An engineered attenuated salmonella vnp-snase, and preparation method and application thereof

Abstract

This invention discloses an engineered attenuated Salmonella VNP-SNase, its preparation method, and its applications, including methods and compositions for treating various solid tumors or metastatic tumors. Specifically, engineered attenuated Salmonella capable of expressing and secreting SNase is recruited to the tumor microenvironment through tumor targeting, specifically degrading intratumoral neutrophil extracellular traps (NETs), weakening the killing effect of NETs on Salmonella, increasing its intratumoral colonization, and simultaneously improving the tumor-promoting and immunosuppressive effects caused by NETs, ​​while effectively preventing bacteria from off-target into normal organs. By releasing SNase to activate / regulate the immune system and directly / indirectly inhibit tumors, it can act as an effective immune effector.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the preparation and application of engineered attenuated Salmonella. Background Technology

[0002] Since Dr. William Coley first attempted to treat cancer patients with heat-inactivated Gram-positive bacteria (streptococci) and Gram-negative bacteria (Salmonella myxoides) in the late 19th century, an increasing number of microorganisms have been developed and modified for the treatment of cancer (Coley WB et al., 1991, Clinical Orthopedics and Related Research, 262(262):3-11). These microorganisms, often referred to as "oncolytic bacteria," include Salmonella, Listeria, and Escherichia coli. Due to their facultative anaerobic properties and the unique characteristics of the tumor microenvironment, including the hypoxic environment, immunosuppressive environment, and nutrients released by a large number of necrotic cells within the tumor, they can achieve efficient colonization at the tumor site after injection (GURBATRI CR et al., 2020, Science translational medicine, 12(530); ZHOUS et al., 2018, Nature reviews Cancer, 18(12): 727-43; SUH S et al., 2019, Advanced science (Weinheim, Baden-Wurttemberg, Germany), 6(3): 1801309.). However, due to their toxicity, these oncolytic bacteria often cause varying degrees of liver and spleen damage in the body. For example, VNP20009, an attenuated strain of Salmonella Typhimurium (hereinafter referred to as VNP), has attracted widespread attention due to its lower toxicity and promising preclinical antitumor effects (Clairmont C et al., 2000, Journal of Infectious Diseases, 181: 1996-2002). VNP's antitumor activity is accompanied by a significant increase in neutrophil infiltration in the tumor microenvironment (TME) (Wu, L et al., 2024, Advanced Materials, 36: 36). Similar phenomena have been observed in preclinical and clinical studies using other oncolytic bacteria, such as attenuated Listeria monocytogenes and Escherichia coli Nissle 1917, both of which have reported large accumulations of neutrophils within the TME (Westphal, K et al., 2008, Cancer Research, 68, 2952-2960). The role of neutrophils in tumor progression remains highly controversial.

[0003] Neutrophils, as the body's first line of defense against bacterial or fungal infections, exhibit high heterogeneity, which leads to their multifaceted role in tumor progression (Wang, H et al., 2024, Signal transduction and targeted therapy, 9, 235). For example, Finis Guerra et al. reported that they inhibited metastasis by inducing cancer cell death through HGF / MET-dependent nitric oxide (Finis Guerra, V et al., 2015, Nature, 522, 349–353). However, more research points to tumor-promoting effects: in a RAS-driven zebrafish model, neutrophils secrete PGE2, accelerating cancer cell proliferation (Antonio, N et al., 2015, EMBO J, 34, 2219–2236); in undifferentiated thyroid carcinoma, tumor-associated neutrophils maintain their vitality and release NETs through mitochondrial metabolic remodeling, promoting tumor growth and exacerbating immunosuppression in the microenvironment (Cristinziano, L et al., 2020, J. Immunol. 204, 1362–1372); numerous studies have also confirmed that NET formation significantly worsens prognosis. Neutrophils can phagocytose and kill bacteria by releasing extracellular neutrophil traps (NETs) (Brinkmann, V et al., 2004, Science). ， 303, 1532-1535).

[0004] Based on this, the present invention proposes a VNP genetic engineering strategy—to construct an engineered strain that can secrete anti-NETs enzymes in situ within tumors. After comprehensive comparison of molecular weight, folding compatibility, and clinical precedents, a 17 kDa Staphylococcus aureus nuclease (SNase) without disulfide bonds became an ideal candidate: it is not only smaller and less immunogenic than the 31 kDa DNase I, but it is also naturally killed by Staphylococcus aureus escaping NETs, ​​and its mechanism has been "tested in real-world applications".

[0005] The VNP-SNase integrated "live bacteria-enzyme" platform possesses three unique advantages: 1) tumor-selective chemotaxis, ensuring that SNase accumulates only in hypoxic lesions; 2) self-amplifying kinetics, allowing for continuous release of therapeutic doses with bacterial proliferation after a single administration; and 3) dynamic adaptation to microenvironmental changes, adjusting enzyme expression levels in real time. This invention simultaneously addresses the two major challenges of improving the safety and therapeutic efficacy of existing attenuated Salmonella, meeting the industry's need for a low-toxicity, highly effective, and economical composition for treating solid tumors. Summary of the Invention

[0006] The purpose of this invention is to address the current treatment challenges posed by oncolytic bacteria by providing an engineered attenuated Salmonella vector targeting extracellular neutrophil traps (NETs) for tumor-targeted therapy. This technology aims to: 1) reduce the infiltration of tumor-promoting neutrophils in the microenvironment and enhance intratumoral colonization of VNPs; and 2) allow the engineered attenuated Salmonella released and proliferating within the tumor to secrete SNase, thereby mitigating NETs-induced immunosuppression, enhancing antitumor immune responses, and improving tumor suppression effects.

[0007] To achieve the above-mentioned application objectives, the technical solution adopted in this application is as follows: Firstly, this application provides an engineered attenuated Salmonella VNP-SNase.

[0008] Secondly, this application provides a method for preparing engineered attenuated Salmonella VNP-SNase.

[0009] Thirdly, this application provides the use of engineered attenuated Salmonella in the preparation of drugs for treating tumors.

[0010] Fourthly, this application provides the application of engineered attenuated Salmonella in the preparation of drugs that remodel the tumor microenvironment.

[0011] Fifthly, this application provides a pharmaceutical composition.

[0012] Sixthly, this application provides a method for degrading NETs in the tumor microenvironment.

[0013] The first aspect of this application provides an engineered attenuated Salmonella VNP-SNase, which contains an exogenous nuclease coding sequence and is capable of secreting and expressing the exogenous nuclease SNase.

[0014] Furthermore, the engineered attenuated Salmonella VNP-SNase contains an exogenous nuclease coding sequence linked to a constitutive promoter, and the nuclease contains a secretory signal peptide, enabling the engineered bacteria to constitutively express and secrete Staphylococcus aureus nuclease SNase. The nucleotide sequence of the exogenous nuclease coding sequence is shown in SEQ ID NO:1. The promoter is a constitutive promoter or an inducible promoter. The constitutive promoter is J23100, J23112, or J23150. The nucleotide sequence of the J23150 promoter is shown in SEQ ID NO:2, the nucleotide sequence of the J23112 promoter is shown in SEQ ID NO:3, and the nucleotide sequence of the J23110 promoter is shown in SEQ ID NO:4. The inducible promoter includes pLuxI, nirB, and padh promoters. The nucleotide sequence of the pLuxI promoter is shown in SEQ ID NO:5, and the nucleotide sequence of the nirB promoter is shown in SEQ ID NO:5. As shown in NO:6, the nucleotide sequence of the padh promoter is derived from US970861262.

