Oncolytic viruses reshaping the tumor microenvironment and their applications

Abstract

The present application relates to oncolytic viruses reshaping tumor microenvironment and application thereof. Specifically, the present application provides a novel engineered oncolytic virus carrying a coding gene of a fusion protein of dominant negative transforming growth factor-beta type II receptor (DN TGF-beta RII) and IL-10 and anti-PD-1 antibody, which can simultaneously play the effects of regulating tumor microenvironment and activating immune cells, and the action molecules have a prolonged half-life, significantly improving the anti-tumor effect of the engineered oncolytic virus. Experimental results show that the engineered oncolytic virus of the present application exhibits excellent tumor inhibition effect on various tumors, and has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to oncolytic viruses that reshape the tumor microenvironment and their applications. Background Technology

[0002] Traditional cancer treatments (including surgery, radiotherapy, and chemotherapy) have significant drawbacks, such as substantial side effects and a high recurrence rate. Oncolytic virus therapy, as a novel immunotherapy approach, works through a dual mechanism of "direct oncolysis + immune activation," making it a hot research topic in the field of cancer treatment.

[0003] Adenovirus (ADV) is a non-enveloped double-stranded DNA virus with advantages such as high infectivity, broad tissue tropism (capable of infecting various tumor cells), and easy genome modification, making it a classic vector for oncolytic viruses. However, wild-type ADV exhibits strong cytotoxicity to normal cells (such as hepatocytes and respiratory epithelial cells) and poor selectivity for tumor cells, which significantly limits its clinical application.

[0004] The development of engineered oncolytic adenoviruses (ADVs) stems from the natural characteristics of adenoviruses and breakthroughs in genetic engineering technology. Its aim is to address the shortcomings of traditional oncolytic viruses, such as poor targeting, low safety, and limited immune activation capabilities. With the continuous development of gene editing technologies such as CRISPR / Cas9 and homologous recombination, researchers can precisely modify the ADV genome, for example, by deleting pathogenic genes, inserting immune-regulating genes, and modifying target receptors, thereby endowing it with characteristics such as tumor-selective replication, immune activation, and targeted delivery. In 2005, my country approved recombinant human type 5 ADV (Ankerui), becoming the world's first marketed oncolytic adenovirus drug, marking the formal entry of engineered ADVs into the clinical application stage.

[0005] However, the immunosuppressive barrier of the tumor microenvironment limits the efficacy of oncolytic viruses. Solid tumors typically have a highly immunosuppressive microenvironment. After entering the tumor, the anti-tumor immune response induced by oncolytic viruses is often rapidly suppressed or depleted by this microenvironment, resulting in their inability to effectively activate systemic and sustained anti-tumor immunity, and thus limiting their control over distant metastases.

[0006] Therefore, there is an urgent need in this field to develop engineered oncolytic viruses that have the ability to reshape the tumor microenvironment, thereby enhancing their tumor-suppressing effects. Summary of the Invention

[0007] The purpose of this invention is to provide engineered oncolytic viruses with the ability to reshape the tumor microenvironment, as well as their preparation and application.

[0008] In a first aspect of the invention, a recombinant oncolytic virus is provided, the genome of which contains an exogenous expression cassette containing a coding sequence of an exogenous protein selected from the group consisting of: dominant-negative transforming growth factor-βII receptor (DN TGF-βRII), interleukin-10 (IL-10) or an active fragment thereof, PD-1 / PD-L1 inhibitors, or combinations thereof.

[0009] In another preferred embodiment, the PD-1 / PD-L1 inhibitor is an anti-PD-1 antibody.

[0010] In another preferred embodiment, the exogenous protein includes a fusion protein of IL-10 and an anti-PD-1 antibody.

[0011] In another preferred embodiment, the exogenous protein comprises: (1) a dominant-negative transforming growth factor-βII receptor (DNTGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody.

[0012] In another preferred embodiment, the exogenous protein is: (1) DN TGF-βRII, and (2) a fusion protein of IL-10 and anti-PD-1 antibody.

[0013] In another preferred embodiment, the DN TGF-βRII is a membrane-bound DN TGF-βRII.

[0014] In another preferred embodiment, the membrane-bound DN TGF-βRII comprises an extracellular region, a transmembrane region, and a truncated intracellular region.

[0015] In another preferred embodiment, the amino acid sequence of the extracellular region of the DN TGF-βRII is shown in SEQ ID NO: 5.

[0016] In another preferred embodiment, the amino acid sequence of the transmembrane region of the DN TGF-βRII is shown in SEQ ID NO: 6.

[0017] In another preferred embodiment, the amino acid sequence of the truncated intracellular region of the DN TGF-βRII is shown in SEQ ID NO:7.

[0018] In another preferred embodiment, the amino acid sequence of the DN TGF-βRII is as shown in SEQ ID NO: 14 or 15.

[0019] In another preferred embodiment, the amino acid sequence of the IL-10 is shown in SEQ ID NO: 9.

[0020] In another preferred embodiment, the anti-PD-1 antibody is pembrolizumab, nivolumab, retifanlimab, sintilimab, toripalimab, camrelizumab, tislelizumab, penpulimab, zimberelimab, pucotenlimab, or serplulimab.

[0021] In another preferred embodiment, the anti-PD-1 antibody is an anti-PD-1 single-chain antibody (scFv).

[0022] In another preferred embodiment, the sequence of the anti-PD-1 single-chain antibody includes: a heavy chain variable region (VH), a light chain variable region (VL), and a linker Linker1 located between VH and VL.

[0023] In another preferred embodiment, the VL sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID NO: 11.

[0024] In another preferred embodiment, the VH sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID NO: 12.

[0025] In another preferred embodiment, the sequence of the bit Linker1 is (G4S)n, where n is an integer selected from 2 to 6; preferably, n=3.

[0026] In another preferred embodiment, the amino acid sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID NO: 16.

[0027] In another preferred embodiment, in the fusion protein of IL-10 and anti-PD-1 antibody, IL-10 and anti-PD-1 single-chain antibody are linked by linker 2.

[0028] In another preferred embodiment, the fusion protein of IL-10 and anti-PD-1 antibody comprises, from the N-terminus to the C-terminus: IL-10, linker 2, and anti-PD-1 single-chain antibody; or the structure from the N-terminus to the C-terminus comprises: anti-PD-1 single-chain antibody, linker 2, and IL-10.

