable formulations may include viruses and inhibitors in sterile medium. The formulation may be in fluid, gel, patch or solid form. The formulation may be provided to a subject or medical professional.

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

In further embodiments of the invention, the oncolytic virus may be administered intratumorally, or prior to and after surgical removal of the tumor.

The following examples are provided by way of illustration and not by way of limitation.

Examples

All constructs were produced using standard recombinant techniques, including chemical synthesis.

Example 1

Schematic of exemplary OHSV Carrier

Fig. 1A and 1B provide exemplary schematic diagrams of representative oHSV vectors.

Example 2

Exemplary constructs

In this example, various constructs and sequences thereof are presented.

hVG001-1-2 comprises a modified ICP34.5 region (FIG. 2; SEQ ID NO. 572), a modified UL54 promoter-regulatory region (FIG. 3; SEQ ID NO. 573), the insertion of a PD-L1 blocker in the gene region between UL3 and UL4 (FIG. 4; SEQ ID NO. 574), and a modified Terminal Repeat (TR) region carrying expression cassettes encoding IL-12, IL-15 and IL-15 receptor alpha subunits (FIG. 5; SEQ ID NO. 575). The virus also has a modified and partially deleted ICP34.5 region.

mVG001-1-2 is functionally identical to human version hVG001-1-2, except mVG001-1-2 carries a mouse version of IL-12 and a mouse PD-L1 blocker in the same location on the viral genome, wherein hVG001-1-2 carries a human IL-12 and a human PD-L1 blocker.

Example 3

Abbreviations used in the examples that follow

TF-Fc: PD-L1 blocking peptides (TF) fused to Fc and used to construct VG 001-1-2.

IL-TF-Fc: plasmids carrying IL-12, IL-15 and PD-L1 blockers.

HSV-345: ICP 34.5-deleted virus.

OS-ICP 27-11: ICP 34.5-deleted virus having Oct4/Sox2 binding site and surviving promoter (OS) inserted in promoter-regulatory region of ICP27 (OS-ICP 27) not used for constructing VG 001-1-2.

OS-ICP27 5-7: viruses with ICP 34.5-deletion that was not used to construct the OS-ICP27 mutation of VG 001-1-2.

NO-ICP27 1-4-4 (also referred to as NO-ICP 27-145): an ICP 34.5-deleted virus having NF-kB response element and Oct4/Sox2 binding site (NO) inserted into the promoter-regulatory region of ICP27 (NO-ICP 27) which is not used for constructing VG001-1-2 at 145bp upstream of the transcription initiation site of ICP 27.

NO-ICP27 5-2-2 (also referred to as NO-ICP 27-99): an ICP 34.5-deleted virus having an NF-kB response element and an Oct4/Sox2 binding site (NO) inserted into the promoter-regulatory region of ICP27 (NO-ICP 27) which is not used for constructing VG001-1-2, 99bp upstream of the transcription initiation site of ICP 27.

VG001-1.7 (also referred to as HSV 1-VG 001-1.7): the backbone (backbone) virus used to construct VG001-1-2 (NO-ICP 27 1-4-4 mutant carrying an exogenous promoter and poly (A) flanking (flank) has a null MCS in the deleted terminal repeat region of the viral genome, which is then used to insert the IL-12/IL-15 expression cassette).

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

VG001-1215h (also referred to as VG001-1-2-1215 h); VG001 carrying human IL-12 and human IL-15.

VG001-PLBh (also referred to as VG 001-1-2-PLBh): VG001 carrying the human PD-L1 blocker inserted into the intergenic region between UL3 and UL 4.

8-8-15RA1-PDL1b: VG001 carrying the human IL-15 and human PD-L1 blockers.

VG001-1-2-1215PLBm (also known as mVG 001-1-2): VG001 carrying mouse IL-12, human IL-15 and mouse PD-L1 blocker.

VG001-1-2-1215PLBh (also referred to as hVG001-1-2 or VG 001-1-2): VG001 carrying the human IL-12, human IL-15 and human PD-L1 blockers.

Example 4

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

In this example, western blot analysis and ELISA expression of IL-12 expression are shown.

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

FIGS. 6B-6C show up-regulation of human IL-12 production following VG001-1-2-1215PLBh virus infection. LS174T or H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI=1) for 48 hours. Infected cell supernatants were harvested and bound to 96-well immunoMaxisorp flat bottom plates coated with anti-human IL-12 capture antibodies. Binding was detected via biotinylated anti-human IL-12 antibody, avidin-horseradish peroxidase (HRP), and 3,3', 5' -Tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450nm using a plate reader. The concentration of human IL-12 in the cultured supernatant was calculated based on the human IL-12 standard curve.

Example 5

Expression of IL-15 after infection of cells with HVG001-1-2

In this example, western blot analysis and ELISA expression of IL-15 expression are shown.

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

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

Example 6

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

In this example, western blot analysis and ELISA expression of IgG4 expression are shown.

FIG. 8A shows Western blot analysis results after VG001-1-2-1215PLBh virus infection. H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (moi=1) for 24 hours. Cell lysates were prepared, loaded onto 12% SDS-PAGE gels, and transferred to PVDF membrane. The membranes were subjected to imprinting analysis with HRP-conjugated anti-human IgG antibodies and images were detected and analyzed using the Bio-Rad ImageLab system.

FIGS. 8B-8C show that human PD-L1 blocker (fused to human Fc region) production is up-regulated following VG001-1-2-1215PLBh virus infection. LS174T or H460 tumor cells were infected with VG001-1-2-1215PLB or VG001-1.7 virus (MOI=1) for 48 hours. Infected cell supernatants were harvested and bound to 96-well Immuno Maxisorp flat bottom plates coated with anti-human IgG4 capture antibodies. Binding was detected via biotinylated anti-human IgG4 antibody, avidin-horseradish peroxidase (HRP), and 3,3', 5' -Tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450nm using a plate reader. The concentration of human IL-4 in the cultured supernatant was calculated based on the human IL-4 standard curve.

Example 7

Constructs comprising PD-L1 blocking peptides

PD-L1 blocking peptides were generated using the Ig kappa chain leader (SEQ ID NO: 501). When two or more blocking peptides are present in the same construct, they are linked to Gly-Ser rich sequence (Gly 4 Ser) 3 (SEQ ID NO: 503). The following constructs were prepared.

