Membrane-coated oncolytic virus as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a membrane-coated oncolytic virus as well as a preparation method and application thereof. The oncolytic virus contains a GSDMD N-terminal expression cassette regulated and controlled by a heated shock promoter, and pyroptosis can be induced under exogenous mild thermal stimulation, so that the virus is promoted to be quickly released, and reinfection and amplification of adjacent tumor cells are realized. A bionic nano-vesicle (iNV) formed by a donor cell membrane modified by genetic engineering is used as an outer layer to coat a genetically engineered oncolytic virus (GOV) to obtain a membrane-coated oncolytic virus iNV-GOV, so that the membrane-coated oncolytic virus iNV-GOV simultaneously has immunocompatibility and active tumor recognition capability, is suitable for solid tumors such as pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (IHCC) and the like, and can be used for preparing an anti-tumor drug. And the tumor volume can be obviously reduced in mouse in-vivo model experiments.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a membrane-coated oncolytic virus, its preparation method, and its application. Background Technology

[0002] Oncolytic viruses (OVs) are a class of replicable viruses capable of infecting both normal and tumor cells. They selectively replicate within tumor cells and induce anti-tumor responses. The first approved oncolytic drug based on herpesvirus (HSV) (Imlygic) primarily relies on intratumoral injection, limiting its efficacy in deep or metastatic lesions. To overcome accessibility issues, clinical and translational research is shifting towards systemic delivery (especially for HSV and adenovirus AdV). However, previous studies have identified two core obstacles: first, the rapid clearance of pre-existing / induced antiviral neutralizing antibodies (NAbs) mediated by the complement-coagulation-reticuloendothelial system (RES); second, while increasing the dose can enhance tumor delivery, it significantly increases systemic toxicity and the risk of death, making it difficult to balance efficacy and safety. Consequently, various systemic delivery strategies have emerged: live-cell carriers represented by T cells, nanocarriers represented by liposomes / cell membranes, and biomimetic carriers, among others.

[0003] Besides systemic delivery, the heterogeneity of intratumoral infection and expansion efficiency also severely hinders overall efficacy: OV often infects only specific subsets of tumor cells, while in vivo antiviral responses further weaken the cascade effect of reinfection and expansion; attempts have been made in vitro and animal experiments to combine OV with immunosuppressants (such as cyclophosphamide, rapamycin, and HDAC inhibitors) to enhance infection, but animal model conversion has not been ideal. Meanwhile, pyroptosis, a type of programmed inflammatory cell death, involves the N-terminus of the Gasdermin family (GSDMD / GSDME) being cleaved to create pores on the membrane, inducing lytic death and immune activation. Studies have reported on the use of viral vectors encoding GSDM family members for antitumor treatment. However, non-selective or persistently high expression of GSDMD... NT It may damage normal tissues, thus requiring tumor cell-specific / controllable expression to balance efficacy and safety. Recent light / ultrasound-induced gene regulation systems (such as placing effector genes under heat shock promoters and using ultrasound and heat to achieve spatiotemporally controllable expression) have provided a feasible path to achieve tumor-induced lytic death and enhanced OV reinfection. However, a universal solution is still lacking in achieving stable, amplifiable, and OV-compatible expression control in vivo. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an oncolytic virus containing a GSDMD N-terminal expression cassette regulated by a heat shock promoter. This cassette can induce pyroptosis under exogenous mild heat stimulation, thereby promoting rapid viral release and enabling reinfection and amplification of adjacent tumor cells. Furthermore, the genetically engineered oncolytic virus (GOV) is coated with biomimetic nanovesicles (iNVs) formed from genetically engineered donor cell membranes, thus simultaneously endowing it with immunocompatibility and active tumor recognition capabilities.

[0005] The present invention provides an oncolytic virus containing an expression cassette that regulates the expression of the GSDMD gene under heat shock promoter.

[0006] In some embodiments, the heat shock promoter includes promoters HSP70 and HSPA6. In some specific embodiments, the sequence of the heat shock promoter is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0007] In some embodiments, the oncolytic virus is a replicable oncolytic virus of adenovirus or herpesvirus.

[0008] In some implementations, the GSDMD includes the GSDMD N-terminal sequence.

[0009] This invention also provides a membrane-coated oncolytic virus, which is obtained by coating the oncolytic virus in a cell membrane; In some embodiments, the cell membrane is the cell membrane of cells that weaken or eliminate HLA-I / II presentation. In some specific embodiments, the cells that weaken or eliminate HLA-I / II presentation are cells with the B2M and CIITA genes knocked out.

[0010] In some embodiments, the cell membrane is the cell membrane of the cell into which the immune regulatory molecule has been knocked in. In some embodiments, the immune regulatory molecule includes HLA-E and CD47. In some embodiments, the knock-in is achieved using a CRISPR-Cas system and homology-directed repair (HDR) to knock in HLA-E / CD47.

[0011] In some embodiments, the cell membrane is the cell membrane of a cell whose genome has integrated the CAR gene. In some specific embodiments, the target of the CAR protein is selected from at least one of HER2, CLDN18.2, GPC3, and PSCA.

[0012] In some embodiments, the CAR gene is integrated into the genome via stable transfection or a viral vector, and its expression and directionality on the membrane surface are verified by flow cytometry.

[0013] In some embodiments, the membrane modification can also be performed by post-decorating the CAR or ligand through lipid insertion or other methods to enhance targeting.

[0014] This invention also provides a method for preparing the membrane-coated oncolytic virus, comprising the following steps: (1) Infect cells with oncolytic virus and culture for 12–72 h to obtain intracellular or membrane-bound virus; (2) After infection, cells are subjected to gentle cell relaxation and membrane separation, and then extruded into shape, so that the virus is embedded or coated by membrane vesicles during the extrusion / reconstruction process to obtain membrane-coated oncolytic virus; In some embodiments, the method for preparing the membrane-coated oncolytic virus further includes removing free virus from the uncoated membrane via density gradient / gel filtration / immunocapture. In some embodiments, the removal of free virus employs a combination of anti-Ad5 (or corresponding virus) antibody magnetic bead immunocapture and gel filtration. In some implementations, the infection multiplicity (MOI) of the infection in step (1) is 0.1 to 10; In some embodiments, step (2) extrusion includes extrusion molding in an isotonic buffer system or short-range ultrasonication to obtain membrane-coated oncolytic viruses with a particle size of 100–250 nm.

