Oncolytic HSV-1 clinical isolates, directionally evolved strains, infectious clones and their uses

Abstract

This invention relates to oncolytic HSV-1 clinical isolates, directionally evolved strains, infectious clones, and their uses. Specifically, it provides isolated HSV-1 clinical virus strains that exhibit significantly superior tumor cell-killing ability compared to commercially available HSV-1 strains. It also provides evolved strains obtained by directionally evolving the clinical virus strain, infectious clones that carry the genetic information of the virus, and uses of the active substance for tumor control and as an exogenous gene vector.

Description

[Technical Field]

[0001] This invention belongs to the fields of biotechnology and medicine, particularly in the field of oncology. Specifically, this invention relates to newly isolated and / or directionally evolved HSV clinical virus strains, infectious clones, and their uses, which have excellent oncolytic properties. [Background technology]

[0002] Since the advent of molecular biology, viruses have gradually transformed from pathogenic microorganisms into vectors with a wide range of applications in the biopharmaceutical field. The ability of viruses to precisely invade specific tissue cells and efficiently express foreign genes within those cells is unmatched by other vectors such as lipid nanoparticles (LNPs), dendrimers, polymers, and exosomes. Supported by these characteristics, viral vectors are seeing increasing applications in gene therapy and cancer treatment. Among these, oncolytic viruses, which have developed based on the high specificity of viruses against cancer cells, represent a particularly promising hotspot in the current field of cancer treatment.

[0003] For decades, viruses belonging to more than a dozen different families have been modified into oncolytic viruses. Among these, herpes simplex virus (HSV) has attracted the most attention due to its characteristics such as a high amount of foreign gene loading, broad-spectrum killing ability against cancer cells, and reliable genetic stability, making it one of the oncolytic viruses with the most projects reaching clinical trials. Currently, T-Vec (talimogene laherparepvec), the only oncolytic virus approved and marketed by the FDA for the treatment of melanoma, is derived from a modified HSV oncolytic virus, and Delytact (teserpaturev / G47Δ), which is conditionally marketed in Japan for the treatment of glioblastoma, is also an HSV oncolytic virus. From the above, the value of HSV as an oncolytic virus is clear.

[0004] Common modifications to confer tumor-selective replication and killing ability to HSV include, but are not limited to, the deletion of non-essential genes such as ICP34.5, ICP6, and US47, with the deletion of the neurotoxic gene ICP34.5 being the most widely used. Deleting these non-essential genes reduces the toxicity of HSV to cancer cells, but the reduction in its killing ability to normal tissue cells is even greater. As a result, it becomes possible to achieve cancer tissue killing at doses that do not kill normal tissue cells, creating a so-called "treatment window." Furthermore, the released viral antigens and cancer cell antigens activate the immune system, enabling sustained killing of tumors.

[0005] However, while HSV oncolytic viruses are already on the market and many have progressed to the clinical research stage, the majority use laboratory strains such as strain F and strain 17+ that have been passaged in the laboratory for decades. These strains have undergone several mutations and have adapted to infecting and killing cells under artificial culture conditions in the laboratory, but their applicability to cancer tissue in clinical settings is not guaranteed. For example, laboratory strains are all passaged in rapidly dividing cell lines, and these cells basically double in number every two days, but the division rate of tumor cells in actual patients is extremely slow, and it may take one to two months for them to double. Also, in the laboratory, viruses are cultured and amplified in two-dimensional monolayer cells, but in reality, viruses need to replicate and spread within three-dimensional spherical tumor tissue. All of these crucial differences between clinical application and laboratory conditions mean that relying on laboratory strains inherently impairs the development and application of oncolytic viruses. For this reason, clinical strains directly isolated from clinical samples have gradually been used in the development of oncolytic viruses. For example, T-Vec was developed using the clinical isolate JS-1.

[0006] However, clinical strains also have drawbacks. For example, directly isolated clinical strains have evolved over long periods within normal human tissue cells, particularly nerve cells, and are adapted for cell killing in three-dimensional tissues, but their ability to kill cancer cells may not be optimal. Moreover, diversity exists among clinical strains, so a single clinical strain randomly isolated may not be suitable for oncolytic virus development. Therefore, it is necessary to isolate clinical strains and screen them to ensure the acquisition of superior clinical strains for subsequent oncolytic virus development.

[0007] The HSV genome is extremely large, reaching approximately 150kb, and late-stage modifications amount to a maximum of only 10kb of it. This means that in oncolytic virus development, only a small number of genes, accounting for less than 10% of the total genes, can be modified to change or improve its oncolytic properties, while the remaining 90% of HSV genes remain unmodifiable. These unmodified genes also determine a significant portion of the oncolytic properties, and this "innate oncolytic property" depends on the selected protovirus strain. For this reason, the selection of the initial HSV strain in oncolytic virus development is one of the most important steps in the process. [Overview of the project]

[0008] The strain related to this application overcomes the shortage of existing laboratory strains and conventional clinical strains. By screening multiple clinical strains, the strain with the strongest cancer cell killing ability is obtained, and further, the oncolytic properties are further improved through methods such as directional evolution within cancer cells, providing an oncolytic-enhancing HSV clinical strain.

[0009] This invention relates not only to the virus strain, but also to reverse genetics systems constructed based on the strain (including systems obtained using BAC technology and homologous recombination technology), as well as oncolytic viruses, vaccine strains, and vaccine vectors obtained by modifying the strain.

[0010] In some aspects of this application, isolated HSV-1 clinical virus strains are provided.

[0011] In some embodiments, the present application provides an isolated HSV-1 clinical virus strain named C1-0803-1-1-1, with deposit number CCTCC NO:V202351 at the China Typical Culture Depository Center (CCTCC).

[0012] In some aspects of this application, isolated HSV-1 clinical virus evolution strains are provided.

[0013] In some embodiments, the clinical viral evolution strain is obtained by directionally evolving the clinical viral strain of the present application in cancer cells. In some embodiments, the clinical viral strain is named C1-0803-1-1-1 and has a deposit number at CCTCC NO:V202351. In some embodiments, the present application provides an HSV-1 clinical viral evolution strain named C1-31110 and has a deposit number at CCTCC NO:V202335.

[0014] In some embodiments, the viral evolution strains of the present invention are obtained through isolation, purification, and amplification. In some embodiments, obtaining the viral evolution strains includes: serial passage of a clinical viral strain (e.g., 3 to 10 times, e.g., 5 times); screening the serially passaged viral strains based on the plaque phenotype after viral infection of Vero cells; isolating viral strains in which the formed plaques are single and larger than those formed by other viral strains; and optionally purifying the isolated viral strains.

[0015] In some aspects of the present application, an infectious clone is provided, which comprises a viral genome derived from a clinical viral strain or viral evolution strain of the present application and a bacterial artificial chromosome (BAC) clone vector backbone sequence inserted into the viral genome.

[0016] In some embodiments, the infectious clone of the present application comprises viral genomic DNA (e.g., the insertion site is viral genome UL37 / UL38) into which a BAC clone vector backbone sequence (e.g., pBeloBAC11-CMV-eGFP backbone sequence) is inserted.

[0017] In some embodiments, the plasmid map of the infectious clone of the present application is shown in FIG. 6. In some embodiments, an infectious clone named CB720 in the present application is provided, and the deposit number at CCTCC is CCTCC NO: V202352.

[0018] In some aspects of the present application, an induced virus strain is provided that is derived from the clinical virus strain, virus evolved strain or infectious clone of the present application, and the induced virus strain is obtained by genetically modifying the clinical virus strain, virus evolved strain or infectious clone.

[0019] In some embodiments, the genetic modification to the clinical virus strain, virus evolved strain or infectious clone includes, but is not limited to, one or more selected from the following group: (1) reverse genetic modification (e.g., modification to the viral genome of the infectious clone); (2) homologous recombination; and (3) modification by the Cre / LoxP site-specific recombination system.

