CN117126811A - Neutrophil loaded with attenuated salmonella and preparation method and application thereof - Google Patents
Neutrophil loaded with attenuated salmonella and preparation method and application thereof Download PDFInfo
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- CN117126811A CN117126811A CN202310943859.5A CN202310943859A CN117126811A CN 117126811 A CN117126811 A CN 117126811A CN 202310943859 A CN202310943859 A CN 202310943859A CN 117126811 A CN117126811 A CN 117126811A
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- attenuated salmonella
- loaded
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- neutrophils
- neutrophil
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/42—Salmonella
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The application discloses a neutral granulocyte loaded with attenuated salmonella and a preparation method and application thereof, belonging to the technical field of biology. The application proves that the vascular clearance of neutrophils is a key factor affecting the tumor implantation rate of oncolytic bacteria, namely attenuated salmonella typhimurium. The application proves that neutrophils can be recruited to a tumor microenvironment after being loaded with oncolytic bacteria, so that the tumor implantation rate and tumor targeting of the bacteria are effectively improved, and the anti-tumor immune response can be induced by directly killing tumor cells and releasing intracellular oncolytic bacteria, thereby effectively inhibiting the lung metastasis progress of melanoma.
Description
Technical Field
The application relates to the technical field of biology, in particular to a neutral granulocyte loaded with attenuated salmonella, and a preparation method and application thereof.
Background
Attenuated salmonella typhimurium strain VNP20009 (abbreviated as VNP) lacks purI and msbB genes to reduce toxic side effects and septic shock, and is one of the most potential oncolytic strains. Compared with wild salmonella typhimurium, the attenuated salmonella VNP20009 has a significant ability to inhibit tumors but has a degree of side effects. Attenuated salmonella VNP20009 titers in tumors were about 1000-fold higher than in other organs (The Journal of Infectious Diseases 2000,181,1996-2002.). In phase I clinical trials of attenuated salmonella VNP20009, most melanoma patients have no significant side effects, except for the highest dose group, demonstrating their safety. However, only in the highest dose group, colonization of the tumor tissue by attenuated salmonella VNP20009 was observed (Journal of Clinical Oncology 2002,20,142-152.). Tumor colonization rate by oncolytic bacteria is the most important factor in tumor treatment. Therefore, how to balance the therapeutic effect and safety of oncolytic bacteria is a problem to be solved.
In order to improve the therapeutic effect of salmonella, many excellent studies have been reported, such as shRNA carrying a gene critical for tumor metastasis (J Exp Clin Cancer Res 2016,35,107;Oncotarget 2016,7,12), expression of effector proteins such as interferon-gamma protein or cytotoxic protein (Eur J Cancer 2017,70,48-61), and loading of 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 viable measure to increase oncolytic colonization in tumors. It has been reported that cell membrane encapsulation of bacteria reduces the immunogenicity of the bacteria and alleviates the vascular clearance of the bacteria, thereby enhancing tumor targeting and safety (Nature Communication 2019,10,3452.).
There is evidence that VNP20009 colonizes the tumor core area by vascular penetration, suggesting that rapid clearance of VNP20009 in the blood may lead to tumor dysplasia of VNP20009 (J Control Release 2015,199,180-9;Cancer Gene Ther 2011,18,457-66.). Westphal work demonstrates this hypothesis and reports that depletion of host neutrophils leads to increased bacterial titers in tumors (Cancer Res 2008,68,2952-60.). There are also studies reporting that enhanced tumor therapeutic effects of VNP20009 can be observed by depletion of host neutrophils (theranosics 2017,7,2250-2260.). Neutrophils can be loaded with drug by phagocytosis, and release drug by cell lysis after recruitment to the tumor area (Adv mate 2018,30,e1706245;Adv Mater 2020,32,e2003598.).
Disclosure of Invention
How to load attenuated salmonella by utilizing neutrophils so as to reduce immunogenicity of bacteria, relieve vascular clearance of the bacteria and enhance targeting and safety of tumors; at the same time, can avoid the inherent bacteria removal characteristic of neutrophils, and can the loaded attenuated salmonella not be removed and still keep the activity? The application aims to provide a neutrophil loaded with attenuated salmonella, a preparation method and application thereof, which can effectively promote the tumor implantation rate and tumor targeting of bacteria, and can effectively inhibit melanoma by directly killing tumor cells and releasing intracellular oncolytic bacteria to induce anti-tumor immune response.