[0015] Furthermore, the attenuated Salmonella is attenuated Salmonella Typhimurium strain VNP20009 and its derivative strains, including but not limited to the aforementioned strains that have been authorized or are pending invention patents (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; patent application numbers 2025102995737, 202411906671.4, 202411906670.X, 202210181929.9, Acta Pharmaceutica Sinica B2021, 11(10):3165-3177; phoP / phoQ, etc.).

[0016] Furthermore, the secretory signal peptide is the pelB signal peptide, and the nucleotide sequence of the pelB signal peptide is shown in SEQ ID NO:7.

[0017] Furthermore, the 5' end of the nuclease coding sequence also includes a tag coding sequence and / or the engineered bacteria also includes a plasmid anti-loss element, the nucleotide sequence of which is shown in SEQ ID NO:8, and the nucleotide sequence of which is shown in SEQ ID NO:9.

[0018] The second aspect of this application provides a method for preparing engineered attenuated Salmonella VNP-SNase, comprising the following steps: (1) Construct a recombinant plasmid containing the nuclease coding sequence, the constitutive promoter, and the secretory signal peptide; (2) Transform the recombinant plasmid into attenuated Salmonella competent cells; (3) Screen positive clones to obtain engineered attenuated Salmonella VNP-SNase that is stably expressed and secreted.

[0019] Furthermore, the recombinant plasmid also includes a plasmid loss prevention element and / or the 5' end of the nuclease coding sequence contains an HA tag coding sequence. The nucleotide sequence of the plasmid loss prevention element is shown in SEQ ID NO:8, and the nucleotide sequence of the HA tag coding sequence is shown in SEQ ID NO:9.

[0020] The third aspect of this application provides the use of engineered attenuated Salmonella in the preparation of a drug for treating tumors.

[0021] Furthermore, the tumor is selected from melanoma, solid tumor, or metastatic tumor.

[0022] The fourth aspect of this application provides the use of engineered attenuated Salmonella in the preparation of drugs that remodel the tumor microenvironment. The engineered attenuated Salmonella remodels the tumor microenvironment by more strongly inhibiting angiogenesis, alleviating immunosuppression, significantly downregulating the protein levels of Ccl-3, Ccl-4, Cxcl2, Csf3r, S100a8, IL-17 and IL-6, and enhancing anti-tumor immunity.

[0023] The fifth aspect of this application provides a pharmaceutical composition comprising engineered attenuated Salmonella and a pharmaceutically acceptable carrier.

[0024] The sixth aspect of this application provides a method for degrading NETs in the tumor microenvironment, comprising administering engineered attenuated Salmonella VNP-SNase or a pharmaceutical composition to a subject.

[0025] The seventh aspect of this application concerns the use of engineered attenuated Salmonella in the preparation of therapeutic agents for combination with other tumor immunotherapy drugs or treatment methods. Examples include sustained antitumor immune effects. Given these unique advantages, this invention anticipates broad biomedical applications for this strategy in cancer treatment. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Explanation of abbreviations in the attached diagram: VNP: Attenuated Salmonella Typhimurium VNP20009; SNase: Staphylococcus aureus nuclease; VNP-SNase: VNP carrying the SNase gene plasmid; VNP-NC: VNP carrying an empty vector plasmid; NETs: Neutrophil extracellular traps; DNaseI: Deoxyribonuclease, a positive control for degrading NETs.

[0028] Figure 1 This is a schematic diagram of the pJ23100-SNase plasmid carried by the VNP-SNase engineered bacteria of the present invention.

[0029] Figure 2 This invention provides an agarose gel electrophoresis analysis of the target gene SNase and a linearization diagram of the Pth-23 vector. M represents the DNA marker; SNase is the DNA fragment of the target gene SNase; M represents the DNA marker; and Pth-23 is the DNA fragment of the plasmid vector carrying the target gene.

[0030] Figure 3 This invention provides a Western blotting diagram to verify the extracellular secretion of VNP-SNase. M represents the protein marker; VNP-SNase: After culturing the VNP-SNase strain in liquid LB medium to the logarithmic growth phase, the supernatant was collected by centrifugation, concentrated by ultrafiltration, and then Western blotting was used to detect the secretory expression of SNase in the supernatant. VNP: After culturing the VNP strain in liquid LB medium to the logarithmic growth phase, the supernatant was collected by centrifugation, concentrated by ultrafiltration, and then Western blotting was used to detect the secretory expression of SNase in the supernatant, serving as a control group.

[0031] Figure 4 This is a bacterial growth curve of VNP-SNase according to the present invention. The concentrations of VNP and VNP-SNase in the logarithmic growth phase were adjusted to OD. 600 =0.1, added to a 96-well plate, and added an antibiotic of appropriate resistance to the plate. After incubation for 28 h, bacterial growth curves were plotted (n=5).

[0032] Figure 5The image shows the expression of VNP-SNase verified by SDS-PAGE in this invention. M (Marker), lane 1 (VNP-SNase bacterial lysate sample), lane 2 (VNP bacterial lysate sample) (left 1); Escherichia coli BL21-pET228-His-SNase was constructed and induced to express by IPTG. M (Marker), lanes 1-3 (un-IPTG induced bacterial lysate samples), lanes 4-6 (IPTG induced bacterial lysate samples) (left 2); After induction with IPTG at 18°C ​​for 8 hours, the cells were sonicated, centrifuged to obtain bacterial supernatant, and purified by elution with different concentrations of imidazole using a nickel column. M (Marker), lane 1 (flow-through buffer), lane 2 (20 mM imidazole elution buffer), lane 3 (70 mM imidazole elution buffer), lane 4 (100 mM imidazole elution buffer), lane 5 (200 mM imidazole elution buffer) (left 3); N: 200 mM imidazole elution buffer collected. mM imidazole elution buffer, desalted, freeze-dried, and purity verified by SDS-PAGE; O: SNase can degrade DNA, M (DNA Marker), lane 1 (genomic DNA of mouse B16F10 cell line), lane 2 (genomic DNA + SNase) (left 4).

[0033] Figure 6 The diagram shows the DNA or RNA degradation capabilities of the SNase of this invention. M (DNA Marker), lane 1 (5 μg of genomic RNA from mouse B16F10 cell line), lane 2 (5 μg RNA + SNase) (left); M (DNA Marker), lane 1 (4 μg of genomic DNA from mouse B16F10 cell line), lane 2 (4 μg DNA + SNase) (right).

[0034] Figure 7 This is an immunofluorescence assay for VNP-SNase degradation of NETs, ​​as presented in this invention. Mouse bone marrow neutrophils were extracted and co-incubated with VNP at an MOI of 100:1 for 6 h. VNP was used as the control group. The effect of VNP-SNase on the NET backbone was observed by DNA staining (Sytox Green). The scale bar is 50 μm (n=5).

[0035] Figure 8 The image shows the morphological effects of VNP-SNase on NETs detected by scanning electron microscopy according to the present invention. The scale bar is 5 μm. The experiment was divided into a control group, a SNase protein group, a VNP group, and a VNP-SNase group.