[0020] In some embodiments, the genetic modification to the clinical virus strain, virus evolved strain or infectious clone includes, but is not limited to, one or more selected from the following group: Deletion of non-essential genes, such as ICP34.5, ICP6, ICP47, the gene encoding functional glycoprotein H, the gene encoding functional thymidine kinase; modifying the promoter of essential genes to replace it with a tumor-specific promoter to confer tumor selectivity for the virus to replicate only within cancer cells; modifying structural proteins such as gB, gD, gH of the virus to insert a domain targeting tumor-associated antigen (TAA) (scFv, VHH or other domains with specific binding ability) to specifically bind and infect cancer cells expressing TAA; and / or Introduction of heterologous genes that improve immune response and / or tumor suppression function (e.g., genes encoding immunostimulatory peptides (GM-CSF, cytokines or chemokines (CCL5, CCL20, CCL21), RNATES, B7.1, B7.2, IL-12, IL-15, HSP70), genes encoding prodrug-activating proteins (e.g., nitroreductase, cytochrome p450), genes encoding tumor suppressor peptides or tumor suppressor proteins (e.g., p53, TRAIL, anti-PD-1 antibody, endostatin), bispecific antibodies or immune killer cell engagers (T cell engagers, NK cell engagers)); and / or Introduction of heterologous genes (e.g., genes related to ECM degradation (hyaluronidase)) that improve virus replication and spread within tumors or modify the tumor microenvironment.

[0021] In some aspects of the present application, products including clinical virus strains, virus evolved strains, infectious clones and / or derived virus strains of the present application are provided.

[0022] In some embodiments, the products of the present application are used as pharmaceuticals or viral vector vaccines for the prevention and / or treatment of tumors, materials for the manufacture and / or research and development of pharmaceuticals or vaccines, and / or used as foreign gene vectors.

[0023] In some aspects of this application, the clinical virus strains, viral evolution strains, infectious clones and / or induced virus strains of this application are provided for use in pharmaceuticals or viral vector vaccines used for the prevention and / or treatment of tumors, or in the manufacture of materials used in the manufacture and / or research and development of pharmaceuticals or vaccines.

[0024] In some aspects of the present application, a method is provided for the prevention and / or treatment of a tumor, the method comprising administering a prophylactic and / or therapeutically effective dose of the clinical viral strain, viral evolutionary strain, infectious clone and / or induced viral strain of the present application to a subject of interest.

[0025] In some aspects of the present application, a method for producing antitumor-directed evolutionary HSV-1 virus strains is provided, the method comprising performing antitumor-directed evolutionary screening on the HSV-1 clinical virus strains of the present application.

[0026] In some aspects of the present application, a method for producing infectious clones is provided, which comprises providing a viral genome of the HSV-1 clinical virus strain and / or viral evolution strain of the present application and inserting a bacterial artificial chromosome (BAC) clone vector skeleton.

[0027] In some aspects of the present application, further methods are provided for producing recombinant viruses, the methods comprising further recombinant modification of the HSV-1 clinical virus strain, viral evolution strain and / or infectious clone of the present application.

[0028] In some embodiments of the present application, the tumor is selected from solid tumors or non-solid tumors. For example, the tumor includes, but is not limited to, breast cancer, liver cancer, throat cancer, lung cancer, and malignant glioma.

[0029] Those skilled in the art can combine the aforementioned technical solutions and technical features as they see fit without departing from the concept and scope of the invention. Other aspects of the invention will be obvious to those skilled in the art based on what is disclosed herein.

[0030] The present invention is further described below with reference to the accompanying drawings, and these expressions are intended solely to illustrate embodiments of the invention and not to limit the scope of the invention. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 shows infection of Vero cells with the isolated C1-0803-1-1-1 clinical virus strain: Figure 1A: Photograph of cytopathic effect (CPE) induced in Vero cells infected with the C1-0803-1-1-1 virus, taken with a Nikon camera under a 10x objective lens; Figure 1B: Photograph of plaques in Vero cells infected with the C1-0803-1-1-1 virus, taken with the GelDoc Imaging System. [Figure 2] Figure 2 shows the results of the evaluation of the killing capacity of clinical strains: Figure 2A: Comparison of the 72-hour killing capacity of isolated clinical strains C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0809-4-1-1 and the commercially available strain HSV-1 VR-733 (ATCC) against the human breast cancer cell line MCF7; Figure 2B: Comparison of the 48-hour killing capacity of isolated clinical strains C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0809-4-1-1 and the commercially available strain HSV-1 VR-733 (ATCC) against the human liver cancer cell line Hep3B. In the figure, significance analysis was performed using a t-test to compare the lethality of the clinical strain C1-0803-1-1-1 and the commercially available strain HSV-1 VR-733 (ATCC). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005, and **** indicates p<0.001. [Figure 3]Figure 3 shows infection of Vero cells by a strain selected during the process of directional evolution (C1-31110 directional evolution strain). Figure 3A: Photograph of cytopathic effect (CPE) induced in Vero cells infected with the C1-31110 virus. This image was taken with a Nikon camera under a 10x objective lens; Figure 3B: Photograph of plaques in Vero cells infected with the C1-31110 virus. This image was taken with the GelDoc Imaging System. [Figure 4] Figure 4 shows the results of the evaluation of the lethality of the evolved strains: Figure 4A: Comparison of 48-hour lethality of isolated evolved strains C1-31110, C1-51110, clinical strain C1-0803-1-1-1, and commercial strain HSV-1 VR-733 against human pharyngeal squamous cell carcinoma cell line FADU; Figure 4B: Comparison of 48-hour lethality of isolated evolved strains C1-31110, C1-51110, clinical strain C1-0803-1-1-1, and commercial strain HSV-1 VR-733 against human non-small cell lung cancer cell line NCI-H358; Figure 4C: Comparison of 48-hour lethality of isolated evolved strains C1-31110, C1-51110, clinical strain C1-0803-1-1-1, and commercial strain HSV-1 VR-733 against human embryonic lung fibroblast cell line MRC5. In the figure, a significance analysis was performed using a t-test to examine the lethality of the C1-31110 evolved strain and the commercially available HSV-1 VR733 strain. ns indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005, and **** indicates p<0.001. [Figure 5]Figure 5A: Comparison of 48-hour spheroid killing capacity of isolated evolutionary strain C1-31110, clinical strain C1-0803-1-1-1, and commercial strain HSV-1 VR-733 against malignant glioblastoma cells U87-MG 3D cultured at MOI=0.5, 1, 3, or 5. Each numbered box and the boxes to its left and right constitute a pair, with each pair representing the same MOI, and each MOI being duplicated three times. The figure consists of a series of 3D spheroid images taken with a high-content imaging device (Perkin Elmer Operetta CLS); Figure 5B: Comparison of 48-hour spheroid volume of isolated evolutionary strain C1-31110, clinical strain C1-0803-1-1-1, and commercial strain HSV-1 VR-733 against malignant glioblastoma cells U87-MG 3D cultured at MOI=0.5, 1, 3, or 5. In the figure, significance analysis was performed using a t-test, where ns indicates no significant difference, * indicates p<0.05, *** indicates p<0.005, and **** indicates p<0.001. [Figure 6] Figure 6 shows the plasmid map of infectious clones by the evolved strain BAC, specifically the map of the CB720 virus strain.

[0032] Deposit Information The following biological materials described in this application have been deposited with an international depositary organization for biological material samples certified by the State Intellectual Property Administration of China, and the specific deposit information is as follows: JPEG2026519708000001.jpg165160 [Modes for carrying out the invention]

[0033] Through extensive and in-depth research, the applicant isolated HSV-1 clinical virus strains from clinical samples of patients who developed diseases due to HSV-1 infection, and obtained specific clinical virus strains with high killing activity against tumor cells by performing viral plaque screening on these clinical virus strains; furthermore, after serial passage of the specific clinical virus strains and screening for antitumor activity, a directional evolutionary strain with excellent oncolytic properties was obtained; and, using reverse genetics techniques, the applicant also obtained an infectious clone that stably loads oncolytic virus genomic information. Thus, the applicant provides a promising new material for the development of HSV-based oncolytic viruses and has broad and excellent prospects in the prevention and / or treatment of tumors and the development of related pharmaceuticals.