[0029] In another preferred embodiment, the Linker2 is a flexible connector; preferably, the connector L is a flexible connector containing G and S.

[0030] In another preferred embodiment, the sequence of the connector L is shown in SEQ ID NO: 10.

[0031] In another preferred embodiment, the full-length amino acid sequence of the fusion protein of IL-10 and anti-PD-1 antibody is shown in SEQ ID NO: 17.

[0032] In another preferred embodiment, the exogenous expression cassette contains a tumor-specific promoter.

[0033] In another preferred embodiment, the tumor-specific promoter is selected from the group consisting of: Survivin promoter, hTERT promoter, AFP promoter, GFAP promoter, Nestin promoter, or combinations thereof.

[0034] In another preferred embodiment, the coding sequence of the exogenous protein is located in the same exogenous expression cassette or in different exogenous expression cassettes; preferably, it is located in the same exogenous expression cassette.

[0035] In another preferred embodiment, the exogenous expression cassette has a structure as shown in Formula I: PE-L1-S-C1-L2-C2-T (Formula I) In the formula, Each "-" can be used independently as a key or connector; P contains a tumor-specific promoter; E is the coding sequence for the E1A protein; L1 is the coding sequence for a peptide that is absent or linked; S represents the coding sequence of the signal peptide or no signal peptide; C1 is the coding sequence of DN TGF-βRII; L2 is the coding sequence for a peptide that is absent or linked; C2 is the coding sequence for the fusion protein of IL-10 and anti-PD-1 antibody; T represents no or a termination element.

[0036] In another preferred embodiment, L1 and L2 are each independently coding sequences of the linker peptide; preferably, the linker peptide is a self-cleaving linker peptide.

[0037] In another preferred embodiment, the self-cleaving linker peptide includes: P2A, T2A, E2A, and F2A.

[0038] In another preferred embodiment, the amino acid sequence of P2A is shown in SEQ ID NO: 3; and the amino acid sequence of T2A is shown in SEQ ID NO: 8.

[0039] In another preferred embodiment, S is a signal peptide; preferably, it is a TGF-βRII signal peptide; preferably, the sequence of the TGF-βRII signal peptide is as shown in SEQ ID NO: 4.

[0040] In another preferred embodiment, T comprises a PolyA element.

[0041] In another preferred embodiment, the oncolytic virus is selected from the group consisting of: adenovirus, vaccinia virus, reovirus, measles virus, herpes simplex virus, parvovirus, chicken anemia virus, measles virus, Coxsackie virus, vesicular stomatitis virus, and Newcastle disease virus.

[0042] In another preferred embodiment, the oncolytic virus is an adenovirus.

[0043] In another preferred embodiment, the oncolytic virus is a modified adenovirus.

[0044] In another preferred embodiment, the adenovirus includes AD5 adenovirus.

[0045] In another preferred embodiment, the modified adenovirus has the pathogenic gene deleted.

[0046] In another preferred embodiment, the pathogenic gene includes E3 and E1B.

[0047] In a second aspect of the invention, a vector is provided comprising an exogenous expression cassette, the exogenous expression cassette comprising a promoter and a coding sequence of an exogenous protein, the exogenous protein being selected from the group consisting of: dominant-negative transforming growth factor-βII receptor (DN TGF-βRII), interleukin-10 (IL-10) or an active fragment thereof, PD-1 / PD-L1 inhibitors, or combinations thereof; wherein the promoter is a tumor-specific promoter.

[0048] In another preferred embodiment, the exogenous protein comprises: (1) a dominant-negative transforming growth factor-βII receptor (DNTGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody.

[0049] In another preferred embodiment, the exogenous protein is: (1) DN TGF-βRII, and (2) a fusion protein of IL-10 and anti-PD-1 antibody.

[0050] In another preferred embodiment, the exogenous protein is defined as described in the first aspect of the invention.

[0051] In another preferred embodiment, the tumor-specific promoter is selected from the group consisting of: Survivin promoter, hTERT promoter, AFP promoter, GFAP promoter, Nestin promoter, or combinations thereof.

[0052] In another preferred embodiment, the exogenous expression cassette has a structure as shown in Formula I: PE-L1-S-C1-L2-C2-T (Formula I) In the formula, Each "-" can be used independently as a key or connector; P contains a tumor-specific promoter; E is the coding sequence for the E1A protein; L1 is the coding sequence for a peptide that is absent or linked; S represents the coding sequence of the signal peptide or no signal peptide; C1 is the coding sequence of DN TGF-βRII; L2 is the coding sequence for a peptide that is absent or linked; C2 is the coding sequence for the fusion protein of IL-10 and anti-PD-1 antibody; T represents no or a termination element.

[0053] In another preferred embodiment, the signal peptide is the TGF-βRII signal peptide.

[0054] In another preferred embodiment, the vector is a non-viral vector or a viral vector.

[0055] In another preferred embodiment, the vector is a plasmid.

[0056] In a third aspect of the invention, a method for preparing a recombinant oncolytic virus as described in the first aspect of the invention is provided, characterized by comprising the steps of: S1. Co-transfecting a backbone plasmid and a transfer plasmid into a host cell; wherein the backbone plasmid contains a viral genome, and the transfer plasmid contains a coding sequence for a foreign protein selected from the group consisting of: dominant-negative transforming growth factor-β II receptor (DN TGF-βRII), interleukin-10 (IL-10) or its active fragment, PD-1 / PD-L1 inhibitors, or combinations thereof; S2. Cultivate the host cells; and S3. Recombinant virus was isolated from the culture.

[0057] In another preferred embodiment, the exogenous protein comprises: (1) a dominant-negative transforming growth factor-βII receptor (DNTGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody.

[0058] In another preferred embodiment, the exogenous protein is: (1) DN TGF-βRII, and (2) a fusion protein of IL-10 and anti-PD-1 antibody.

[0059] In another preferred embodiment, the exogenous protein is defined as described in the first aspect of the invention.

[0060] In another preferred embodiment, the backbone plasmid contains all genes of other adenoviruses with the pathogenic gene deleted.

[0061] In another preferred embodiment, the exogenous protein is as described above.

[0062] In another preferred embodiment, the host cell is a HEK293 cell.