[0015] The present invention also provides a pharmaceutical composition comprising the oncolytic virus or the membrane-coated oncolytic virus described above.

[0016] In some embodiments, the pharmaceutical composition further includes a PD-1 / PD-L1 inhibitor or a chemotherapeutic agent. In some specific embodiments, the chemotherapeutic agent includes gemcitabine.

[0017] This invention provides the use of the oncolytic virus or the membrane-coated oncolytic virus, and the pharmaceutical composition thereof, in the preparation of drugs for treating cancer; In some embodiments, the cancer includes pancreatic ductal adenocarcinoma, hepatocellular carcinoma, or intrahepatic bile duct carcinoma.

[0018] In some embodiments, the drug is administered via intravenous or intraperitoneal administration.

[0019] In some embodiments, after drug administration, mild thermal induction with medical ultrasound or high-intensity focused ultrasound (HIFU) is applied to the tumor region, causing the N-terminus of GSDMD (GSDMD) regulated by the HSP promoter to be activated. NT It is expressed in tumor cells, inducing pyroptosis and rapidly releasing viral particles.

[0020] In some embodiments, the ultrasound induction satisfies any of the following parameters: tissue temperature of 40–43°C, sound intensity of 0.8–1.5 W / cm², 2–4 cycles of 5 min on / 5 min off pulses, total duration of 10–30 min, and can be repeated according to the treatment window.

[0021] The present invention also provides the use of the membrane-coated oncolytic virus in combination with PD-1 / PD-L1 inhibitors or chemotherapeutic drugs in the preparation of drugs for treating cancer.

[0022] In some embodiments, the chemotherapy drug includes gemcitabine.

[0023] In some embodiments, the cancer includes pancreatic ductal adenocarcinoma, hepatocellular carcinoma, or intrahepatic bile duct carcinoma.

[0024] In some embodiments, when the membrane-coated oncolytic virus is used in combination with an immune checkpoint inhibitor (PD-1 / PD-L1) or a chemotherapy drug (gemcitabine), the membrane-coated oncolytic virus is administered first and then activated by ultrasound, and the immune checkpoint inhibitor or chemotherapy drug is administered within ±24 h before and after ultrasound activation.

[0025] the term: GSDMD is the gene encoding Gasdermin D (a member of the Gasdermin family D), located on human chromosome 8q24.3, and is one of the core genes regulating pyroptosis. The GSDMD protein it encodes is a key molecule mediating inflammatory cell death, playing a central role in innate immunity, infection defense, and various diseases (such as inflammation, tumors, and autoimmune diseases). The N-terminal domain (GSDMD) produced after the GSDMD protein is activated (primarily by cleavage by Caspase family proteases)... NT GSDMD is a key functional unit that mediates its core function—"cell perforation and pyroptosis initiation"—and is also the core carrier for GSDMD to exert its biological effects.

[0026] The “CMV” promoter is one of the most commonly used strong promoters in genetic engineering. Its full name is Cytomegalovirus Immediate Early Promoter.

[0027] The beneficial effects of this invention are: (1) The oncolytic virus of the present invention contains a GSDMD N-terminal expression cassette regulated by a heat shock promoter, which can induce pyroptosis under exogenous mild heat stimulation, thereby promoting rapid release of the virus and enabling reinfection and amplification of adjacent tumor cells. Using biomimetic nanovesicles (iNV) formed by genetically engineered donor cell membranes as the outer layer, the genetically engineered oncolytic virus (GOV) is coated to obtain membrane-coated oncolytic virus iNV-GOV, which simultaneously possesses immunocompatibility and active tumor recognition capabilities. It is suitable for solid tumors such as pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC), and intrahepatic cholangiocarcinoma (IHCC), and can significantly reduce tumor volume in mouse in vivo model experiments.

[0028] (2) The combination of membrane-coated oncolytic virus iNV-GOV with PD-1 / PD-L1 inhibitor / gemcitabine can further enhance tumor suppression and survival benefits, and maintain effective delivery and efficacy even in the presence of neutralizing antibodies.

[0029] (3) The integrated infection-separation-extrusion process and release criteria for loading / stability / escape capability of the present invention can achieve controllable particle size (100–250 nm) and high loading (≥1×10⁻⁶). 8 VP / 2 μg membrane protein), serum / shear-stable membrane-coated oncolytic virus, facilitating GMP scale-up and batch-to-batch consistency. Attached Figure Description

[0030] Figure 1 A is a schematic diagram of the HSPA6 promoter structure, showing the upstream predicted heat shock element (HSE) and the location of the HSPA6 open reading frame (ORF), as well as the connection construction of the two promoter fragments (HSP-1 and HSP-2) sites; B is a comparison of the expression readout (RLU) of different HSP-Gluc constructs derived from the HSPA6 and HSP70 sites after US-mediated thermal activation in Capan-1 cells.

[0031] Figure 2 The setup and temperature control (approximately 42 °C) for the US pulse train are illustrated, and the supernatant is collected after 12 h to compare the effects of different pulses on the Gluc signal.

[0032] Figure 3 Confocal micrographs of live / dead cells after ultrasound activation of pyroptosis protein expression: input dose 20 VP / cell; ultrasound conditions 1.2 W / cm², 15 min pulse (5 min on / 5 min off, 3 cycles); scale bar shown in the figure.

[0033] Figure 4 For the results of Western blotting, OV, GOV and GOV US Full length of GSDMD after processing (GSDMD) FL) and cutting N-end (GSDMD) NT The expression of ).

