WO2025026111A1 - Neutrophil loaded with attenuated salmonella and preparation method therefor and use thereof - Google Patents

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

Neutrophil loaded with attenuated Salmonella and preparation method and application thereof Technical Field

The invention relates to the field of biotechnology, and in particular to a neutrophil loaded with attenuated Salmonella, and a preparation method and application thereof.

Background Art

The attenuated Salmonella typhimurium VNP20009 (VNP for short) strain has lost the purI and msbB genes to reduce toxic side effects and septic shock, and is one of the most promising oncolytic strains. Compared with wild-type Salmonella typhimurium, the attenuated Salmonella VNP20009 has a significant ability to inhibit tumors but has a certain degree of side effects. The titer of attenuated Salmonella VNP20009 in tumors is about 1000 times that of other organs (The Journal of Infectious Diseases 2000, 181, 1996–2002.). In the Phase I clinical trial of attenuated Salmonella VNP20009, except for the highest dose group, most melanoma patients had no obvious side effects, proving its safety. However, only in the highest dose group was attenuated Salmonella VNP20009 observed to colonize tumor tissue (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.

In order to improve the therapeutic effect 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 protein (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 oncolytic bacteria colonization in tumors. It is reported that cell membrane encapsulation of bacteria can reduce bacterial immunogenicity and alleviate bacterial vascular clearance, thereby enhancing tumor targeting and safety (Nature Communication 2019, 10, 3452.).

There is evidence that VNP20009 colonizes the core area of the tumor through vascular penetration, which indicates that The rapid clearance of VNP20009 from the blood may lead to poor tumor colonization of VNP20009 (J Control Release 2015, 199, 180-9; Cancer Gene Ther 2011, 18, 457-66.). This hypothesis is confirmed by Westphal's work, which reports that depletion of host neutrophils leads to increased bacterial titers in tumors (Cancer Res 2008, 68, 2952-60.). Other studies have reported that by depleting host neutrophils, VNP20009-enhanced tumor therapeutic effects can be observed (Theranostics 2017, 7, 2250-2260.). Neutrophils can load drugs by phagocytosis and release drugs by cell lysis after being recruited to the tumor area (Adv Mater 2018, 30, e1706245; Adv Mater 2020, 32, e2003598.).

Summary of the invention

How to use neutrophils to load attenuated Salmonella to reduce the immunogenicity of bacteria, alleviate the vascular clearance rate of bacteria, and enhance tumor targeting and safety; at the same time, avoid the inherent bacterial clearance characteristics of neutrophils, which causes the loaded attenuated Salmonella to remain active without being cleared? The purpose of the present invention is to provide a neutrophil loaded with attenuated Salmonella and its preparation method and application, which effectively improves the tumor colonization rate and tumor targeting of bacteria, and can directly kill tumor cells and release intracellular oncolytic bacteria to induce anti-tumor immune response, effectively inhibiting melanoma.

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 recombinant engineered strains derived therefrom, and wherein the recombinant engineered strain is a strain that does not carry or carries an expression plasmid that expresses an exogenous gene of 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 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 efficacy to VNP20009 and can be combined with biological drugs, gene therapy, chemotherapy, and traditional Chinese medicine for treatment.

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

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 tracing protein LuxCDABE gene.

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.

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.

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, co-incubating 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.

The in vivo distribution of neutrophils loaded with attenuated Salmonella and the evaluation and tracing method of tumor colonization rate of the present invention comprise the following steps: using Cy5.5-labeled neutrophils and attenuated Salmonella loaded with neutrophils to sustainably express fluorescent protein or luciferase tracer protein, and evaluating the in vivo distribution and level of neutrophils loaded with attenuated Salmonella by tissue plating and in vivo imaging technology.

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.

Furthermore, neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune responses to inhibit the progression of melanoma lung metastasis.

The invention discloses 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.

Beneficial effects: The present invention provides a highly tumor-targeted, safe, convenient, low-cost, and new oncolytic bacteria delivery method, that is, using neutrophils for loading, which can greatly increase the tumor targeting and safety of oncolytic bacteria while improving the tumor treatment effect of oncolytic bacteria, and can directly kill tumors. It can damage tumor cells and activate the body's anti-tumor immunity to inhibit tumor progression, thus showing good prospects for tumor treatment.

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

(1) The present invention proves that neutrophils are a key factor affecting tumor colonization by oncolytic bacteria attenuated Salmonella.

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

(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.

