Neutrophil loaded with attenuated salmonella and preparation method therefor and use thereof

Abstract

A neutrophil loaded with attenuated salmonella and a preparation method therefor and a use thereof, relating to the technical field of biology. Vascular clearance by a neutrophil is a key factor affecting the tumor colonization rate of oncolytic bacteria, i.e., attenuated salmonella typhimurium. The neutrophil can be recruited to a tumor microenvironment after being loaded with the oncolytic bacteria, so that the tumor colonization rate and tumor targeting performance of the bacteria are effectively improved. The neutrophil can directly kill tumor cells and release intracellular oncolytic bacteria to induce an anti-tumor immune response, thereby effectively inhibiting the progression of pulmonary metastases from melanoma.

Description

Neutrophils loaded with attenuated Salmonella, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to neutrophils loaded with attenuated Salmonella, and a preparation method and application thereof. Background Art

[0002] The attenuated Salmonella typhimurium VNP20009 (VNP), which has deleted the purI and msbB genes to reduce toxic side effects and septic shock, is one of the most promising oncolytic strains. Compared to wild-type Salmonella typhimurium, attenuated Salmonella VNP20009 has significant tumor suppression capabilities, but with some side effects. The titer of attenuated Salmonella VNP20009 in tumors is approximately 1000 times higher than in other organs (The Journal of Infectious Diseases 2000, 181, 1996–2002). In a Phase I clinical trial of attenuated Salmonella VNP20009, most melanoma patients, except those in the highest-dose group, experienced no significant side effects, demonstrating its safety. However, tumor colonization of attenuated Salmonella VNP20009 was observed only in the highest-dose group (Journal of Clinical Oncology 2002, 20, 142-152). The tumor colonization rate of oncolytic bacteria is the most important factor in tumor treatment. Therefore, how to balance the therapeutic effect and safety of oncolytic bacteria is an urgent problem to be solved.

[0003] To improve the therapeutic efficacy of Salmonella, many excellent studies have been reported, such as carrying shRNA targeting key genes for tumor metastasis (J Exp Clin Cancer Res 2016, 35, 107; Oncotarget 2016, 7, 12.), expressing effector proteins such as interferon-γ protein or cytotoxic proteins (Eur J Cancer 2017, 70, 48-61.), and carrying plasmids encoding tumor-specific antigens (Scientific Reports 2016, 6, 34178; Biotechnol Bioeng 2016, 113, 2698-2711.), or covalently bound photosensitizers or cytotoxic biomaterials (Biomaterials 2019, 214, 119226; ACS Nano 2018, 12, 5995-6005.). Recent studies have shown that bacterial immunogenicity shielding is a feasible measure to increase the colonization of oncolytic bacteria in tumors. It is reported that cell membrane-encapsulated bacteria can reduce the immunogenicity of bacteria and alleviate the vascular clearance rate of bacteria, thereby enhancing tumor targeting and safety (Nature Communication 2019, 10, 3452.).

[0004] There is evidence that VNP20009 colonizes the core of tumors by penetrating blood vessels, suggesting that rapid clearance of VNP20009 from the blood may contribute to its poor tumor colonization (J Control Release 2015, 199, 180-9; Cancer Gene Ther 2011, 18, 457-66). Westphal's work supports this hypothesis, reporting that depletion of host neutrophils leads to increased bacterial titers in tumors (Cancer Res 2008, 68, 2952-60). Other studies have reported that depleting host neutrophils can enhance the tumor therapeutic effect of VNP20009 (Theranostics 2017, 7, 2250-2260). Neutrophils can be loaded with drugs through phagocytosis and released through cell lysis after being recruited to the tumor area (Adv Mater 2018, 30, e1706245; Adv Mater 2020, 32, e2003598.).