[0036] Figure 9This is a diagram illustrating the degradation of extracellular DNA in the tumor microenvironment by VNP-SNase according to the present invention. Each mouse carrying a B16F10 tumor was intraperitoneally injected with 5 × 10⁻⁶ cells / mL. 210 Total extracellular DNA (n=3) in tumor tissue on day 9 after U bacteria treatment. The experiment was divided into PBS group, VNP group, and VNP-SNase group.

[0037] Figure 10 This is an agarose gel electrophoresis analysis of extracellular DNA fragmentation in tumor tissue after bacterial treatment according to the present invention (n=3).

[0038] Figure 11 This invention provides an enzyme-linked immunosorbent assay (ELISA) to analyze the level of the extracellular neutrophil trap (NETs) marker MPO-DNA in tumor tissue. Tumor tissue from mice treated with bacteria was ground, digested, and the supernatant was used to detect changes in the NETs marker MPO-DNA (n=3).

[0039] Figure 12 The figure shows the effect of SNase protein on NETs-induced B16F10 cell proliferation (n=5) detected by CCK-8 assay according to the present invention. Cells were treated with NETs culture medium, and 1 μg of SNase protein was added simultaneously. The proliferation of B16F10 cells was examined at different time points during co-culture.

[0040] Figure 13 The figure shows the effect of SNase on NETs-induced wound healing in B16F10 cells using a scratch assay according to the present invention (n=5). Clear and vertical wounds were drawn in 12-well plates containing B16F10 cells using a pipette tip. The cells were treated with NETs culture medium, and 1 μg of SNase protein was added to each well. The wound healing was observed under a microscope at different time points during co-culture.

[0041] Figure 14 This figure shows the effect of SNase protein on NETs-induced B16F10 cell migration (n=5) detected by the Trans-well assay of the present invention. 500 B16F10 cells were added to the upper chamber of the Trans-well apparatus, and NETs culture medium was placed in the lower chamber. 1 μg of SNase was added simultaneously. After fixation and crystal violet staining, the effect of SNase protein on NETs-induced B16F10 cell migration was observed under a microscope.

[0042] Figure 15The figure shows the effect of the SNase protein of this invention on NETs-induced B16F10 cell colony formation (n=5). 1000 B16F10 cells were seeded into each well of a 6-well plate. After complete adhesion, the medium was replaced with NETs medium, and 1 μg of SNase was added. After 7 days of culture, the cells were fixed, stained with crystal violet, and the effect of the SNase protein on NETs-induced B16F10 cell colony formation was observed under a microscope.

[0043] Figure 16 This is a schematic diagram illustrating the in vivo tumor-suppressing effect of VNP-SNase according to the present invention. VNP and VNP-SNase (5.0 × 10⁻⁶) were administered via intraperitoneal injection. 5 CFU / mouse and SNase (100 μg / mouse) were used to treat subcutaneous melanoma in C526L / 6J mice.

[0044] Figure 17 The images show the changes in tumor growth in tumor-bearing mice after treatment with different drugs according to the present invention. (Left) and a tumor photograph (Right). 1. PBS; 2. SNase; 3. VNP; 4. VNP+SNase; 5. VNP-SNase (n=7).

[0045] Figure 18 This is a time-dosing graph of tumor volume in tumor-bearing mice after treatment with different drugs according to the present invention. The calculation formula is TDT = t × log2 / log (Vt / V0), where t represents the number of days between two tumor volume measurements, Vt represents the tumor volume of the mouse on day t, and V0 represents the initial tumor volume of the mouse.

[0046] Figure 19 This figure shows the changes in survival time of tumor-bearing mice after treatment with different drugs according to the present invention. The Log-rank (Mantel–Cox) test was used.

[0047] Figure 20 The figure shows the content (n=7) of the NETs markers CitH3-DNA and MPO-DNA in serum for quantitative detection by ELISA according to the present invention.

[0048] Figure 21 This is a graph showing the relative mRNA expression (n=5) of NETs-related marker genes in tumor tissue after bacterial treatment according to the present invention. The target genes detected include Ltf, Padi4, Mpo, Elane, and MMP-9.

[0049] Figure 22 This image shows the formation of tumor NETs in tumor tissue after bacterial treatment, as detected by immunofluorescence according to the present invention. The scale bar is 100 μm (n=5). The detection indicators are MPO and CitH3.

[0050] Figure 23 This is a weight diagram of the major organs of the mice in this invention. On the last day of the experiment, the mice were dissected and their organs were collected to compare the differences in organ weight among the groups of mice (n=7).

[0051] Figure 24 This is a graph showing the number of VNPs in the major organs of mice according to the present invention. On the day of mouse dissection, the organs were weighed, ground, diluted in an appropriate ratio, spread on LB solid culture medium resistant to Cannab-R-N-T, inverted for culture, and counted (n=3).

[0052] Figure 25 This figure shows the Western blot analysis (n=3) of the angiogenesis-related gene expression in HUVEs after treatment with NETs combined with SNase. The assay indicators included changes in the PI3K-AKT pathway and its downstream angiogenesis-related genes ANG1, ANG2, and iNOS.

[0053] Figure 26 The present invention provides an immunohistochemical method for monitoring CD31 in tumor tissue. + The diagram is presented at a scale of 100 μm (n=5).

[0054] Figure 27 This is a graph showing the expression of angiogenesis-related proteins in tumors using immunofluorescence monitoring, with a scale bar of 200 μm (n=5). The indicators include ANG1, ANG2, and iNOS.

[0055] Figure 28 This is a diagram illustrating the reduction of B16F10 metastasis in the lungs of mice treated with VNP-SNase according to the present invention. The treatment involved injecting 2 × 10⁻⁶ saturates into the tail vein of each mouse. 5 A lung metastasis model was constructed using 10 B16F10 cells. On the fourth day of modeling, each mouse was injected via the tail vein with 1×10⁻⁶ B16F10 cells. 5 CFU of bacteria were used to treat mice, and the lung metastases and lung weight of mice were measured (n=7).

[0056] Figure 29 The images show representative lung metastases of the present invention (top), H&E scans (bottom), and quantitative analysis of B16F10 lung metastases (n=7).

[0057] Figure 30 The figure shows the overall survival rate (n=9) of B16F10 lung metastases in mice after VNP-SNase treatment according to the present invention. The Log-rank (Mantel–Cox) test was used.

[0058] Figure 31 This is an immunofluorescence image of NETs in lung tumors according to the present invention; the scale bar is 100 μm (n=5).

[0059] Figure 32 This is an immunohistochemical staining image of MMP-9 in tumors according to the present invention. The scale bar is 100 μm (n=5).

[0060] Figure 33 This is a comparison of the number of tumor-infiltrating T cells on day 9 after treatment in the B16F10 tumor model of the present invention. 1. PBS group; 2. SNase group; 3. VNP group and 4. VNP-SNase group (n = 5).

[0061] Figure 34 The diagram shows the MFI (molecular fibrillation index) of CD8⁺ T cell intracellular cytotoxic molecules GZMB and PRF, and the MFI (n=5) of exhaustion markers Tim-3 and PD-1.

[0062] Figure 35 This figure shows the Trans-well assay of the chemotactic effect of NETs on neutrophils (n = 5) according to the present invention. Mouse bone marrow neutrophils were sorted and seeded at 100,000 per well in the upper layer of the chamber. NET culture medium and SNase protein were added to the lower chamber, and the chemotactic effect of neutrophils in the upper chamber was observed.