(4) The present invention combines cell therapy and bacterial therapy, and a single administration can achieve good therapeutic effects, providing a convenient and low-cost combined tumor therapy. At the same time, the present invention can also load two or more oncolytic bacteria at the same time.

(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

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 paying any creative work.

Description of the abbreviations in the accompanying drawings:

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.

FIG1 is a graph showing the percentage of neutrophils in all immune cells in the blood after injection of attenuated Salmonella of the present invention;

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.

FIG3 is a flow cytometric analysis of the purity of peritoneal neutrophils in the present invention.

FIG. 4 is a fluorescent image of neutrophils loaded with attenuated Salmonella of the present invention.

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

FIG. 6 shows the survival ability of neutrophils loaded with attenuated Salmonella of the present invention.

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

FIG. 8 shows the rate of release of the attenuated Salmonella of the present invention from neutrophils.

FIG. 9 shows that neutrophils loaded with attenuated Salmonella of the present invention promote apoptosis of tumor cells.

FIG. 10 shows that the neutrophils loaded with attenuated Salmonella of the present invention inhibit tumor cell proliferation.

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

FIG. 12 shows the cell status of neutrophils loaded with attenuated Salmonella detected by real-time fluorescence quantitative PCR of the present invention.

FIG. 13 is a photograph of in vivo imaging of mouse organs after drug administration of the present invention.

FIG. 14 shows the in vivo organ distribution and levels of the melanoma lung metastasis model of the present invention.

FIG. 15 shows the organ distribution and levels of the subcutaneous melanoma model of the present invention.

FIG. 16 shows the number of lung tumor metastases after systemic administration of the present invention.

FIG. 17 shows the changes in body weight of tumor-bearing mice after systemic administration of the present invention.

FIG. 18 shows organ weights and spleen photographs of tumor-bearing mice after systemic administration of the present invention.

FIG. 19 is the HE staining results of other normal organ tissues of tumor-bearing mice after systemic administration of the present invention.

FIG. 20 shows the levels of serum biochemical indices in tumor-bearing mice after systemic administration of the present invention.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present 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 the present application and are not used to limit the present application.

In this application, the term "and/or" describes the association relationship between associated objects, indicating that there may be three A relationship, such as A and/or B, can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character "/" generally indicates that the objects before and after are in an "or" relationship.

In this application, "at least one" means one or more, and "plurality" 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, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be single or multiple, respectively.

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, and 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.

The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" 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 other meanings.

The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

The present invention mainly relates to a method for constructing neutrophils loaded with attenuated Salmonella and its application in anti-tumor treatment. On the one hand, the present invention proves that neutrophils are a key factor affecting tumor colonization of oncolytic bacteria including attenuated Salmonella; on the other hand, the present invention develops a method for constructing neutrophils loaded with attenuated Salmonella and its application in anti-tumor treatment. A neutrophil delivery system for bacteria has shown potential for anti-tumor therapy.

In response to the limitations in 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 response, thereby effectively inhibiting melanoma lung metastasis.

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 is an expression plasmid that does not carry or carries an exogenous gene expressing 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 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 efficacy to VNP20009 and can be combined with biological drugs, gene therapy, chemotherapy, and traditional Chinese medicine for treatment.

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 skeleton that can express interfering RNA.

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

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.

The expression plasmid 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 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.

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 shows potential for anti-tumor treatment, and the system describes an efficient, highly tumor-targeted, convenient and low-cost oncolytic bacteria delivery strategy, which can significantly increase the tumor colonization rate and level, thereby achieving a balance between the therapeutic effect and safety of oncolytic bacteria.

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

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.

The 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, and the cells were treated with 50-100 μg/mL gentamicin for 30-60 minutes to kill extracellular bacteria; then the 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.

The 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 by tissue plating and in vivo imaging technology.

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, and an intravenous injection preparation or a spray.

Neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune responses to inhibit the progression of melanoma lung metastasis.

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

The use of neutrophils loaded with attenuated Salmonella in tumor treatment described in the present invention is characterized in that premature exposure of blood vessels of attenuated Salmonella and clearance by the immune system are avoided, thereby increasing the tumor targeting of attenuated Salmonella, and increasing the safety of attenuated Salmonella by reducing the infiltration of attenuated Salmonella into normal tissues and organs; the attenuated Salmonella delivery system described in the present invention can effectively maintain the balance between the therapeutic effect and safety of oncolytic bacteria, thereby expanding the application range of oncolytic bacteria.