In order to solve the problems in the prior art, the application provides the following technical scheme: the application relates to a neutrophil loaded with attenuated salmonella, which comprises neutrophil and oncolytic bacteria, wherein the oncolytic bacteria are attenuated salmonella typhimurium VNP20009 and derived recombinant engineering strains thereof, and the recombinant engineering strains are strains which do not carry or carry expression plasmids for expressing therapeutic genes and trace genes exogenous genes. Attenuated salmonella VNP20009, including the strains of the previously filed application patents (ZL 201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B2021,11 (10): 3165-3177; phop/phoQ, etc.), have all been experimentally demonstrated to have similar tumor targeting and antitumor efficacy, and the engineered strains have lower toxicity than VNP 20009. The modified strains have similar curative effects to VNP20009 and can be used for combination treatment with biological drugs, gene therapy, chemotherapy and traditional Chinese medicines.
Further, therapeutic or tracer effect genes that may be expressed in attenuated salmonella typhimurium VNP20009 include at least one of an immune checkpoint blocker antibody gene, a cytokine gene, an angiogenesis inhibitor gene, an apoptosis-type anti-tumor gene, or an interfering plasmid; the interference plasmid which can be carried by the attenuated salmonella typhimurium VNP20009 comprises an interference plasmid which takes pRNA-U6.1 as a framework and can express interference RNA.
Further, the gene having the tracer effect may be expressed in attenuated salmonella typhimurium VNP20009, including at least one of red fluorescent protein RFP gene, green fluorescent protein GFP gene, and luciferase tracer protein LuxCDABE gene.
Furthermore, the nucleotide sequence of the red fluorescent protein RFP gene is shown as SEQ ID No.1, the nucleotide sequence of the green fluorescent protein GFP gene is shown as SEQ ID No.2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown as SEQ ID No. 3.
Further, the expression plasmid carried by the attenuated salmonella typhimurium VNP20009 comprises a constitutive strong promoter J23100, a PelB extracellular protein secretion signal peptide and a Flag tag, wherein the nucleotide sequence of the constitutive strong promoter J23100 is shown as SEQ ID NO.4, and the nucleotide sequence of the PelB extracellular protein secretion signal peptide is shown as SEQ ID NO. 5.
The preparation method of the attenuated salmonella-loaded neutrophil comprises the following steps: extraction of 5% starch broth stimulated celiac neutrophils, and VNP20009 as attenuated Salmonella according to cell 100:1 and then treating the cells with 50-100 mug/mL gentamicin for 30-60 minutes to kill extracellular bacteria after 0.5-2.5 hours of co-incubation, thereby preparing the neutral granulocytes loaded with the attenuated salmonella.
The application relates to an in-vivo distribution and tumor implantation rate evaluation and tracing method of neutrophils loaded with attenuated salmonella, which comprises the following steps: the in vivo distribution and levels of the attenuated salmonella-loaded neutrophils were assessed by tissue plating and in vivo imaging techniques using cy 5.5-labeled neutrophils and the attenuated salmonella-loaded neutrophils for sustained expression of fluorescent protein or luciferase tracer protein.
The application relates to an application of attenuated salmonella-loaded neutrophils in preparing an anti-tumor pharmaceutical preparation, wherein the pharmaceutical preparation comprises at least one of an oral preparation, an injection preparation and an intravenous injection preparation or a spray.
Further, the attenuated salmonella-loaded neutrophils can directly kill tumor cells or activate an anti-tumor immune response to inhibit the progression of melanoma lung metastasis.
The application relates to an application of neutrophils loaded with attenuated salmonella in combination with other antitumor drugs in preparation of antitumor drugs, such as antibody drugs, chemotherapeutics and the like.
The beneficial effects are that: the application provides a high tumor targeting, safe, convenient, low-cost and brand-new delivery mode of oncolytic bacteria, namely neutrophil loading, which can greatly increase tumor targeting and safety of oncolytic bacteria while improving tumor treatment effect of oncolytic bacteria, and can inhibit tumor progression by directly killing tumor cells and activating organism anti-tumor immunity, thus showing good tumor treatment prospect.
Compared with the prior art, the application has the following advantages:
(1) The application proves that the neutrophil is a key factor affecting the tumor colonization of the oncolytic bacteria attenuated salmonella.