[0034] Figure 5 This is a quantitative result of lactate dehydrogenase (LDH) release in cell supernatant on day 6.

[0035] Figure 6 A schematic diagram of the compartment metastasis experiment (top) and confocal images / quantification (bottom): GOV particles released by ultrasound-activated tumor cells (TRITC-labeled) in the external cavity can infect untreated tumor cells in the internal cavity, demonstrating reinfection capability.

[0036] Figure 7 The iNV-GOV preparation process is illustrated as follows: Genetically engineered 293 donor cells are infected with oncolytic virus to obtain virus-containing donor cells, and their membrane nanovesicles are separated and combined with the virus to form iNV-GOV.

[0037] Figure 8 The particle size distributions of GOV, iNV, and iNV-GOV were determined by dynamic light scattering (DLS).

[0038] Figure 9 To detect B2M, CIITA, HLA-E, CD47, E1A, and membrane / vesicle-related markers (such as ALIX and Na) in different treatment groups using immunoblotting + ,K + Expression of ATPase, etc.

[0039] Figure 10 Transmission electron microscopy (TEM) images and anti-Ad5 gold-labeled immunoelectron microscopy: comparing the morphology and surface antigenicity of GOV and iNV-GOV.

[0040] Figure 11 To quantify iNV-GOV using dot blot and to quantify the viral load on the surface of nanovesicles using qPCR.

[0041] Figure 12 The results show the virus stability test under different conditions. In A, the iNV-GOV signal showed no significant change at 4°C or 37°C, with or without serum, indicating that the virus did not leak from the vesicles. In B, the GOV and iNV-GOV signal intensities were similar under static conditions at 37°C or oscillation at 100 rpm, indicating that the vesicle structure was stable.

[0042] Figure 13 Flow cytometry was used to detect the activation levels of T cells and NK cells in PBMCs. In this study, A represents the detection of CD69 when PMA was used as a positive control. + CD3 +T cell percentage; when B is α-GalCer as a positive control, CD69 was detected. + CD56 + NK cell ratio.

[0043] Figure 14 This is an analysis of the results of co-culturing iNV-GOV with PDO and T cells from different patient sources. In this figure, A represents cells showing CD8+. + Distribution of T cells to Tregs; B shows the CD8 ratio. + Flow cytometry quantitative analysis of the proportion of IFN-γ positive cells in T cells.

[0044] Figure 15 For confocal microscopy imaging and quantification: to evaluate the killing effect of iNV-GOV on PDO (Calcein-AM labeling of surviving organoids, TO-PRO-3 labeling of dead cells / regions).

[0045] Figure 16 A represents human tumor-associated macrophages (hTAMs, hCD11b) in tumor tissue on day 12. + CD68 + The M1 / M2 ratio (M1-hCD86) + / M2-hCD206 + Quantitative analysis of ) ; B is Treg (hFoxp3 + ) accounts for hCD4 + T cell ratio.

[0046] Figure 17 A represents activated hCD8 in tumor tissue. + hIFN-γ in T cells + The expression ratio of hCD8 in tumor tissue; B represents the activated hCD8 expression ratio in tumor tissue. + hGZMB in T cells + The proportion of expression.

[0047] Figure 18 To obtain human cells derived from the spleen, IFN-γ ELISA was performed after in vitro restimulation with tumor antigens to demonstrate the induction level of tumor antigen-specific effector T cells.

[0048] Figure 19 This study presents the results of systemic administration of iNV-GOV to HubBMC-NCG-PDX pancreatic cancer mice, followed by ultrasound-activated pyroptosis protein expression in the tumor region for the treatment of orthotopic pancreatic cancer. In the figures, A represents tumor volume and H&E section data for each group; B represents changes in survival curves.

[0049] Figure 20 To investigate the effects of iNV-GOV on HuHSC-NCG-PDX mice USResults of combined treatment with anti-PD-1 antibody; where A shows changes in tumor volume and H&E histological sections in each group; and B shows changes in survival curves.

[0050] Figure 21 To investigate the effects of iNV-GOV on HuHSC-NCG-PDX mice US Results of combined treatment with gemcitabine; where A shows changes in tumor volume and H&E histological sections in each group; and B shows changes in survival curves.

[0051] Figure 22 For cross-tumor validation (HCC-PDX): Tumor volume and survival curve results in each group after intravenous administration of iNV-GOV and US activation in the HubBMC-NCG model.

[0052] Figure 23 For cross-tumor validation (IHCC-PDX): Tumor volume and survival curve results in each group after intravenous administration of iNV-GOV and US activation in the HubBMC-NCG model. Detailed Implementation

[0053] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0054] Unless otherwise stated, all percentages (%) are weight-volume percentages (w / v%); temperatures are in °C; and particle sizes are the volume-weighted median values ​​of dynamic light scattering (DLS). Those skilled in the art can make conventional modifications to the implementation conditions without departing from the spirit of this invention.

[0055] Example 1: Screening for heat shock gene switches 1. Carrier Construction The promoters HSP70 (nucleotide sequence SEQ ID NO: 1) and HSPA6 promoters HSP-1 (nucleotide sequence SEQ ID NO: 2) and HSP-2 (nucleotide sequence SEQ ID NO: 3) were selected and ligated to the luciferase gene Gluc to obtain the expression cassette HSP-Gluc. Then, the expression cassette HSP-Gluc was ligated to the pGluc-Basic (Miaoling Bio P0788) vector to obtain a recombinant vector containing the expression cassette HSP-Gluc.