[0063] In a fourth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (i) the recombinant oncolytic virus as described in the first aspect of the invention; and (ii) Pharmaceutically acceptable carriers.

[0064] In another preferred embodiment, the pharmaceutically acceptable carrier includes excipients and diluents.

[0065] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0066] In another preferred embodiment, the pharmaceutical composition is administered via intratumoral, intravenous, or intraperitoneal injection.

[0067] In a fifth aspect of the invention, the use of the recombinant oncolytic virus as described in the first aspect of the invention, or the vector as described in the second aspect of the invention, is provided for the preparation of a medicament for treating tumors or cancer.

[0068] In another preferred embodiment, the tumor or cancer includes solid tumors and hematomas.

[0069] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: gastric cancer, colorectal cancer, cervical cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, glioblastoma, renal cell carcinoma, pancreatic cancer, glioma, mesothelioma, or combinations thereof.

[0070] In a sixth aspect of the invention, a method for treating tumors or cancer is provided, comprising the steps of administering to a subject in need a therapeutically effective amount of a recombinant oncolytic virus as described in the first aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention.

[0071] In another preferred embodiment, the subject in need is a human or a non-human mammal.

[0072] In another preferred embodiment, the non-human mammals include monkeys, orangutans, cattle, pigs, dogs, sheep, rabbits, or mice.

[0073] In another preferred embodiment, the desired object is a mouse or a human.

[0074] In another preferred embodiment, the administration is intratumoral, intravenous, or intraperitoneal.

[0075] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0076] Figure 1 A schematic diagram of the oncolytic virus molecular design is shown.

[0077] Figure 2 The viral replication kinetics curve is shown.

[0078] Figure 3 The expression level of TGF-β receptor was detected.

[0079] Figure 4 The results showed the detection of IL-10 expression levels.

[0080] Figure 5 The results show the percentage of cell viability (CCK-8) and cytotoxicity (LDH) assays.

[0081] Figure 6 The curves showing the changes in tumor volume in mice with different tumor models are displayed. Detailed Implementation

[0082] Through extensive and in-depth research, the inventors have developed a novel engineered oncolytic adenovirus (ADV). Through engineering, the oncolytic virus carries the encoding genes for a fusion protein of dominant-negative transforming growth factor-β type II receptor (DN TGF-βRII) and IL-10 with an anti-PD-1 antibody. After the engineered ADV of this invention enters tumor tissue and infects tumor cells, the genes it carries are specifically regulated and expressed within the tumor cells by tumor-specific promoters. DN TGF-βRII is fixed to the tumor cell membrane surface through its transmembrane region and truncated intracellular segment, inhibiting the immunosuppressive and tumor-promoting functions of TGF-β. The fusion protein of IL-10 with an anti-PD-1 antibody (e.g., pembrolizumab single-chain antibody) exhibits multiple tumor-suppressive functions and has an extended in vivo half-life, improving stability and bioavailability. In vivo experiments show that the fusion protein of DN TGF-βRII and IL-10 with pembrolizumab single-chain antibody carried by the ADV demonstrates an unexpected and significant synergistic tumor-suppressive effect. This invention is based on these findings.

[0083] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0084] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0085] Oncolytic virus As used in this article, the term "oncolytic virus" refers to a virus that can selectively replicate in tumor cells in vitro or in vivo and slow tumor cell growth or induce tumor cell death, while having little or no effect on normal cells.

[0086] In some embodiments, the oncolytic virus contains a viral genome packaged in a viral particle (or viral unit) and is infectious (i.e., capable of infecting and entering a host cell or subject). In some embodiments, the oncolytic virus may be a DNA virus or an RNA virus and may be in any suitable form, such as a DNA viral vector, an RNA viral vector, or a viral particle.

[0087] As used herein, selective replication refers to a significantly higher replication rate of oncolytic viruses in tumor cells compared to non-tumor cells (e.g., healthy cells). In some embodiments, the oncolytic viruses of the present invention can selectively replicate in the following cells: AGS (gastric cancer), HCT-116 (colorectal cancer), HELA (cervical cancer), and HCC827 (non-small cell lung cancer), but are not limited thereto.

[0088] The oncolytic virus of the present invention can be derived from adenovirus, vaccinia virus, reovirus, measles virus, herpes simplex virus, parvovirus, chicken anemia virus, measles virus, Coxsackie virus, vesicular stomatitis virus, Newcastle disease virus, but is not limited thereto. In a preferred embodiment, the oncolytic virus of the present invention is derived from adenovirus.

[0089] Adenovirus is a non-enveloped, double-stranded DNA virus with a genome size of approximately 36-38 kb and a broad natural affinity for epithelial cells. The virus has an icosahedral structure, with its core genome encapsulated by capsid proteins. In nature, adenovirus infection in humans often causes self-limiting respiratory, conjunctival, or gastrointestinal diseases. However, due to its genome not integrating into the host chromosome, its broad host range, ease of genetic engineering manipulation, and ability to accommodate large exogenous gene fragments, it has become one of the most commonly used viral vector platforms for gene therapy and oncolytic virus therapy.

[0090] In this invention, the oncolytic virus may be a modified or altered oncolytic virus. As used herein, the terms "modified oncolytic virus" or "altered oncolytic virus" refer to an oncolytic virus that has been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins. In some embodiments, the modified oncolytic viruses provided herein are genetically altered through the deletion and / or addition of nucleic acid sequences.

[0091] In some embodiments, the modified oncolytic virus of this disclosure is attenuated. In some embodiments, the modified oncolytic virus exhibits reduced or undetectable virulence in normal cells (e.g., healthy cells) compared to its wild-type counterpart.

[0092] The recombinant oncolytic virus of the present invention This invention provides a recombinant oncolytic virus with a foreign protein in its genome, preferably a recombinant oncolytic adenovirus, wherein the foreign protein is selected from the group consisting of: dominant-negative transforming growth factor-βII receptor (DN TGF-βRII), interleukin-10 (IL-10), anti-PD-1 antibody, or combinations thereof. In this invention, the terms "recombinant oncolytic adenovirus," "engineered oncolytic adenovirus," "recombinant ADV," and "engineered ADV" are used interchangeably.

[0093] In a preferred embodiment, the recombinant oncolytic adenovirus of the present invention simultaneously introduces DN TGF-βRII, IL-10, and an anti-PD-1 antibody. In a preferred embodiment, the IL-10 and anti-PD-1 antibody are provided in the form of a fusion protein.