TF only: METDTLLLWVLLLWVPGSTGTAHPSPSPRSAGQF (SEQ ID NO: 537);

ET+TF：

METDTLLLWVLLLWVPGSTGEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF(SEQ ID NO:538)；

YT+TF：

METDTLLLWVLLLWVPGSTGYYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQF(SEQ ID NO:539)；

mouse TF: METDTLLLWVLLLWVPGSTGTRYPSPSPKPEGRF (SEQ ID NO: 540);

mouse wt+tf:

METDTLLLWVLLLWVPGSTGWNRLSPSNQTGGGGSGGGGSGGGGSTRYPSPSPKPEGRF(SEQ ID NO:541)。

Triple TF+ET：

METDTLLLWVLLLWVPGSTGTAHPSPSPRSAGQFTAHPSPSPRSAGQFTAHPSPSPRSAGQFGGGGSGGGGSGGGGSEYRMSPSNQTEYRMSPSNQTEYRMSPSNQT(SEQ ID NO:542)

METDTLLLWVLLLWVPGSTGEYRMSPSNQTEYRMSPSNQTEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFTAHPSPSPRSAGQFTAHPSPSPRSAGQF(SEQ ID NO:543)。

additional constructs were made using the IL-2 signal sequence (MYRMQLLSCIALSLALVTNS (SEQ ID NO: 502), and either the human IgG4 Fc region (with hinge region) (SEQ ID NO: 544) or the murine IgG1 Fc region (with hinge region) (SEQ ID NO: 545):

TF only:

MYRMQLLSCIALSLALVTNSTAHPSPSPRSAGQFISAMVRSPPCPSCPAPEFLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK

PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR

EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:546)

ET+TF：

MYRMQLLSCIALSLALVTNSEYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFISAMVRSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:547)

YT+TF：

MYRMQLLSCIALSLALVTNSYYRMSPSNQTGGGGSGGGGSGGGGSTAHPSPSPRSAGQFISAMVRSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:548)

mouse TF:

MYRMQLLSCIALSLALVTNSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:549)

mouse wt+tf:

MYRMQLLSCIALSLALVTNSWNRLSPSNQTGGGGSGGGGSGGGGSTRYPSPSPKPEGRFISAMVRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFP EDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:550)。

example 8

Constructs comprising IL-15 and IL-15Rα under the control of a bidirectional CMV promoter

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

In construct 1, the bi-CMV promoter drives expression of the Sushi domains of IL-15Rα and IL-15 (FIG. 9, SEQ ID No. 557).

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

In construct 3, the bi-CMV promoter drives the expression of IL-15-K5 and IL-15Rα Sushi domain-E5 (FIG. 11, SEQ ID No. 559).

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).

Example 9

Constructs comprising IL-15 and IL-15Rα genes under the control of EF1 α promoter

In this example, various constructs were generated to express IL-15 and IL-15Rα in polycistronic transcripts under the control of the EF 1. Alpha. Promoter (SEQ ID NO: 551). IL-15 and IL-15Rα are linked by an exemplary IRES sequence (SEQ ID NO: 552).

In construct 1, the EF 1. Alpha. Promoter controls the expression of the IL-15-IRES-IL-15R. Alpha. Sushi domain (FIG. 13, SEQ ID No. 561).

In construct 2, the EF 1. Alpha. Promoter controls the expression of IL-15-IRES-IL-15R. Alpha. Variant 4 (FIG. 14, SEQ ID No. 562).

In construct 3, the EF 1. Alpha. Promoter controls the expression of IL-15K 5-IRES-IL-15R. Alpha. Sushi domain E5 (FIG. 15, SEQ ID No. 563).

In construct 4, the EF 1. Alpha. Promoter controls the expression of IL-15K 5-IRES-IL-15R. Alpha. Variant 4E5 (FIG. 16, SEQ ID No. 564).

Example 10

Constructs comprising IL-12, IL-15 and IL-15Rα genes under the control of CMV promoters

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

In construct 1, the CMV promoter (SEQ ID NO: 553) controls

Expression of IL-12-p2A-IL-15-p2A-IL-15Rα Sushi domain (FIG. 17,SEQ ID Nos.565,569).

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).

In construct 3, CMV initiates control of expression of IL-12-p2A-IL-15K5-p2A-IL-15Rα Sushi domain E5 (FIG. 19,SEQ ID Nos.567,571).

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).

Example 11

Constructs comprising a PD-L1 blocker interposed between UL3 and UL4

In this example, 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; the region between bases 676-829, UL3 and UL4 and upstream of the PD-L1 blocker cassette; the region PD-L1 between bases 830-833:UL3 and UL4 blocks downstream of the cassette; nucleotide 834-1433 UL4 coding sequence.

Example 12

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

Recombinant human PD-L1 Fc protein was coated on the bottom of 96-well flat bottom plates overnight at 4 ℃. After coating the plates overnight, different PD-L1 blockers were added to each well of the plates and incubated for 2 hours at room temperature, followed by the addition of recombinant human PD-1Fc 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 addition of TMB substrate. The color development was measured by a microplate reader at a wavelength of 450 nm. Percent inhibition was calculated by comparison to non-synthetic peptide. FIG. 21 shows the percent inhibition of peptides ET, ET+TF, YT, YT+TF, TW, TW+TF, WT, WT+TF and TF produced at two different concentrations (3. Mu.M and 10. Mu.M). At 10 μm, the inhibition range is about 22% to about 48%.

Example 13

Enhancement of cytotoxicity against tumor cells by blocking PD-L1 blocking peptide binding

Human Peripheral Blood Mononuclear Cells (PBMC) were stimulated with anti-CD 3 antibody + human IL-2 for 24 hours and then incubated with different synthetic PD-L1 blockers and calcein-AM labeled target cells for 4 hours. Cells from the culture supernatant were harvested after 4 hours of incubation and the released calcein-AM fluorescence was measured by a microplate reader. The percent cytotoxicity was calculated based on the following formula: [ (sample reading-minimum release)/(maximum release-minimum release) ]. Times.100.

Fig. 22A-22B show the results for four different tumor cells: h460, U87, LS147T and MDA-MB-231 cells. On some tumor cells, cytotoxicity was statistically significantly increased for all peptides except TF.

Example 14

Synergistic effects of IL-12 and IL-15 on cytokine production

Human PBMC were incubated with medium control, IL-12 only, IL-15RA only, or combined IL-12, IL-15, and IL-15Rα1+ neutralizing anti-IL-12 or anti-IL-15 antibodies for 48 hours. The cultured cell supernatants were harvested for measurement of human ifnγ and tnfα production by ELISA.

Fig. 23A and 23B show the results of cytokine production. The combination of IL-12 and IL-15R alpha 1 resulted in a statistically significant increase in the cytokines human IFN gamma and TNF alpha. anti-IL-12 antibodies inhibit their production.