(2) Attenuated salmonella is rapidly engulfed and cleared by neutrophils after injection into the body. According to the application, the preparation method of the neutrophil loaded with the attenuated salmonella is provided, so that the attenuated salmonella is loaded in the neutrophil; and temporarily protects the premature exposure and elimination of attenuated salmonella, an effective strategy is invented that can achieve a balance between the therapeutic effect and safety of oncolytic bacteria.
(3) According to the application, the primary neutrophils are used for loading the attenuated salmonella, the delivery system can greatly increase the intratumoral positioning rate and the intratumoral positioning quantity of the attenuated salmonella, and reduce the infiltration of the attenuated salmonella to normal tissues, so that the safety of the attenuated salmonella can be obviously improved.
(4) The application combines cell therapy and bacterial therapy, can realize good treatment effect by single administration, and provides a convenient and low-cost tumor combined therapy. Meanwhile, the application can load two or more oncolytic bacteria at the same time.
(5) The neutral granulocyte loaded with the attenuated salmonella can directly promote tumor tissue necrosis so as to exert good anti-tumor capability; the neutrophil granulocyte loaded with attenuated salmonella can activate anti-tumor immune response so as to promote tumor apoptosis and realize tumor treatment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
Brief description of the drawings:
VNP: attenuated mice injured salmonella VNP20009; RFP: red fluorescent protein; EGFP: green fluorescent protein; VNP-RFP: VNP carries RFP gene plasmid; VNP-EGFP: VNP carries EGFP gene plasmid; NEs: primary neutrophils in mice; NEs (VNP): neutrophils loaded with VNP; VNP-Lvx: VNP carries LuxCDABE gene plasmid.
FIG. 1 shows the percentage of neutrophils in blood to all immune cells after injection of attenuated salmonella of the present application;
FIG. 2 shows that the vascular clearance of neutrophils is a key factor affecting the tumor implantation rate of oncolytic bacteria, namely attenuated Salmonella typhimurium, and the in vivo distribution of attenuated Salmonella is detected by injecting the attenuated Salmonella after the antibody depletes the neutrophils in tumor-bearing mice.
FIG. 3 shows the purity of the neutrophils in the abdominal cavity by flow cytometry according to the present application.
FIG. 4 is a fluorescent image of an attenuated Salmonella-loaded neutrophil of the present application.
FIG. 5 is a graph showing the detection of the activity of the attenuated Salmonella of the present application in neutrophil cells.
FIG. 6 shows the ability of the attenuated Salmonella-loaded neutrophil viability of the present application.
FIG. 7 shows chemotactic ability of Salmonella attenuated-loaded neutrophils according to the application.
FIG. 8 is a graph showing the intracellular release rate of the attenuated salmonella of the present application from neutrophils.
FIG. 9 shows that the attenuated Salmonella-loaded neutrophils of the present application promote apoptosis of tumor cells.
FIG. 10 shows the inhibition of tumor cell proliferation by attenuated Salmonella-loaded neutrophils of the present application.
FIG. 11 shows nitric oxide and reactive oxygen species levels of Salmonella attenuated loaded neutrophils according to the application.
FIG. 12 shows the real-time fluorescent quantitative PCR detection of the cell status of the attenuated Salmonella-loaded neutrophils according to the application.
FIG. 13 is a photograph showing the vital signs of organs of mice after administration of the present application.
FIG. 14 shows in vivo organ distribution and level of melanoma lung metastasis model of the present application.
FIG. 15 shows in vivo organ distribution and level of a subcutaneous model of melanoma according to the application
FIG. 16 is a graph showing the number of lung tumor metastases following systemic administration of the present application.
Figure 17 shows the body weight change of tumor-bearing mice following systemic administration according to the application.
FIG. 18 is a photograph of organ weights and spleens of tumor-bearing mice after systemic administration of the present application.
FIG. 19 shows HE staining of other normal organ tissues of tumor-bearing mice after systemic administration according to the application.
FIG. 20 shows serum biochemical levels of tumor-bearing mice following systemic administration according to the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, in the description of the embodiment of the application, the mass can be a mass unit which is well known in the chemical industry field such as mu g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
The application mainly relates to a construction method of neutrophils loaded with attenuated salmonella and an anti-tumor therapeutic application. In one aspect, the present application demonstrates that neutrophils are a key factor in tumor colonization by oncolytic bacteria including attenuated salmonella; in another aspect, the present application exploits a delivery system for attenuated salmonella-loaded neutrophils that demonstrates potential for anti-tumor therapy.