[0063] Figure 36 The figure shows the relative expression (n = 5) of neutrophil chemotaxis-related gene mRNAs in tumor tissues detected by real-time quantitative PCR according to the present invention. The indicators detected include Ccl-3, Ccl-4, Ccl-6, Csf3r, C20r1, S100a8 and S100a9.

[0064] Figure 37 The figure shows the levels of neutrophil chemotactic proteins in mouse serum detected by ELISA (n=7). The indicators detected include Ccl-3, Ccl-4, Cxcl2, and Csf3r.

[0065] Figure 38 This is a schematic diagram illustrating the use of the VNP-SNase therapy of the present invention for tumor immunotherapy.

[0066] VNP treatment induces neutrophil recruitment mapping in the tumor microenvironment (TME) and promotes the formation of extracellular neutrophil traps (NETs). However, these NETs may counteract the therapeutic effects of VNP by promoting tumor growth, metastasis, angiogenesis, and T cell exhaustion. The engineered VNP strain VNP-SNase overcomes this limitation by degrading NETs in the TME and blocking NETs-mediated effects, thereby enhancing the antitumor efficacy of VNP. Detailed Implementation

[0067] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed.

[0068] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0069] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Here, A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0070] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c are single or multiple.

[0071] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0072] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0073] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application refers to units of mass known in the chemical industry, such as µg, mg, g, and kg.

[0074] The first aspect of this application provides an engineered attenuated Salmonella VNP-SNase, which contains an exogenous nuclease coding sequence and is capable of secreting and expressing the exogenous nuclease SNase.

[0075] The engineered attenuated Salmonella VNP-SNase contains an exogenous nuclease coding sequence, which is linked to a promoter. The nuclease contains a secretion signal peptide, enabling the engineered bacteria to constitutively express and secrete Staphylococcus aureus nuclease SNase. The nucleotide sequence of the exogenous nuclease coding sequence is shown in SEQ ID NO:1. The promoter is a constitutive or inducible promoter. The constitutive promoter is J23100, J23112, or J23150. The nucleotide sequence of the J23150 promoter is shown in SEQ ID NO:2, the nucleotide sequence of the J23112 promoter is shown in SEQ ID NO:3, and the nucleotide sequence of the J23110 promoter is shown in SEQ ID NO:4. The inducible promoter includes pLuxI, nirB, and padh promoters. The nucleotide sequence of the pLuxI promoter is shown in SEQ ID NO:5, and the nucleotide sequence of the nirB promoter is shown in SEQ ID NO:5. As shown in NO:6, the nucleotide sequence of the padh promoter is derived from US970861262.

[0076] In some embodiments, the attenuated Salmonella is attenuated Salmonella Typhimurium strain VNP20009 and its derivative strains, including but not limited to the strains that have been granted or are for which invention patents have been applied (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; patent application numbers 2025102995737, 202411906671.4, 202411906670.X, 202210181929.9, Acta Pharmaceutica Sinica B 2021, 11(10):3165-3177; phoP / phoQ, etc.).

[0077] In some embodiments, the secretory signal peptide is the pelB signal peptide, the nucleotide sequence of which is shown in SEQ ID NO:7.

[0078] In some embodiments, the 5' end of the nuclease coding sequence further includes a tag coding sequence and / or the engineered bacteria further includes a plasmid anti-loss element, the nucleotide sequence of which is shown in SEQ ID NO:8, and the nucleotide sequence of which is shown in SEQ ID NO:9.

[0079] The second aspect of this application provides a method for preparing engineered attenuated Salmonella VNP-SNase, comprising the following steps: (1) Construct a recombinant plasmid containing the nuclease coding sequence, the constitutive promoter, and the secretory signal peptide; (2) Transform the recombinant plasmid into attenuated Salmonella competent cells; (3) Screen positive clones to obtain engineered attenuated Salmonella VNP-SNase that is stably expressed and secreted.

[0080] In some embodiments, the recombinant plasmid further comprises a plasmid loss prevention element and / or the 5' end of the nuclease coding sequence comprises an HA tag coding sequence. The nucleotide sequence of the plasmid loss prevention element is shown in SEQ ID NO:8, and the nucleotide sequence of the HA tag coding sequence is shown in SEQ ID NO:9.

[0081] The third aspect of this application provides the use of engineered attenuated Salmonella in the preparation of a medicament for treating tumors.

[0082] In some embodiments, the tumor is selected from melanoma, solid tumor, or metastatic tumor.

[0083] The fourth aspect of this application provides the application of engineered attenuated Salmonella in the preparation of drugs that remodel the tumor microenvironment. The engineered attenuated Salmonella remodels the tumor microenvironment by more strongly inhibiting angiogenesis, alleviating immunosuppression, significantly downregulating the protein levels of Ccl-3, Ccl-4, Cxcl2, Csf3r, S100a8, IL-17 and IL-6, and enhancing anti-tumor immunity.

[0084] The fifth aspect of this application provides a pharmaceutical composition comprising engineered attenuated Salmonella and a pharmaceutically acceptable carrier.

[0085] The sixth aspect of this application provides a method for degrading NETs in the tumor microenvironment, comprising administering engineered attenuated Salmonella VNP-SNase or a pharmaceutical composition to a subject.

[0086] The seventh aspect of this application concerns the use of engineered attenuated Salmonella in the preparation of therapeutic agents in combination with other tumor immunotherapy drugs or treatment methods.

[0087] Example 1 This invention discloses an engineered attenuated Salmonella VNP-SNase, comprising an exogenous nuclease coding sequence linked to a promoter, and the nuclease containing a secretory signal peptide, enabling the engineered bacterium to constitutively express and secrete Staphylococcus aureus nuclease SNase. The nucleotide sequence of the exogenous nuclease coding sequence is shown in SEQ ID NO:1. The promoter is a constitutive promoter or an inducible promoter. The constitutive promoter is a J23100 promoter, or a J23112 promoter, or a J23150 promoter. The nucleotide sequence of the J23150 promoter is shown in SEQ ID NO:2, the nucleotide sequence of the J23112 promoter is shown in SEQ ID NO:3, and the nucleotide sequence of the J23110 promoter is shown in SEQ ID NO:4. The inducible promoter includes pLuxI, nirB, and padh promoters. The nucleotide sequence of the pLuxI promoter is shown in SEQ ID NO:4. As shown in NO:5, the nucleotide sequence of the nirB promoter is shown in SEQ ID NO:6, and the nucleotide sequence of the padh promoter is derived from US970861262. Although the levels of expression and secretion of SNase by the above-mentioned constitutive or inducible promoters vary in different VNPs and their derived strains, they can all produce similar antitumor therapeutic effects. Therefore, this invention uses the J23150 constitutive promoter, which has representative effects, as an example to demonstrate a typical case.

[0088] The attenuated Salmonella strains are attenuated Salmonella Typhimurium strain VNP20009 and its derivative strains, including but not limited to the strains that have been authorized or are applying for invention patents (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; patent application numbers 2025102995737, 202411906671.4, 202411906670.X, 202210181929.9, Acta Pharmaceutica Sinica B2021, 11(10):3165-3177; phoP / phoQ, etc.). Although the constitutive or inducible promoters expressed and secreted SNase at levels that are basically similar in different VNPs and their derivative strains, and produced similar antitumor effects, this invention takes the chassis strain of attenuated Salmonella Typhimurium VNP20009 as an example as a typical case demonstration.