Example 15

Synergistic effect of IL-12 and IL-15 on tumor cells in cytotoxicity

Human PBMC were incubated with tumor target cells and medium control, IL-12 alone, IL-15RA alone, or combined IL-12, IL-15, and IL-15RA1+ neutralizing anti-IL-12 or anti-IL-15 antibodies for 24 hours. The cultured cell supernatants were harvested for measurement of cytotoxicity by LDH. The percent cytotoxicity was calculated based on the following formula: [ (sample reading-minimum release)/(maximum release-minimum release) ]. Times.100.

FIGS. 24A and 24B show that IL-12 and IL-15 together increase cytotoxicity in a statistically significant manner. On MDA-MB-231 cell lines, the addition of anti-IL-12 or IL-15 antibodies significantly reduced this effect.

Example 16

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

In the present embodiment, 3×10 will be ⁴ Individual H460 or LS174T tumor cells were seeded in each well to 96-well plates and incubated overnight at 37 ℃. The following day, the inoculated cells were infected with VG001-1.7backbone, 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 evaluated (FIGS. 25A-C). Then 3X 10 ⁵ Human PBMCs were added to the cultures and co-cultured for 24 hours to assess cytotoxicity by LDH assay (fig. 25D) or for 48 hours to assess human IFNg production by ELISA (fig. 25E). For cytotoxicity assays, the percent cytotoxicity was calculated based on the following formula: [ (actual reading-minimum release)/(maximum release-minimum release) ]X 100%. The supernatant harvested from tumor cells incubated with medium alone served as minimal release, while the supernatant harvested from tumor cells incubated with lysis buffer served as maximal release.

Example 17

In vitro efficacy of various constructs

Fig. 26A-26D show the results of in vitro assays of the various constructs.

FIGS. 26A-26B show the results of transfecting cells with IL-TF-Fc plasmid carrying IL-12, IL-15 and PD-L1 blockers. In FIGS. 26A-26B, different tumor cell lines were transfected with IL-TF-Fc plasmid DNA for 24 hours and human PBMC were then added to the culture. Cell supernatants were harvested after 24 hours for quantitative analysis of cytotoxicity by LDH assay (fig. 26A) and after 48 hours for detection of human IFNg production by ELISA assay (fig. 26B).

FIGS. 26C-26D show the results of infection of cells with various mutant viruses including hVG 001-1-2. The virally encoded IL12, IL15 and PD-L1 blockers synergistically increase IFNg production and cytotoxicity. H460 tumor cells were seeded into each of the 96-well plates and incubated overnight at 37 ℃. The next day, the vaccinated cells were infected with the indicated viruses at moi=1 for 24 hours. Human PBMCs were then added to the cultures and incubated for 24 hours to assess cytotoxicity by LDH assay (fig. 26C), or 48 hours to assess human IFNg production by ELISA (fig. 26D). For cytotoxicity assays, the percent cytotoxicity was calculated based on the following formula: [ (actual reading-minimum release)/(maximum release-minimum release) ]. Times.100%. The supernatant harvested from tumor cells incubated with medium alone served as minimal release, while the supernatant harvested from tumor cells incubated with lysis buffer served as maximal release.

In FIGS. 27A-27E, a set of 9 different human tumor cell lines (+Vero cells) were infected with VG001-1-2-1212PLBh (VG 001-1-2 h) and HSV-345 virus at MOI 0, 0.04, 0.2, 1 and 5. Cell viability was quantitatively analyzed 48 hours after infection using the MTT assay.

FIGS. 28A-28J show the results of in vitro assays of the various constructs. FIGS. 28A-28E show the results of cell viability assays of mVG001-1-2 and HSV-345 performed on mouse tumor cell lines and Vero cell lines; FIGS. 28F-28J show characterization of transgene expression after CT26 mouse tumor cell infection mVG001-1-2 or VG 001-1.7.

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

In FIGS. 28F-28J, 3×10 will be shown ⁴ Individual CT26 tumor cells were seeded into each well of a 96-well plate and incubated overnight at 37 ℃. The following day, the inoculated cells were infected with VG001-1.7 backup 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 3X 10 from Balb/c mice ⁵ Individual 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)]X 100%. The supernatant harvested from tumor cells incubated with medium alone served as minimal release, while the supernatant harvested from tumor cells incubated with lysis buffer served as maximal release.

In FIGS. 29A-29E, 3×10 will be shown ⁴ Individual H460, LS174T or UMUC3 tumor cells were seeded into each well of a 96-well plate and incubated overnight at 37 ℃. The next day, the inoculated cells were infected with VG001-1.7 backup 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. Then 3X 10 ⁵ The individual BMCs were added to the cultures 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 percent cytotoxicity was calculated based on the following formula: [ (actual reading-minimum release)/(maximum release-minimum release) ]X 100%. The supernatant harvested from tumor cells incubated with medium alone served as minimal release, while the supernatant harvested from tumor cells incubated with lysis buffer served as maximal release.

In FIGS. 30A-30G, the anti-tumor effect of VG001-1-2-1215PLBh (hVG 001-1-2) virus was evaluated in various human cancer cells including U87, MCF7, H460, LNCaP, LS174T, MDA, and PC3 at 72 hours post-infection and MOI of 0 to 5. The percent cell survival was quantitatively analyzed by MTT assay. Among all human tumor cell lines tested, VG001-1-2-1215PLBh virus exhibited potent T cell killing ability.

Example 18

In vivo efficacy of VG001-1-2 viral constructs

In FIGS. 31A-31B, 5 intratumoral injections were performed for a total of 1X 10 in A20 murine B-cell lymphoma tumor-bearing BALB/c mice ⁷ PFU/mouse VG001-1-2-1215PLBm (mVG 001-1-2) virus or VG001-1.7back bone virus or injected with PBS (vector control). Tumor size measurements were made after a specified number of injections. Mice treated with VG001-1-2-1215PLBm showed significant tumor volume compared to mice treated with PBS (P<0.05 A) decrease.

In FIGS. 31C-31D, 5 intratumoral injections were performed for a total of 5X 10 in CT26 colon cancer tumor-bearing BALB/C mice ⁶ PFU/mouse VG001-1-2-1215PLBm (mVG 001-1-2) virus or VG001-1.7back bone virus or injected with PBS (vector control). Tumor size measurements were made after a specified number of injections. Mice treated with VG001-1-2-1215PLBm showed significant tumor volume compared to mice treated with PBS (P<0.05 A) decrease.