[0034] In some embodiments of the present invention, the HSV-1 clinical strain and directionally evolved isolate virus of the present invention are used in the production of oncolytic viruses. In some embodiments of the present invention, the HSV-1 directionally evolved clinical isolate virus of the present invention is used in the production of therapeutic or prophylactic pharmaceuticals or vaccines. In some embodiments of the present invention, the HSV-1 directionally evolved clinical isolate virus of the present invention is used in the production of vaccine vectors.

[0035] In some embodiments, the present invention includes an oncolytic virus comprising the HSV-1 clinical strain or directional evolutionary isolate virus of the present invention. In some embodiments, the present invention includes an oncolytic virus obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a therapeutic or prophylactic HSV-1 vaccine comprising the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a therapeutic or prophylactic HSV-1 vaccine obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a pharmaceutical composition comprising an oncolytic virus obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a pharmaceutical composition comprising a vaccine strain obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a pharmaceutical composition comprising a vaccine obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention. In some embodiments, the present invention includes a pharmaceutical composition comprising a vaccine obtained by modifying the HSV-1 directional evolutionary clinical isolate virus of the present invention.

[0036] In other embodiments, the present invention includes a reverse genetics system constructed based on the HSV-1 directional evolution clinical isolate virus of the present invention. In some embodiments, the present invention includes infectious clones constructed based on BAC and homologous recombination techniques for the HSV-1 directional evolution clinical isolate virus of the present invention. In some embodiments, infectious clones of the HSV-1 directional evolution clinical isolate by BAC do not undergo foreign sequence deletions in multiple consecutive passages.

[0037] In some embodiments, the HSV-1 directional evolution clinical isolate virus of the present invention has a large plaque phenotype and cell membrane fusion ability. In some embodiments, compared to commercially available strains, the HSV-1 directional evolution clinical isolate virus of the present invention exhibits significantly enhanced killing ability against tumor cells under the same MOI. In some embodiments, compared to commercially available strains, the HSV-1 directional evolution clinical isolate virus of the present invention can effectively kill tumor cells in 3D cell spheroids that more closely resemble in vivo tumor structures.

[0038] All numerical ranges provided herein are intended to explicitly include all numerical values ​​between the endpoints of the range and the numerical ranges between them. Features mentioned in the invention or in the examples can be combined. All features disclosed herein can be used in combination with any configuration, and each feature disclosed herein can be replaced with any alternative function that can provide the same, equivalent, or similar purpose. Thus, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar functions.

[0039] As used herein, “contains,” “has,” or “includes” includes “contains,” “mainly consists of,” “basically consists of,” and “consists of”; “mainly consists of,” “basically consists of,” and “consists of” belong to the sub-containments of “contains,” “has,” or “includes.”

[0040] Isolated HSV-1 clinical virus strains and their evolutionary strains In some aspects of this application, isolated HSV-1 clinical oncolytic virus strains are provided.

[0041] In some embodiments, the HSV-1 virus is obtained by isolating herpes samples from individuals infected with herpes simplex virus, and the virus is isolated from, for example, lip and facial blister tissue exudate obtained from patients with oral herpes.

[0042] In some embodiments, plaque screening is performed on the virus, and the resulting high-titer strains are isolated, purified, amplified, and identified. In some embodiments, the selected clinical strains have significantly superior tumor cell-killing ability against multiple tumor cells compared to commercially available strains (e.g., HSV-1 VR733).

[0043] In some embodiments, the isolated HSV-1 clinical oncolytic virus strain was named C1-0803-1-1-1, exhibiting a viral titer on Vero cells nearly twice that of the commercially available HSV-1 strain VR733, and possessing significantly enhanced cytotoxic activity against multiple tumor cell lines.

[0044] In some embodiments of the present application, isolated HSV-1 clinical virus evolution strains are provided. In some embodiments, these strains are obtained by directionally evolving the clinical virus strains of the present application in cancer cells.

[0045] In some embodiments, obtaining the viral evolution strains of the present invention includes: serially passage the clinical oncolytic virus strains of the present invention (e.g., 3 to 20 times, e.g., 10, 8, 5 passages); screening the virus strains by plaque phenotype after viral infection of Vero cells to select virus strains in which the formed plaques are single and larger than those formed by other virus strains; and isolating and purifying the selected virus strains.

[0046] Through directional evolution as described in this invention, a specific HSV-1 clinical virus evolutionary strain named C1-31110 was obtained, which exhibits extremely significant cytotoxic activity against multiple tumor cells (even under low conditions of 0.01 MOI). Furthermore, this directional evolutionary strain also exhibits highly efficient cytotoxic activity against 3D morphological tumor cells.

[0047] Reverse genetics system In some aspects of this application, a reverse genetics system obtained by reverse genetics techniques is also provided. As used herein, the term “reverse genetics” refers to the rescue of live viruses or similar virus-like substances in cultured cells or susceptible hosts using viral genetic material. Genetic material capable of rescuing viruses is called an “infectious clone,” and generally contains a cDNA copy of the entire viral genome in a bacterial plasmid, with the cDNA itself or the RNA obtained by in vitro transcription from the cDNA being infectious. Viral reverse genetics systems can effectively study the structure and function of viral genes and virus-host interactions in vivo by directionally modifying the viral genome sequence and detecting the phenotype of the rescued, artificially modified virus. Reverse genetics systems and techniques allow for various modifications or alterations of the viral genome at the DNA level, and the effects of these genetic manipulations can be determined from the phenotypic changes when the virus is subsequently rescued. This enables the study of viral genome expression control, molecular mechanisms of viral pathogenicity, etc., and also makes it possible to obtain reduced-toxicity strains and develop novel vaccines.

[0048] The infectious clone of the present invention may include a viral genome derived from the clinical viral strain or viral evolution strain of the present invention and a bacterial artificial chromosome (BAC) clone vector skeleton inserted into the viral genome. For example, the infectious clone of the present invention includes viral genomic DNA (e.g., insertion site is viral genome UL37 / UL38) into which a BAC clone vector skeleton sequence (e.g., pBeloBAC11-CMV-eGFP skeleton sequence) is inserted.

[0049] In some embodiments, the plasmid map of the infectious clone of the present invention is shown in Figure 6. In some embodiments, the present invention provides an infectious clone named CB720, in which the exogenous gene loaded onto the clone may be stably present in the genome.

[0050] Further genetic modification of clinical virus strains, evolutionary strains, and infectious clones The clinical virus strains, evolved strains, and infectious clones of this application may further include or undergo genetic modification. For example, one or more genetic modifications selected from the following group may be performed on the clinical virus strain, the viral evolved strain, or the infectious clone: ​​(1) reverse genetic modification (e.g., modification of the viral genome of the infectious clone); (2) homologous recombination; and (3) modification by the Cre / LoxP site-directed recombination system.

[0051] In some embodiments, the gene modifications include, but are not limited to, one or more selected from the following groups: deletion of non-essential genes, e.g., ICP34.5, ICP6, ICP47, functional glycoprotein H coding genes, functional thymidine kinase coding genes; modification of the promoter of an essential gene to replace it with a tumor-specific promoter to confer tumor selectivity to the virus, allowing it to replicate only in cancer cells; modification of viral structural proteins such as gB, gD, gH to insert a domain that targets tumor-associated antigens (TAAs) (scFv, VHH, or other domains with specific binding ability) to allow the virus to specifically bind to and infect cancer cells expressing TAAs; and / or heterologous genes that improve immune response and / or tumor suppressor function. Introduction of genes encoding immunostimulatory peptides (GM-CSF, cytokines or chemokines (CCL5, CCL20, CCL21), RNATES, B7.1, B7.2, IL-12, IL-15, HSP70), prodrug-activated proteins (e.g., nitroreductase, cytochrome p450), tumor suppressor peptides or tumor suppressor proteins (e.g., p53, TRAIL, anti-PD-1 antibody, endostatin), bispecific antibodies, or immune killer cell engagers (T cell engagers, NK cell engagers)); and / or introduction of heterologous genes (e.g., ECM degradation-related genes (hyaluronidase)) that improve viral replication and spread within tumors or modify the tumor microenvironment.