[0005] Summary of the Invention

[0006] How can neutrophils be loaded with attenuated Salmonella to reduce bacterial immunogenicity, mitigate bacterial clearance from blood vessels, and enhance tumor targeting and safety? At the same time, how can this be achieved while avoiding the inherent bacterial clearance properties of neutrophils, which can prevent the loaded attenuated Salmonella from being cleared and remaining active? The present invention aims to provide neutrophils loaded with attenuated Salmonella, as well as their preparation and use, to effectively enhance bacterial tumor colonization and tumor targeting. These neutrophils can effectively inhibit melanoma by directly killing tumor cells and releasing intracellular oncolytic bacteria to induce anti-tumor immune responses.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions: a neutrophil loaded with attenuated Salmonella of the present invention, wherein the neutrophil loaded with attenuated Salmonella comprises neutrophils and oncolytic bacteria, wherein the oncolytic bacteria are attenuated Salmonella typhimurium VNP20009 and a recombinant engineered strain derived therefrom, and the recombinant engineered strain is a strain that does not carry or carries an expression plasmid expressing an exogenous therapeutic gene or a tracer gene. Attenuated Salmonella VNP20009, including the aforementioned strains for which invention patents have been applied (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta Pharmaceutica Sinica B 2021, 11(10): 3165-3177; phoP / phoQ, etc.), have all been experimentally proven to have similar tumor targeting and anti-tumor efficacy, and the above-mentioned modified strains have lower toxicity than VNP20009. These modified strains have similar therapeutic effects as VNP20009 and can be combined with biological drugs, gene therapy, chemotherapy, and traditional Chinese medicine.

[0008] Furthermore, the therapeutic or tracing effect genes that can be expressed in attenuated Salmonella typhimurium VNP20009 include at least one of immune checkpoint blocker antibody genes, cytokine genes, angiogenesis inhibitor genes, apoptosis anti-tumor genes or interfering plasmids; the interfering plasmid that can be carried by attenuated Salmonella typhimurium VNP20009 includes an interfering plasmid with pRNA-U6.1 as the backbone that can express interfering RNA.

[0009] Furthermore, the genes with a tracing effect that can be expressed in the attenuated Salmonella typhimurium VNP20009 include at least one of a red fluorescent protein RFP gene, a green fluorescent protein GFP gene, and a luciferase tracer protein LuxCDABE gene.

[0010] Furthermore, the nucleotide sequence of the red fluorescent protein RFP gene is shown in SEQ ID No. 1, the nucleotide sequence of the green fluorescent protein GFP gene is shown in SEQ ID No. 2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown in SEQ ID No. 3.

[0011] Furthermore, the expression plasmid that can be carried by attenuated Salmonella typhimurium VNP20009 includes a constitutive strong promoter J23100, a PelB extracellular protein secretion signal peptide, and a Flag tag. The nucleotide sequence of the constitutive strong promoter J23100 is shown in SEQ ID NO.4, and the nucleotide sequence of the PelB extracellular protein secretion signal peptide is shown in SEQ ID NO.5.

[0012] The method for preparing neutrophils loaded with attenuated Salmonella of the present invention comprises the following steps: extracting peritoneal neutrophils stimulated with 5% starch broth, incubating them with VNP20009 at a ratio of attenuated Salmonella:cells of 100:1 for 0.5-2.5 hours, and then treating the cells with 50-100 μg / mL gentamicin for 30-60 minutes to kill extracellular bacteria, thereby preparing neutrophils loaded with attenuated Salmonella.

[0013] The in vivo distribution of neutrophils loaded with attenuated Salmonella and the evaluation and tracing method of the tumor colonization rate of the present invention include the following steps: using Cy5.5-labeled neutrophils and neutrophil-loaded attenuated Salmonella that can sustainably express fluorescent protein or luciferase tracer protein, and evaluating the in vivo distribution and level of neutrophils loaded with attenuated Salmonella through tissue plating and in vivo imaging technology.

[0014] The invention discloses an application of neutrophils loaded with attenuated Salmonella in the preparation of anti-tumor pharmaceutical preparations, wherein the pharmaceutical preparations include at least one of oral preparations, injection preparations, intravenous injection preparations or sprays.

[0015] Furthermore, neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune responses, thereby inhibiting the progression of melanoma lung metastasis.

[0016] The present invention provides an application of neutrophils loaded with attenuated Salmonella in combination with other anti-tumor drugs in the preparation of anti-tumor drugs, such as antibody drugs, chemotherapy drugs, etc.