Aiming at the limitation of application of oncolytic bacteria including attenuated salmonella, the application provides a technology for preparing a neutrophil carrier capable of loading oncolytic bacteria, neutrophils can be recruited to a tumor microenvironment after loading VNP20009, so that the tumor implantation rate and tumor targeting of the oncolytic bacteria are effectively improved, and the technology can effectively inhibit melanoma lung metastasis by directly killing tumor cells and releasing intracellular oncolytic bacteria to induce an anti-tumor immune response.
The first aspect of the embodiment of the application provides a neutrophil loaded with oncolytic bacteria including attenuated salmonella, wherein the neutrophil loaded with the oncolytic bacteria is primary neutrophil, the oncolytic bacteria is attenuated salmonella VNP20009 and a recombinant engineering strain derived from the same, and the recombinant engineering strain is an expression plasmid which does not carry or carry exogenous genes for expressing therapeutic genes and tracer genes. Attenuated salmonella VNP20009, including the strains of the previously filed application patents (ZL 201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B2021,11 (10): 3165-3177; phop/phoQ, etc.), have all been experimentally demonstrated to have similar tumor targeting and antitumor efficacy, and the engineered strains have lower toxicity than VNP 20009. The modified strains have similar curative effects to VNP20009 and can be used for combination treatment with biological drugs, gene therapy, chemotherapy and traditional Chinese medicines.
The therapeutic or tracer effect gene that can be expressed in attenuated salmonella VNP20009 includes at least one of an immune checkpoint blocker antibody gene, a cytokine gene, an angiogenesis inhibitor gene, an apoptosis-type anti-tumor gene, or an interfering plasmid; the interference plasmid which can be carried by the attenuated salmonella typhimurium VNP20009 comprises an interference plasmid which takes pRNA-U6.1 as a framework and can express interference RNA.
The gene with tracing effect can be expressed in attenuated salmonella typhimurium VNP20009 and comprises at least one of red fluorescent protein RFP gene, green fluorescent protein GFP gene and luciferase tracing protein LuxCDABE gene.
The nucleotide sequence of the red fluorescent protein RFP gene is shown as SEQ ID No.1, the nucleotide sequence of the green fluorescent protein GFP gene is shown as SEQ ID No.2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown as SEQ ID No. 3.
The expression plasmid carried by attenuated salmonella typhimurium VNP20009 comprises a constitutive strong promoter J23100, a PelB extracellular protein secretion signal peptide and a Flag tag, wherein the nucleotide sequence of the constitutive strong promoter J23100 is shown as SEQ ID NO.4, and the nucleotide sequence of the PelB extracellular protein secretion signal peptide is shown as SEQ ID NO. 5.
The application builds a neutral granulocyte delivery system loaded with attenuated salmonella by taking attenuated salmonella VNP20009 as a basic strain and combining cell drug-loading technology, wherein the attenuated salmonella VNP20009 comprises but is not limited to strains (ZL 201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B2021,11 (10): 3165-3177; phoP/phoQ, and the like) of the prior patent application, and the delivery system shows potential of anti-tumor treatment, and describes a high-efficiency, high-tumor targeting, convenient and low-cost oncolytic bacteria delivery strategy, so that the tumor colonization rate and level can be remarkably increased, and the balance between the therapeutic effect and the safety of oncolytic bacteria is realized.
In a second aspect, the embodiment of the application provides a method for preparing neutrophils loaded with attenuated salmonella, comprising the following steps:
wild mice were stimulated with 5% nutritional starch broth for 4-6 hours and then were aspirated for peritoneal effusion, and the cells obtained after percoll gradient centrifugation were examined by flow cytometry for 90% of the cells as neutrophils.
Incubating the modified or unmodified VNP20009 with celiac neutrophils for 0.5, 1.5, 2.5 hours in a ratio of 100:1 of attenuated salmonella to cells, treating the cells with 50-100 μg/mL gentamicin for 30-60 minutes to kill extracellular bacteria; cells were then treated with 1% triton x-100 and incubated on ice for 1-2 hours to lyse cells to free bacteria, and plating after dilution counted the number of bacteria and bacterial activity in neutrophil cells.
In a third aspect, the present application provides a method for evaluating and tracing in vivo distribution and tumor implantation rate of neutrophils loaded with attenuated salmonella, comprising the steps of: the in vivo distribution and levels of the attenuated salmonella-loaded neutrophils were assessed by tissue plating and in vivo imaging techniques using cy 5.5-labeled neutrophils and the attenuated salmonella-loaded neutrophils for sustained expression of fluorescent protein or luciferase tracer protein.