[0089] The secretory signal peptide is the pelB signal peptide, and the nucleotide sequence of the pelB signal peptide is shown in SEQ ID NO:7.

[0090] The 5' end of the nuclease coding sequence also contains a tag coding sequence and / or the engineered bacteria also contains a plasmid anti-loss element, the nucleotide sequence of which is shown in SEQ ID NO:8, and the nucleotide sequence of which is shown in SEQ ID NO:9.

[0091] Example 2 The present invention discloses a method for preparing engineered attenuated Salmonella VNP-SNase, comprising the following steps: (1) Construct a recombinant plasmid containing the nuclease coding sequence, the constitutive promoter, and the secretory signal peptide; (2) Transform the recombinant plasmid into attenuated Salmonella competent cells; (3) Screen positive clones to obtain engineered attenuated Salmonella VNP-SNase that is stably expressed and secreted.

[0092] The recombinant plasmid further comprises a plasmid loss prevention element and / or the 5' end of the nuclease coding sequence contains an HA tag coding sequence. The nucleotide sequence of the plasmid loss prevention element is shown in SEQ ID NO:8, and the nucleotide sequence of the HA tag coding sequence is shown in SEQ ID NO:9. Example 3

[0093] The present invention relates to the application of an engineered attenuated Salmonella strain in the preparation of a drug for treating tumors.

[0094] The tumor is selected from melanoma, solid tumor, or metastatic tumor.

[0095] Example 4 The present invention provides a pharmaceutical composition comprising engineered attenuated Salmonella and a pharmaceutically acceptable carrier.

[0096] Example 5 The present invention provides a method for degrading NETs in the tumor microenvironment, comprising administering engineered attenuated Salmonella VNP-SNase or a pharmaceutical composition to a subject.

[0097] Example 6 Method for constructing secretory engineered attenuated Salmonella VNP-SNase The VNP20009 strain VNP-SNase, expressing SNase, was constructed by electroporation based on a specific plasmid. To facilitate observation and stability of experimental results, the constitutive strong promoter J23100 and the plasmid loss prevention element AT were used in the plasmid. The SNase uses the secretion signal peptide pelB to assist in the extracellular secretion effect after protein expression, and an HA tag is attached to the N-terminus of the SNase for subsequent protein detection and analysis. An empty vector plasmid containing the inserted SNase was used as a control group, and the corresponding transformed strain was designated VNP-NC.

[0098] The VNP-SNase strain was cultured in 40 mL LB medium supplemented with kanamycin until OD500 was reached. 600 Between 0.6 and 0.8. The solution was then centrifuged at 5000 rpm and 4°C for 10 minutes, and the supernatant and precipitate were collected. The precipitate was resuspended in 2 mL PBS, heated in a dry bath at 110°C for 20 minutes to disrupt the bacteria and release proteins, and then centrifuged at 13000 rpm for 10 minutes. The total protein from the bacteria was contained in the supernatant. Total protein was collected from the bacterial LB medium supernatant obtained in the first step according to the trichloroacetic acid (TCA) protein precipitation method. In short, the supernatant was transferred to a 50 mL centrifuge tube and centrifuged at 15,000 g, 4°C for 10 minutes. The supernatant was transferred to a new 50 mL centrifuge tube, 10% TCA was added, vortexed to mix thoroughly, and incubated on ice for 30 minutes. The mixture was then centrifuged again at 7,000 g, 4°C for 20 minutes, the precipitate was resuspended in 300 mL PBS, and transferred to a 1.5 mL sterile EP tube. Add 1.2 ml of pre-chilled acetone (pre-set at -20°C), centrifuge at 17,000 g, 4°C for 20 min. Discard the supernatant, add 300 ml of PBS again, and repeat the above operation. Discard the supernatant, add 40 ml of PBS to resuspend, and obtain the total protein secreted by the bacteria in the supernatant. Add the centrifuged bacterial cells to a loading buffer and boil at 100°C for 10 minutes to obtain the total bacterial protein. The collected total protein was analyzed using Western blotting (WB) to detect the production and secretion of the target protein.

[0099] Example 7 VNP-induced neutrophil formation (NETs): Neutrophils sorted by magnetic beads were adjusted to an appropriate density (1×10⁶ cells / mL) using antibiotic-free RPMI-1640 medium. 5Neutrophils (500 μL / 100 μg) were added to 24-well plates pre-filled with cell slides and incubated at 37°C in a 5% CO2 incubator for 4 h. The experimental group received 100 μL of VNP-RFP (neutrophils:VNP-RFP = 1:10), while the control group received 100 μL of PBS buffer. Cells were incubated at 37°C in a 5% CO2 incubator for 6 h. At the end of the incubation, gentamicin at a final concentration of 50 μg / mL was added to each well for sterilization for 1 h. The culture medium in the 24-well plates was discarded, and the cell slides were transferred to another 24-well plate. The plates were gently washed once with PBS, the PBS was discarded, and 200 μL of 4% paraformaldehyde was added for fixation for 20 min. The fixative in the 24-well plates was discarded, and the plates were gently washed once with PBS, the PBS was discarded. 300 μL of PBS was added again, and 0.3 μL of Sytox Green (1 μM) was added in the dark. The mixture was gently mixed and incubated for 20 min. Discard the staining solution, wash three times with PBS for 1 min each time, remove the smears, and add 10 μL of anti-fluorescence quencher to each slide. Mount the slides, allow them to air dry, and then observe and photograph them under a microscope. Take the supernatant of the co-cultured cell culture medium, centrifuge at 100,000 rpm for 20 min, collect the supernatant, and use ELISA to detect the content of the NETs marker MPO-DNA. This supernatant will be used for the NETs-treated group in subsequent cell experiments. Example 8

[0100] SNase reverses the effects of NETs on the proliferation, migration, and colony formation of B16F10 cells. I) Effect of CCK-8 assay on SNase on NETs-induced B16F10 cell proliferation 500 B16F10 cells per well were seeded in a 96-well plate and incubated overnight at 37°C with 5% CO2 to ensure complete adhesion. Four groups were established: a Ctrl group, a SNase group (1 μg), a NETs group, and a NETs / SNase group. Cell viability was assessed using CCK-8 assays at 0, 24, 48, 72, and 96 h. CCK-8 was prepared by adding 10% CCK-8 stock solution to 90% blank medium and mixing thoroughly. The medium in each well was discarded using a multipipeline, and 100 μL of CCK-8 working solution was added to each well, mixed thoroughly, and the 96-well plate was incubated for 2 h. Cell viability was calculated by measuring the absorbance at OD450 using a microplate reader.

[0101] II) Effect of SNase on NETs-induced wound healing in B16F10 cells during scratch assay 1×10 per hole 5B16F10 cells were seeded in 12-well plates and cultured at 37°C in a 5% CO2 incubator for 12 hours. The next day, using a sterile 200 μL pipette tip, vertical lines were streaked against the 12-well plate cap, ensuring the pipette tip was perpendicular to the plate and not tilted. Immediately after streaking, the cells were washed three times with PBS for 5 minutes each time to remove any cells that had fallen into the wells. Separate groups were established: a Ctrl group, a SNase group (1 μg), a NETs group, and a NETs combined with SNase group. Cells were incubated statically at 37°C in a 5% CO2 incubator for 48 hours. After incubation, the wells were observed under a microscope to monitor wound healing and photographed. The distance ratio between scratches was calculated using ImageJ software. Cell migration rate = (initial cell distance - cell distance at time t) / initial intercellular distance.