[0017] Beneficial effects: The present invention provides a highly tumor-targeted, safe, convenient, low-cost, and novel method for delivering oncolytic bacteria, namely, using neutrophils for loading. While improving the tumor treatment effect of oncolytic bacteria, it can greatly increase the tumor targeting and safety of oncolytic bacteria. It can inhibit tumor progression by directly killing tumor cells and activating the body's anti-tumor immunity, and therefore shows good prospects for tumor treatment.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The present invention demonstrates that neutrophils are a key factor affecting tumor colonization by the oncolytic bacterium attenuated Salmonella.

[0020] (2) After being injected into the body, attenuated Salmonella is rapidly phagocytosed and cleared by neutrophils. The present invention invents a method for preparing neutrophils loaded with attenuated Salmonella, loading the attenuated Salmonella into neutrophils; and can temporarily protect the attenuated Salmonella from premature exposure and clearance, thus inventing an effective strategy for maintaining a balance between the therapeutic effect and safety of oncolytic bacteria.

[0021] (3) The present invention uses primary neutrophils to load attenuated Salmonella. This delivery system can greatly increase the rate and number of attenuated Salmonella colonization in tumors and reduce the infiltration of attenuated Salmonella into normal tissues, thereby significantly improving the safety of attenuated Salmonella.

[0022] (4) The present invention combines cell therapy and bacterial therapy, achieving good therapeutic effects with a single administration, providing a convenient and inexpensive combined tumor therapy. Furthermore, the present invention can also simultaneously load two or more oncolytic bacteria.

[0023] (5) The neutrophils loaded with attenuated Salmonella of the present invention can directly promote tumor tissue necrosis, thereby exerting good anti-tumor ability; the neutrophils loaded with attenuated Salmonella of the present invention can activate anti-tumor immune response, thereby promoting tumor apoptosis and achieving tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Description of the abbreviations of the accompanying drawings:

[0026] VNP: attenuated mouse Salmonella VNP20009; RFP: red fluorescent protein; EGFP: green fluorescent protein; VNP-RFP: VNP carrying RFP gene plasmid; VNP-EGFP: VNP carrying EGFP gene plasmid; NEs: primary mouse neutrophils; NEs(VNP): neutrophils loaded with VNP; VNP-Lvx: VNP carrying LuxCDABE gene plasmid.

[0027] FIG1 shows the percentage of neutrophils in the blood to all immune cells after injection of attenuated Salmonella of the present invention;

[0028] FIG2 shows the present invention's confirmation that neutrophil vascular clearance is a key factor affecting the tumor colonization rate of oncolytic bacteria, namely, attenuated Salmonella typhimurium. After antibody depletion of neutrophils in tumor-bearing mice, attenuated Salmonella was injected and the distribution of attenuated Salmonella in the body was detected by plating.

[0029] FIG3 is a flow cytometry analysis of the purity of peritoneal neutrophils in the present invention.

[0030] FIG4 is a fluorescence image of neutrophils loaded with attenuated Salmonella of the present invention.

[0031] FIG5 is a graph showing the intracellular activity detection of the attenuated Salmonella of the present invention in neutrophils.

[0032] FIG6 shows the survival ability of neutrophils loaded with attenuated Salmonella of the present invention.

[0033] FIG. 7 shows the chemotactic ability of neutrophils loaded with attenuated Salmonella of the present invention.

[0034] FIG8 shows the rate of release of the attenuated Salmonella of the present invention from neutrophils.

[0035] FIG9 shows that neutrophils loaded with attenuated Salmonella of the present invention promote apoptosis of tumor cells.

[0036] FIG10 shows that the neutrophils loaded with attenuated Salmonella of the present invention inhibit tumor cell proliferation.

[0037] FIG. 11 shows the nitric oxide and reactive oxygen species levels of neutrophils loaded with attenuated Salmonella of the present invention.

[0038] FIG12 shows the cell status of neutrophils loaded with attenuated Salmonella detected by real-time fluorescence quantitative PCR of the present invention.

[0039] FIG13 is an in vivo imaging photograph of mouse organs after drug administration of the present invention.

[0040] FIG14 shows the organ distribution and levels of the melanoma lung metastasis model in vivo.

[0041] FIG15 shows the organ distribution and levels of the subcutaneous melanoma model of the present invention.