According to a fourth aspect of the embodiment of the application, there is provided an application of attenuated salmonella-loaded neutrophils in preparing an anti-tumor pharmaceutical preparation, wherein the pharmaceutical preparation comprises at least one of an oral preparation, an injection preparation and an intravenous injection preparation or a spray.
The neutrophils loaded with attenuated salmonella can directly kill tumor cells or activate an anti-tumor immune response to inhibit the progression of melanoma lung metastasis.
The application relates to an application of neutrophils loaded with attenuated salmonella in combination with other antitumor drugs in preparation of antitumor drugs.
The application of the neutrophils loaded with the attenuated salmonella in tumor treatment is characterized in that the premature exposure of blood vessels of the attenuated salmonella and the elimination of the attenuated salmonella by an immune system are avoided, so that the tumor targeting of the attenuated salmonella is improved, and the safety of the attenuated salmonella is improved by reducing the infiltration of the attenuated salmonella to normal tissues and organs; the attenuated salmonella delivery system of the present application can effectively maintain the balance of the oncolytic bacteria in the treatment effect and the safety thereof, thereby expanding the application range of oncolytic bacteria.
Example 1
Construction and evaluation of attenuated salmonella-loaded neutrophils
Obtaining celiac neutrophils: the neutrophils are primary peritoneal neutrophils of the mice, and 1ml of nutrient broth is injected subcutaneously into the mice 4-6 hours before the separation of the peritoneal neutrophils to stimulate the maturation of the peritoneal neutrophils. Separating neutrophils by using a Percoll gradient, and identifying by using a flow cytometer;
co-incubation of bacteria with neutrophils: at 5% CO 2 Cells were cultured at 37℃to give a molar concentration of 100:1 with neutrophils for 0.5, 1.5 or 2.5 hours. Then, 50-100 mug/mL gentamicin sulfate is added to the culture medium for 1 hour to kill bacteria on the surface of the neutrophil; to measure the efficiency of the loading VNP of neutrophils, the proportion of VNP-RFP positivity in neutrophils was detected with a flow cytometer; to assess intracellular bacterial activity, 1% Triton X-100 treatment was usedCells were incubated on ice for 1-2 hours to lyse the cells to free bacteria, and the number of bacteria and bacterial activity in neutrophil cells were counted by plating after dilution.
Neutrophil release attenuated salmonella detection: the neutrophils loaded with attenuated salmonella are respectively combined with tumor cells B16F1, B16F10 and mouse embryo fibroblast MEF at 37 ℃ and 5% CO 2 The incubator was co-cultured for 0.5h, 1h, 2h, 4h, the supernatant was collected, and the number of attenuated salmonella released was counted on an LB plate.
Example 2
Neutral granulocyte state characterization of attenuated salmonella loading
Reactive oxygen and nitric oxide level detection: after loading the neutrophils with attenuated salmonella for 0.5h, 1h, 5h or 2.5h, detecting the neutrophils with ROS detection kit (S0033S, biyun) and DAF-FM DA (S0019, biyun) and NO, and performing experimental operation according to the specification of the kit;
neutrophil chemotaxis assay: neutrophil chemotaxis employs a Transwell with a pore size of 3 μm. The lower chamber was filled with neutrophil chemokine fMLP (10 nM) or preset 1X 10 6 Macrophages per well, 5X 10 cells per well of the upper chamber 5 And (3) co-culturing the neutrophils for 2 hours. The lower chamber supernatant was collected and the neutrophil count and chemotaxis ratio were counted.
Cell proliferation and apoptosis assays. Proliferation of tumor cells after administration was examined by the B16F10-GFP method, and the B16F10 cells were used at 1X 10 per well 5 Is seeded in 24-well plates and measured every 12h with a miniature plate reader (488 nm to 510 nm) to calculate the proliferation rate of B16F10 cells. Apoptosis rate of B16F10 cells was stained with Annexin V/PI. 1 μl Annexin V-APC (1 mg/ml) and 1 μl PI-PE (PI, 1 mg/ml) with 1×10 6 Cells were incubated in binding buffer for 30 min at 4 ℃. Stained cells were analyzed by flow cytometry. Results were statistically analyzed using flowjovx software.