In fig. 31E-31G, ohv treatment of xenograft human prostate tumors in mice was evaluated. LNCaP human prostate tumor cells were transplanted into the right lower abdomen of twelve mice. 35 days after implantation, 6 animals of the randomly selected group were twice intratumorally injected for a total of 5×10 ⁷ VG001-1-2-1215PLBh of PFU/mouse

(hVG 001-1-2) virus, while the remaining 6 animals served as carrier controls and were injected twice with equal volumes of PBS. Tumor size was measured using two different methods. Caliper measurements were expressed as fold-changes in tumor volume at a given time point compared to tumor volume at either virus or PBS (FIG. 31E). Tumor-bearing mice treated with VG001-1-2-1215PLBh virus showed significant tumor atrophy, with tumor size decreasing by more than 50% at the end of 15 days, while mice treated with vehicle showed an approximately 3-fold increase in tumor volume over the same period of time. Tumor growth was also monitored using a whole animal bioluminescence imaging system (IVIS Imaging System; xenogen, mountain View, calif.). The signal intensity was quantitatively analyzed as the sum of all photons detected per second (fig. 31F). Quantitative imaging of tumor growth with IVIS showed an even more significant reduction in tumor size in the ohv-treated animals compared to PBS-treated controls, with fluorescence subsidence to undetectable levels 50 days after tumor implantation (fig. 31G; two vehicle controls on the left, two ohv-treated mice on the right).

Example 19

Replication of HVG001-1-2 in cell lines

The growth curves and cytotoxic expression in FIGS. 32A-32C, 33A-33D, and 34A-34E show hVG001-1-2 viral replication and parent HSV-345 virus. These expressions also show that the virus does not grow in mouse tumor cell lines, but HSV-1 is known to grow poorly in mouse cells, as compared to human cell lines.

Example 20

Evaluation of Virus modification

Human PBMC were stimulated with medium alone, recombinant IL-12 alone, recombinant IL-15 alone, or IL-15/IL-15RA1 complex with or without IL-12 (6 mg/ml) or IL-12+ different forms of neutralizing antibodies (0.5 mg/ml) for 48 hours. The cultured supernatants were then harvested for human IFNg and human TNFa production was determined using ELISA as shown in fig. 35A and 35B.

To assess cytotoxicity against tumor cells, calcein-AM-labeled tumor cells were incubated with stimulated human PBMCs for 24 hours. Supernatants were harvested for measurement of released fluorescence. Supernatants harvested from calcein-labeled tumor cells were incubated with medium used only for minimal release, and supernatants harvested from calcein-labeled tumor cells were incubated with lysis buffer used for maximal release. Percent cytotoxicity was calculated based on the following formula: [ (actual reading-minimum release)/(maximum release-minimum release) ]. Times.100%. The cytotoxicity results of the U87 tumor cells are shown in FIG. 35C, and the cytotoxicity results of the MDA-MB-231 tumor cells are shown in FIG. 35D.

Example 21

In vitro efficient expression

Human Peripheral Blood Mononuclear Cells (PBMCs) were stimulated with medium alone, recombinant IL-12 alone, recombinant IL-15 alone, or 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 for 48 hours. The cultured supernatants were then harvested for human IFNg and human TNFa production as determined by ELISA as shown in fig. 36A and 36B.

To assess cytotoxicity against tumor cells, 1×10 was used ⁴ Individual calcein-AM-labeled tumor cells and 1 x 10 ⁵ The individual stimulated human PBMCs were incubated for 24 hours. Supernatants were harvested for measurement of released fluorescence. Supernatants from calcein-labeled tumor cells were incubated with medium used as minimal release, and supernatants from calcein-labeled tumor cells were incubated with lysis buffer used as maximal release. Calculating the percent cytotoxicity based on the formula: [ (actual reading-minimum release)/(maximum release-minimum release)]X 100%. FIG. 36C shows the cytotoxicity results of U87 tumor cells, and FIG. 36D shows the results of MDA-MB-231 tumor cells.

Example 22

Tumor cells infected with VG001-1-2h produced human IL-12, human IL-15/IL15Ra and human IgG4

Briefly, LNCaP cells were transplanted into nude mice and received injections of vehicle, ICP27-, or VG001-1-2h virus. Serum and tumor samples were harvested 120 hours after injection and evaluated for production of human IL-12, human IL-15/IL-15Ra and human IgG4 by ELISA. The results are shown in fig. 37A.

Fadu cells were transplanted into nude mice and received vehicle, HSV 1-VG 001-1.7 or VG001-1-2h virus injections. Tumor samples were harvested 24 hours after injection and evaluated for human IL-12, human IL-15/IL-15Ra and human IgG4 production by ELISA. The results are shown in fig. 37B.

Example 23

Effect of VG001-1-2m in immune response

CT26 colon cancer cells were implanted into balb/c mice and received injections of 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. The results are shown in fig. 38A-38C.

Example 24

Another exemplary construct

In this example, constructs were further engineered, in particular the US12 (ICP 47) promoter region flanking the IL12-IL15-IL15RA1 expression cassette, to propose another construct and its sequence.

VG001-1-2 comprises a modified ICP34.5 region (FIG. 40; SEQ ID NO. 599), a modified UL54 promoter-regulatory region (FIG. 41; SEQ ID NO. 596), insertion of a PD-L1 blocker in the gene region between UL3 and UL4 (FIG. 42; SEQ ID NO. 589), and a modified Terminal Repeat (TR) region carrying expression cassettes encoding IL-12, IL-15 and IL-15 receptor alpha subunits (IL 12-IL15-IL15RA 1) (FIG. 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, IL15RA1, self-cleaving ligation peptide P2A upstream of self-cleaving ligation peptide P2A, respectively.

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

The promoter region of US12 (ICP 47) flanking the IL12-IL15-IL15RA1 expression cassette carried by VG001-1-2 was modified to construct versions of the various flanking US12 (ICP 47) promoters: versions of short (S; SEQ ID No. 583), medium (M; SEQ ID No. 584), long (long, L; SEQ ID No. 585), survivin (SEQ ID No. 586) and the like, and corresponding VG001-1-2 may be specifically classified into VG001-1-2 (S), VG001-1-2 (M), VG001-1-2 (L), VG001-1-2 (survivin) and the like according to the version of the flanking ICP47 promoter. In the examples that follow, VG001-1-2 refers to VG001-1-2 (M), i.e., the vector-borne IL12-IL15-IL15RA1 expression cassette is flanked by the medium (M) version of the ICP47 promoter, unless otherwise indicated. The structure of the VG001-1-2 vector is shown in FIG. 70.

Example 25

Cytotoxicity of VG001-1-2

As shown in FIG. 56A, U87, H460, MCF-7, LS174T, MDA-MB-231, etc. human cancer cell monolayers were infected with VG001-1-2 virus (MOI 0.04, 0.2, 1, 5), and the percent cell survival was quantified by MTT assay 72 hours post infection to assess cytotoxicity of VG 001-1-2. As shown in FIG. 56B, four mouse tumor cell lines B16-F10, 4T1, CT26 and A20 were infected with mVG001-1-2 virus (MOI 0, 0.04, 0.2, 1, 5) and cell viability was quantified by MTT assay 72 hours after infection.