Example 1

Construction and evaluation of neutrophils loaded with attenuated Salmonella

Acquisition of peritoneal neutrophils: The neutrophils of the present invention are primary peritoneal neutrophils of mice. 4-6 hours before the separation of peritoneal neutrophils, mice are subcutaneously injected with 1 ml of nutrient broth to stimulate the maturation of peritoneal neutrophils. Neutrophils are separated using Percoll gradient and identified by flow cytometry;

Co-incubation of bacteria with neutrophils: Cells were cultured at 5% CO ₂ 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; in order to measure the efficiency of VNP loading of neutrophils, the proportion of VNP-RFP positive in neutrophils was detected by flow cytometry; in order to evaluate the activity of intracellular bacteria, cells were treated with 1% TritonX-100, incubated on ice for 1-2 hours to lyse the cells and release the bacteria, and the number of intracellular bacteria in neutrophils and bacterial activity were counted after dilution.

Detection of attenuated Salmonella released by neutrophils: Neutrophils loaded with attenuated Salmonella were co-cultured with tumor cells B16F1, B16F10 and mouse embryonic fibroblasts MEF in an incubator at 37°C and 5% _CO2 for 0.5h, 1h, 2h, and 4h, respectively. The supernatant was collected and the number of released attenuated Salmonella was counted on LB plates.

Example 2

Characterization of the state of neutrophils loaded with attenuated Salmonella

Detection of reactive oxygen species and nitric oxide levels: After neutrophils were loaded with attenuated Salmonella for 0.5 h, 1 h, 5 h, or 2.5 h, ROS detection kit (S0033S, Bio-Tech) and DAF-FM DA (S0019, Bio-Tech) were used to detect neutrophil ROS and NO. The experimental operation was carried out according to the instructions of the kit.

Neutrophil chemotaxis assay: Neutrophil chemotaxis was performed using a Transwell with a pore size of 3 μm. Neutrophil chemotactic factor fMLP (10 nM) or 1×10 ⁶ macrophages/well were added to the upper chamber, and 5×10 ⁵ neutrophils were inoculated per well in the upper chamber for 2 hours. The supernatant in the lower chamber was collected to count the number of neutrophils and the chemotactic ratio.

Cell proliferation and apoptosis detection. The proliferation of tumor cells after drug administration was detected by the B16F10-GFP method. B16F10 cells were seeded in 24-well plates at a density of 1×10 ⁵ per well and measured once every 12 hours using a microplate reader (488nm～510nm) to calculate the proliferation rate of B16F10 cells. The apoptosis rate of B16F10 cells was stained using Annexin V/PI. 1μl Annexin V-APC (1mg/ml) and 1μl PI-PE (PI, 1mg/ml) were incubated with 1×10 ⁶ cells in binding buffer for 30 minutes at 4°C. The stained cells were analyzed by flow cytometry. The results were statistically analyzed using FlowJo VX software.

Real-time fluorescence quantitative PCR to detect neutrophil status: Total RNA was extracted using TRIzol reagent. According to the instructions, the cDNA template was reverse transcribed using the TUYOBO cDNA synthesis kit. The relative gene expression level was detected using the one-step RT-PCR SYBR Green kit (Novozymes).

Example 3

Identification of the therapeutic effect of neutrophils loaded with attenuated Salmonella on tumors

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

Grouping of tumor-bearing mice and administration of bacteria: B16F10 mouse melanoma-bearing mice were randomly divided into 4 groups and injected with 3×10 ⁶ CFU of bacteria and 1×10 ⁶ neutrophils loaded with attenuated Salmonella via tail vein.

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 in the mice.

Example 4

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

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

Construction of lung metastasis melanoma tumor model: B16F10 mouse melanoma cells were cultured in DMEM. After culturing to the exponential growth phase with cell culture medium, the cells were digested with trypsin and centrifuged at 1000 rpm, 4°C for 3 minutes to collect the cells. After washing twice with PBS, the cells were counted and the final cell concentration was 1×10 ⁶ cells/mL. Each C57BL/6 mouse was inoculated with 1×10 ⁵ cells/mouse.

After administration at different time points, the mice were killed, and the various organ tissues of the tumor-bearing mice, including tumors, liver, spleen, kidney, lung and heart, were collected under a sterile environment. They were weighed and placed in 2 mL PBS for tissue homogenization to lyse the cells and release bacteria. According to the different bacterial titers in the tissues, the appropriate bacterial solution was obtained according to different dilution gradients, and spread on LB plates containing kanamycin, and then inverted and cultured in a 37°C bacterial incubator for 12 hours, and the colonies were counted to compare and analyze the tissue distribution of wild-type attenuated Salmonella and NE (VNP) in tumor-bearing mice.