Real-time fluorescent quantitative PCR detection of neutrophil status: total RNA was extracted with TRIzol reagent. According to the instructions, TUYOBO cDNA synthesis kit reverse transcription of cDNA template. The relative gene expression level was detected using one-step RT-PCR SYBR Green kit (Norpraise).
Example 3
Identification of tumor therapeutic effect of attenuated salmonella-loaded neutrophils
Constructing a lung metastasis melanoma tumor model: the B16F10 mouse melanoma cells are cultured to the exponential phase of growth by using DMEM cell culture medium, then digested by pancreatin, 1000rpm,4 ℃, collected by centrifugation for 3 minutes, washed 2 times with PBS, and then subjected to cell count, and finally the final cell concentration is 1×10 6 And each mL. 1X 10 Vaccination with each C57BL/6 mouse 5 And/or just.
Tumor bearing mice were grouped and bacteria were dosed: the mice bearing melanoma of B16F10 mice were randomly divided into 4 groups and injected 3X 10 by tail vein 6 CFU bacteria and 1×10 bacteria 6 And (3) loading the neutral granulocytes of the attenuated salmonella.
The change in body weight was recorded daily, and after one week the mice were dissected to obtain individual organs to count the number of pulmonary metastases in the mice.
Example 4
Investigation of distribution of neutrophils loaded with attenuated salmonella in tumor-bearing mice
Constructing a subcutaneous melanoma tumor model: the B16F10 mouse melanoma cells are cultured to the exponential phase of growth by using DMEM cell culture medium, then digested by pancreatin, 1000rpm,4 ℃, collected by centrifugation for 3 minutes, washed 2 times with PBS, and then subjected to cell count, and finally the final cell concentration is 1×10 6 And each mL. 1X 10 Vaccination with each C57BL/6 mouse 5 Experiments were performed when the tumor volume of mice grew to about 100mm 3.
Constructing a lung metastasis melanoma tumor model: the B16F10 mouse melanoma cells are cultured to the exponential phase of growth by using DMEM cell culture medium, then digested by pancreatin, 1000rpm,4 ℃, collected by centrifugation for 3 minutes, washed 2 times with PBS, and then subjected to cell count, and finally the final cell concentration is 1×10 6 And each mL. 1X 10 Vaccination with each C57BL/6 mouse 5 And/or just.
After various time points of administration, mice were sacrificed, and individual organ tissues of tumor-bearing mice, including tumor, liver, spleen, kidney, lung and heart, were collected in a sterile environment, weighed and placed in 2mL PBS for tissue homogenization to lyse cells and release bacteria. According to different bacterial titer in tissues, obtaining proper bacterial liquid according to different dilution gradients, coating on an LB plate containing the kanamycin, then inversely culturing for 12 hours in a bacterial incubator at 37 ℃ and counting colonies, thereby comparing and analyzing the distribution of wild attenuated salmonella and NE (VNP) in vivo tissues of tumor-bearing mice.
In vivo imaging and immunofluorescence section detection of distribution of neutrophils loaded with attenuated salmonella in vivo, fluorescence protein or luciferase tracer protein can be continuously expressed by using neutrophils marked by Cy5.5 and attenuated salmonella loaded with neutrophils, and tumor-bearing mice are respectively injected with 3×10 6 CFU bacteria and 1×106 attenuated salmonella-loaded neutrophils, the in vivo distribution and levels of attenuated salmonella-loaded neutrophils were assessed by in vivo imaging techniques and immunofluorescent staining.
Histopathological and blood biochemical analysis: after allowing the blood to stand at room temperature for 30 minutes, the supernatant was carefully aspirated at 4℃at 3000 rpm for 15 minutes. Freezing and preserving serum obtained from blood at-80deg.C until biochemical index of blood is measured; h & E staining for blood biochemical analysis, tumor, heart, liver, spleen, lung and kidney sections and fluorescent immunostaining for tumor sections were prepared by the wuhan service biosystems.
Test example 1
According to the application, the neutrophil depletion model proves that the blood vessel clearance of the neutrophil is a key factor influencing the tumor implantation rate of oncolytic bacteria, namely attenuated salmonella, namely the tumor implantation rate of the attenuated salmonella is obviously improved after the neutrophil depletion (figure 2).
Test example 2
The purity proportion of the primary neutrophils extracted from the mice is more than 90 percent (figure 3), and the fluorescence photo can prove that the neutrophils can carry attenuated salmonella (figure 4); and attenuated salmonella can maintain more than 30% of activity in neutrophil cells (fig. 5); neutrophils can remain active for more than about 80% after 2 hours of phagocytosis of attenuated salmonella, thus, neutrophils guarantee activity and chemotactic capacity before recruitment to the tumor area (fig. 6, 7); neutrophils can release more attenuated salmonella under induction of high malignancy (B16F 10) (fig. 8).