[0052] As used herein, “deletion” or “removal” refers to the absence of some or all of a nucleotide sequence. Deletion of specific fragments in a nucleotide sequence can be achieved using common techniques in this art; for example, see “Molecular Cloning Experiment Manual” (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989). This publication describes how deleting ICP34.5, ICP6, and ICP47 in a viral vector improves the antitumor specificity of the recombinant virus and enhances its oncolytic ability.

[0053] Recombinant viruses containing a foreign target gene can be constructed by inserting a gene encoding a target protein, peptide, or fragment into a virus or viral vector. Insertion of a foreign target gene into the viral genome can also be performed according to known methods described in the literature (e.g., Graham FL et al., Proc. Natl. Acad. Sci. USA 91: 8802-8806 (1994), Miyake S. et al., Proc. Natl. Acad. Sci. USA 93: 1320-1324 (1996)).

[0054] In some embodiments, the exogenous target gene may include coding sequences for one or more antitumor molecules and / or antitumor effect-enhancing molecules. In some embodiments, one or more other antitumor molecules and / or antitumor effect-enhancing molecules include, but are not limited to,: coding sequences for immune checkpoint (e.g., PD1, PDL1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA) inhibitors, coding sequences for cytokines (e.g., IL-5, IL-2, IL-6, IL-24, GM-CSF, TNF-α, IFN-γ), suicide genes (e.g., multi-substrate deoxynucleoside kinase Dm-DNK, thymidine kinase (TK), cytosine deaminase (CD), and tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL)), etc.

[0055] Products and Methods The present invention further provides products (e.g., pharmaceuticals or kits) comprising, however, an effective amount of the HSV-1 clinical virus strains, evolutionary strains and / or infectious clones of this specification and a pharmaceutically or immunologically acceptable carrier. Where used herein, the terms “active substance” or “active substance of the present invention” may be used interchangeably to refer to the HSV-1 clinical virus strains, evolutionary strains, infectious clones and / or recombinant viruses of this application.

[0056] In preferred embodiments, the product can be used for the prevention or treatment of tumors. Tumors may be solid or non-solid tumors and include, but are not limited to, bladder cancer, liver cancer, lung cancer, breast cancer, melanoma, ovarian cancer, prostate cancer, kidney cancer, intestinal cancer, head and neck cancer, skin cancer, pancreatic cancer, colorectal cancer, squamous cell carcinoma, mesothelioma, hematological malignancies (e.g., leukemia), and nervous system malignancies (e.g., glioblastoma, neuroblastoma).

[0057] As used herein, the terms “contains” or “has” include “contains,” “substantially consists of,” and “consists of.” As used herein, a “pharmaceutically acceptable” ingredient is a substance that is applicable to humans and / or animals, does not cause excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio. As used herein, the term “effective amount” is an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0058] As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier used for administering therapeutic agents, and includes various excipients and diluents. The term refers to several pharmaceutical carriers that are not essential active ingredients themselves but are not excessively toxic after administration. Suitable carriers are well known to those skilled in the art. A comprehensive study of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0059] The composition may include pharmaceutically acceptable carriers, such as liquids, water, brine, glycerol, and ethanol. Furthermore, these carriers may also include auxiliary substances, such as fillers, disintegrants, lubricants, flow enhancers, foaming agents, wetting agents or emulsifiers, flavorings, and pH buffering agents. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous medium, typically at a pH of about 5–8, preferably about 6–8.

[0060] The active substance in the composition of the present invention constitutes 0.001 to 99.9% by weight of the total weight of the composition; preferably 1 to 95% by weight, more preferably 5 to 90% by weight, and still more preferably 10 to 80% by weight. The remainder consists of pharmaceutically acceptable carriers and their additives.

[0061] As used herein, the term “unit dosage form” refers to the preparation of the compositions of the present invention into dosage forms required for single-dose administration to facilitate administration, and includes, but is not limited to, a variety of solid preparations (such as tablets), liquid preparations, capsules, and sustained-release preparations.

[0062] In some embodiments of this disclosure, the composition is in a single dosage form or a multi-dosage form, and the content of the active substance therein is 10 6 -10 14 PFU / agent, for example 10 7 -10 12 PFU / agent, 10 8 -10 12It is a PFU / agent. In some embodiments of this disclosure, the composition disclosed is administered daily, every other day, every three days, weekly, every two weeks, every three weeks, monthly, every two months, or every six months, for example, 1 to 6 doses.

[0063] It should be understood that the effective dose of the active substance used may vary depending on the severity of the condition of the subject being administered or treated. The specific circumstances are determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, and desired effect), within the scope of a skilled physician's judgment.

[0064] The pharmaceuticals or active substances described herein may be in solid form (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets), liquid form (e.g., solutions), or other suitable form. The following routes of administration may be used: (1) direct injection, e.g., intravenous injection or perfusion, intraperitoneal injection, intratumoral injection, intracranial injection, etc.; (2) a method of linking recombinant viruses to transferrin / poly-L-lysine complexes to enhance their biological effects; (3) a method of complexing viruses with positively charged lipids; (4) a method of embedding recombinant adenoviruses in lipid bodies; (5) a method of transfecting viruses into carrier cells.

[0065] Furthermore, the compositions of the present invention may also contain other active substances used for the improvement and treatment of tumors. In some embodiments, the other active substances include, but are not limited to, one or more of the following: monoclonal antibody drugs, e.g., rituximab, trastuzumab, bevacizumab; cytotoxic agents, e.g., alkylating agents, mechloretamine, platinum-based mixtures, methotrexate, 5-FU, cytarabine, gemcitabine, actinomycin D, doxorubicin, irinotecan, topotecan, hydroxycamptothecin, paclitaxel, docetaxel, vincristine, navelbine, podophyllotoxins, homohalingtonin, etc.; and bioresponse modifiers, e.g., interferon, interleukin, thymosine, etc.

[0066] The active substances described herein can be used in combination with each other, and can also be used in combination with other pharmaceuticals and therapeutic means, for example, recombinant viruses can be used in combination with one or more therapies selected from the following groups: immunotherapy, gene therapy, chemotherapy, radiotherapy, and surgery. [Examples]

[0067] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are not intended to limit the scope of the present application, but are merely illustrative. Those skilled in the art can make appropriate modifications and variations to the present invention, all of which fall within the scope of the present invention.

[0068] The experimental methods in the following examples, which do not specify particular conditions, are usually carried out according to the standard conditions described in J. Sambrook et al., "Molecular Cloning: Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989)," or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts refer to weight.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those known to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this application. Preferred embodiments and materials described herein are for illustrative purposes only.

[0070] Example 1. Clinical sample collection 1: After washing the wounds of volunteers with oral herpes with saline solution, approximately 10 μl of tissue exudate was collected from the volunteers' lips and facial blisters using a sterile swab; 2: The swab was placed in a pre-prepared virus collection tube, and the virus collection tube containing the swab was frozen and stored in a -80°C freezer for virus culture and isolation; 3. After collection, the volunteers' wounds were disinfected with iodine to reduce the risk of viral infection.

[0071] Example 2. Isolation of clinical strains Small-scale amplification of clinical virus strains 1: Vero cells (African green monkey kidney cells, SAILY BIO) on a T75 plate are digested with trypsin, and after cell digestion, they are counted. The Vero cells are then transferred to a 6-well plate, with 10 cells per well. 6 The seeds were seeded individually and incubated overnight in a cell culture incubator at 37°C and 5% CO2. 2: After culturing the cells for one day, observation confirmed that the cells were a monolayer without voids; the virus collection tubes, which had been frozen and stored in a -80°C freezer, were removed and the virus samples were dissolved in a 37°C water bath; The cell supernatant was aspirated from a 3:6 well plate, 4 ml of lysed virus sample was pipetteed into the 6 well plate to infect the cells, and the 6 well plate was cultured in a cell culture incubator at 37°C and 5% CO2. The 6-well plates were gently mixed every 4:15 minutes to ensure more uniform virus infection of the Vero cells; 5. After 2 hours, the viral supernatant was aspirated and removed, 2 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was returned to a cell culture incubator at 37°C and 5% CO2 for incubation. 6. Morphological changes in virus-infected cells were observed, and when the majority of cells were infected, the supernatant and cells were collected in a 15 ml centrifuge tube and frozen in a -80°C freezer for reserve.