[0042] FIG16 shows the number of lung tumor metastases after systemic administration of the present invention.

[0043] FIG17 shows the changes in body weight of tumor-bearing mice after systemic administration of the present invention.

[0044] FIG18 shows organ weights and spleen photos of tumor-bearing mice after systemic administration of the present invention.

[0045] FIG19 shows the HE staining results of other normal organ tissues of tumor-bearing mice after systemic administration of the present invention.

[0046] FIG20 shows the levels of serum biochemical indicators in tumor-bearing mice after systemic administration of the present invention. DETAILED DESCRIPTION

[0047] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0048] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0049] 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 refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0050] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can 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 the present application.

[0051] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0052] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass in the examples of this application may be μg, mg, g, kg, etc., which are mass units known in the chemical industry.

[0053] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0054] The present invention primarily relates to a method for constructing neutrophils loaded with attenuated Salmonella and their application in anti-tumor therapy. On the one hand, the present invention demonstrates that neutrophils are a key factor influencing tumor colonization by oncolytic bacteria, including attenuated Salmonella. On the other hand, the present invention develops a delivery system for neutrophils loaded with attenuated Salmonella, which demonstrates potential for anti-tumor therapy.

[0055] In response to the limitations of the application of oncolytic bacteria, including attenuated Salmonella, the present invention provides a technology for preparing neutrophil carriers that can carry oncolytic bacteria. After being loaded with VNP20009, neutrophils can be recruited to the tumor microenvironment, effectively improving the tumor colonization rate and tumor targeting of oncolytic bacteria. It can directly kill tumor cells and release intracellular oncolytic bacteria to induce anti-tumor immune responses, thereby effectively inhibiting melanoma lung metastasis.

[0056] In a first aspect, an embodiment of the present application provides a neutrophil loaded with oncolytic bacteria including attenuated Salmonella. The neutrophil loaded with oncolytic bacteria is a primary neutrophil. The oncolytic bacteria are attenuated Salmonella VNP20009 and a recombinant engineered strain derived therefrom. The recombinant engineered strain does not carry or carries an expression plasmid that expresses an exogenous gene for a therapeutic gene or a tracer gene. Attenuated Salmonella VNP20009, including the aforementioned strains for which invention patents have been applied (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta Pharmaceutica Sinica B 2021, 11(10): 3165-3177; phoP / phoQ, etc.), have all been experimentally proven to have similar tumor targeting and anti-tumor efficacy, and the above-mentioned modified strains have lower toxicity than VNP20009. These modified strains have similar therapeutic effects as VNP20009 and can be combined with biological drugs, gene therapy, chemotherapy, and traditional Chinese medicine.

[0057] The therapeutic or tracing effect genes that can be expressed in attenuated Salmonella VNP20009 include at least one of immune checkpoint blocker antibody genes, cytokine genes, angiogenesis inhibitor genes, apoptosis anti-tumor genes or interfering plasmids; the interfering plasmid that can be carried by attenuated Salmonella typhimurium VNP20009 includes an interfering plasmid with pRNA-U6.1 as the backbone that can express interfering RNA.

[0058] The genes with a tracing effect that can be expressed in the attenuated Salmonella typhimurium VNP20009 include at least one of a red fluorescent protein RFP gene, a green fluorescent protein GFP gene, and a luciferase tracer protein LuxCDABE gene.

[0059] The nucleotide sequence of the red fluorescent protein RFP gene is shown in SEQ ID No. 1, the nucleotide sequence of the green fluorescent protein GFP gene is shown in SEQ ID No. 2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown in SEQ ID No. 3.

[0060] The expression plasmid that can be carried by attenuated Salmonella typhimurium VNP20009 includes a constitutive strong promoter J23100, a PeIB extracellular protein secretion signal peptide, and a Flag tag. The nucleotide sequence of the constitutive strong promoter J23100 is shown in SEQ ID NO.4, and the nucleotide sequence of the PeIB extracellular protein secretion signal peptide is shown in SEQ ID NO.5.