Example 26

In vitro characterization of IL12, IL15 and PD-L1 blockers of VG001-1-2 expression

As shown in FIG. 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 immunoblotting (left panel) and ELISA (right panel). FIG. 57A shows the expression of human IL-2, FIG. 57B shows the expression of human IL-5, and FIG. 57C shows the expression of PD-L1 blocker.

Example 27

In vitro characterization of PD-L1 blockers of VG001-1-2 expression

As shown in fig. 58, supernatant containing tf+fc peptide harvested from VG001-1-2 infected 293FT cells was mixed with recombinant human PD-1Fc and bound to human PD-L1 Fc coated 96-well immune Maxisorp flat bottom plates. Binding was detected by biotinylated anti-PD-1 antibody, streptavidin-horseradish peroxidase (HRP) and 3,3', 5' -Tetramethylbenzidine (TMB) substrate. Absorbance measurements were collected at 450nm using a plate reader. The percent increase (%) of human PD-1/PD-L1 inhibition was compared to the peptide-free sample.

Example 28

In vitro characterization of IL12 expressed by VG001-1-2

FIG. 59 shows the results of a cell-based assay of TF+Fc peptide treatment. Activation with PHA 1. Mu.g/ml and PMA 50ng/ml 5X 10 ⁴ Jurkat T cells and 1X 10 ⁵ Together with the PD-L1-expressing tumor cells, the cells were mixed with the supernatant containing the PD-L1 blocking peptide and incubated at 37℃for 48 hours. After 48 hours, cell culture supernatants were harvestedIL-2 production by Jurkat T cells was determined by IL-2 ELISA.

Example 29

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

U87 human glioma cells and MDA-MB-231 human breast cancer cells overexpressing IL12, IL15/IL15RA, or coexpression of IL12 and IL15/IL15RA were treated with human PBMC.

FIG. 60A shows the measurement of cytokine IFN-. Gamma.and TNF-. Alpha.production by ELISA.

Fig. 60B shows cytotoxicity induced by LDH assay of immune cells.

Fig. 60C shows PBMC activation by synergy of virally encoded IL12, IL15 and PD-L1 blocker: h460 tumor cells were seeded into each well of a 96-well plate and incubated overnight at 37 ℃ and then infected with the VG001-1-2 virus with moi=1 for 24 hours and incubated with human PBMC for 48 hours. IFN-gamma production was quantified by ELISA. The viruses tested included HSV 1-VG001-1.7 (no immunomodulator), HSV 1-VG001-1.7-PDL1b (PD-L1 blocker), HSV 1-VG 001-1.7-15RA1 (IL 15/IL15 RA), HSV 1-VG 001-1.7-RA1-PDL1b (IL 15/IL15RA+PD-L1 blocker), HSV 1-VG001-1.7-h1215 (IL 12) +IL15/IL15 RA) and VG001-1-2 (IL 12+IL15/IL15RA+PD-L1 blocker).

Fig. 60D shows that PHA-activated human PBMCs (n=4) were incubated for 48 hours with recombinant human PD-L1 protein and supernatant from VG001-1-2 infected cells. Antibody-mediated neutralization of IL-1 and/or IL-15 is performed in conjunction with depletion of PD-L1 blockers. Co-incubation with supernatant from uninfected cells served as negative control. Human IFN-gamma production was assessed by ELISA.

Example 30

In vivo efficacy of VG001-1-2 intratumoral vaccination

FIG. 61A shows the efficacy of VG001-1-2 in the U87 human glioblastoma model: u87 cells were implanted into the lower flank of 7 nude mice, and then injected intratumorally 1X 10 twice at 2-day intervals ⁷ VG001-1-2 from PFU/mouse or vector control.

Fig. 61B shows immune-mediated remote clearance of non-injected distal tumors: implantation of A20 cells into the flank of immunocompetent miceTwo sides. mVG001-1-2 or HSV1-VG 001-1.7 backbone virus was expressed as 5X 10 ⁶ The PFU/mouse/day dose was injected into tumors on only one side for 5 consecutive days. 16 mice were treated with mVG001-1-2 and 6 mice were treated with HSV1-VG 001-1.7. Compared to 0/6 HSV1-VG 001-1.7 treated mice, 6/16 mVG001-1-2 treated mice experienced complete tumor regression on both sides.

Fig. 61C shows that mice treated with mVG001-1-2 were protected from CT26 tumor re-challenge: CT26 cells were implanted into the lower flank of 16 immunocompetent BALB/C mice, with 8 animals randomly assigned to the mVG001-1-2 treatment group and 8 animals assigned to the vehicle control group. At 21 days post tumor implantation, all 8 vehicle control animals died from tumor burden. No animals in the mVG001-1-2 treated group died from the tumor, but 4 animals in the group died due to non-tumor related conditions. The 4 mice surviving in mVG001-1-2 treated group were re-implanted with CT26 cells at the same location 90 days after injection.

FIG. 61D shows tumor size 7 days after CT26 re-challenge in mVG001-1-2 treated animals compared to age-matched control mice not treated with mVG 001-1-2.

Example 31

Gene expression and T cell Activity in tumors treated with MVG001-1-2

As shown in FIG. 62, BALB/c mice were implanted with CT26 tumor cells, followed by 5X 10 for 5 consecutive days ⁶ PFU/mouse/day doses were injected multiple times mVG001-1-2, HSV1-VG 001-1.7 or PBS control. Mouse IFN-. Gamma.ELISPot.assays were performed on spleen cells collected on days 5, 7 and 9 post-injection. The quantitative results are plotted in the right panel, with 2 mice per group.

Example 32

Effect of MVG001-1-2 treatment on intratumoral lymphocyte populations

As shown in FIG. 63A, BALB/c mice received CT26 cell implantation, and after 8 days, PBS, HSV1-VG 001-1.7 backbone, or mVG001-1-2 virus were injected 5 times. Tumors were harvested 24 or 120 hours after the last injection. The percentage of different T cell subsets (fig. 63B) and immunosuppressive cells (fig. 63C) within the tumor mass was analyzed by flow cytometry. FIG. 63D shows immunohistochemical analysis of excised CT26 tumor sections treated with PBS control (vector) or mVG001-1-2 with monoclonal antibodies to CD3 and perforin and polyclonal antibodies to HSV-1.