[0072] Isolation and purification of clinical virus strains 1: Based on the titer of small-scale amplified clinical strain viruses, the viruses were gradient-diluted with 2% DMEM to prepare 500 μl of virus dilution containing 1, 5, or 10 infectious virus particles; Observe the density of Vero cells in a 2:6 well plate (10 per well the day before). 6Individual seeds were seeded and cultured overnight in a 37°C, 5% CO2 cell culture incubator. When the Vero cells formed a monolayer with no intercellular spaces, the supernatant was removed, 500 μl of virus dilution was added to the cells, and the 6-well plates were cultured in a 37°C, 5% CO2 cell culture incubator. The 6-well plates were gently mixed every 3:15 minutes to ensure the virus was more uniformly adsorbed and infected the Vero cells; 4. After 1 hour and 15 minutes, 1 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was incubated in a 37°C, 5% CO2 cell culture incubator for a further 2 hours. 5. After 2 hours, discard the viral culture supernatant, add 500 μl of DMEM medium to each well to cover the cells, add 3 ml of methylcellulose on top to fix the virus, and return the 6-well plate to a 37°C, 5% CO2 cell culture incubator to continue culturing; 6. After culturing the virus for 3 days, visible viral CPE was observed, and 2 ml of neutral red was added to the cells in each well, and the cells were cultured overnight in an incubator. 7. The number of plaques in each well was observed on a white plate, and plaques were picked from the well with the fewest plaques. The virus isolation process was repeated 3-4 times, and purification was considered complete when a virus with only a single plaque in one well was selected.

[0073] The above steps yielded clinical virus strains C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, and C1-0809-4-1-1. Of these, the plaque of the clinical virus strain C1-0803-1-1-1 is shown in Figure 1. The plaque phenotypes of the other three isolated virus strains were also similar to those of C1-0803-1-1-1, except that Figure 1A is a CPE image of Vero cells infected with the C1-0803-1-1-1 (CCTCC NO: V202351) virus, and Figure 1B is a plaque image of Vero cells infected with the C1-0803-1-1-1 virus.

[0074] As a result, a clinical virus strain with an infection and killing effect on Vero cells was isolated and purified by the above method.

[0075] Example 3. Amplification and titer measurement of strains Virus amplification 1: Vero cells on a T75 plate were digested with trypsin, the cells were counted, and then 3×10 6 cells per plate were seeded onto a new T75 plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2; 2: 48 hours after overnight culture, the Vero cells on the T75 plate became a monolayer with no gaps between cells. The cells on one T75 plate were digested with trypsin, and the number of cells was designated as a; 3: When the clinical strain viruses of C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0809-4-1-1 and the commercially available virus HSV-1 VR-733 (ATCC) were used to infect Vero cells on a T75 plate at MOI = 0.02 PFU / cell, and the virus titer was b (PFU / ml), the required virus volume V (ml) for one plate was V = a×0.02 / b, and the final volume at the time of cell infection for each T75 plate was 5 ml; 4: The cell supernatant on the T75 plate was aspirated and removed, 5 ml of DMEM containing 2% FBS with the required amount of virus was added to infect the Vero cells, and the T75 plate after virus infection was placed in a cell culture incubator at 37°C and 5% CO2 for culture; 5: The T75 plate was gently shaken every 15 minutes to ensure that the virus adsorbed and infected the Vero cells uniformly; 6: After 1 hour and 15 minutes, the cell supernatant containing the virus was removed, 18 ml of medium was added to the cells on each T75 plate, and the cell plate was cultured in a cell culture incubator at 37°C and 5% CO2; 7: After overnight culture in the incubator, the state of virus-infected cells was observed under a microscope. After approximately 100% of the cells showed the CPE morphology, the cells were collected using a cell scraper, and the supernatant containing the cells was collected into a 50 ml centrifuge tube and stored frozen at -80°C.

[0076] potency measurement 1: Based on the estimated viral titer, the virus was gradient diluted with 2% DMEM to prepare a 500 μl viral dilution containing 30 to 100 infectious viral particles; Observe the density of Vero cells in a 2:6 well plate (10 per well the day before). 6 After seeding, the Vero cells were cultured overnight in a 37°C, 5% CO2 cell culture incubator. If the Vero cells formed a monolayer with no intercellular spaces, the supernatant was removed, 500 μl of virus dilution was added to each well, and the 6-well plates were cultured in a 37°C, 5% CO2 cell culture incubator. The 6-well plates were gently mixed every 3:15 minutes to ensure the virus was more uniformly adsorbed and infected the Vero cells; 4. After 1 hour and 15 minutes, 1 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was incubated in a 37°C, 5% CO2 cell culture incubator for a further 2 hours. 5. After 2 hours, discard the viral culture supernatant, add 500 μl of DMEM medium to each well to cover the cells, add 3 ml of methylcellulose on top to fix the virus, and return the 6-well plate to a 37°C, 5% CO2 cell culture incubator to continue culturing; 6: After culturing the virus for 3 days, visible viral CPE was observed. The viral culture supernatant was removed, and 300 μl of crystal violet staining solution containing fixative formalin was added to each well and stained at room temperature for 30 minutes. 7. After staining, the crystal violet stain was removed, and the cells were washed three times with PBS to completely remove the crystal violet stain and clarify the viral plaques. 8. The number of viral plaques in each well was counted on a white plate, and the viral titer (PFU / ml) was calculated as a × b × 1000 / 500 based on the viral dilution a and the number of viral particles b.

[0077] The viral titers of each clinically isolated virus strain (C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0803-4-1-1) and commercially available HSV-1 VR733 are shown in the table below: JPEG2026519708000002.jpg50160

[0078] The above results indicate that the screening method of this application yielded clinical virus strains with viral titers equivalent to or nearly double that of existing commercially available HSV-1 (i.e., C1-0803-1-1-1, CCTCC NO:V202351).

[0079] Example 4. Evaluation of the lethality of clinical strains 1: Tumor cells such as MCF7 (human breast cancer cells) and Hep3B (human liver cancer cells) on a T75 plate are digested with trypsin, the cells are counted, and then the tumor cells are transferred to a 96-well plate at a rate of 1 × 10⁶ per well. 4 The cells were seeded at 100 μl and cultured overnight in a cell culture incubator at 37°C and 5% CO2. 2: After overnight culture, the confluence of tumor cells on a 96-well plate reached approximately 90% after 24 hours; the tumor cells in one well were digested with trypsin and the cells were counted, and this number was denoted as 'a'. 3: Each clinical virus strain obtained by the methods of Examples 1 and 2 (C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0809-4-1-1, and HSV-1 VR733) was used to infect tumor cells on a 96-well plate at MOI = 10, 1, 0.1, or 0.01 PFU / cell. When the viral titer was b (PFU / ml), the required viral load V (ml) per well was V = a × 10 / b (for MOI = 10 PFU / cell), and the final volume of each well in the 96-well plate upon cell infection was 100 μl (three wells were duplicated). 4. At 24, 48, and 72 hours after viral infection, 20 μl of CCK8 detection reagent (enhanced CCK-8 reagent kit, Biyuntian, C0042) was added to each well and incubated in a 37°C incubator for approximately 2 hours (the specific incubation time was determined according to the change in the color of the culture medium). The OD value at 450 nm of the culture medium supernatant was measured, and cell viability was calculated (blank wells and cell wells were set up).

[0080] The results of evaluating the killing ability of isolated clinical strains C1-0803-1-1-1, C1-0803-4-1-2, C1-0809-1-1-1, C1-0809-4-1-1, and the commercially available strain HSV-1 VR-733 (ATCC) against tumor cells such as MCF7 and Hep3B are shown in Figures 2A and 2B, respectively. Compared with the commercially available strain HSV-1 VR733, clinical strain C1-0803-1-1-1 shows a remarkably enhanced killing ability against tumor cells under the same MOI.