Test example 3
According to the application, tumor cell apoptosis and proliferation experiments prove that the neutrophils loaded with the attenuated salmonella have stronger tumor inhibition capability (figures 9 and 10); neutrophils loaded with attenuated salmonella have stronger nitric oxide and active oxygen levels (fig. 11); the cell status of the attenuated salmonella-loaded neutrophils was detected by real-time fluorescent quantitative PCR, which indicated that the attenuated salmonella induced polarization of the neutrophils toward an anti-tumor morphology (fig. 12).
Test example 4
According to the application, the in-vivo distribution of the neutrophils loaded with the attenuated salmonella is detected by labeling the neutrophils with the Cy5.5 and loading the attenuated salmonella expressing fluorescein, and experimental results show that: neutrophils were more enriched in the lung (fig. 13). The in vivo distribution results of the lung metastasis tumor model show that: the neutrophil loaded with the attenuated salmonella significantly improves the lung enrichment of the attenuated salmonella, prolongs the distribution time of the attenuated salmonella in the lung, simultaneously significantly reduces the infiltration of the attenuated salmonella in the spleen and the heart, and improves the tumor targeting (figure 14). The subcutaneous tumor model results showed that: the attenuated salmonella-loaded neutrophils reduced the attenuated salmonella titer of other normal organs, and the bacterial titer of tumor tissue remained unchanged (fig. 15). Neutrophils loaded with attenuated salmonella significantly reduced tumor lung metastasis (fig. 16). The safety evaluation result of the system is unexpected, and the neutrophils loaded with the attenuated salmonella can remarkably relieve toxic and side effects caused by the 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 (fig. 19); and the biochemical detection result of the serum of the tumor-bearing mice after treatment also proves that the system can improve the safety of oncolytic bacteria (figure 20).
The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be understood by those skilled in the art that the present application is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the application, and various changes and modifications may be made therein without departing from the spirit and scope of the application, the scope of which is defined in the appended claims, specification and their equivalents.
Claims (10)
1. A salmonella-attenuated-loaded neutrophil, comprising: the neutrophils loaded with the attenuated salmonella comprise neutrophils and oncolytic bacteria, wherein the oncolytic bacteria are attenuated salmonella typhimurium VNP20009 and recombinant engineering strains derived from the attenuated salmonella typhimurium, and the recombinant engineering strains are strains which do not carry or carry expression plasmids for expressing therapeutic genes and trace genes exogenous genes.
2. The neutrophil-loaded recombinant attenuated salmonella engineering bacterium of claim 1, wherein: genes which can express therapeutic or tracing effects in attenuated salmonella VNP20009 and recombinant engineering strains derived therefrom include at least one of immune checkpoint blocker antibody genes, cytokine genes, angiogenesis inhibitor genes, apoptosis-type antitumor genes and interfering plasmids; the interference plasmid which can be carried by the attenuated salmonella typhimurium VNP20009 comprises an interference plasmid which takes pRNA-U6.1 as a framework and can express interference RNA.
3. The neutrophil-loaded recombinant attenuated salmonella engineering bacterium of claim 2, wherein: the gene capable of expressing the tracing effect in the attenuated salmonella typhimurium VNP20009 comprises 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 engineering bacterium of claim 3, wherein: the nucleotide sequence of the red fluorescent protein RFP gene is shown as SEQ ID No.1, the nucleotide sequence of the green fluorescent protein GFP gene is shown as SEQ ID No.2, and the nucleotide sequence of the luciferase tracer protein LuxCDABE gene is shown as SEQ ID No. 3.
5. The neutrophil-loaded recombinant engineered attenuated salmonella engineering bacterium of claim 2, wherein: the expression plasmid carried by the attenuated salmonella VNP20009 comprises a constitutive strong promoter J23100, a PelB extracellular protein secretion signal peptide and a Flag tag, wherein the nucleotide sequence of the constitutive strong promoter J23100 is shown as SEQ ID NO.4, and the nucleotide sequence of the PelB extracellular protein secretion signal peptide is shown as SEQ ID NO. 5.