[0061] The present invention uses attenuated Salmonella VNP20009, including but not limited to the aforementioned strains for which invention patents have been applied (ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta Pharmaceutica Sinica B 2021, 11(10): 3165-3177; phoP / phoQ, etc.) as the basic strain, and combines cell drug loading technology to construct a neutrophil delivery system loaded with attenuated Salmonella. The delivery system demonstrates potential for anti-tumor treatment, and the system describes an efficient, highly tumor-targeted, convenient, and low-cost oncolytic bacteria delivery strategy that can significantly increase the tumor colonization rate and level, thereby achieving a balance between the therapeutic effect and safety of oncolytic bacteria.

[0062] A second aspect of the present invention provides a method for preparing neutrophils loaded with attenuated Salmonella, comprising the following steps:

[0063] Wild-type mice were stimulated with 5% nutrient starch broth for 4-6 hours, and then peritoneal fluid was aspirated. Cells obtained after Percoll gradient centrifugation were detected by flow cytometry, and 90% of the cells were neutrophils.

[0064] Modified or unmodified VNP20009 was incubated with peritoneal neutrophils at a ratio of attenuated Salmonella:cells of 100:1 for 0.5, 1.5, and 2.5 hours. The cells were treated with 50-100 μg / mL gentamicin for 30-60 minutes to kill extracellular bacteria. The cells were then treated with 1% TritonX-100 and incubated on ice for 1-2 hours to lyse the cells and release the bacteria. After dilution, the number of intracellular bacteria in neutrophils and the bacterial activity were counted.

[0065] A third aspect of the embodiments of the present application provides a method for evaluating and tracing the in vivo distribution of neutrophils loaded with attenuated Salmonella and the tumor colonization rate, comprising the following steps: using Cy5.5-labeled neutrophils and neutrophil-loaded attenuated Salmonella that can sustainably express fluorescent protein or luciferase tracer protein, and evaluating the in vivo distribution and level of neutrophils loaded with attenuated Salmonella through tissue plating and in vivo imaging technology.

[0066] A fourth aspect of the embodiments of the present application provides a use of neutrophils loaded with attenuated Salmonella in the preparation of an anti-tumor pharmaceutical preparation, wherein the pharmaceutical preparation includes at least one of an oral preparation, an injection preparation, an intravenous injection preparation, or a spray.

[0067] Neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune responses, thereby inhibiting the progression of melanoma lung metastasis.

[0068] The present invention discloses an application of neutrophils loaded with attenuated Salmonella in combination with other anti-tumor drugs in the preparation of anti-tumor drugs.

[0069] The use of neutrophils loaded with attenuated Salmonella in tumor treatment described in the present invention is characterized by avoiding premature vascular exposure and clearance of the attenuated Salmonella by the immune system, thereby increasing the tumor targeting of the attenuated Salmonella and increasing the safety of the attenuated Salmonella by reducing the infiltration of the attenuated Salmonella into normal tissues and organs. The attenuated Salmonella delivery system described in the present invention can effectively maintain a balance between the therapeutic effect and safety of oncolytic bacteria, thereby expanding the scope of application of oncolytic bacteria.

[0070] Example 1

[0071] Construction and evaluation of neutrophils loaded with attenuated Salmonella

[0072] Acquisition of peritoneal neutrophils: The neutrophils used in this study are primary mouse peritoneal neutrophils. Four to six hours prior to isolation, mice are subcutaneously injected with 1 ml of nutrient broth to stimulate maturation of peritoneal neutrophils. Neutrophils are separated using a Percoll gradient and identified by flow cytometry.

[0073] Co-incubation of bacteria with neutrophils: Cells were cultured under 5% CO2 and 37°C, and attenuated Salmonella at a molar concentration of 100:1 was incubated with neutrophils for 0.5, 1.5, or 2.5 hours. Then, 50-100 μg / mL gentamicin sulfate was added to the culture medium for 1 hour to kill bacteria on the surface of neutrophils. To measure the efficiency of VNP loading by neutrophils, the proportion of VNP-RFP-positive neutrophils was detected by flow cytometry. To assess the activity of intracellular bacteria, cells were treated with 1% TritonX-100 and incubated on ice for 1-2 hours to lyse the cells and release the bacteria. After dilution, the number of intracellular bacteria in neutrophils and the bacterial activity were counted on the plate.