[0102] III) Trans-well assay of the effect of SNase on NETs-induced B16F10 cell migration B16F10 cells in the logarithmic growth phase were digested with trypsin, resuspended in serum-free medium, and the cell density was adjusted to 5 × 10⁶ cells / year. 4 Cells / mL. 200 μL of B16F10 cell suspension was added to the upper chamber of the Trans-well. The lower chamber of the Trans-well was used to establish Ctrl, SNase (1 μg), NETs, ​​and NETs combined with SNase groups, respectively. The chambers were placed in 24-well plates and incubated at 37°C in a 5% CO2 cell culture incubator for 48 h. The Trans-well chambers were removed, the culture medium in the inner chamber was discarded, and the chambers were transferred to the desired 24-well plates. 1 mL of 4% paraformaldehyde was added to the inner chamber, and the cells were fixed at room temperature for 30 min. After cell fixation, the fixative was discarded, and 1 mL of 1% crystal violet staining solution was added to the inner chamber, and staining was performed for 30 min. The crystal violet staining solution was recovered, and each chamber was washed three times with 500 μL of PBS for 1 min each time. The inner surface of the chambers was wiped with a cotton swab to remove unmigrated or invasive B16F10 cells. The chambers were air-dried, observed and photographed under an optical microscope, and the data were analyzed using ImageJ.

[0103] IV) Colony formation assay to detect the effect of SNase on NETs-induced B16F10 cell colony formation. B16F10 cells were digested with trypsin and prepared into single-cell suspensions, at a concentration of 1 × 10⁶ cells per well. 3B16F10 cells were seeded into 6-well plates and cultured in a 37°C, 5% CO2 incubator. After cell adhesion, Ctrl, SNase (1 μg), NETs, ​​and NETs combined with SNase groups were established. The culture medium was changed every day. After 7 days of culture, the presence of obvious colonies was observed under a microscope. When visible cell clusters appeared in the culture dish, the culture medium was discarded, and the cells were carefully washed once with PBS for 1 min. The PBS was discarded, and 2 mL of 4% paraformaldehyde fixative was added to each well. The cells were fixed at room temperature for 30 min, and the fixative was discarded. 2 mL of 0.1% crystal violet staining solution was added to each well, and the cells were stained for 30 min. The crystal violet staining solution was recovered, and the cells were gently washed three times with PBS for 3 min each time. The 6-well plates were air-dried at room temperature and photographed under an optical microscope. Cell counting was performed using ImageJ.

[0104] Example 9

[0105] I) Intratumoral titer detection: To detect the dynamic distribution of intracellular bacteria in vivo, 100 µL of PBS and SNase (100 μg) were administered intraperitoneally (5 × 10⁻⁶). 5 VNP or 5 × 10 5 VNP-SNase was introduced into B16F10 tumor-bearing mice. Mice were sacrificed at the designed time points, and various organs and tumor tissues were collected. After homogenization using a tissue homogenizer, 0.3% Triton X-100 was added, and the cells were incubated at room temperature for 15-20 min to perforate and release VNPs, with the cells being inverted and shaken several times during this process. After appropriate dilution, the samples were plated onto cannabinoid LB agar plates for analysis to determine the VNP titers in each organ. Changes in tumor / spleen targeting and tumor / liver targeting were calculated and compared.

[0106] II) Histopathology, complete blood count and blood biochemistry analysis: Blood was collected via enucleation for a complete blood count (CBC). After allowing the blood to stand at room temperature for 30 minutes, it was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was carefully aspirated. The serum obtained from the blood was frozen and stored at -80°C until the determination of blood biochemical indicators and ELISA. Routine blood tests, blood biochemical analyses, H&E staining of tumor, heart, liver, spleen, lung, and kidney sections, and fluorescent immunostaining of macrophages in tumor sections were prepared by Wuhan Service Biotechnology Co., Ltd.

[0107] Example 10

[0108] I) Evaluation of the effect of inhibiting melanoma solid tumors: An orthotopic melanoma-bearing mouse model was established. Specifically, 6-8 week old female C526L / 6 mice were used as experimental animals. Mice were randomly assigned to different groups. 100 mL of cells (containing 2 × 10⁶ cells per 100 mL) were inoculated under the right forelimb axilla of the mice. 5 (C526L / 6, B16F10) was used to induce tumor formation in mice. The tumors were allowed to grow to approximately 80-160 mm. 3 Subsequently, SNase (100 μg) was injected intraperitoneally every other day, while VNP and VNP-SNase (5.0 × 10⁻⁶ μg per mouse) were administered. 210 U), administered via a single intraperitoneal injection. All strains used in intraperitoneal injection were activated twice prior to injection. Tumor volume was measured 3-4 times weekly using calipers, calculated using the formula V = length × width. 2 ×0.52 was used. Tumor size was measured at specific intervals in specific groups of mice and calculated to plot tumor growth curves. Values ​​are expressed as Mean ± SEM. To plot survival curves, tumor-bearing mice were monitored daily and sacrificed upon observation of adverse reactions or reaching the human endpoint (tumor weight equal to 10% of mouse body weight). For tumor tissue, immunohistochemistry was used to detect changes in the vascular marker CD31, immunofluorescence was used to detect the formation of intratumoral NETs (MPO, CitH3), and qPCR was used to detect changes in the transcriptional levels of NETs-related genes (Padi4, MPO, Elane, MMP-9).

[0109] II) Evaluation of the effect of inhibiting melanoma lung metastasis: For experimental lung metastases, 1×10⁻⁶ PBS was administered via tail vein. 6 B16F10 cells were injected into mice. Treatment began on day 6, and mice were sacrificed on day 18. The extent of lung metastasis was determined and compared by observation and lesion counting, and the total surface area of ​​the tumor was analyzed using ImageJ. Example 11

[0110] Analysis of the mechanism of tumor suppression

[0111] Tumor-bearing mice were euthanized, and the tumors were dissected. Cells were then cultured for 30 minutes in digestive medium (10 U / mL collagenase I and 400 U / mL collagenase IV, both diluted in HBSS). Cell clusters were removed through a 40 μm cell filter to obtain a single-cell suspension. Cells were stained with immobilizable active dyes (BD, 564407) and incubated at room temperature in the dark for 10–15 minutes, followed by staining with the following anti-mouse antibodies: CD45-PE-Cy7 (clone 30-F11), CD12-PE (clone M1 / 70), Ly6G-BV510 (clone 18), CD3e-FITC (clone 145-711), and CD8-APC (clone 53-6.7). Blood was collected from the retroorbital sinus of mice, incubated at room temperature for 1 hour, and then centrifuged at 4°C and 3000 rpm to obtain serum. Changes in NETs markers in serum were analyzed using the CBA kit (BD, 560485) according to the product instructions. All flow cytometry analyses were performed on a BD Canto II flow cytometer. H&E staining of mouse tumors and fluorescent immunostaining of neutrophils on tumor sections were prepared by Servicebio (Wuhan).