Example 33

Minimal dose of human IL12 or IL15/IL15RA on immune cell function effect

As shown in FIG. 64, human IL-12 harvested from the supernatant of transfected 293FT cells was co-cultured with PHA-stimulated human PBMC for 48 hours. Human IFN-gamma production was assessed by ELISA. As shown in fig. 64B, 293FT cells were transfected with plasmids expressing human IL12 or human IL15/IL15RA for 48 hours, the supernatants were harvested and co-cultured with PHA-stimulated human PBMCs for 48 hours, and cell proliferation was assessed by MTT assay.

Example 34

Biodistribution of viruses

As shown in FIGS. 65A and 65B, 5X 10 nude mice bearing LS174T tumor ⁷ Intratumoral injection of VG001-1-2 was performed at a dose of PFU/mouse/day. Mice were euthanized at various time points and genomic DNA was isolated from these organs and qPCR was performed to quantify viral copy number using IL15RA1 gene.

Example 35

qPCR measurement

Mouse CT26 tumor cells were implanted into mice by daily injection of mVG001-1-2, control virus HSV1-VG 001-1.7 (total 5X 10) ⁷ pfu) or PBS. Tumors were harvested 24 and 48 hours after the last virus injection, RNA was isolated and purified, and then RNA was isolated and purified using the Mouse Innate from Qiagen&Adaptive Immune ResponsesR RT2 Profiler TM PCR Array Gene expression profiling was performed. As shown in FIG. 66, differential expression of innate and adaptive immune-related genes in tumors treated with mVG001-1-2 compared to tumors treated with HSV1-VG 001-1.7 and PBS. Overexpression of the indicated targets was verified by RT-qPCR.

Example 36

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

To understand the effect of promoter engineered VG001-1-2 viruses on cells, cells were infected with VG001-1-2 and wild type (VG 001-1-2-1215PLBh, ICP47 promoter not engineered) viruses, respectively, under identical conditions.

Specifically: 293T cells were plated in 6-well plates (1X 106 cells/well) and incubated in DMEM medium supplemented with 10% serum. After the cells had grown to a monolayer, 1×106pfu (moi=1) of the following viruses or blank culture medium were added to each well:

VG001-1-2 Virus (2 wells)

b. Wild type virus (2 holes)

c. Non-infected control with culture medium alone (2 wells)

Cells were cultured overnight after virus infection, then digested with 750ul trypsin, added with 1ml DMEM, transferred to flow tubes, centrifuged (1500 rpm,5 min), the supernatant removed and washed with 2ml PBS, repeated washing once, and PBS removed. Each of the above groups a-c had 1 well of antibody against MHC (histocompatibility complex) protein (Anti-Hu HLA-ABC) suspended cells with 100ul of PBS+2% FBS and 2ul of antibody. In addition, 100ul PBS+2% FBS was added to each 1 well, and no antibody was added. After incubation in the dark for 1 hour, the antibody was removed, the cells were washed with 3ml PBS+2% FBS, centrifuged (1500 RPM,5 min), and the majority of the cell washes were discarded for flow cytometry. The results are shown in FIG. 67.

As can be seen from FIG. 67, infection with wild type HSV-1 virus resulted in a decrease in the expression of MHC proteins on the cell surface. Unexpectedly, cells infected with VG001-1-2 virus had MHC expression levels even further below cells infected with wild-type HSV-1 virus. Thus, compared with the wild type virus, by modifying the ICP47 promoter, the VG001-1-2 virus can avoid immune cell attack in infected cells for a longer time after the cells are infected, so that the exogenous gene can be better replicated and expressed.

Example 37 exploratory test of oncolytic Virus stabilizers

In order to optimize the medium in which oncolytic viruses were stored, different formulations of the medium were tested. A batch of VG001-1-2 virus samples was prepared and the virus was preserved according to the formulation and conditions of Table 1.

TABLE 1 Medium storing different formulations of viruses

Samples were prepared according to Table 1, placed in a 37℃incubator, and after 48 hours, the samples were transferred to a-80℃refrigerator until virus titer detection was performed. Wherein, the sample G8 is taken out of the refrigerator at the temperature of minus 80 ℃ before titration, and the original virus and other seven groups of samples are subjected to virus titration simultaneously after dilution. The results of G8 are reference criteria for determining the stability of other groups of virus samples.

Vero cells were plated at 8X 10 per well ⁵ Is inoculated into a 6-well plate, 3ml of the cell is placed in each well, and is cultured for 24 hours in a carbon dioxide incubator at 37 ℃. The virus samples were subjected to gradient dilution according to the protocol of table 2.

TABLE 2 Virus titration dilution procedure

Six well plates, which had been replaced with no FBS medium, were removed from the 37 degree incubator, medium was aspirated, 1ml of diluent was added to each well, and 4 wells (2 six well plates/sample) were made per sample. After 1ml of virus dilution per well was added, the mixture was placed in a 37-degree incubator, virus was adsorbed for 60 minutes, the virus liquid was aspirated, 2 ml/well of MEM (no FBS) culture solution and methylcellulose (1.5%) were added, and the mixture was placed in the 37-degree incubator to be cultured for 96 hours.

After 96 hours, the cover was discarded, 1ml of 4% glutaraldehyde solution was added to each well, left at room temperature for 30 minutes, then dyed with 2% crystal violet dye, 1 ml/well, dyed at room temperature for 15 minutes, rinsed with demineralized water, and dried. After the six-well plate was air-dried, white spots were formed due to uncolored dead cells caused by viruses, and the spots were counted under a medical film viewer.

And (3) calculating results: taking the number of plaques in the wells of 20-150 as titer counts, taking the average and multiplying the average by the virus dilution, e.g. the average number of plaques in the wells of tube 6 is 20, i.e. the virus titer is 20X 10 ⁶ I.e. 2.0 x 107pfu/ml. Above-mentionedThe samples were each replicated in triplicate. The titer of the virus was calculated from the plaque results and the results of G1-G7 were compared to G8 to determine the most stable protectant for the virus.

The results of measuring the virus titer of each group are shown in fig. 68. The test results showed that the viral activity at 37℃as stored in formulation G5 remained good and was essentially indistinguishable from the activity of the virus stored at-80 ℃. While increasing sucrose is generally believed to increase the stability of the virus solution, it is unexpected that HSV-1 oncolytic viruses, in the presence of sucrose, are detrimental to maintaining viral activity, whether glycerol is present or not.

Example 38 stability exploratory study of proteins in mouse serum

VG001-1-2 carries a human PD-L1 blocking peptide (TF), and the C-terminus of the PD-L1 blocking peptide may comprise an Fc sequence. The Fc may be derived from one of the IgG subclasses, and the following experiments were performed in order to understand the stability of various fusion proteins formed by fusion of TF ends to Fc sequences of different origins.