[0074] Neutrophil release of attenuated Salmonella detection: Neutrophils loaded with attenuated Salmonella were co-cultured with tumor cells B16F1, B16F10 and mouse embryonic fibroblasts MEF in a 37°C, 5% CO2 incubator for 0.5 h, 1 h, 2 h, and 4 h, respectively. The supernatant was collected and the number of attenuated Salmonella released was counted on LB plates.

[0075] Example 2

[0076] Characterization of the status of neutrophils loaded with attenuated Salmonella

[0077] Detection of reactive oxygen species and nitric oxide levels: Neutrophils were loaded with attenuated Salmonella for 0.5 h, 1 h, 5 h, or 2.5 h. Neutrophil ROS and NO were detected using a ROS detection kit (S0033S, Beyotime) and DAF-FM DA (S0019, Beyotime). The experimental procedures were performed according to the kit instructions.

[0078] Neutrophil chemotaxis assay: Neutrophil chemotaxis was performed using a Transwell with a pore size of 3 μm. Neutrophil chemotaxis factor fMLP (10 nM) or 1×10 6 Macrophages were seeded in each well of the upper chamber at 5×10 5 Neutrophils were co-cultured for 2 hours. The supernatant of the lower chamber was collected and the number of neutrophils and chemotaxis ratio were counted.

[0079] Cell proliferation and apoptosis detection. The proliferation of tumor cells after drug administration was detected by B16F10-GFP method. B16F10 cells were plated at 1×10 5 The cells were seeded at a density of 100 μg / ml in a 24-well plate and measured every 12 h using a microplate reader (488 nm to 510 nm) to calculate the proliferation rate of B16F10 cells. The apoptosis rate of B16F10 cells was stained using Annexin V / PI. 1 μl Annexin V-APC (1 mg / ml) and 1 μl PI-PE (1 mg / ml) were mixed with 1×10 6 Cells were incubated in binding buffer for 30 minutes at 4°C. Stained cells were analyzed by flow cytometry. Statistical analysis was performed using FlowJo VX software.

[0080] Neutrophil status was assessed by real-time quantitative PCR: Total RNA was extracted using TRIzol reagent. The cDNA template was reverse transcribed using the TUYOBO cDNA Synthesis Kit according to the manufacturer's instructions. Relative gene expression levels were determined using the One-Step RT-PCR SYBR Green Kit (Novagen).

[0081] Example 3

[0082] Evaluation of the therapeutic effect of neutrophils loaded with attenuated Salmonella on tumors

[0083] Construction of lung metastasis melanoma tumor model: B16F10 mouse melanoma cells were cultured in DMEM cell culture medium until the exponential growth phase and then digested with trypsin. The cells were collected by centrifugation at 1000 rpm and 4°C for 3 minutes, washed twice with PBS, and then counted. The final cell concentration was 1×10 6 Each C57BL / 6 mouse was inoculated with 1×10 5 Pieces / pieces.

[0084] Grouping of tumor-bearing mice and administration of bacteria: Mice bearing B16F10 mouse melanoma were randomly divided into 4 groups and injected with 3×10 6 CFU bacteria and 1 × 10 6 Neutrophils loaded with attenuated Salmonella enterica serovar Typhimurium.

[0085] Body weight changes were recorded every day, and one week later, the mice were dissected to obtain various organs to count the number of lung metastases.

[0086] Example 4

[0087] Study on the distribution of neutrophils loaded with attenuated Salmonella in tumor-bearing mice

[0088] Construction of subcutaneous melanoma tumor model: B16F10 mouse melanoma cells were cultured in DMEM cell culture medium until the exponential growth phase and then digested with trypsin. The cells were collected by centrifugation at 1000 rpm and 4°C for 3 minutes, washed twice with PBS, and then counted. The final cell concentration was 1×10 6 Each C57BL / 6 mouse was inoculated with 1×10 5 The experiment was performed when the tumor volume of mice grew to about 100 mm3.

[0089] Construction of lung metastasis melanoma tumor model: B16F10 mouse melanoma cells were cultured in DMEM cell culture medium until the exponential growth phase and then digested with trypsin. The cells were collected by centrifugation at 1000 rpm and 4°C for 3 minutes, washed twice with PBS, and then counted. The final cell concentration was 1×10 6 Each C57BL / 6 mouse was inoculated with 1×10 5 Pieces / pieces.