[0112] Example 12

[0113] The method for preparing engineered attenuated Salmonella according to this invention involves constructing VNP20009 (hereinafter referred to as VNP-SNase) that stably expresses SNase via electroporation based on a specific plasmid. To make the experimental results easier to observe and more stable, the plasmid uses the constitutive strong promoter J23150 and the plasmid loss prevention element AT, and an HA tag is attached to the N-terminus of the SNase for subsequent detection. This invention first examines the expression and secretion of SNase by VNP-SNase. After successfully constructing and obtaining the relevant plasmid ( Figure 1 Two engineered strains were randomly selected after electroporation and cultured in liquid LB medium until the growth plateau phase. Total protein was collected from the bacterial pellet and the liquid medium, and the SNase content in these proteins was detected by Western blotting. The results showed that plasmid electroporation was successful, and the strains effectively expressed SNase protein and secreted it into the supernatant. Figure 2 ).

[0114] Example 13

[0115] The functional verification method for SNase expression by engineered attenuated Salmonella as described in this invention is as follows: First, this invention successfully constructed a SNase expression system. Under the induction of IPTG at 18°C, SNase protein expression was successfully induced. Through large-scale culture of the expression system, followed by ultrasonic disruption of the bacterial cells and purification using Ni-affinity chromatography, it was found that the SNase protein could be eluted in 100 and 200 mM imidazole elution buffers. Furthermore, the eluent was dialyzed to remove salt, and after ultrafiltration concentration, SNase protein with a purity greater than 90% was obtained. Figure 5 Next, this invention investigated the ability of SNase to degrade nucleic acids. Nucleic acid was extracted from B16F10 cells using a cell DNA and RNA extraction kit, and incubated with SNase protein in a suitable buffer for 10 min in vitro. Agarose gel electrophoresis results showed that SNase possessed the ability to degrade nucleic acids. Figure 6 In summary, this invention successfully constructed a secretory VNP-SNase strain with good growth, obtained high-purity SNase protein through purification, and exhibited strong nucleic acid degradation ability. Example 14

[0116] The detection method for the specific degradation of NETs by engineered attenuated Salmonella described in this invention involves co-culturing bacteria with neutrophils in vitro. Immunofluorescence results show that the VNP group formed a large number of NETs, ​​and VNP-SNase can significantly degrade the DNA backbone of NETs and destroy their network structure. Figure 7 The scanning electron microscope results also yielded the same conclusion. Figure 8 As an enzyme capable of degrading DNA, SNase may also degrade DNA from other sources in the tumor microenvironment (TME), such as DNA released from necrotic tumor cells or extracellular DNA from immune cells. This is because VNP can indeed induce tumor cell apoptosis, releasing the genome of apoptotic cells into the tumor microenvironment. To address these concerns, this invention extracts and purifies extracellular DNA from tumor tissue treated with VNP or VNP-SNase. Quantitative analysis shows that, compared to VNP, VNP-SNase slightly reduces the total amount of extracellular DNA per unit tumor mass (…). Figure 9 ), and moderately enhanced DNA fragmentation ( Figure 10 Notably, detection of the neutrophil extracellular trap (NETs) specific marker MPO-DNA showed that VNP-SNase significantly reduced extracellular MPO-DNA levels in tumor tissue. Figure 11 This provides strong evidence for the specific degradation effect of VNP-SNase on NETs. Example 15 The method for detecting the attenuation of tumor progression-promoting effects of engineered attenuated Salmonella secreted and expressed SNase by NETs, ​​as described in this invention, involves co-culturing B16F10 cells with SNase, NETs, ​​or a combination of SNase and NETs, ​​and then detecting the function of SNase in vitro. Compared with the PBS group, the SNase monotherapy group had little effect on the growth and proliferation of B16F10 cells, while NETs significantly promoted the proliferation of B16F10 cells. Figure 12 ),migrate( Figure 13 , Figure 14 ) and settlement formation ability ( Figure 15 The addition of SNase alleviated the promoting effect of NETs on the proliferation, migration and invasion of B16F10 cells.

[0117] Example 16 The therapeutic effect detection method of the engineered attenuated Salmonella described in this invention is used to evaluate the antitumor efficacy of VNP-SNase in a mouse subcutaneous melanoma model constructed in this invention. Tumor growth curves show that, compared with the PBS group, SNase alone had no significant effect on tumor progression, while VNP exhibited strong antitumor activity. Figure 16 Compared to wild-type VNP, VNP-SNase and the combined use of VNP and exogenous SNase showed stronger antitumor effects. Figure 17 Among them, engineered VNP-SNase has superior therapeutic effects, manifested in delaying tumor growth, reducing tumor weight, and significantly prolonging tumor doubling time. Figure 18 ) and mouse lifespan ( Figure 19 ).

[0118] Example 17 The detection method for engineered attenuated Salmonella-specific degradation of intratumoral NETs described in this invention involves ELISA detection of changes in NET-related markers CitH3-DNA and MPO-DNA in mouse serum. Compared with the PBS group, the levels of NET markers in the serum of mice in the VNP group were significantly increased. Compared with wild-type VNP, the combined use of VNP-SNase and VNP with exogenous SNase showed stronger NET degradation ability, with the engineered VNP-SNase showing better results. Figure 20 RT-PCR and immunofluorescence analysis showed that, in tumor tissues, VNP-SNase significantly reduced NET formation compared to the VNP or VNP combined with SNase group. This reduction was confirmed by the downregulation of NET markers, including Padi4, Elane, Mpo, Mmp9, and Ltf, as well as a decrease in NET signaling. Figure 21 , Figure 22VNP-SNase enhances the antitumor efficacy of VNPs by targeting and degrading NETs, ​​while also improving the safety of bacterial therapy. Figure 23 ) and intratumoral colonization ( Figure 24 ).

[0119] The detection method for inhibiting angiogenesis using engineered attenuated Salmonella described in this invention involves investigating the effects of NETs combined with SNase on angiogenesis-related genes in HUVECs in vitro. Western blot results showed that NETs upregulated the expression of angiogenesis-related genes through the VEGFR-PI3K-AKT signaling pathway, and the addition of SNase reversed the pro-angiogenic effect of NETs. Figure 25 Considering that VNP possesses the ability to inhibit angiogenesis and that SNase can reverse the pro-angiogenic effect of NETs, ​​the present invention further performed immunohistochemical and immunofluorescence detection on tumor tissues. The immunohistochemical results indicated that VNP, VNP combined with SNase, and VNP-SNase all significantly inhibited the expression of CD31 in tumor tissues, and VNP-SNase showed a better effect in inhibiting angiogenesis. Figure 26 Meanwhile, immunofluorescence results showed that VNP, VNP combined with SNase, and VNP-SNase upregulated the expression of the pro-angiogenic gene ANG2 and downregulated the expression of the anti-angiogenic genes ANG1 and iNOS, and VNP-SNase showed a stronger anti-angiogenic effect. Figure 27 ).