Cell culture

RPMI-1640 medium containing diabody and 10% foetal calf serum was used for 293 cells in the presence of 5% CO ₂ Is cultured in an incubator at 37 ℃. When the cell density reached about 80%, digestion with trypsin was performed to 1: subculturing was performed at a ratio of 3-4.

Plasmid extraction: for VG001-1-2, sequences encoding IgG1 Fc or IgG4 Fc fragments were inserted at the ends of their PD-L1 encoding regions, respectively, resulting in plasmids VG001-1-2-Fc1 and VG001-1-2-Fc4, which were capable of expressing PD-L1 fused to IgG1 Fc and PD-L1 fused to IgG4 Fc, respectively. The two plasmids were transformed into E.coli BL21 by heat shock, cultured on LB+AMP bacterial plates overnight at 37℃to pick up single colonies, and placed in LB+AMP liquid medium (2-3 ml) at 37℃in a shaking table (150 rpm/min) overnight to culture.

Plasmids were extracted from the above-mentioned culture and turbid medium, labeled Fc-1 and Fc-4, and the date of extraction.

On agarose electrophoresis, the quality of the extracted plasmid was identified, and the concentration of plasmid DNA was detected with a DNA concentration detector.

2.3 plasmid transfection

293 cells were plated in 6-well plates and placed in a cell incubator overnight for adherent culture.

The two plasmids were mixed with plasmid transfection reagent and serum-free medium according to the plasmid concentration and the method of the instructions for plasmid transfection. The culture medium was changed to 6% FBS serum after addition to 293 cells and incubation for 4 hours and continued for 48 hours.

After plasmid transfection, the supernatant (2 ml) was taken at 48 hours.

All samples supernatant samples were frozen in a-80 ℃ refrigerator.

2.4 mixing of proteins with serum

The above transfected supernatant (2 mL) and mouse serum were taken out from-80℃and, after thawing on ice, samples were prepared according to the formulation of Table 3.

Table 3: configuration of mixed samples

After the samples are prepared, the sample group G01/G02 is transferred to a refrigerator at the temperature of minus 20 ℃ for preservation, other groups are placed in an incubator at the temperature of 37 ℃, the sample group G1/G2 is transferred to a refrigerator at the temperature of minus 20 ℃ for preservation on the 14 th day, and the sample group G3/G4 is transferred to a refrigerator at the temperature of minus 20 ℃ for preservation on the 28 th day. And after the collection of all samples is finished, ELISA detection is carried out.

ELISA detection of samples

The samples were thawed on ice. 300ul of each sample was removed, ELISA assays were performed, 100ul of each well, and three replicates of each sample were performed. The protein content of each sample was calculated according to the instructions of the kit.

1) Coating ELISA plates with antibodies in the kit, overnight at 4 ℃;

2) Washing the coated ELISA plate twice by using washing liquid in the kit, and absorbing liquid in the dry hole by using absorbent paper for each washing;

3) Adding a sealing liquid, and sealing for 2 hours at room temperature;

4) Washing twice with washing liquid, adding the prepared standard curve, the samples and the Assay buffer, and repeating each sample for 2 times;

5) The sealing plate film is used for preventing the liquid in the hole from evaporating, and the hole is placed at room temperature for 2 hours for incubation;

6) After the incubation is finished, washing four times by using a washing liquid, and airing the liquid in the hole by using water absorbing paper each time;

7) According to the requirements of the kit, adding 100 ul/hole of detection antibody, sealing the plates, and incubating for 1 hour at room temperature;

8) After the incubation is finished, washing four times by using a washing liquid, and airing the liquid in the hole by using water absorbing paper each time;

9) 100ul of substrate is added to each hole, and the mixture is incubated for 15 minutes at room temperature in a dark place;

10 100ul of stop solution is added into each hole, and the mixture is incubated for 10 to 20 minutes at room temperature;

11 Debugging ELISA enzyme label instrument, and reading the plate at the wavelengths of 450nm and 570 nm;

12 Data were analyzed as required by the kit.

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, resulting in the stability of IgG1Fc and IgG4Fc fusion proteins in mouse serum (fig. 69).

As can be seen from the test results, the TF fused to IgG1Fc is not as stable in serum as the IgG4Fc fused structure, contrary to conventional belief. After 28 days, the ability of TF-IgG4Fc fusion proteins to bind to PD-L1 was significantly higher compared to TF-IgG1Fc fusion proteins, with a statistically significant difference (p=0.01).

The following are other exemplary embodiments of the disclosure:

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

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

3) The HSV vector of embodiment 2, wherein the nucleic acid sequence encodes a polypeptide selected from the group consisting of

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

4) The HSV vector of embodiments 1-3, wherein one or more IRES sequences are located between the coding sequences of IL12, IL15 and IL15 receptor alpha subunits. In a preferred embodiment, IL12 is murine or human sequence, IL15 is human sequence, and the IL15 receptor alpha subunit is human sequence.

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

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

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

8) The HSV vector of any of embodiments 1-7, wherein each of IL15 and IL15 receptor alpha subunit follows a nucleic acid sequence encoding Lys5 or Glu 5. In a preferred embodiment, IL12 is murine or human sequence, IL15 is human sequence, and IL15 receptor alpha subunit is human sequence.

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

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

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

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

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

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

15 The HSV vector of embodiment 10), wherein the sequence encoding the PD-L1 blocking peptide is inserted between viral genes, e.g., between UL3 and UL4 viral genes, between UL50 and UL51 genes, and/or between US1 and US 2.

16 The HSV vector of any of embodiments 1-15, further comprising NFkB and OCT4/SOX2 enhancing elements in the ICP4 or ICP27 regulatory region.

17 The HSV vector of any of embodiments 1-16, wherein the ICP34.5 gene is deleted.

18 The HSV vector of any one of embodiments 1-17, wherein the expression cassette comprises at least one bidirectional CMV promoter.

19 The HSV vector of any one of embodiments 1-18, wherein the expression cassette comprises at least one cellular promoter.

20 The HSV vector of any one of embodiments 1-19, wherein the expression cassette of the IL12/IL15 receptor alpha subunit is inserted into an internal repeat region or terminal repeat region, wherein the original viral sequence is replaced with the expression cassette.

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

22 The HSV vector of any one of embodiments 1-21, wherein the ICP34.5 gene is regulated by a 3' utr comprising a target sequence of a miRNA that is underexpressed in a tumor cell.

23 A pharmaceutical composition comprising the HSV vector of any of embodiments 1-22, and a pharmaceutically acceptable carrier.