[0081] Example 5. Directional evolution Continuous passage of viruses Observe the density of Vero cells in a 1:6 well plate (10 per well the day before). 6 Individual seeds were seeded and cultured overnight in a 37°C, 5% CO2 cell culture incubator. Once the spaces between cells were gone, the cell supernatant was removed, 1 μl of C1-0803-1-1-1 virus and 500 μl of 2% FBS DMEM were added to one well of a 6-well plate, and the 6-well plate was placed in a 37°C, 5% CO2 cell culture incubator to continue culturing. The 6-well plates were gently mixed every 2:15 minutes to ensure that the virus adsorbed and infected the Vero cells more uniformly; 3. After 1 hour and 15 minutes, 1 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was incubated in a 37°C, 5% CO2 cell culture incubator for a further 2 hours. Then, the supernatant was removed, 2 ml of DMEM containing 2% inactivated serum was added, and the culture was continued in a 37°C, 5% CO2 cell culture incubator. 4. The progression of the viral infection was observed, and the virus was collected when the infection reached 100% (approximately 48 hours); 5. The recovered virus was subjected to three freeze-thaw cycles at -80°C / 37°C, followed by centrifugation at 3000 rpm and 4°C for 10 minutes. The supernatant was collected and stored frozen at -80°C for use in subsequent viral infections. The above procedure steps were repeated to pass the virus through blind culling five times, and finally, the virus was screened and isolated.

[0082] Screening and isolation of evolved strains Plaque purification was performed using P5 generation viruses that had been blind-passed 1:5 times, and the virus was diluted to a specific ratio. 500 μl of the virus dilution was then placed on Vero cells in a 6-well plate (10 per well the day before). 6 The cells were seeded and incubated overnight in a 37°C, 5% CO2 cell culture incubator. The number of infective virus particles in each well was adjusted to 1, 5, 10, or 100, and the cells were incubated in a 37°C, 5% CO2 cell culture incubator. The 6-well plates were gently mixed every 2:15 minutes to ensure that the virus adsorbed and infected the Vero cells more uniformly; 3. After 1 hour and 15 minutes, 1 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was incubated in a 37°C, 5% CO2 cell culture incubator for a further 2 hours. 4. After 2 hours, discard the viral culture supernatant, add 500 μl of DMEM medium to each well to cover the cells, add 3 ml of methylcellulose on top to fix the virus, and return the 6-well plate to a 37°C, 5% CO2 cell culture incubator to continue culturing; 5. After culturing the viruses for 48 hours, 2 ml of neutral red was added to the cells in each well and stained overnight; the following day, the staining of the viruses was observed, and viruses exhibiting a single plaque in each well and a relatively large plaque phenotype were selected. The above procedure steps were repeated until a virus strain with a giant plaque phenotype and cell membrane fusion ability was isolated.

[0083] Directional evolution strains C1-31110 and C1-51110 were obtained, and the viral plaque of the directional evolution strain C1-31110 is shown in Figure 3. These results indicate that directional evolution yielded the virus C1-31110 (CCTCC NO:V202335), which possesses a large plaque phenotype and cell membrane fusion ability.

[0084] Example 6. Amplification and titer measurement of evolutionary virus strains Virus amplification 1: Digest Vero cells on a T75 plate with trypsin, count the cells, and then transfer 3 × 10⁶ Vero cells per plate to a new T75 plate. 6 The seeds were seeded individually and cultured overnight in a cell culture incubator at 37°C and 5% CO2. 2: After overnight culture, 48 hours later, the Vero cells on the T75 plate formed a monolayer with no intercellular spaces. The cells from one T75 plate were digested with trypsin, and the number of cells was denoted as 'a'. 3: When Vero cells on a T75 plate were infected with the virus at an MOI of 0.02 PFU / cell, and the viral titer was b (PFU / ml), the required viral volume V (ml) per plate was V = a × 0.02 / b, and the final volume of cells infected on each T75 plate was 5 ml. 4: The cell supernatant on the T75 plate was aspirated and removed, and 5 ml of DMEM containing 2% FBS with the required amount of virus was added to infect the Vero cells. The T75 plate after virus infection was cultured in a cell culture incubator at 37°C and 5% CO2. The T75 plate was gently shaken every 5:15 minutes to ensure that the virus adsorbed and infected the Vero cells uniformly; 6. After 1 hour and 15 minutes, the cell supernatant containing the virus was removed, 18 ml of culture medium was added to the cells in each T75 plate, and the cell plates were cultured in a cell culture incubator at 37°C and 5% CO2. 7. After culturing in an incubator overnight, the condition of the virus-infected cells was observed under a microscope. Once approximately 100% of the cells exhibited the CPE morphology, the cells were collected using a cell scraper, and the supernatant containing the cells was collected in a 50 ml centrifuge tube and frozen at -80°C.

[0085] potency measurement 1: Based on the estimated viral titer, the virus was gradient diluted with 2% DMEM to prepare a 500 μl viral dilution containing 30 to 100 infectious viral particles; Observe the density of Vero cells in a 2:6 well plate (10 per well the day before). 6 After seeding, the Vero cells were cultured overnight in a 37°C, 5% CO2 cell culture incubator. If the Vero cells formed a monolayer with no intercellular spaces, the supernatant was removed, 500 μl of virus dilution was added to each well, and the 6-well plates were cultured in a 37°C, 5% CO2 cell culture incubator. The 6-well plates were gently mixed every 3:15 minutes to ensure the virus was more uniformly adsorbed and infected the Vero cells; 4. After 1 hour and 15 minutes, 1 ml of DMEM containing 2% inactivated serum was added to each well, and the 6-well plate was incubated in a 37°C, 5% CO2 cell culture incubator for a further 2 hours. 5. After 2 hours, discard the viral culture supernatant, add 500 μl of DMEM medium to each well to cover the cells, add 3 ml of methylcellulose on top to fix the virus, and return the 6-well plate to a 37°C, 5% CO2 cell culture incubator to continue culturing; 6: After culturing the virus for 3 days, visible viral CPE was observed. The viral culture supernatant was removed, and 300 μl of crystal violet staining solution containing fixative formalin was added to each well and stained at room temperature for 30 minutes. 7. After staining, the crystal violet stain was removed, and the cells were washed three times with PBS to completely remove the crystal violet stain and clarify the viral plaques. 8. The number of viral plaques in each well was counted on a white plate, and the viral titer (PFU / ml) was calculated as a × b × 1000 / 500 based on the viral dilution a and the number of viral particles b.

[0086] The results showed that the viral titer of the directional evolution strain C1-31110 was 2.11E8 PFU / ml, and the viral titer of C1-51110 was 2.5E9 PFU / ml.

[0087] Example 7. Testing the lethality of evolved strains. 1: Tumor cells such as FADU (human pharyngeal squamous cell carcinoma cells) and NCI-H358 (human non-small cell lung cancer cells) and MRC5 cells on a T75 plate were digested with trypsin, and the cells were counted. Then, FADU cells, NCI-H358 cells and other tumor cells and MRC5 cells were placed in a 96-well plate at a rate of 1 × 10⁶ cells per well. 4 The cells were seeded at 100 μl and cultured overnight in a cell culture incubator at 37°C and 5% CO2. 2: After overnight culture, the confluence of tumor cells on a 96-well plate reached approximately 90% after 24 hours; the tumor cells in one well were digested with trypsin and the cells were counted, and this number was denoted as 'a'. 3: When the virus was used to infect each tumor cell and MRC5 cell on a 96-well plate at MOI = 3, 1, 0.1, 0.05, or 0.01 PFU / cell, and the viral titer was b (PFU / ml), the required viral load V (ml) per well was V = a × 10 / b (for MOI = 10 PFU / cell), and the final volume of each well in the 96-well plate upon cell infection was 100 μl (three wells were duplicated). 4. 24, 48, or 72 hours after viral infection, 20 μl of CCK8 detection reagent was added to each well and incubated in a 37°C incubator for approximately 2 hours (the specific incubation time was determined based on the color change of the culture medium). The OD value of the culture medium supernatant at 450 nm was measured, and cell viability was calculated (blank wells and cell wells were set up).