[0094] As used herein, the dominant-negative transforming growth factor-β type II receptor (DN TGF-βRII) is a functionally inactive mutant lacking an intracellular kinase domain. It competitively binds to TGF-β ligands, blocking their interaction with the wild-type receptor, thereby specifically inhibiting downstream Smad2 / 3 phosphorylation of TGF-β. In this invention, the introduction of the DN TGF-βRII molecule allows it to bind to TGF-β in the tumor microenvironment, thereby exerting the following functions: enhancing immune cell activity and restoring CD8+. + T cells exert cytotoxicity and promote NK cell infiltration, inhibit Treg cell function, and reshape the immune-activated tumor microenvironment; they also inhibit tumor cell invasion and metastasis, reverse epithelial-mesenchymal transition, inhibit tumor angiogenesis, and reduce fibrosis.

[0095] As used in this article, interleukin-10 (IL-10) is a cytokine that plays a central role in immune regulation. IL-10 can target and regulate the immunosuppressive state of the tumor microenvironment, inhibit Treg expansion and MDSC activation, while promoting the maturation of NK cells and dendritic cells, enhancing antigen presentation efficiency. In addition, IL-10 can directly activate CD8 through metabolic reprogramming. + It promotes oxidative phosphorylation of T cells, enhancing their proliferative capacity and cytotoxic effects.

[0096] As used in this article, anti-PD-1 antibodies generally refer to blocking anti-PD-1 antibodies, which specifically block the binding of PD-1 to PD-L1 / PD-L2, thereby relieving inhibitory signals on the surface of T cells and restoring depleted CD8+. + T-cell effector function. In this invention, the specific type of anti-PD-1 antibody is not limited, for example, antibodies known in the art that block PD-1 binding to ligands. In a preferred embodiment, the anti-PD-1 antibody of this invention is pembrolizumab.

[0097] In a preferred embodiment, the anti-PD-1 antibody of the present invention is an anti-PD-1 single-chain fragment variable (scFv). In this invention, the term "single-chain antibody" refers to a genetically engineered antibody fragment composed of a variable region (VH) and a variable region (VL) of an immunoglobulin heavy chain covalently linked by a flexible polypeptide linker to form a single polypeptide chain. Its core structural feature is that the linker enables the VH and VL to spontaneously fold and correctly pair in space, forming a functional monovalent domain with antigen-binding activity. In this invention, the structure of the single-chain antibody from the N-terminus to the C-terminus can be VH-linker-VL or VL-linker-VH, as long as its antigen-binding specificity is preserved. Furthermore, unless otherwise explicitly defined, the single-chain antibody described in this invention encompasses all possible linker lengths and compositions, as well as, optionally, interchain disulfide bond modifications introduced to improve stability.

[0098] In a preferred embodiment of the present invention, IL-10 and the anti-PD-1 antibody are provided in the form of a fusion protein. While allowing IL-10 and the anti-PD-1 antibody to function individually, the half-life of IL-10 can be extended, thereby improving stability and bioavailability. As used herein, a fusion protein refers to a single polypeptide chain produced by artificially linking nucleic acid sequences encoding two or more different functional proteins using genetic engineering techniques and expressing them within the same open reading frame. In this invention, the linking method of IL-10 and the anti-PD-1 antibody is not limited; for example, they can be directly linked or linked via a linker. In a preferred embodiment, IL-10 and the anti-PD-1 antibody are linked via a flexible linker; preferably, the flexible linker is a linker rich in G and S; more preferably, the sequence of the flexible linker is: GGSGGGSEGGGSEGGGSEGGGSEGGGGS (SEQ ID NO: 10). Alternatively, those skilled in the art can choose other flexible linkers, such as the (G4S)3 linker.

[0099] In a preferred embodiment, the IL-10 and anti-PD-1 antibody fusion protein of the present invention is an IL-10 and anti-PD-1 single-chain antibody fusion protein. The structure of the IL-10 and anti-PD-1 single-chain antibody fusion protein from the N-terminus to the C-terminus of the present invention can be either IL-10-linker-anti-PD-1 single-chain antibody or anti-PD-1 single-chain antibody-linker-IL-10. Preferably, the full-length amino acid sequence of the IL-10 and anti-PD-1 single-chain antibody fusion protein is shown in SEQ ID NO: 17.

[0100] In this invention, a method for preparing the recombinant oncolytic adenovirus of the present invention and a vector (e.g., plasmid) for preparing the virus are also provided.

[0101] The exogenous expression cassette in this invention refers to an expression cassette containing the target gene of this invention, wherein the target gene includes a gene encoding a fusion protein of DN TGF-βRII and IL-10 with an anti-PD-1 antibody. In a preferred embodiment, the target gene is driven by a tumor-specific promoter. Preferably, the tumor-specific promoter includes, but is not limited to, the Survivin promoter and the hTERT promoter.

[0102] In a preferred embodiment, the exogenous expression cassette of the present invention has a structure as shown in Formula I: PE-L1-S-C1-L2-C2-T (Formula I) In the formula, Each "-" can be used independently as a key or connector; P contains a tumor-specific promoter; E is the coding sequence for the E1A protein; L1 is the coding sequence for a peptide that is absent or linked; S represents the coding sequence of the signal peptide or no signal peptide; C1 is the coding sequence for membrane-bound DN TGF-βRII; L2 is the coding sequence for a peptide that is absent or linked; C2 is the coding sequence for the fusion protein of IL-10 and anti-PD-1 antibody; T represents no or a termination element.

[0103] In a preferred embodiment, the exogenous expression cassette is included in the vector (e.g., plasmid) used to prepare or package the recombinant oncolytic adenovirus of the present invention.

[0104] The recombinant oncolytic adenovirus of the present invention can be packaged using methods known in the art. In a preferred embodiment, the packaging method includes co-transfecting a backbone plasmid and a transfer plasmid into a host cell; wherein the backbone plasmid contains a viral genome and the transfer plasmid contains a foreign expression cassette; and after culturing the host cell, the recombinant virus is isolated from the culture.