[0056] >HSP70 nucleotide sequence (SEQ ID NO: 1): CACTCTGGCCTCTGATTGGTCCAAGGAAGGCTGGGGGGCAGGACGGGAGGCGAAAACCCTGGAATATTCCCGACCTGGCAGCCTCATCGAGCTCGGTGATTGGCTCAGAAGGGAAAAGGCGGGTCTCCGTGACGACTTATAAAAGCCCAGGGGCAAGCGGTCCGGATAACGGCTAGCCTGAGGAGCTGCTGCGACAGTCCACTACCTTTTTCGAGAGTGACTCCCGTTGTCCCAAGGCTTCCCAGAGCGAACCTGTGCGGCTGCAGGCACCGGCGCGTCGAGTTTCCGGCGTCCGGAAGGACCGAGCTCTTCTCGCGGATCCAGTGTTCCGTTTCCAG >HSP-1 nucleotide sequence (SEQ ID NO: 2): HSP-2 nucleotide sequence (SEQ ID NO: 3): AATTCTACCACTGAACCACCAATGCTACTGTCAGCTAAAGACCTGCAGTATTGTCTCTTAAAGCTCACTATCTCTGGCCATTCACTAAGGAACCAGGCACCGTCTTAAATCGCGGTTTGGAAAATATTTTGTTCAAGATAAAACTGTTTTAAGATATACGTGTATATATCTTATATATCTGTATTCGCATG GTAACATATCTTCGGCCTTCCTGAGCCGCTGGGCTCTCAGCGGCCCTCCAAGGCAGCCCGCAGGCCCCTGTGTGCCTCAGGGATCCGACCTCCCACAGCCCCGGGGAGACCTTGCCTCTAAAGTTGCTGCTTTTGCAGCCTCTGCCACAACCGCGCGTCCTCAGAGCCAGCCCGGAGGAGCTAGAACCTTCC CCGCATTTCTTTCAGCAGCCTGAGTCAGAGGCGGGCTGGCCTGGCGTAGCCGCCCAGCCTCGCGGCTCATGCCCCGATCTGCCCGAACCTTCTCCCGGGGTCAGCGCCGCGCCGCGCCACCCGGCTGAGTCAGCCCGGGCGGGCGAGAGGCTCTCAACTGGGCGGGAAGGTGCGGGAAGGTGCGGAAAGGT TCGCGAAAGTTCGCGGCGGCGGGGTCGGGTGAGGCGCAAAAGGATAAAAAGCCGGTGGAAGCGGAGCTGAGCAGATCCGAGCCGGGCTGGCTGCAGAGAAACCGCAGGGAGAGCCTCACTGCTGAGCGCCCCTCGACGGCGGAGCGGCAGCAGCCTCCGTGGCCTCCAGCATCCGACAAGAAGCTTCAGCC 2. HSP-Gluc Screening and Activation Parameters The recombinant vector was transiently transduced into Capan-1 cells, and the induction intensity and background values ​​of different heat shock promoter HSP elements under mild and hot conditions in diagnostic ultrasound (US) were compared; the US pulse train settings included: (1) Maintain the temperature at approximately 42°C using online temperature control with a pulse rate of 1.2 W / cm² and a total pulse duration of 5 min; (2) The online temperature control maintains approximately 42°C by pulses of 1.2 W / cm², 5 min on / 5 min off × 2, for a total of 10 min. (3) The online temperature control maintains approximately 42°C by pulses of 1.2 W / cm², 5 min on / 5 min off × 3, for a total of 15 min. (4) Maintain the online temperature at approximately 42°C with a pulse of 1.2 W / cm² and a total of 15 min.

[0057] The supernatant luminescence intensity (RLU) was collected at 6–12 h, and stability was assessed. Results are as follows: Figure 1 As shown, HSP-2 background is low and activated gene expression is high; Figure 2 As shown, the US pulse column setting (3) has high activation efficiency. Therefore, the subsequent virus construction selects the HSP-2 promoter and selects (3) for the US pulse column setting.

[0058] Example 2 Construction of genetically engineered oncolytic virus (GOV) 1. Carrier Construction GSDMD NT The N-terminal domain of the GSDMD gene and the self-cleaving peptide sequence T2A were ligated into the pcDNA3.1-mCherry vector (Miaoling Biotechnology; P34727) to obtain pcDNA3.1-GSDMD. NT -T2A-mCherry, then the HSPA6 (HSP-2) gene promoter fragment is ligated to pcDNA3.1-GSDMD NT On the -T2A-mCherry vector, a gene containing the expression cassette HSP(HSP-2)→GSDMD was constructed. NT The recombinant plasmid.

[0059] Using a similar approach, the HSP-2 gene promoter fragment and the CMV promoter fragment were ligated into pcDNA3.1-GSDMD, respectively. FL -T2A-mCherry and pcDNA3.1-GSDMD NT On the -T2A-mCherry vector, expression cassettes HSP-2→GSDMD were constructed respectively. FL CMV→GSDMD NT The recombinant plasmid was used as a control, in which GSDMD FL This is the full-length sequence of the GSDMD gene.