[0090] At different time points after dosing, mice were sacrificed, and various organ tissues from tumor-bearing mice, including tumors, liver, spleen, kidneys, lungs, and hearts, were collected under a sterile environment. These tissues were weighed and homogenized in 2 mL of PBS to lyse the cells and release the bacteria. Based on the bacterial titer in the tissues, appropriate bacterial suspensions were obtained using different dilution gradients. These suspensions were then plated onto LB plates containing kanamycin and incubated in an inverted incubator at 37°C for 12 hours. Colony counts were performed to compare the tissue distribution of wild-type attenuated Salmonella and NE(VNP) in tumor-bearing mice.

[0091] In vivo imaging and immunofluorescence sections were used to detect the distribution of neutrophils loaded with attenuated Salmonella in vivo. Cy5.5-labeled neutrophils and neutrophil-loaded attenuated Salmonella that could sustainably express fluorescent protein or luciferase tracer protein were used. Tumor-bearing mice were injected with 3×10 6 CFU bacteria and 1×106 neutrophils loaded with attenuated Salmonella were used to evaluate the in vivo distribution and levels of neutrophils loaded with attenuated Salmonella by in vivo imaging technology and immunofluorescence staining.

[0092] Histopathology and blood biochemical analysis: After the blood was allowed to rest 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 at -80°C until the determination of blood biochemical parameters. Blood biochemical analysis, H&E staining of tumor, heart, liver, spleen, lung, and kidney sections, and fluorescent immunostaining of tumor sections were prepared by Wuhan Service Biological Company.

[0093] Test Example 1

[0094] The present invention uses a neutrophil depletion model to demonstrate that neutrophil vascular clearance is a key factor affecting the tumor colonization rate of oncolytic bacteria, namely attenuated Salmonella. That is, the tumor colonization rate of attenuated Salmonella is significantly increased after neutrophil depletion (Figure 2).

[0095] Test Example 2

[0096] The purity of primary mouse neutrophils extracted by the present invention is greater than 90% (Figure 3), and fluorescent photography demonstrates that the neutrophils can be loaded with attenuated Salmonella (Figure 4). Furthermore, the attenuated Salmonella can maintain over 30% activity within the neutrophils (Figure 5). Neutrophils can maintain over approximately 80% activity after phagocytizing the attenuated Salmonella for two hours, thereby maintaining neutrophil activity and chemotactic ability before being recruited to the tumor area (Figures 6 and 7). Neutrophils can release more attenuated Salmonella when induced by a highly malignant tumor (B16F10) (Figure 8).

[0097] Test Example 3

[0098] The present invention demonstrated through tumor cell apoptosis and proliferation experiments that neutrophils loaded with attenuated Salmonella had stronger tumor suppression ability (Figures 9 and 10); neutrophils loaded with attenuated Salmonella had higher levels of nitric oxide and reactive oxygen species (Figure 11); and real-time fluorescence quantitative PCR was used to detect the cellular state of neutrophils loaded with attenuated Salmonella, and the results showed that attenuated Salmonella could induce neutrophil polarization toward an anti-tumor morphology (Figure 12).

[0099] Test Example 4

[0100] The present invention detects the in vivo distribution of neutrophils loaded with attenuated Salmonella by labeling neutrophils with Cy5.5 and loading attenuated Salmonella expressing fluorescein. The experimental results show that neutrophils are more enriched in the lungs (Figure 13). The in vivo distribution results of the lung metastasis tumor model show that neutrophils loaded with attenuated Salmonella significantly increase the lung enrichment of attenuated Salmonella and prolong the distribution time of attenuated Salmonella in the lungs, while significantly reducing the infiltration of attenuated Salmonella in the spleen and heart, and improving tumor targeting (Figure 14). The results of the subcutaneous tumor model show that neutrophils loaded with attenuated Salmonella can reduce the titer of attenuated Salmonella in other normal organs, and the bacterial titer of tumor tissue remains unchanged (Figure 15). Neutrophils loaded with attenuated Salmonella can significantly reduce tumor lung metastasis lesions (Figure 16). The safety evaluation results of this system were unexpected. Neutrophils loaded with attenuated Salmonella could significantly alleviate the toxic side effects caused by attenuated Salmonella, such as weight loss and hepatosplenomegaly (Figures 17 and 18). HE staining results of other normal tissues showed that organ damage was also significantly alleviated (Figure 19). The serum biochemical test results of tumor-bearing mice after treatment also proved that this system can improve the safety of oncolytic bacteria (Figure 20).