[0105] The recombinant oncolytic adenovirus of this invention has multiple functions. First, DN TGF-βRII can inhibit the immunosuppressive and tumor-promoting functions of TGF-β by binding to TGF-β in the tumor microenvironment without activating downstream intracellular signaling. Second, the combination of IL-10 and Pembrolizumab single-chain antibody fusion protein can dually activate CD8. + The fusion protein enhances the killing effect of T cells on tumor cells, inhibits tumor angiogenesis, and cuts off the tumor's nutrient supply. Furthermore, it prolongs the in vivo half-life of IL-10, improves its stability and bioavailability, and reduces the frequency of administration. In vitro and in vivo experiments have demonstrated that the recombinant oncolytic adenovirus of this invention has a significant tumor-inhibiting effect.

[0106] application The present invention also provides a pharmaceutical composition comprising the recombinant oncolytic adenovirus of the present invention as an active ingredient, which can be used to inhibit and / or treat tumors.

[0107] The pharmaceutical composition of the present invention comprises: an effective amount of the recombinant oncolytic virus of the present invention, and a pharmaceutically acceptable vector.

[0108] Typically, the recombinant oncolytic virus or active ingredient combination of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

[0109] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injectables, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of this invention can also be formulated as sustained-release formulations.

[0110] Furthermore, the recombinant oncolytic virus of the present invention can also be used in conjunction with other therapeutic agents (such as antitumor agents or chemotherapeutic agents).

[0111] When using the pharmaceutical composition, a safe and effective amount of the recombinant oncolytic virus or active ingredient of the present invention is applied to mammals.

[0112] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0113] It should be understood that the effective amount of each active ingredient (or its formulation) in the pharmaceutical composition of the present invention may vary depending on the administration method and the severity of the tumor. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; tumor severity, patient weight, patient immune status, route of administration, etc.

[0114] There are no particular limitations on the administration method of the pharmaceutical composition described in this invention. Representative examples include (but are not limited to): intratumoral injection, intraperitoneal injection, and intravenous injection.

[0115] The beneficial effects of this invention include: 1. In the engineered ADV of this invention, the DN TGF-βRII molecule is introduced to bind to TGF-β in the tumor microenvironment, thereby enhancing the activity of immune cells and restoring CD8. + T cells exert cytotoxicity and promote NK cell infiltration, inhibit Treg cell function, and reshape the immune-activated tumor microenvironment; they also inhibit tumor cell invasion and metastasis, reverse epithelial-mesenchymal transition, inhibit tumor angiogenesis, and reduce fibrosis.

[0116] 2. In the engineered ADV of this invention, an IL-10 fusion protein with an anti-PD-1 antibody is introduced to promote CD8. + T cell activation, proliferation, and cytotoxicity can be mitigated by blocking the PD-1 / PD-L1 pathway, thus relieving the inhibition of T cell activation. Furthermore, the fusion protein can prolong the half-life of IL-10 and anti-PD-1 antibodies in vivo, improving their stability and bioavailability, and reducing the frequency of administration.

[0117] 3. In the engineered ADV of the present invention, the simultaneous expression of DN TGF-βRII and IL-10 with the anti-PD-1 antibody fusion protein exhibits a synergistic tumor-suppressing effect, which is superior to the sum of the effects of ADV expressed alone, and shows excellent inhibitory effects on a variety of tumors in vivo.

[0118] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0119] Example 1: Molecular Design and Preparation of Oncolytic Viruses 1. Molecular design of oncolytic viruses according to Figure 1 The schematic diagram shown illustrates four molecular constructs designed and named 1#, 2#, 3#, and 4#.

[0120] 2. Oncolytic virus molecular construction, packaging, and titer detection 2.1 Oncolytic virus molecular construction The desired target gene sequence was obtained from NCBI and amplified by PCR. The oncolytic virus transfer plasmid (pDC316-mCMV-EGFP) used in the experiment was purchased from Fenghui Biotechnology. The target gene fragment and plasmid were double-digested with Hpy188Ⅰ and EcoRI restriction enzymes, respectively, and the digested target gene was ligated to the linearized plasmid. The ligation product was transformed into TOP10 competent cells, and positive clones were screened and identified after culture. Correctly identified recombinant plasmids were extracted, purified, and used for subsequent virus packaging experiments. Plasmids containing different target sequences were named plasmids #1, #2, #3, and #4, respectively.

[0121] 2.2 Oncolytic virus packaging Healthy HEK293 cells were collected, digested, and counted. 4E6 cells / 10ml were evenly seeded into 10cm dishes. After 24 hours, using the Lipofectamine 3000 kit, 7.5μg each of plasmids #1, #2, #3, and #4, and the pBHGlox-E1,3cre DH plasmid (which deletes the E1,3 gene and all other genes of AD5 adenovirus, while expressing cre recombinase) were transiently transfected into HEK293 cells. The cells were labeled and incubated at 37℃. After 5 hours, cell status was observed, all liquid in the 10cm dishes was aspirated, and 10ml of fresh DMEM medium was added. The dishes were then incubated at 37℃. Cell status was observed daily, and replenishment was performed every other day. On Day 10, all HEK293 cells detached. The cell supernatant and cells were collected, subjected to three freeze-thaw cycles, and centrifuged at 1000rpm for 5 minutes at 4℃. The supernatant was filtered through a 0.22μm filter and stored at -80℃.

[0122] 2.3 Titer Detection Healthy HEK293 cells were digested, counted, and adjusted to 3E5 cells / 2mL. These cells were then seeded into 6-well cell culture plates and incubated at 37°C with 5% CO2 for 12 hours until 80%-90% of the cells formed a monolayer. ADV oncolytic virus stock solution was serially diluted 10-fold with serum-free DMEM, resulting in 10 dilutions. 1mL of each virus dilution was mixed with 1mL of fresh medium and added to the corresponding well of the 6-well plate (one dilution per well, 3 replicates). A negative control (virus-free medium) and a positive control (ADV sample with a known titer) were also included. The 6-well plate was incubated at 37°C with 5% CO2 for 2 hours. The virus dilution was aspirated from the wells, and the cells were gently washed twice with PBS. 1.5mL of 1.25% low-melting-point agarose was added and gently covered onto the cell layer. The plate was incubated at 37°C for 30 minutes. Return the 6-well plate to the 37℃, 5% CO2 incubator and continue culturing, observing plaque formation daily. After plaque formation, stain with neutral red staining solution for 10 minutes. Rinse off excess dye with distilled water and observe the red plaques under a microscope. Count the number of plaques at each dilution and calculate PFU / mL using the formula: PFU / mL = (number of plaques × dilution factor) / inoculum volume (mL).