[0060] GSDMD FL Amino acid sequence (SEQ ID NO: 4): MPSAFEKVVKNVIKEVSGSRGDLIPVDSLRNSTSFRPYCLLNRKFSSSRFWKPRYSCVNLSIKDILEPSAPEPEPECFGSFKVSDVVDGNIQGRVMLSGMGEGKISGGAAVSDSSSASMNVCILRVTQKTWETMQHERHLQQPENKILQQLRSRGDDLFVVTEVLQTKEEVQITEVHSQEGSGQFTLPGALCLKGEGKGHQSRKKMVTIPAGSILAFRVAQLLIGSKWDILLVSDEKQRTFEPSSGDRKAVGQRHHGLNVLAALCSIGKQLSLLSDGIDEEELIEAADFQGLYAEVKACSSELESLEMELRQQILVNIGKILQDQPSMEALEASLGQGLCSGGQVEPLDGPAGCILECLVLDSGELVPELAAPIFYLLGALAVLSETQQQLLAKALETTVLSKQLELVKHVLEQSTPWQEQSSVSLPTVLLGDCWDEKNPTWVLLEECGLRLQVESPQVHWEPTSLIPTSALYASLFLLSSLGQKPC >GSDMD FL Nucleotide sequence (SEQ ID NO: 5): >GSDMD NT Amino acid sequence (SEQ ID NO: 6): MPSAFEKVVKNVIKEVSGSRGDLIPVDSLRNSTSFRPYCLLNRKFSSSRFWKPRYSCVNLSIKDILEPSAPEPEPECFGSFKVSDVVDGNIQGRVMLSGMGEGKISGGAAVSDSSSASMNVCILRVTQKTWETMQHERHLQQPENKILQQLRSRGDDLFVVTEVLQTKEEVQITEVHSQEGSGQFTLPGALCLKGEGKGHQSRKKMVTIPAGSILAFRVAQLLIGSKWDILLVSDEKQRTFEPSSGDRKAVGQRHHGLNVLAALCSIGKQLSLLSD >GSDMD NT Nucleotide sequence (SEQ ID NO: 7): atgccatcggcctttgagaaagtggtcaagaatgtgatcaaggaggtaagcggcagcagaggcgatctcattccggtggacagcctgcggaactccaccagcttcaggccctactgccttctgaacaggaaattttcaagctcaaggttctggaaaccccgttattcatgtgtcaacctgtcaatcaaggacatcctggagcccagtgctccagaaccagaaccggagtgttttggctccttcaaagtctctgatgtcgtcgatgggaacattcagggcagagtgatgttgtcaggcatgggagaagggaaaatttctggtggggctgcagtgtctgacagttccagtgcctccatgaatgtgtgtatactgcgtgtgactcagaagacctgggagaccatgcagcatgaaaggcaccttcagcagcctgagaacaaaatcctgcaacagcttcggagtcgtggggatgacctgtttgtggtgaccgaggtgctgcagacaaaggaggaagtgcagatcactgaggtccacagccaagagggctcaggccagtttacgctgcctggagctttatgcttgaagggtgaaggcaagggccaccaaagccggaagaagatggtgaccattcctgcaggcagcatcctggcattccgagtggcccaactgcttattggctctaaatgggatatccttctcgtctcagatgagaaacagaggacctttgagccctcctcaggtgacagaaaagcagtgggccagaggcaccatggcctcaatgtgcttgctgcgctttgttccatcggaaagcagctcagtctcctgtcagat 2. GOV construction Insert the hTERT-E1A gene fragment and the above-mentioned HSP-2→GSDMD NT expression cassette into plasmid pDC315 (Miaoling Biology; P1429) to construct the recombinant shuttle plasmid pDC315-hTERT-E1A-HSP-GSDMD NT .

[0061] The recombinant shuttle plasmid was then co-transfected with the Ad5 AdMax backbone plasmid pBHGlox(delta)E13Cre (Miaoling Biotechnology; P0883) into 293 cells for virus packaging. After cell lysis, the viral fluid was collected, amplified in HeLa cells, and purified using a commercially available chromatography purification kit. Finally, dosage calibration was performed using virus particle count / cell count (VP / Cell) or multiple of infection (MOI) to obtain a high-purity recombinant oncolytic virus (GOV) formulation.

[0062] hTERT-E1A nucleotide sequence (SEQ ID NO: 8): 3. GOV infects tumor cells and induces pyroptosis. Human pancreatic cancer cells Capan-1 (ATCC-derived) were used for infection at 10–20 VP / cell. Activation was performed via ultrasound (US) on day 3 post-infection. The control group received no activation or received oral veneer (OV) without GSDMD. NT ).

[0063] After US activation, live / dead cells were stained with Calcein-AM / TO-PRO-3. Calcein-AM labeled live cells (green), and TO-PRO-3 labeled dead cells (red). The results are as follows: Figure 3 As shown, when GSDMD NT During the expression, almost all cancer cells died within 60 minutes.

[0064] Cells were collected on day 4, and GSDMD was detected by immunoblotting. NT or GSDMD FL The result is as follows Figure 4 As shown, only GSDMD cells infected with OV or GOV were found. FL Background expression (due to the fact that oncolytic adenovirus infection itself induces cell lysis and endogenous GSDMD activation, GSDMD derived from host cells can also be detected). FL (background expression), without GSDMD NT The expression indicates that GSDMD is visible after US activation. NT Express.

[0065] Cell supernatant was collected on day 6, and LDH release was measured. The results are as follows: Figure 5 As shown, GOV US The LDH release in the group was significantly higher than that in the control group and other groups, indicating increased pyroptosis.

[0066] 4. GOV reinfection aptitude test A dual-chamber culture apparatus (outer and inner chambers) was used to verify the "release-reinfection" capability of GOV. First, TRITC-labeled cancer cells in the outer chamber were inoculated with GOV and cultured for 72 hours as the initial source of infection. Subsequently, the outer chamber cells were activated by sonication, and fresh culture medium was added at 76 hours to connect the inner and outer chambers, allowing viral particles to diffuse through membrane pores. After further culture for 120 hours, the inner chamber cells were collected for confocal microscopy and flow cytometry analysis. Results are as follows: Figure 6 As shown, the ultrasound activation group (GOV) US The results showed a significant enhancement of viral marker signal (GFP) in the luminal cells, which was significantly higher than that in the non-activated group (GOV). This indicates that GOV can be released from the external cavity and reinfect neighboring cells after being activated by ultrasound, thus achieving a cascade reinfection effect.

[0067] Example 3 Preparation of engineered membrane-coated oncolytic virus (iNV-GOV) 1. Donor cell immunotherapy like Figure 7 As shown, in wild-type HEK293 cells (HLA-I + HLA-II + The B2M and CIITA were knocked out using CRISPR-Cas (to weaken or remove HLA-I / II presentation), while HLA-E and CD47 were knocked into the homology-directed repair (HDR) sites to reduce NK / phagocytic clearance.

[0068] Then, the CAR gene was introduced into cells using the lentiviral vector Lenti-CAR, allowing the CAR gene to integrate into the cell genome. This enabled the cells to stably express the extracellular recognition domain and transmembrane anchoring structure of the chimeric antigen receptor (CAR) on the cell membrane surface. The target site of the CAR protein was selected from at least one of HER2, CLDN18.2, GPC3, and PSCA, resulting in genetically engineered HEK293 donor cells (HLA-I). - HLA-II - ICD47 + HLA-E + CAR + The phenotypes were verified by flow cytometry and immunoblotting.