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

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

All patents, publications, scientific articles, websites, and other documents and materials cited or referred to herein represent the level of skill of those 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 collectively incorporated herein to the same extent as if fully set forth herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information in any such patents, publications, scientific articles, websites, electronic information, and other references or documents.

The written description of this patent includes all claims. Furthermore, all claims, including all original claims and all claims in any and all priority documents, are fully incorporated by reference into the written description section of the specification, applicant reserves the right to actually incorporate the written description or any other section of this application, any and all such claims. Thus, for example, in no event should a patent be construed as claiming that no written description of the claims is provided for by claims that do not specify the exact terms of the claims in the original document of the written description of the patent.

The claims are to be interpreted in accordance with the law. However, in any event, any claim or any portion thereof is not to be construed as having lost any equivalent right and does not form a part of the prior art during this patent application, although it is stated or believed that it is easy or difficult to interpret any claim or portion thereof.

All features disclosed in the present specification may be combined arbitrarily. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Accordingly, from the foregoing, while specific non-limiting embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Other aspects, advantages, and modifications are included within the scope of the following claims, and the invention is not limited to the following claims.

The specific methods and compositions described herein represent preferred, non-limiting embodiments and are exemplary and are not intended to limit the scope of the invention. Those skilled in the art will, upon reading this specification, produce other objects, aspects and embodiments, which are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily appreciated by those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each of the examples herein, the terms "comprising," "including," "containing," and the like are to be construed as being extended and non-limiting in a non-limiting embodiment or example of the invention. The methods and processes appropriately set forth herein may be carried out in a different order of steps, and they are not necessarily limited to the order of steps shown herein or in the claims.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Therefore, it should be understood that while the present invention has been specifically disclosed by various non-limiting embodiments and/or preferred non-limiting embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope as defined by the appended claims.

The present invention has been described broadly and generically herein. Each of the narrower species and subspecies that fall within the present disclosure also form part of the invention. This includes the generic description of the invention with the proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically recited herein.

It should also be understood that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, the terms "X and/or Y" mean "X" or "Y" or both "X" and "Y," and the letter "s" following a noun denotes both plural and singular of that noun. Furthermore, features or aspects of the invention are described in terms of Markush groups, and those skilled in the art will recognize that the invention is encompassed and therefore also described in terms of any individual member of the Markush group and any subgroup of any member, and that the applicant reserves the right to modify the application or claims, especially for any individual member or subgroup of any member of the Markush group.

Other non-limiting embodiments are included in the following claims. This patent is not to be construed as limited to the specific embodiments or non-limiting embodiments or methods specifically and/or explicitly disclosed herein. In no event should a patent be construed as limited by any statement made by any examiner or any other official or employee of the patent and trademark office unless the statement is specifically adopted by the applicant in the written reply file and is not otherwise limited or reserved.

Claims (15)

1. A herpes simplex virus HSV vector comprising an expression cassette encoding IL12, IL15 and IL15 receptor alpha subunits, wherein the expression cassette is flanked by a modified ICP47 promoter;

wherein the IL15 receptor alpha 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),

wherein the herpes simplex virus HSV vector still further comprises an expression cassette encoding one or more PD-L1 blocking peptides, and a sequence encoding an IgG4 Fc region, wherein upon expression, the IgG4 Fc region is linked to the 3' -end of the PD-L1 blocking peptide;

wherein the herpes simplex virus HSV vector further comprises a modified ICP34.5 region.

2. The herpes simplex virus HSV vector of claim 1, wherein the sequence of the modified ICP47 promoter comprises SEQ ID No.584.

3. The herpes simplex virus HSV vector of claim 1, wherein the nucleic acid sequence encoding the self-cleaving peptide 2A is in-frame between the coding sequences of IL12, IL15 and IL15 receptor alpha subunit.

4. The herpes simplex virus HSV vector of claim 3, wherein the self-cleaving peptide 2A has an amino acid sequence of: VKQTLNFDLLKLAGDVESNPGP, QCTNYALLKLAGDVESNPGP, ATNF-SLLKQAGDVEENPGP, HYAGYFADLLIHDIETNPGP, GIFNAHYAGYFADLLIHDIETNPGP, KAVRGYHADYYKQRLIHDVEMNPGP, GATNFSLLKLAGDVELNPGP, EGRGSLLTCGDVEENPGP, AARQMLLLLSGDVETNPGP, FLRKRTQLLMSGDVESNPGP, GSWTDILLLLSGDVETNPGP, TRAEUEDELIRAGIESNPGP, AKFQIDKILISGDVELNPGP, SKFQIDKILISGDIELNPGP, SSIIRTKMLVSGDVEENPGP or CDAQRQKLLLSGDIEQNPGP.

5. The herpes simplex virus HSV vector of claim 1, wherein one or more IRES sequences are located between the coding sequences of IL12, IL15 and IL15 receptor alpha subunits.

6. The herpes simplex virus HSV vector of claim 1, wherein the IL15 and IL15 receptor alpha subunits are expressed by a bi-directional promoter.

7. The herpes simplex virus HSV vector of claim 1, wherein the IL15 and IL15 receptor alpha subunit each follow a nucleic acid sequence encoding Lys5 or Glu 5.

8. The herpes simplex virus HSV vector of claim 1, wherein the expression cassette comprising IL12, IL15 and IL15 receptor alpha subunit is inserted into an internal repeat region of HSV or a terminal repeat region of the HSV genome.

9. The herpes simplex virus HSV vector of claim 1, wherein the expression cassette of the PD-L1 blocking peptide is inserted between UL3 and UL4 of an HSV virus gene.

10. The herpes simplex virus HSV vector of any of claims 1-9, wherein NFkB and OCT4/SOX2 enhancing elements are further included in the ICP4 or ICP27 regulatory region of the herpes simplex virus HSV vector.

11. The herpes simplex virus HSV vector of any of claims 1-9, wherein the ICP34.5 gene portion of the herpes simplex virus HSV vector is deleted or non-functional.

12. A pharmaceutical composition comprising the herpes simplex virus HSV vector of any one of claims 1-11, and a pharmaceutically acceptable carrier.

13. Use of a herpes simplex virus HSV vector according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 12, in the manufacture of a medicament for the treatment of cancer.

14. The use according to claim 13, wherein the cancer is selected from liver cancer, stomach cancer, intestinal cancer, lung cancer, breast cancer, nasopharyngeal cancer, head and neck cancer, bladder cancer, colon cancer, rectal cancer, kidney cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma, burkitt's lymphoma, acute or chronic myelogenous leukemia, melanoma, endometrial cancer, head and neck cancer, glioblastoma, osteosarcoma leukemia, lymphoma, myeloma, and sarcoma.

15. The use 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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