In vivo imaging and immunofluorescence sections were used to detect the in vivo distribution of neutrophils loaded with attenuated Salmonella. Neutrophils labeled with Cy5.5 and neutrophils loaded with attenuated Salmonella could continuously express fluorescent protein or luciferase tracer protein. Tumor-bearing mice were injected with 3× ¹⁰⁶ CFU of bacteria and 1×106 neutrophils loaded with attenuated Salmonella, respectively. The in vivo distribution and level of neutrophils loaded with attenuated Salmonella were evaluated by in vivo imaging technology and immunofluorescence staining.

Histopathology and blood biochemical analysis: After the blood was allowed to stand at room temperature for 30 minutes, it was centrifuged at 4 degrees and 3000 rpm for 15 minutes, and the supernatant serum was carefully aspirated. The serum obtained from the blood was frozen and stored at -80°C until the blood biochemical indicators were determined; 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 Biology Company.

Test Example 1

The present invention uses a neutrophil depletion model to demonstrate that vascular clearance of neutrophils 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 ( FIG. 2 ).

Test Example 2

The purity ratio of primary mouse neutrophils extracted by the present invention is greater than 90% (Figure 3), and fluorescent photos can prove that neutrophils can be loaded with attenuated Salmonella (Figure 4); and attenuated Salmonella can maintain more than 30% activity in neutrophils (Figure 5); neutrophils can maintain activity at more than about 80% after phagocytizing attenuated Salmonella for 2 hours, so that neutrophils are guaranteed to have activity and chemotaxis before being recruited to the tumor area (Figures 6 and 7); neutrophils can release more attenuated Salmonella under the induction of highly malignant tumors (B16F10) (Figure 8).

Test Example 3

The present invention proves through tumor cell apoptosis and proliferation experiments that neutrophils loaded with attenuated Salmonella have stronger tumor inhibition ability (Figures 9 and 10); neutrophils loaded with attenuated Salmonella have stronger nitric oxide and reactive oxygen levels (Figure 11); the cell state of neutrophils loaded with attenuated Salmonella is detected by real-time fluorescence quantitative PCR, and the results show that attenuated Salmonella can induce neutrophils to polarize toward an anti-tumor morphology (Figure 12).

Test Example 4

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).

The above shows and describes 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, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims (10)

A neutrophil loaded with attenuated Salmonella, characterized in that: the neutrophil loaded with attenuated Salmonella includes 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 that express exogenous genes such as therapeutic genes and tracer genes. The neutrophil-loaded recombinant attenuated Salmonella engineered bacteria described in claim 1 is characterized in that: genes that can express 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 backbone that can express interfering RNA. The neutrophil-loaded recombinant attenuated Salmonella engineered bacteria according to claim 2 is characterized in that the gene that can be expressed in the 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 tracer protein LuxCDABE gene. The neutrophil-loaded recombinant engineered attenuated Salmonella engineered bacteria according to claim 3 is 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. The neutrophil-loaded recombinant engineered attenuated Salmonella engineered bacteria according to claim 2 is characterized in that: the expression plasmid that can be carried by the attenuated Salmonella 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. The method for preparing neutrophils loaded with attenuated Salmonella according to claim 1 is characterized in that it comprises the following steps: extracting neutrophils stimulated with 5% starch broth, incubating with attenuated Salmonella 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 obtaining neutrophils loaded with attenuated Salmonella. Granulocytes. The method for evaluating and tracing the in vivo distribution of neutrophils loaded with attenuated Salmonella and the tumor colonization rate described in claim 1 is characterized by comprising 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 the lysed tissue and then plating to count the number of bacteria. The use of neutrophils loaded with attenuated Salmonella in claim 1 in the preparation of an anti-tumor pharmaceutical preparation is characterized in that the pharmaceutical preparation comprises at least one of an oral preparation, an injection preparation, and an intravenous injection preparation or a spray. The use according to claim 8 is characterized in that neutrophils loaded with attenuated Salmonella can directly kill tumor cells or activate anti-tumor immune response to inhibit tumor progression. Use of the neutrophils loaded with attenuated Salmonella as claimed in claim 1 in combination with other anti-tumor drugs in the preparation of anti-tumor drugs.