[0120] Example 18 The detection method for the anti-tumor metastasis effect of engineered attenuated Salmonella described in this invention is the effect of VNP-SNase on B16F10 lung metastasis. Next, this invention investigated the anti-B16F10 metastasis ability of VNP-SNase. Compared with the VNP group, the lung weight of mice in the VNP-SNase group was reduced (…). Figure 28 Meanwhile, lung metastasis counts and H&E staining results indicated that although SNase monotherapy had no significant effect on melanoma lung metastasis, VNP and VNP-SNase showed significant anti-mouse melanoma lung metastasis effects, with VNP-SNase being more effective. Figure 29 Finally, VNP-SNase significantly prolonged the survival time of mice. Figure 30 Immunofluorescence results showed that, compared with the VNP group, VNP-SNase significantly reduced the level of intratumoral NETs (). Figure 31 Furthermore, compared to the VNP group, the VNP-SNase group showed a significant decrease in serum NETs markers (including Mpo-DNA and CitH3-DNA). Figure 32Given that MMP-9 is a component of NETs and is associated with tumor metastasis, this invention detected MMP-9 levels in serum and lung tissue. Results showed that MMP-9 levels were significantly decreased in the VNP-SNase group (…). Figure 32 ).

[0121] Example 19 The detection method for enhancing anti-tumor immunity by loading engineered attenuated Salmonella as described in this invention involves detecting changes in T cells in the immune microenvironment on day 9 using flow cytometry. The results indicate that the VNP group showed a significant decrease in the number of CD4⁺ and CD8⁺ T cells due to NETs (Nety-Telepathies). Figure 33 The expression of CD8⁺ T cell exhaustion markers PD-1 and Tim-3 was increased, while the expression of functional molecules GZMB and PRF was decreased. Figure 34 VNP-SNase reversed the aforementioned inhibitory changes. Figure 34 In summary, VNP-induced PD-L1⁺ TANs inhibited CD8⁺ T cell killing function through the PD-1 / PD-L1 axis on day 9, while VNP-SNase effectively alleviated this immunosuppression.

[0122] Example 20 The method described in this invention for detecting the reduced neutrophil infiltration in the tumor microenvironment by loading engineered attenuated Salmonella involves using a transwell assay to investigate the recruitment effect of NETs on neutrophils in the upper chamber. The results indicate that NETs can significantly promote neutrophil recruitment, and the addition of SNase can improve this phenomenon. Figure 35 In addition, this invention detected the expression of genes related to neutrophil chemotaxis in tumor tissues. RT-PCR results showed that VNP significantly upregulated the expression of Ccl-3, Ccl-4, Ccl-6, Csf3r, C20r1, S100a8, and S100a9, while VNP-SNase significantly downregulated these genes. Figure 36 Serological testing further confirmed that the protein levels of Ccl-3, Ccl-4, Cxcl2, Csf3r, S100a8, IL-17, and IL-6 were significantly reduced in the VNP-SNase group. Figure 37 ).

[0123] Because the engineered attenuated Salmonella of this patent invention can remodel the tumor microenvironment, including more strongly inhibiting tumor angiogenesis, alleviating tumor immunosuppression, and significantly downregulating the levels of Ccl-3, Ccl-4, Cxcl2, Csf3r, S100a8, IL-17, and IL-6 proteins, thereby enhancing anti-tumor immunity, according to existing theories, the engineered attenuated Salmonella of this patent invention can be used in combination with other existing tumor immunotherapy drugs or treatment methods to prepare combination therapy drugs, thereby increasing the sensitivity of tumors to existing tumor immunotherapy drugs or treatment methods.

[0124] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. An engineered attenuated Salmonella VNP-SNase, characterized in that: The engineered attenuated Salmonella VNP-SNase contains an exogenous nuclease coding sequence and is capable of secreting and expressing the exogenous nuclease SNase.

2. The engineered attenuated Salmonella VNP-SNase according to claim 1, characterized in that: The attenuated Salmonella VNP-SNase contains an exogenous nuclease coding sequence linked to a promoter, and the nuclease contains a secretion signal peptide, enabling the engineered bacteria to express and secrete Staphylococcus aureus nuclease SNase. The nucleotide sequence of the exogenous nuclease coding sequence is shown in SEQ ID NO:

1. The promoter is a constitutive promoter or an inducible promoter. The constitutive promoter is J23150, J23112, or J23110 promoter. The nucleotide sequence of the J23150 promoter is shown in SEQ ID NO:2, the nucleotide sequence of the J23112 promoter is shown in SEQ ID NO:3, and the nucleotide sequence of the J23110 promoter is shown in SEQ ID NO:

4. The inducible promoter includes pLuxI, nirB, and padh promoters. The nucleotide sequence of the pLuxI promoter is shown in SEQ ID NO:5, and the nucleotide sequence of the nirB promoter is shown in SEQ ID NO:

5. As shown in NO:6, the nucleotide sequence of the padh promoter is derived from US970861262.

3. The engineered attenuated Salmonella VNP-SNase according to claim 1, characterized in that: The attenuated Salmonella strain is attenuated Salmonella Typhimurium strain VNP20009 and its derivative strains, including but not limited to the strains that have been authorized or are for which invention patents have been applied.

4. The engineered attenuated Salmonella VNP-SNase according to claim 1, characterized in that: The secretory signal peptide is the pelB signal peptide, and the nucleotide sequence of the pelB signal peptide is shown in SEQ ID NO:

7.

5. The engineered attenuated Salmonella VNP-SNase according to claim 1, characterized in that: The 5' end of the nuclease coding sequence further includes a tag coding sequence and / or the engineered bacteria further includes a plasmid anti-loss element, the nucleotide sequence of which is shown in SEQ ID NO:8, and the nucleotide sequence of which is shown in SEQ ID NO:

9.

6. A method for preparing engineered attenuated Salmonella VNP-SNase according to any one of claims 1-5, characterized in that... Includes the following steps: (1) Construct a recombinant plasmid containing the nuclease coding sequence, the promoter, and the secretion signal peptide; (2) Transform the recombinant plasmid into attenuated Salmonella competent cells; (3) Screen positive clones to obtain engineered attenuated Salmonella VNP-SNase that is stably expressed and secreted.

7. The preparation method according to claim 6, characterized in that: The recombinant plasmid further includes a plasmid loss prevention element and / or the 5' end of the nuclease coding sequence contains an HA tag coding sequence, the nucleotide sequence of the plasmid loss prevention element is shown in SEQ ID NO:8, and the nucleotide sequence of the HA tag coding sequence is shown in SEQ ID NO:

9.

8. The use of the engineered attenuated Salmonella according to any one of claims 1-7 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, characterized in that: The tumor is selected from melanoma, solid tumor, or metastatic tumor.

10. The application according to claim 8, characterized in that: The engineered attenuated Salmonella works by degrading extracellular neutrophil traps (NETs) in the tumor microenvironment.

11. The application according to claim 8, characterized in that: The engineered attenuated Salmonella remodels the tumor microenvironment, including more strongly inhibiting tumor angiogenesis, alleviating tumor immunosuppression, significantly downregulating the protein levels of Ccl-3, Ccl-4, Cxcl2, Csf3r, S100a8, IL-17 and IL-6, and enhancing anti-tumor immunity.

12. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the engineered attenuated Salmonella as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

13. A method for degrading NETs in the tumor microenvironment, characterized in that: This includes administering to a subject the engineered attenuated Salmonella VNP-SNase as described in any one of claims 1-5 or the pharmaceutical composition as described in claim 12.

14. The application according to claim 8 in the preparation of a therapeutic agent in combination with other existing tumor immunotherapy drugs or treatment methods.

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