[0123] 3. Detection of viral replication dynamics Healthy HELA cells were digested, counted, and adjusted to 2E5 cells / 2mL. These cells were then seeded into 6-well cell culture plates and incubated at 37°C with 5% CO2 for 12 hours, until 80%-90% of the cells formed a monolayer. The ADV oncolytic virus stock solution was serially diluted 10-fold with serum-free DMEM, resulting in 10 dilutions. 1mL of each virus dilution was mixed with 1mL of fresh medium and added to the corresponding well of the 6-well plate (one dilution per well, 3 replicates). A negative control (virus-free medium) and a positive control (ADV sample with a known titer) were also included. The 6-well plate was incubated at 37°C with 5% CO2 for 2 hours. The virus dilution was aspirated from the wells, and the cells were gently washed twice with PBS. 1.5mL of 1.25% low-melting-point agarose was added and gently covered onto the cell layer. The plate was incubated at 37°C for 30 minutes. Return the 6-well plate to the 37℃, 5% CO2 incubator and continue culturing, observing plaque formation daily. After plaque formation, stain with neutral red staining solution for 10 minutes. Rinse off excess dye with distilled water and observe the red plaques under a microscope. Titration calculation: Count the number of plaques at each dilution. Calculate PFU / mL using the formula: PFU / mL = (number of plaques × dilution factor) / inoculum volume (mL).

[0124] The results are as follows Figure 2 As shown, there was no significant difference in the replication kinetics of ADV viruses 1#, 2#, 3#, and 4#, indicating that the engineering modification did not affect the replication ability of ADV viruses in tumor cells.

[0125] Example 2: Molecular expression detection 1. DN TGF-β RII detection Healthy HEK293 cells were collected, digested, counted, and adjusted to a concentration of 5E5 cells / mL. These cells were then seeded into 24-well cell culture plates and incubated at 37°C with 5% CO2 for 18 hours, until 80%-90% of the cells formed a monolayer. HEK293 cells were infected with ADV at an MOI of 5. The 24-well plates were then placed in a 37°C, 5% CO2 incubator for 2 hours to allow adsorption. The virus dilution solution was removed from the wells and replaced with fresh DMEM medium. After 48 hours, cells were digested and collected for flow cytometry analysis. 5 × 10⁶ cells were collected from each group. 5Centrifuge cells at 400g for 5 minutes at room temperature. Discard the supernatant, resuspend the cells in 1 ml of 2% FBS PBS, and centrifuge at 400g for 5 minutes. Discard the supernatant, resuspend the cells in 50 µl of 2% FBS PBS, then add Fc block to block Fc receptors, and incubate at room temperature for 10 minutes. Transfer half of the cells from the control group as a blank control. Add primary antibody (APC anti-human TGF-β Receptor II Antibody, Biolegend, 399706) to the antibody group cells, incubate at room temperature for 15 minutes, then centrifuge at 400g for 5 minutes. Discard the supernatant, resuspend the cells in 1 ml of 2% FBS PBS, and centrifuge at 400g for 5 minutes. Discard the supernatant, add 50 µl of 7-AAD dye prepared with 2% FBS PBS to the antibody group cells, and incubate at room temperature for 7 minutes. Add 1 ml of 2% FBS PBS, and centrifuge at 400g for 5 minutes. Discard the supernatant, add 200 µl of PBS containing 2% FBS to resuspend the cells, and then perform the analysis.

[0126] The results are as follows Figure 3 As shown, after infection of 293 cells with ADV #1 and #2, flow cytometry analysis revealed that almost 100% of the cells expressed TGF-β receptors, while normal 293 cells and 293 cells infected with ADV #3 and #4 did not express TGF-β receptors. This result confirms that engineered ADVs can induce infected cells to express DN TGF-β RII and successfully anchor to the cell membrane.

[0127] 2. Detection of IL-10-Pembrolizumab fusion protein Healthy AGS, HCT-116, HELA, and HCC827 cells were digested, counted, and adjusted to 5E5 cells / mL. They were then seeded into 24-well cell culture plates and incubated at 37°C with 5% CO2 for 18 hours until 80%-90% of the cells formed a monolayer. Different tumor cells were infected with ADV at MOI=5. The 24-well plates were placed in a 37°C, 5% CO2 incubator for 2 hours for adsorption. The virus dilution solution in the wells was removed, and fresh DMEM medium was replaced. After 48 hours, the cell supernatant was collected for ELISA detection. The kit used was a pre-prepared human IL-10 ELISA kit (Dakeway, 1111002). All reagents and consumables were provided with the kit, and the procedure was performed according to the kit instructions. Sample addition: Diluted cytokine standards were added to the standard wells at 100 µl / well. The samples were diluted with dilution buffer R (1×) and added to the sample wells at 100 µl / well. Cover with sealing film and incubate at room temperature for 2 hours. Wash plate: Discard the liquid in the wells, add 1× washing buffer (300 µl / well); let stand for 1 minute, then discard the liquid in the wells. Repeat 3 times, drying on filter paper each time. Add detection antibody: Add biotinylated antibody working solution (100 µl / well). Cover with sealing film and incubate at room temperature for 1 hour. Repeat washing. Add enzyme: Add streptavidin-HRP working solution (100 µl / well). Cover with sealing film and incubate at room temperature (18-25℃) for 30 minutes. Repeat washing. Color development: Add TMB (100 µl / well), incubate at room temperature in the dark for 5-30 minutes, stopping the reaction based on the intensity of the color in the wells (dark blue). Usually, 10-20 minutes of color development achieves good results. Stop the reaction: Quickly add stop solution (100 µl / well) to stop the reaction. Read the OD value at 450 nm using a microplate reader. Four-parameter method for data analysis.

[0128] The results are as follows Figure 4 As shown, after infection with ADV #1 and #3, high concentrations of IL-10 were detected by ELISA in all four types of tumor cells, while no IL-10 expression was detected in the control group tumor cells and the four types of tumor cells infected with ADV #2 and #4. These experimental results demonstrate that engineered ADV can express and secrete the IL-10-Pembrolizumab fusion protein within tumor cells.