[0069] 2. Membrane separation and forming (1) Place the donor cells in a hypotonic buffer solution to relax and rupture the cells; (2) Differential centrifugation and density gradient centrifugation were performed sequentially to separate and purify membrane components, with nuclease and protease inhibitors added during the process; (3) The membrane components were refolded in an isotonic buffer system and extruded or subjected to short-range ultrasonication to obtain membrane nanovesicles (iNVs) with a particle size of 100–250 nm; iNVs with a particle size of about 150–190 nm were obtained by extruding 5–15 times with a pore size of 200 nm. (4) Conduct quality control, including testing at least particle size / polydispersity index, zeta potential, and membrane protein markers (Na+). + ,K + -ATPase, ALIX / TSG101), endotoxin and residual nucleic acid; these parameters ensure that vesicles retain donor membrane transmembrane / outward protein orientation and are scalable reproducible.

[0070] The results are as follows Figure 8 As shown, the vesicle particle size exhibits a single-peak distribution, with a particle size of approximately 150 nm.

[0071] 3. Loading of iNV-GOV GOV oncolytic adenovirus was infected in donor cells at an MOI of 0.1–10 and cultured for 12–72 h to allow the virus to replicate intracellularly and acquire host membrane components. Following cytochalasin B-induced lysis, GOV-loaded vesicles were obtained. A membrane extrusion step was used to embed or coat the virus within the membrane vesicles during remodeling, resulting in nanoscale engineered membrane-coated oncolytic virus (iNV-GOV). This iNV-GOV carries CD47, HLA-E, and CAR molecules on its surface, exhibiting immune stealth and targeted recognition properties. Free virus and uncomposite membranes were removed via density gradient / gel filtration / immunocapture to obtain purified iNV-GOV.

[0072] Immunoblotting detection of B2M, CIITA, HLA-E, CD47, E1A, and membrane / vesicle-related markers (such as ALIX and Na) in wild-type HEK 293 cells, donor cells, iNV-GOV, and iNV. + ,K + The expression of ATPase, etc. was observed. The results were as follows: Figure 9 As shown, in gene-edited donor cells, the expression signals of B2M and CIITA genes were completely absent, indicating successful knockout of the HLA-I and HLA-II pathways. Meanwhile, the expression of HLA-E and CD47 proteins was significantly enhanced, confirming the successful knock-in of immune regulatory molecules. Furthermore, adenovirus-specific E1A protein signaling was detected in iNV-GOV, while no obvious banding was observed in iNV, indicating that iNV-GOV contains viral components of GOV. Membrane / vesicle marker proteins ALIX, FLOT1, and Na... + ,K + -ATPase was detected in both iNV-GOV and iNV, suggesting that both have typical cell membrane origin characteristics.

[0073] The complex structure of GOV and iNV-GOV was detected using transmission electron microscopy combined with anti-adenovirus type 5 (Ad5) antibody gold nanoparticles (Anti-Ad5-Gold). Figure 10 TEM results showed that GOV particles exhibited a typical adenovirus morphology, while iNV-GOV particles were encapsulated by a distinct cell membrane structure. After Anti-Ad5-Gold immunolabeling, numerous gold particle signals were observed on the surface of GOV particles, indicating that their capsid proteins could be specifically recognized. In contrast, almost no gold particles were observed binding on the surface of iNV-GOV particles, demonstrating that the cell membrane coating effectively shielded the viral capsid antigen. These results confirm that the iNV membrane successfully encapsulated the oncolytic virus GOV, achieving immune concealment of the adenovirus capsid.

[0074] The viral load of iNV-GOV was determined using dot blot and real-time quantitative PCR methods, and the GOV copy number in iNV samples with different protein levels was analyzed. Results are as follows: Figure 11 As shown, the viral copy number in iNV-GOV increased linearly with increasing iNV protein content, while no significant signal was detected in the blank iNV group. Further calculations indicated that the viral load was approximately 1 × 10⁻⁶. 8 The saturation value was reached at VP / 2 μg iNV protein. These results indicate that the viral loading of iNV-GOV exhibits good linear correlation and uniformity, demonstrating that the preparation system possesses controllable and stable loading efficiency.

[0075] Example 4: iNV-GOV In Vitro Experiment 1. iNV-GOV stability The iNV-GOV prepared in Example 3 was incubated for 24 h at 37°C and 100 rpm with ±10% human serum added. Viral load and infectivity retention were detected using dot blot and qPCR. Results are as follows: Figure 12 As shown, iNV-GOV can maintain stable viral signal and viral load under the above physiological conditions, indicating that its membrane encapsulation structure has good temperature and serum stability.

[0076] 2. Immunogenicity of iNV-GOV iNV-GOV was co-cultured with human peripheral blood mononuclear cells for 3 days, and CD3 count was measured. + T cells and CD56 + Expression of CD69, an early activation marker of NK cells. Results are as follows: Figure 13 As shown, compared with naked GOV, the activation levels of T cells and NK cells induced by iNV-GOV were significantly reduced, indicating that its immunogenicity was significantly weakened and it has good immune stealth properties, which helps to improve in vivo delivery and safety.

[0077] 3. In vitro immune activation and tumor-killing effects of iNV-GOV Primary cancer cells were isolated from fresh tumor tissue of patients with pancreatic ductal adenocarcinoma (PDAC) and cultured in three dimensions to form pancreatic cancer organoids (PDOs). Subsequently, PDOs from different donors were co-cultured with their matched peripheral blood mononuclear cells, and immune activation and killing effects were evaluated under different treatment conditions such as iNV-GOV±US.