[0088] The results of the evaluation of the killing capacity of the evolved C1-31110 and C1-51110 strains, the clinical strain C1-0803-1-1-1, and the commercially available HSV-1 VR-733 strain against FADU, NCI-H358, and MRC5 cells are shown in Figures 4A, 4B, and 4C, respectively. Compared to the commercially available HSV-1 VR733 strain, the evolved C1-31110 strain showed a remarkably enhanced killing capacity against tumor cells under the same MOI. While the evolved C1-51110 strain and the clinical strain C1-0803-1-1-1 strain also showed some improvement in tumor cell killing capacity, the killing capacity of the evolved C1-31110 strain against embryonic lung fibroblasts (MRC5) did not differ significantly from that of commercially available HSV-1 overall.

[0089] Example 8. Testing the lethality of 3D cells of the evolved cell line. 1: Place 6 × 10⁶ U87-MG cells (glioblastoma cells) in each well of a ULA 96-well plate. 3 Individual MRC5 cells (human embryonic lung fibroblasts, used to form 3D structures through co-culture with cancer cells) 3 × 10 3 Individual cells were inoculated and used in complete culture medium (10% DMEM + 1% PS); 2: Centrifuge 300g at room temperature for 5 minutes, then culture at 37°C and 5% CO2 for 48 hours to form spheroids. 3: Number of spheroid cells: 2 × 10 4 The cells were estimated to be one, and each cell was infected with the C1-31110, C1-0803-1-1-1, and HSV-1 VR733 viruses at MOI = 0.5, 1, 3, or 5 PFU / cell; 4: 3D spheroids were imaged using a high-content imaging system (Perkin Elmer Operetta CLS), and their volume was measured. 5. The viral infection status was photographed daily, and the spheroid volume was tallied 48 hours after infection.

[0090] The results are shown in Figure 5. The results indicate that the directional evolution strain C1-31110 of this application can effectively kill tumor cells in 3D cell spheroids that are more closely related to the in vivo tumor structure, 48 hours after viral infection, compared to HSV-1 VR733.

[0091] Example 9. Construction of infectious clones using BAC of evolved strains 1: Construction and linearization of repair donor DNA The pBeloBAC11 plasmid (Toyoki Biological; ZT178) was used as a clonal vector, and the loxP site and lacZα-inducible expression cassette (vector amplification primers pBAC_HomF (SEQ ID NO:4); pBAC_HomR (SEQ ID NO:5)) originally present in the vector were replaced with a CMV-eGFP expression cassette (template sequence shown in SEQ ID NO:1, amplification primers CMV_HomF (SEQ ID NO:2); bGH_HomR (SEQ ID NO:3)). Transformation was then performed using InFusion ligation (2×EasyGeno recombinant reagent kit, Tiangen, catalog number: VI201-02) to obtain the pBeloBAC11-CMV-eGFP intermediate vector.

[0092] Using the extracted evolutionary strain genomic DNA as a template, two sequences of approximately 1000 bp corresponding to the UL37 gene (primers: 37BAC_loxP_F (SEQ ID NO:6); 37BAC_PacI_R (SEQ ID NO:7)) and the UL38 gene (primers: 38BAC_PacI_F (SEQ ID NO:8); 38BAC_loxp_R (SEQ ID NO:9)) on both sides of the target were amplified and edited as homology arms using long-chain primers, and loxP sequences aligned in the same direction as the PacI cleavage sites were added to both ends using the primers. Furthermore, the homology arms at both ends were ligated by overlap PCR to obtain homology arm sequences having the structure loxP-UL37Arm-PacI-UL38Arm-loxP.

[0093] The obtained homology arm sequences were constructed downstream of the eGFP expression cassette in the intermediate vector (vector amplification primers pBAC-loxP-VF2 (SEQ ID NO:10); pBAC-loxp-VR2 (SEQ ID NO:11)) to obtain pBeloBAC11-CMV-eGFP-37.38 donor clones. After plasmid extraction, the clones were digested with PacI restriction enzymes and purified to obtain linearization repair donor DNA containing the BAC backbone.

[0094] 2: Recombination of the BAC scaffold into clinical strain genomes The day before, 293A cells were placed in 24-well plates in 2x10⁶ rows. 5 Cells were seeded at 500 μl / well and transfected with 1 μg of linearized BAC scaffold repair donor DNA and 1 μg of PX330-sgRNA plasmid (PX330 plasmid: Toyoki Seibutsu-BR613; sgRNA sequence (SEQ ID NO: 12)) using the liposome transfection reagent Lipofectamine 3000 (Invitrogen, catalog number L3000-008). After transfection, cells were cultured in a cell culture incubator at 37°C and 5% CO2, and after 6 hours, the medium was replaced with fresh medium supplemented with SCR7 (MCE, catalog number HY-107845) (final concentration 10 μM). 24 hours after transfection, the cells were infected with the evolutionary strain C1-31110 at MOI=0.01 and cultured in a cell culture incubator at 37°C and 5% CO2 while maintaining a constant concentration of 10 μM SCR7 in the culture system. After 24 hours, the cells and culture supernatant were collected and a recombinant virus P0 generation sample was obtained by repeating freeze-thaw cycles three times at -80°C / 37°C. This sample contained a large amount of wild-type virus and a very small amount of successfully recombinant virus, and further screening and purification are required.

[0095] 3: Crude screening of recombinant viruses Place Vero cells (1 x 10⁶ cells per well) into a 6-well plate. 6Cells were seeded at 1.5 ml / cell, and the recombinant virus obtained in the previous round was gradient-diluted 10-fold and used to infect Vero cells in a 6-well plate. After incubation for 2 hours, the medium was changed to one containing 1% methylcellulose, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2. After approximately 24 hours, viral plaques with GFP fluorescence signals were picked up using a fluorescence microscope, and the next generation of viral samples P were obtained. n+1 The results obtained were as follows: Screening of recombinant viruses in 6-well plates requires multiple rounds, and after reaching the stage where most infected plaques show fluorescence, monoclonal selection of the virus can be performed.

[0096] 4. Monoclonal selection of recombinant viruses 8 x 10⁶ Vero cells per well in a 96-well plate. 3 Cells were seeded at a concentration of 100 μl and cultured overnight in an incubator. Recombinant virus samples purified by crude screening were limitingly diluted 2 to 5 times, and 100 μl was added to each well to infect Vero cells. These cells were then cultured in a cell culture incubator at 37°C and 5% CO2. After approximately 24 hours, fluorescent plaques were observed under a fluorescence microscope, their locations were marked, and the culture was continued until the CPE reached 50%. The virus samples were then collected. During collection, viral monoclones were selected from the group with the highest possible dilution.

[0097] 5. Identification of recombinant viral monoclones Small amounts (approximately 50-100 μl) were taken from the total sample, and DNA was extracted from selected recombinant viral monoclonal samples using a viral DNA extraction kit. Target sites and the BAC backbone were identified and sequenced by PCR. Based on the sequencing results, sequence alignment was performed, and a viral monoclon in which the pBeloBAC11-CMV-eGFP backbone sequence was actually inserted at the UL37 / UL38 site of the viral genome was selected as the BAC-infectious clone of the formally evolved strain, named CB720 (CCTCC NO: V202352). This clone can be amplified, cryopreserved, and used. The plasmid map of the BAC-infectious clone CB720 of the strain is shown in Figure 6.

[0098] 6: Measurement of monoclonal performance -- Genomic stability of exogenous sequences The viral monoclonal selected in Step 5 was designated as SP0 (Successive Passage), and gradient-infected in a 6-well plate pre-seed with Vero cells. The cells were then cultured in a CO2 incubator for 1-2 days. Wells with a mild infection state (cytopathic effect CPE of approximately 10-30%, with each plaque independently dispersed and not yet fused) were selected, and the culture supernatant was collected and designated as SP0. n+1 We obtained the following results. Furthermore, we infected new Vero cells in the same way and performed serial passaging. During serial passaging, we observed the fluorescence of the plaques using a fluorescence microscope and focused on confirming whether or not the fluorescence had disappeared. No disappearance of fluorescence was observed in eight consecutive generations of passaging.