[0129] Example 3: Detection of in vitro tumor-lysing properties Healthy AGS (gastric cancer), HCT-116 (colorectal cancer), HELA (cervical cancer), and HCC827 (non-small cell lung cancer) cells were digested, counted, and adjusted to 3E5 cells / mL. 100 μL was seeded into each well of a 96-well cell culture plate and incubated at 37°C with 5% CO2 for 18 hours until 80%-90% of the cells formed a monolayer. Different tumor cells were infected with ADV at MOIs of 0.01, 0.1, 1, 10, and 100, respectively. The 96-well plates were placed in a 37°C, 5% CO2 incubator for 2 hours for adsorption (during which the bottom of the plate was gently agitated to ensure even virus contact with the cells). The virus dilution solution was aspirated from the wells and replaced with fresh DMEM medium. After 24 hours, the culture medium was aspirated from the wells, and the cell supernatant was centrifuged and used for LDH (Meilun Biotechnology, MA0649) detection. Cells in the wells were washed with PBS, and tumor cell viability was detected by the CCK8 assay (Meilun Biotechnology, MA0218-5).

[0130] The results are as follows Figure 5 As shown in Tables 1 and 2, ADVs #1, #2, #3, and #4 all exhibited significant killing effects on AGS, HCT-116, HELA, and HCC827 tumor cell lines in a dose-dependent manner. There were no inter-group differences in the killing effects of ADVs on tumor cells among them. These experimental results demonstrate that the engineered modification did not affect the infection and lysis capabilities of the ADVs on tumor cells.

[0131] Table 1. Percentage of tumor cells surviving after ADV infection (CCK-8, %) Table 2. Cytotoxicity of ADV-infected tumor cells (LDH, release fold) Example 4: In vivo tumor suppression experiment Healthy AGS, HCT-116, HELA, and HCC827 cells were collected, digested, counted, and then the cell density was adjusted to 1×10⁻⁶. 7 Cells / mL. 100 μL of cell suspension was injected subcutaneously into the back of an immune system-humanized mouse using a syringe. Tumor growth was monitored, and the tumor was allowed to grow to 100 mm. 3 At that time, oncolytic virus was injected intratumorally at a dose of 5 × 10⁻⁶. 7 PFU / 50μL / mouse. After injection of oncolytic virus, tumor volume changes in mice were monitored weekly. The longest diameter (a) and shortest diameter (b) of the tumor were measured with calipers, and the tumor volume was calculated (formula: V=1 / 2×a×b). 2 ).

[0132] The results are shown in Table 3 and Figure 6As shown, ADV 1# significantly inhibited tumor growth in all four different tumor models, demonstrating the best tumor-suppressing effect. ADV 2# and ADV 3# partially inhibited tumor volume, demonstrating a certain tumor-suppressing effect. ADV 4# had the worst effect in inhibiting tumor volume.

[0133] Table 3. Percentage reduction in tumor volume (%) in different tumor models Furthermore, compared to the effect of ADV #4, ADV #1 showed the greatest improvement in tumor volume reduction, exceeding the combined improvement of ADV #2 and ADV #3. This indicates that the engineered ADV, simultaneously expressing membrane-bound DN TGF-βRII and IL-10-Pembrolizumab, produced a synergistic tumor-suppressive effect.

[0134] discuss The core advantage of engineered ADV oncolytic viruses lies in their ability to synergistically enhance precision, safety, and immune activation. Specifically: First, through gene modification (such as inserting tumor-specific promoters like hTERT and survivin), viral replication is regulated by these promoters, thus avoiding infection of normal cells and significantly reducing toxicity. Second, engineered ADVs can activate the immune system by inserting immunomodulatory genes. For example, expressing the fusion protein sPD1 (which blocks the PD-1 / PD-L1 pathway) and CD137L (which activates CD137 co-stimulatory signals) can significantly enhance the infiltration and activity of CD8+ T cells in solid tumor models; expressing Tα1 (an immunomodulatory peptide) can reverse the M2 polarization (pro-tumor phenotype) of tumor-associated macrophages (TAMs), promoting their transformation to the M1 type (anti-tumor phenotype), while simultaneously increasing the infiltration of CD8+ T cells within tumor tissues. Third, the ADV genome has the ability to accommodate multiple exogenous genes (such as immune regulatory genes, target genes, and apoptosis-inducing genes), enabling synergistic effects of multiple anti-tumor mechanisms. For example, inserting an apoptosis-inducing gene (such as Bax) can enhance tumor cell apoptosis; inserting angiogenesis-inhibiting genes (such as VEGF inhibitors) can block the nutrient supply to the tumor. Fourth, by deleting pathogenic genes (such as E1B-55KD and E3-gp19K), the pathogenicity of engineered ADVs is significantly reduced.

[0135] This invention provides a novel engineered adventitious virus (ADV) with synergistic tumor-suppressive capabilities. Firstly, abnormal activation of transforming growth factor-β (TGF-β) signaling in the tumor microenvironment (TME) is a key pathogenic factor, promoting tumor progression through inducing immunosuppression, stromal fibrosis, and vascular abnormalities. In the TME, the DN TGF-βRII effectively reverses TGF-β-mediated regulatory T cell expansion, myeloid suppressor cell activation, and fibroblast activation, alleviating collagen deposition and interstitial hypertension; simultaneously, it enhances CD8... + T cell infiltration and effector function improve tumor immunosuppression.

[0136] Secondly, the tumor microenvironment (TME) often leads to effector T cell exhaustion due to PD-1 / PD-L1 axis activation and immunosuppressive factors (such as Treg and MDSC infiltration), hindering anti-tumor immunity. The IL-10 and Pembrolizumab (anti-PD-1 monoclonal antibody) fusion protein synergistically remodels the TME through a dual mechanism: Pembrolizumab specifically blocks the binding of PD-1 to PD-L1 / PD-L2, relieving inhibitory signals on the T cell surface and restoring exhausted CD8+. + T cell effector function; IL-10 targets and regulates the immunosuppressive state of the TME, inhibiting Treg expansion and MDSC activation, while promoting NK cell and dendritic cell maturation and enhancing antigen presentation efficiency. IL-10 can directly activate CD8 through metabolic reprogramming. + IL-10 promotes oxidative phosphorylation in T cells, enhancing their proliferative capacity and cytotoxic effects. IL-10 and PD-1 signaling have a synergistic effect, jointly regulating CD8+. + T cell function. Blocking PD-1 enhances the effect of IL-10 on CD8. + T cell activation, and IL-10 can also alleviate PD-1-mediated inhibition; the combination of the two can more effectively restore CD8. + The fusion of the two not only retains the precise immune checkpoint blocking ability of Pembrolizumab, but also leverages IL-10 to locally regulate the microenvironment, forming a dual effect of "de-inhibition + active activation," which significantly enhances the intensity of the anti-tumor immune response and provides a new strategy for TME reprogramming.