[0078] Mass spectrometry flow cytometry analysis results are as follows Figure 14 As shown, iNV-GOV US The processing significantly improved CD8 + T cell / Treg cell ratio ( Figure 14 A), and enhanced CD8+ IFN-γ expression level in T cells ( Figure 14 (B) indicates that ultrasound-activated iNV-GOV can effectively promote tumor antigen-specific T cell responses. Further assessment of PDO cell survival was performed using confocal microscopy and Calcein-AM / TO-PRO-3 double staining. Results are as follows... Figure 15 The display shows that iNV-GOV US In the treatment group, the number of dead cells in PDO was significantly increased, and the organoid structure was significantly disrupted, while only limited damage was observed in the control group and other treatment groups. Quantitative results indicate that iNV-GOV US The organoid cell mortality rate was highest in group (P<1×10⁻⁶). -9 This study verified that the system has significant immune activation and tumor killing effects in in vitro organoid models.

[0079] Example 5: In vivo therapeutic effect of iNV-GOV on a mouse model of pancreatic ductal adenocarcinoma (PDAC). 1. Constructing a humanized in situ PDAC-PDX mouse model NCG-immunodeficient mice were selected, and the human immune system was reconstructed using human PBMCs to obtain the HuPBMC-NCG model. From HLA-A2... + Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and hydrodynamically injected with hCSF1, hGM-CSF, and hIL-4 expression plasmids to promote immune reconstitution. Blood samples were collected on day -14 to detect hCD45. + Cells were used to confirm successful reconstruction, resulting in the HuPBMC-NCG mouse model.

[0080] Tumor tissue blocks from PDAC patients were harvested and orthotopic tumor transplantation of the pancreas was performed in a HuPBMC-NCG mouse model to establish a humanized orthotopic PDAC-PDX model.

[0081] 2. Drug administration and activation In the constructed humanized orthotopic PDAC-PDX mouse model, iNV-GOV was administered systemically once on day 0 (administration route: intraperitoneal instillation ip, dose: 1.5 × 10⁻⁶). 10 VP), and then diagnostic ultrasound and ultrasound-guided thermal induction were applied to the tumor area on days 3, 6, and 9, respectively, with the following parameter windows selected: 40-43 ℃, 0.8-1.5 W / cm², 5 min on / 5 min off × 3, total 15 min.

[0082] 3. Immunological assessment On day 12, mouse tumor tissue was collected, and TAM polarization (M1: CD86+; M2: CD206+), Treg (Foxp3+), and CD8+ T cell IFN-γ / GZMB were assessed by flow cytometry / immunohistochemistry. The results are as follows: Figure 16-17 As shown, iNV-GOV US After treatment, the proportion of M1 macrophages significantly increased compared to M2 macrophages, and the release capacity of IFN-γ and granzyme B (GZMB) from CD8+ T cells was also significantly enhanced. Tumor antigen-specific effector T cells in the spleen were detected using ELISApot, and the results were as follows: Figure 18 As shown, iNV-GOV US The treatment can increase the proportion of tumor antigen-specific effector T cells, demonstrating that the therapy can promote antigen cross-presentation.

[0083] 4. Therapeutic Indicators like Figure 19 As shown in Figure A, compared with the control group, iNV-GOV US The tumor volume was significantly reduced in this group, and H&E indicated extensive necrosis in the tumor area and an increased proportion of normal glandular characteristics; such as Figure 19 As shown in B, iNV-GOV US The group showed stronger tumor suppression and survival benefits.

[0084] This embodiment demonstrates that in the humanized in situ PDAC-PDX model, a single systemic administration of iNV-GOV followed by ultrasound activation of pyroptosis expression can effectively reshape the tumor immune microenvironment, enhance tumor antigen-specific T cell responses, and achieve significant tumor growth inhibition, thus verifying the in vivo effectiveness of iNV-GOV and the activation method.

[0085] Example 6: iNV-GOV in combination with other drugs 1. Construction of HuHSC-NCG-PDX mouse model NCG mice were selected and pretreated with sublethal doses of irradiation. Human-derived CD34 was then injected via the tail vein. + Hematopoietic stem cells (CD34) + HSC, 1×10 6 / animal). Raised under sterile conditions for 8-12 weeks. hCD45 was measured by peripheral blood flow cytometry. + Cell proportions confirmed human immune reconstitution, resulting in HuHSC-NCG mice. HLA-A2 was obtained. +Fresh tumor tissue from PDAC patients was fragmented and passaged subcutaneously for 1-3 generations (P1→P3) to establish stable PDX tumor sources. 14 days before drug administration, passaged PDX tumor fragments were implanted into HuHSC-NCG mice under sterile conditions via orthotopic pancreatic transplantation to obtain the HSC humanized orthotopic PDAC-PDX mouse model.

[0086] 2. Combination therapy of iNV-GOV with PD-1 / PD-L1 inhibitors In the HuHSC-NCG-PDX orthotopic pancreatic cancer model, iNV-GOV US Combined with α-PD1, time-series coupling was as follows: iNV-GOV was administered on day 0. US (Administration method: intraperitoneal instillation ip, dose: 1.5 × 10) 10 VP), US activation was administered on days 3, 6, and 9. a-PD1 (purchased from BioXcell, catalog number BE0188) was administered on day 5 via intraperitoneal instillation (ip), at a dose of 200 μg antibody, every 3 days for 4 doses. Twenty-five days after administration, mouse tumor tissue was collected, such as... Figure 20 As shown, in the huHSC-NCG-PDX model, the combined application of iNV-GOV activated by ultrasound and anti-PD-1 antibody produced a synergistic tumor-suppressing effect: the tumor volume in the combined treatment group was close to zero, and "tumor clearance" was observed in 5 / 6 individuals. Histological scores showed a significant increase in normal tissue and a significant decrease in PDAC tissue, and survival was significantly better than that of the same dose of single drug and the same control group. This demonstrates that the iNV-GOV formulation and the administration / activation regimen can be combined with immune checkpoint inhibitors for the treatment of pancreatic cancer.