[0101] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. A neutrophil loaded with attenuated Salmonella, characterized in that: The neutrophils loaded with attenuated Salmonella include neutrophils and oncolytic bacteria, the oncolytic bacteria are attenuated Salmonella typhimurium VNP20009 and recombinant engineered strains derived therefrom, and the recombinant engineered strains are strains that do not carry or carry expression plasmids expressing exogenous genes such as therapeutic genes and tracer genes.

2. The neutrophil-loaded recombinant attenuated Salmonella engineered bacteria according to claim 1, characterized in that: Genes that can be expressed as therapeutic or tracing effects in attenuated Salmonella VNP20009 and its derived recombinant engineered strains include at least one of immune checkpoint blocker antibody genes, cytokine genes, angiogenesis inhibitor genes, apoptosis anti-tumor genes and interfering plasmids; the interfering plasmid that can be carried by attenuated Salmonella typhimurium VNP20009 includes an interfering plasmid with pRNA-U6.1 as the skeleton that can express interfering RNA.

3. The neutrophil-loaded recombinant attenuated Salmonella engineered bacteria according to claim 2, characterized in that: The gene that can be expressed in attenuated Salmonella typhimurium VNP20009 and has a tracing effect includes at least one of a red fluorescent protein RFP gene, a green fluorescent protein GFP gene and a luciferase tracing protein LuxCDABE gene.

4. The neutrophil-loaded recombinant engineered attenuated Salmonella engineered bacteria according to claim 3, characterized in that: The nucleotide sequence of the red fluorescent protein RFP gene is shown in SEQ ID No.1, the nucleotide sequence of the green fluorescent protein GFP gene is shown in SEQ ID No.2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown in SEQ ID No.

3.

5. The neutrophil-loaded recombinant engineered attenuated Salmonella engineered bacteria according to claim 2, characterized in that: The expression plasmid that can be carried by the attenuated Salmonella VNP20009 includes a constitutive strong promoter J23100, a PeIB extracellular protein secretion signal peptide and a Flag tag. The nucleotide sequence of the constitutive strong promoter J23100 is shown in SEQ ID NO.4, and the nucleotide sequence of the PeIB extracellular protein secretion signal peptide is shown in SEQ ID NO.

5.

6. The method for preparing neutrophils loaded with attenuated Salmonella according to claim 1, characterized in that The method comprises the following steps: extracting neutrophils stimulated by 5% starch broth, co-incubating with attenuated Salmonella at a ratio of attenuated Salmonella: cells of 100:1 for 0.5-2.5 hours, treating the cells with 50-100 μg / mL gentamicin for 30-60 minutes to kill extracellular bacteria, and preparing neutrophils loaded with attenuated Salmonella.

7. The method for evaluating and tracing the in vivo distribution of neutrophils loaded with attenuated Salmonella and the tumor colonization rate according to claim 1, characterized in that The method comprises the following steps: using Cy5.5-labeled neutrophils and neutrophils loaded with attenuated Salmonella that can sustainably express fluorescent protein or luciferase tracer protein, and evaluating the in vivo distribution and level of neutrophils loaded with attenuated Salmonella by in vivo imaging technology or by diluting lysed tissue and then plating to count the number of bacteria.

8. The use of neutrophils loaded with attenuated Salmonella according to claim 1 in the preparation of anti-tumor drug preparations, characterized in that: The pharmaceutical preparation includes at least one of an oral preparation, an injection preparation, and an intravenous injection preparation or a spray.

9. The use according to claim 8, characterized in that: Neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune responses to inhibit tumor progression.

10. Use of the neutrophils loaded with attenuated Salmonella according to claim 1 in combination with other anti-tumor drugs in the preparation of anti-tumor drugs.

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