[0137] The engineered ADV of this invention simultaneously addresses the issues of tumor microenvironment immune regulation, immune cell reactivation, and short cytokine half-lives, further enhancing the anti-tumor effect of the engineered ADV. In vivo experimental results show that, compared with the expression of DN TGF-βRII alone (2# ADV) or the expression of IL-10-Pembrolizumab fusion protein alone (3# ADV), the simultaneous expression of DN TGF-βRII and IL-10-Pembrolizumab fusion protein (1# ADV) exhibits a synergistic tumor-suppressive effect, with significant and unexpected results.

[0138] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0139] nucleotide sequence Survivin promoter (SEQ ID NO: 1): Ggattacaggcgtgagccactgcacccggcctgcacgcgttctttgaaagcagtcgaggggggcgctaggtgtgggcagggacgagctggcgcgcgtcgctgggtgcaccgcgaccacgggcagagccacgcggcgggaggactacaactc ccggcacaccccgcgccgccccgcctctactcccagaaggccgcggggggtggaccgcctaagagggcgtgcgctcccgacatgccccgcggcgcgccattaaccgccagatttgaatcgcgggacccgttggcagaggtggcggcggcggc amino acid sequence E1A 32 kDa protein (SEQ ID NO: 2): MRHIICHGGVITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDVTAPEDPNEEAVSQIFPDSVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDEDEEGEEFVL DYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSPVSEPEPEPEPEPEPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIHPVVPLCPIKPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP Q2A (SEQ ID NO: 3): ATNFSLLKQAGDVEENPGP TGF-βRII(SEQ ID NO: 4): MGRGLLRGLWPLHIVLWTRIAS DN TGF-βRII(SEQ ID NO: 5): TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITCICECPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ DN TGF-βRII(SEQ ID NO: 6): VTGISLLPPLGVAISVIIIFY DN TGF-βRII (SEQ ID NO: 7): CYRVNRQQKLSS T2A (SEQ ID NO: 8): EGRGSLLTCGDVEENPGP IL-10(SEQ ID NO: 9): MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN Linker between IL-10 and the single-chain antibody of Pembrolizumab (SEQ ID NO: 10): GGSGGGSEGGGSEGGGSEGGGSEGGGGS Pembrolizumab VL (SEQ ID NO: 11): EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK Pembrolizumab VH (SEQ ID NO: 12): QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS Linker between Pembrolizumab VL and VH (SEQ ID NO: 13): GGGGSGGGGSGGGGS Full-length sequence of membrane-bound DN TGF-βRII (including signal peptide) (SEQ ID NO: 14): MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS Full-length sequence of membrane-bound DN TGF-βRII (excluding signal peptide) (SEQ ID NO: 15): TIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS The full-length sequence of the Pembrolizumab single-chain antibody (SEQ ID NO: 16) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGG GGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS Full-length sequence of the IL-10 and Pembrolizumab single-chain antibody fusion protein (SEQ ID NO:17) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGSEGGGSEGGGSEGGGSEGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS。

Claims

1. A recombinant oncolytic virus, characterized in that, The genome of the virus contains an exogenous expression cassette containing a coding sequence for an exogenous protein, the exogenous protein including: (1) a dominant-negative transforming growth factor-βII receptor (DN TGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody.

2. The recombinant oncolytic virus as described in claim 1, characterized in that, The DN TGF-βRII comprises an extracellular region, a transmembrane region, and a truncated intracellular region, and the amino acid sequence of the truncated intracellular region of the DN TGF-βRII is shown in SEQ ID NO:

7.

3. The recombinant oncolytic virus as described in claim 1, characterized in that, The anti-PD-1 antibody is an anti-PD-1 single-chain antibody. The VL sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID NO: 11, and the VH sequence of the anti-PD-1 single-chain antibody is shown in SEQ ID NO:

12.

4. The recombinant oncolytic virus as described in claim 1, characterized in that, The full-length amino acid sequence of the fusion protein of IL-10 and anti-PD-1 antibody is shown in SEQ ID NO:

17.

5. The recombinant oncolytic virus as described in claim 1, characterized in that, The oncolytic virus is selected from the following group: adenovirus, vaccinia virus, reovirus, measles virus, herpes simplex virus, parvovirus, chicken anemia virus, measles virus, Coxsackie virus, vesicular stomatitis virus, and Newcastle disease virus.

6. A carrier, characterized in that, The vector contains an exogenous expression cassette containing a promoter and a coding sequence for an exogenous protein, the exogenous protein including: (1) a dominant-negative transforming growth factor-βII receptor (DNTGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody; the promoter is a tumor-specific promoter.

7. A method for preparing the recombinant oncolytic virus as described in claim 1, characterized in that, Including the following steps: S1. Co-transfect the backbone plasmid and the transfer plasmid into the host cell; wherein the backbone plasmid contains a viral genome, and the transfer plasmid contains a coding sequence for a foreign protein, the foreign protein including: (1) a dominant-negative transforming growth factor-βII receptor (DN TGF-βRII), and (2) a fusion protein of interleukin-10 (IL-10) and an anti-PD-1 antibody; S2. Cultivate the host cells; and S3. Recombinant virus was isolated from the culture.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the recombinant oncolytic virus as described in claim 1; and (ii) Pharmaceutically acceptable carriers.

9. The use of the recombinant oncolytic virus as described in claim 1, or the vector as described in claim 6, characterized in that, Used to prepare drugs for treating tumors or cancer.

10. The use as described in claim 9, characterized in that, The tumor or cancer is selected from the group consisting of: gastric cancer, colorectal cancer, cervical cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, glioblastoma, renal cell carcinoma, pancreatic cancer, glioma, mesothelioma, or combinations thereof.

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