6. A method for preparing the attenuated salmonella-loaded neutrophil of claim 1, comprising the steps of: neutrophils after 5% starch broth stimulation were extracted, and purified salmonella was purified according to the following: the cells were 100:1 and then treating the cells with 50-100 mug/mL gentamicin for 30-60 minutes to kill extracellular bacteria after 0.5-2.5 hours of co-incubation, thereby preparing the neutral granulocytes loaded with the attenuated salmonella.
7. The method for evaluating and tracing the in vivo distribution of neutrophils loaded with attenuated salmonella and tumor engraftment rate as claimed in claim 1, comprising the steps of: the in vivo distribution and levels of neutrophils loaded with attenuated salmonella were assessed by in vivo imaging techniques, or by plating the number of bacteria after dilution of lysed tissue using cy5.5 labeled neutrophils and loading them with attenuated salmonella that can continuously express fluorescent protein or luciferase tracer protein.
8. Use of the attenuated salmonella-loaded neutrophil of claim 1, for the preparation of an anti-tumor pharmaceutical formulation, wherein: the pharmaceutical formulation includes at least one of an oral formulation, an injectable formulation, and an intravenous formulation or a spray.
9. The use according to claim 8, characterized in that: the neutrophils loaded with attenuated salmonella can directly kill tumor cells or activate an anti-tumor immune response to inhibit tumor progression.
10. The use of the attenuated salmonella-loaded neutrophil granulocyte in combination with other anti-tumor agents of claim 1 for the manufacture of an anti-tumor agent.
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| CN202310943859.5A CN117126811A (en) | 2023-07-30 | 2023-07-30 | Neutrophil loaded with attenuated salmonella and preparation method and application thereof |
| PCT/CN2024/106722 WO2025026111A1 (en) | 2023-07-30 | 2024-07-22 | Neutrophil loaded with attenuated salmonella and preparation method therefor and use thereof |
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| CN118767147A (en) * | 2024-05-27 | 2024-10-15 | 吉林大学 | Drug delivery system for treating malignant solid tumors based on attenuated Salmonella, preparation method and application thereof |
| WO2025026111A1 (en) * | 2023-07-30 | 2025-02-06 | 江苏靶标生物医药研究所有限公司 | Neutrophil loaded with attenuated salmonella and preparation method therefor and use thereof |
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| CN120118818A (en) * | 2025-05-13 | 2025-06-10 | 南昌大学 | A controllable self-destructive engineering bacteria and preparation method thereof |
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| CN1974759B (en) * | 2006-07-26 | 2010-06-09 | 吉林大学 | Attenuated Salmonella Carrying Recombinant Plasmid and Its Application in Antitumor |
| KR101149872B1 (en) * | 2009-04-03 | 2012-06-27 | 대한민국 | Method for Attenuation of Pathogen Using Neutrophils |
| CN105087418B (en) * | 2014-05-16 | 2018-04-03 | 江苏靶标生物医药研究所有限公司 | Salmonella strain for RNA interference in mammalian cells, preparation method and application thereof |
| CN106591365A (en) * | 2016-12-07 | 2017-04-26 | 南昌大学 | Attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method |
| CN108913690B (en) * | 2018-07-11 | 2020-01-14 | 新乡医学院 | PD-1 specific interference sequence, plasmid, attenuated salmonella and application in tumor resistance |
| CN115137830A (en) * | 2022-02-25 | 2022-10-04 | 江苏靶标生物医药研究所有限公司 | Attenuated salmonella and immune checkpoint inhibitor combined medicine and application thereof |
| CN114736861B (en) * | 2022-03-18 | 2024-05-28 | 江苏靶标生物医药研究所有限公司 | Mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and preparation method and application thereof |
| CN114736860B (en) * | 2022-03-18 | 2024-05-17 | 江苏靶标生物医药研究所有限公司 | Monocyte or macrophage loaded with attenuated salmonella and preparation method and application thereof |
| CN117126811A (en) * | 2023-07-30 | 2023-11-28 | 江苏靶标生物医药研究所有限公司 | Neutrophil loaded with attenuated salmonella and preparation method and application thereof |
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| WO2025026111A1 (en) * | 2023-07-30 | 2025-02-06 | 江苏靶标生物医药研究所有限公司 | Neutrophil loaded with attenuated salmonella and preparation method therefor and use thereof |
| CN118767147A (en) * | 2024-05-27 | 2024-10-15 | 吉林大学 | Drug delivery system for treating malignant solid tumors based on attenuated Salmonella, preparation method and application thereof |
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