[0099] 100 μl of supernatant was collected from each generation of SP0, SP1, SP4, and SP8, and DNA samples were extracted using a viral DNA extraction kit. Fragments of UL37Arm (primer UL37_iF3 (SEQ ID NO:13); sopC-iR (SEQ ID NO:14)) and UL38Arm (primer eGFP_BACinf_F (SEQ ID NO:15); UL38-iR3 (SEQ ID NO:16)), which are recombination junctions between the exogenous BAC backbone sequence and the virus's original sequence, were extracted, as well as sopB-repE (primer sopB-iF2 (SEQ ID NO:17); repE-iR1 (SEQ ID NO:18)) and ChlR+ (primer BAC_seque-iF (SEQ ID NO:19); ChlR+_iR3 (SEQ ID NO:19)), which are characteristic of the BAC backbone, and ChlR+ (primer BAC_seque-iF (SEQ ID NO:19); ChlR+_iR3 (SEQ ID NO: PCR identification of fragment NO:20)) revealed no deletion of the exogenous BAC skeletal sequence in eight consecutive passages.

[0100] 7: Functional Test Functional tests confirmed that the evolved BAC-mediated infectious clone CB720 can efficiently rescue viruses and produce recombinant viruses with excellent oncolytic activity, and that the use of the evolved infectious clone CB720 enables convenient and efficient genetic modification.

[0101] All references cited herein are cited and referred to herein so as to be cited alone as references. It should be understood that, based on the foregoing disclosure of this application, a person skilled in the art may make various changes or modifications to this application, and these equivalent forms are also included within the scope defined in the claims attached to this application.

[0102] Note: The sequence numbers, sequence names, and specific sequences of the nucleic acid molecules used in each step of each example are as follows: JPEG2026519708000003.jpg234143 JPEG2026519708000004.jpg233143

Claims

1. An isolated HSV-1 clinical virus evolution strain, named C1-31110, characterized by its deposit number at the China Center for Typical Cultures Depository (CCTCC) being CCTCC NO: V202335.

2. The aforementioned viral evolution strains are obtained by directional evolution of HSV-1 clinical virus strains; and / or The aforementioned clinical virus strains were isolated from the exudate of oral and facial blister tissues of patients with herpes simplex; and / or The aforementioned clinical virus strain is named C1-0803-1-1-1, and its depositary number at the CCTCC is CCTCC NO: V202351; and / or The viral evolution strain in question has undergone isolation, purification, and amplification. The viral evolution strain described in feature 1.

3. The acquisition of the aforementioned viral evolution strain is characterized by comprising: serial passage of a clinical viral strain (e.g., 3 to 10 times, e.g., 5 times); screening the serially passaged viral strains based on the plaque phenotype after viral infection of Vero cells; isolating viral strains in which the formed plaques are single and larger than those formed by other viral strains; and optionally purifying the isolated viral strain.

4. An isolated HSV-1 clinical virus strain, named C1-0803-1-1-1, with the CCTCC storage number being CCTCC NO: V202351.

5. An infectious clone characterized by comprising a clinical viral evolution strain described in any one of claims 1 to 3, or a viral genome derived from a clinical viral strain described in claim 4, and a bacterial artificial chromosome (BAC) clone vector skeleton sequence inserted into the viral genome.

6. The BAC clone vector includes a backbone sequence (e.g., pBeloBAC11-CMV-eGFP backbone sequence) inserted into viral genomic DNA (e.g., insertion site is viral genomic UL37 / UL38); and / or Plasmid maps are shown in Figure 6; and / or It was named CB720, and its deposit number at the CCTCC is CCTCC NO: V202352. An infectious clone as described in claim 5.

7. A clinical viral evolution strain according to any one of claims 1 to 3, a clinical viral strain according to claim 4, or an induceable viral strain derived from an infectious clone according to claim 5 or 6, obtained by performing one or more genetic modifications selected from the following group on the clinical viral strain, the viral evolution strain, or the infectious clone: ​​(1) reverse genetic modification (e.g., modification of the viral genome of the infectious clone); (2) homologous recombination; and (3) modification by a Cre / LoxP site-specific recombination system.

8. The aforementioned genetic modification is Deletion of non-essential genes, e.g., ICP34.5, ICP6, ICP47, functional glycoprotein H coding genes, functional thymidine kinase coding genes; and / or Modifying the promoters of essential genes and replacing them with tumor-specific promoters to confer tumor selectivity to the virus, allowing it to replicate only in cancer cells; and / or Modifying the structural proteins of the virus, such as gB, gD, and gH, to insert domains that target tumor-associated antigens (TAAs) (scFv, VHH, or other domains with specific binding ability), thereby specifically binding and infecting cancer cells that express TAAs; The introduction of heterologous genes that improve immune response and / or tumor suppressor function (e.g., immunostimulatory peptide (GM-CSF), cytokine or chemokine (CCL5, CCL20, CCL21), RNATES, B7.1, B7.2, IL-12, IL-15, HSP70) coding genes, prodrug-activated protein (e.g., nitroreductase, cytochrome p450) coding genes, tumor suppressor peptide or tumor suppressor protein (e.g., p53, TRAIL, anti-PD-1 antibody, endostatin) coding genes, bispecific antibodies or immune killer cell engagers (T cell engagers, NK cell engagers)); and / or The introduction of heterologous genes (e.g., ECM degradation-related genes (hyaluronides)) to improve viral replication and spread within tumors or to modify the tumor microenvironment; An inducement virus strain according to claim 7, characterized by comprising one or more selected from the above.

9. A product characterized by comprising a clinical viral evolution strain described in any one of claims 1 to 3, a clinical viral strain described in claim 4, an infectious clone described in claim 5 or 6, and / or an induced viral strain described in claim 7 or 8.

10. The aforementioned products are pharmaceuticals or viral vector vaccines used for the prevention and / or treatment of tumors, materials used in the manufacture and / or research and development of pharmaceuticals or vaccines, and / or products used as exogenous gene vectors. For example, the tumor is selected from solid tumors such as bladder cancer, liver cancer, lung cancer, breast cancer, melanoma, ovarian cancer, prostate cancer, kidney cancer, intestinal cancer, head and neck cancer, skin cancer, pancreatic cancer, colorectal cancer, squamous cell carcinoma, mesothelioma, myeloma, osteosarcoma, thyroid cancer, cervical cancer, and bile duct cancer; non-solid tumors such as hematological malignancies (e.g., leukemia), lymphoma; and nervous system tumors (e.g., glioblastoma, neuroblastoma); preferably, the tumor is selected from breast cancer, liver cancer, throat cancer, lung cancer, and malignant glioma. The product according to feature 9.

11. Uses of the clinical viral evolution strain described in any one of claims 1 to 3, the clinical viral strain described in claim 4, the infectious clone described in claim 5 or 6, and / or the induced viral strain described in claim 7 or 8, in pharmaceuticals or viral vector vaccines used for the prevention and / or treatment of tumors, materials used for the manufacture and / or research and development of pharmaceuticals or vaccines, and / or in the manufacture of exogenous gene vectors. For example, the tumor is selected from solid tumors such as bladder cancer, liver cancer, lung cancer, breast cancer, melanoma, ovarian cancer, prostate cancer, kidney cancer, intestinal cancer, head and neck cancer, skin cancer, pancreatic cancer, colorectal cancer, squamous cell carcinoma, mesothelioma, myeloma, osteosarcoma, thyroid cancer, cervical cancer, and bile duct cancer; non-solid tumors such as hematological malignancies (e.g., leukemia), lymphoma; and nervous system tumors (e.g., glioblastoma, neuroblastoma); preferably, the tumor is selected from breast cancer, liver cancer, pharyngeal cancer, lung cancer, and malignant glioma.

Images