[0087] 2. iNV-GOV in combination with chemotherapy drugs (GEM) In the HuHSC-NCG-PDX treatment of pancreatic carcinoma in situ, iNV-GOV was administered in combination with gemcitabine (GEM, purchased from MCE, catalog number HY-17026) on day 0. US (Administration method: intraperitoneal instillation ip, dose: 1.5 × 10⁻⁶) 10 VP), US activation was administered on days 3, 6, and 9. GEM was administered on day 5 via intraperitoneal instillation (ip), at a dose of 10 mg / kg, every 3 days for 4 administrations. Tumor tissue was collected from mice 25 days after administration, and the results are as follows... Figure 21 As shown, GEM monotherapy only exhibited limited tumor shrinkage, while iNV-GOV US iNV-GOV, when used alone, significantly reduced tumor volume and decreased adenocarcinoma component. USThe combination of iNV-GOV and GEM showed the most significant tumor suppression. Hematologic and epithelial analysis (H&E) revealed extensive tumor regression and the highest proportion of normal glandular features, with some mice achieving a tumor-free state. Survival analysis also showed that the combination group had the longest survival, significantly better than either single-drug therapy, suggesting that the therapy of iNV-GOV, which is activated by ultrasound to express pyroptosis to promote oncolytic virus amplification and killing, has a synergistic anti-tumor effect and a clear survival benefit with GEM.

[0088] Example 7: Validation of cross-tumor application The same procedure was followed to validate the model in the hepatocellular carcinoma (HCC-PDX) and intrahepatic cholangiocarcinoma (IHCC-PDX) xenograft models: In the orthotopic models of HCC-PDX and IHCC-PDX in HubBMC-NCG mice (the mouse model construction method is as described in Example 5), the following administration and activation protocols were followed: A single intravenous injection (iv) of iNV-GOV (1.5 × 10⁻⁶) was administered on day 0. 10 VP / animal), and gentle ultrasound was applied to the tumor area for thermal induction on days 3, 6, and 9. Results are as follows Figure 22 and Figure 23 As shown, compared with the saline control and the unactivated iNV-GOV, iNV-GOV... US Both PDX models showed significant reduction in tumor volume and prolongation of survival, indicating that the iNV-GOV therapy, which promotes oncolytic virus amplification and killing through ultrasound-activated pyroptosis, has cross-tumor applicability and in vivo antitumor effects.

[0089] Those skilled in the art can make reasonable substitutions or equivalent changes to the US activation parameters, MOI, extrusion orifice size / number of times, buffer system formulation, and combined drug administration sequence of the above embodiments without departing from the spirit and substance of the present invention, and all such equivalent substitutions should fall within the protection scope of the present invention.

Claims

1. An oncolytic virus, characterized in that, The oncolytic virus contains an expression cassette that regulates the expression of the GSDMD gene under the heat shock promoter. Preferably, the heat shock promoter includes promoters HSP70 and HSPA6; More preferably, the sequence of the heat shock promoter is as shown in any one of SEQ ID NO: 1 to SEQ ID NO:

3.

2. The oncolytic virus according to claim 1, characterized in that, The GSDMD includes the GSDMD N-terminal sequence.

3. A membrane-coated oncolytic virus, characterized in that, The membrane-coated oncolytic virus is obtained by coating the oncolytic virus according to any one of claims 1-2 into a cell membrane; Preferably, the cell membrane is a cell membrane that weakens or removes HLA-I / II presenting cells; More preferably, the cells that weaken or remove HLA-I / II presentation are cells with the B2M and CIITA genes knocked out.

4. The membrane-coated oncolytic virus according to claim 3, characterized in that, The cell membrane is the cell membrane of a cell into which immune regulatory molecules have been knocked in; Preferably, the immunomodulatory molecules include HLA-E and CD47.

5. The membrane-coated oncolytic virus according to claim 3, characterized in that, The cell membrane is the cell membrane of a cell whose genome has integrated the CAR gene; Preferably, the target of the CAR protein is selected from at least one of HER2, CLDN18.2, GPC3, and PSCA.

6. The method for preparing membrane-coated oncolytic virus according to any one of claims 3-5, characterized in that, Includes the following steps: (1) Infect cells with oncolytic virus and culture for 12–72 h to obtain intracellular or membrane-bound virus; (2) After infection, cells are subjected to gentle cell relaxation and membrane separation, and then extruded into shape, so that the virus is embedded or coated by membrane vesicles during the extrusion / reconstruction process to obtain membrane-coated oncolytic virus; Preferably, the method for preparing the membrane-coated oncolytic virus further includes removing free virus and uncoated membrane by density gradient / gel filtration / immunocapture; Preferably, the multiplicity of infection (MOI) of the infection in step (1) is 0.1 to 10; Preferably, step (2) extrusion includes extrusion molding in an isotonic buffer system or short-range ultrasound to obtain membrane-coated oncolytic viruses with a particle size of 100–250 nm.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the oncolytic virus according to any one of claims 1-2 or the membrane-coated oncolytic virus according to any one of claims 3-5.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes PD-1 / PD-L1 inhibitors or chemotherapy drugs; Preferably, the chemotherapy drug includes gemcitabine.

9. The use of the oncolytic virus according to any one of claims 1-2 or the membrane-coated oncolytic virus according to any one of claims 3-5, or the pharmaceutical composition according to any one of claims 7-8 in the preparation of a cancer treatment drug; Preferably, the cancer includes pancreatic ductal adenocarcinoma, hepatocellular carcinoma, or intrahepatic bile duct carcinoma.

10. The use of the membrane-coated oncolytic virus according to any one of claims 3-5 in combination with a PD-1 / PD-L1 inhibitor or a chemotherapeutic agent in the preparation of a medicament for treating cancer; Preferably, the chemotherapy drug includes gemcitabine; Preferably, the cancer includes pancreatic ductal adenocarcinoma, hepatocellular carcinoma, or intrahepatic bile duct carcinoma.

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