WO2022257890A1 - 一种经修饰的细菌及其制备方法和应用 - Google Patents

一种经修饰的细菌及其制备方法和应用 Download PDF

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WO2022257890A1
WO2022257890A1 PCT/CN2022/097190 CN2022097190W WO2022257890A1 WO 2022257890 A1 WO2022257890 A1 WO 2022257890A1 CN 2022097190 W CN2022097190 W CN 2022097190W WO 2022257890 A1 WO2022257890 A1 WO 2022257890A1
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bacterium
bacteria
modified
manganese
bacterial
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PCT/CN2022/097190
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English (en)
French (fr)
Inventor
刘庄
彭睿
王晨雅
庄齐
赵琪
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苏州百迈生物医药有限公司
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Priority claimed from CN202111284429.4A external-priority patent/CN116024113A/zh
Application filed by 苏州百迈生物医药有限公司 filed Critical 苏州百迈生物医药有限公司
Priority to CN202280007236.0A priority Critical patent/CN117083372A/zh
Publication of WO2022257890A1 publication Critical patent/WO2022257890A1/zh

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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present application relates to the field of biomedicine, in particular to a modified bacterium and its preparation method and application.
  • Bacterial treatment of tumors has a long history, but its development is slow. It was not until the 1990s that BCG was used in the treatment of human bladder cancer that bacterial therapy was introduced into the public eye.
  • attenuated Salmonella attenuated Attenuated bacteria such as Listeria toxin are used in the treatment of different tumors.
  • Bacterial therapy of tumors is also an extension of immunotherapy. With the stimulation of foreign substances such as bacteria, it triggers an immune response and further inhibits tumor growth.
  • most of the bacteria currently used are live bacteria after attenuation treatment, which still have high risks in clinical use, and the safety window is narrow, and inactivated bacteria cannot achieve the due immune stimulation effect. , the current tumor bacterial therapy is difficult to achieve effective treatment at a safe dose.
  • manganese is also one of the trace elements in the human body.
  • Some patents have gradually tried to explore the application of immune stimulation enhancement.
  • patents CN201610347815.6 and CN201910319344.1 disclose the function of manganese in the STING pathway, and the precipitation of manganese Colloidal manganese or colloidal manganese have the effect of enhancing immunity, which disclosed in the 156 patent that divalent manganese has the function of activating the cGAS-STING pathway, but the immune enhancing effect of divalent manganese is not ideal and it is difficult to obtain a higher concentration of divalent manganese
  • divalent nascent precipitated manganese and colloidal manganese are used to stimulate the immune system, and a stronger immune stimulating effect than divalent manganese ions is obtained, and then the nascent precipitated manganese, colloidal manganese and antigen are simply mixed , a more effective vaccine than this antigen can be obtained.
  • the patent CN201710795715.4 discloses a manganese dioxide nanoparticle, which is loaded with oligonucleotides (CpG) and/or antigens on the surface of the manganese dioxide nanoparticle, and the synergistic immunostimulatory effect, along with the two The degradation of manganese oxide in a weakly acidic environment, and finally manganese ions can be excreted from the body.
  • CpG oligonucleotides
  • manganese ions Using the adjuvant-like function of manganese ions, it can activate the STING pathway, induce the production of type I interferon, regulate the host immune system, activate T cell-specific immune responses, and induce the polarization of macrophages to anti-tumor phenotypes to induce pathogen specificity immune response, thereby further inducing the generation of immune memory.
  • manganese ions act as immune adjuvants, similar to aluminum adjuvants, and an antigen must be added to induce an immune response against this antigen, which is the function of a vaccine.
  • manganese ions are mixed with inactivated bacteria and injected, which mainly produces an immune response against the bacteria to achieve the purpose of preventing bacterial infection; if manganese ions (or other manganese-containing substances) Substance) mixed with the mutated antigen specifically expressed by tumor cells and then injected will produce an immune response against the antigen, which will have an immune attack effect on the tumor cells expressing the antigen, but will not be effective against the tumor cells not expressing the antigen.
  • the application provides a modified bacterium and its preparation method and application.
  • the modified bacterium has one or more of the following advantages: (1)
  • the application provides a modified bacterium that can In order to inactivate bacteria, the metal compound on its surface can be metabolized by decomposing into an ion state, which has better safety; (2) the modified bacteria provided by the application, the insoluble metal compound modified on its surface has the ability to neutralize tumor weak
  • the role of the acidic microenvironment can improve the activity of immune cells in the tumor site, reduce the drug resistance of tumors, and improve the curative effect; (3) the modified bacteria provided by the application can activate immune cells through multi-channel stimulation, induce strong Anti-tumor immune response, and produce immune memory effect to reduce the probability of cancer metastasis and recurrence, is a new type of immune stimulant.
  • the present application provides a modified bacterium, which includes a bacterium body and an insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body.
  • the present application provides a modified bacterium, which includes a bacterium body and an insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body, and the metal compound is modified on the surface of the bacterium body through a deposition reaction.
  • the surface of the bacterial body forms an adhesion layer.
  • the present application provides a modified bacterium, which includes a bacterium body and an insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body, and the metal compound is The content on a single bacterium is about 0.1 ⁇ g to 600 ⁇ g.
  • the bacterial entity is a gram-positive bacterium or a gram-negative bacterium.
  • the bacterial entity is selected from one or more of cocci, bacilli and spirochetes.
  • the coccus is selected from the group consisting of Staphylococcus aureus, Micrococcus urea, Streptococcus pneumoniae, Streptococcus pneumoniae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus agalactiae, One or more of Staphylococcus, Staphylococcus albus, and Staphylococcus citrus.
  • the bacillus is selected from Escherichia coli, Salmonella, Yersinia pestis, Shigella hyigigellar, Pasteurella multocida, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Lactobacillus bifidus, Acetobacter, Corynebacterium one or more of.
  • the spirochete is selected from one or more of Helicobacter pylori, Vibrio comma and Vibrio cholerae.
  • the bacterial entity is selected from Salmonella, Staphylococcus aureus, Escherichia coli, Lactobacillus, attenuated strains of Salmonella, attenuated strains of Staphylococcus aureus, attenuated strains of Escherichia coli and attenuated strains of Lactobacillus one or more of.
  • the bacterial body is a wild-type bacterium, a genetically engineered bacterium and/or an attenuated strain.
  • the bacterial body is a live bacterium or an inactivated bacterium.
  • the live or inactivated bacteria comprise attenuated bacteria.
  • the metal compound is modified to form an adhesion layer on the surface of the bacterial body through physical or chemical combination.
  • said physical association includes electrostatic adsorption and/or partial embedding.
  • the chemical binding includes coupling, chemical bonding, and/or complexing.
  • the metal compound is modified by a deposition reaction to form an adhesion layer on the surface of the bacterial body.
  • the metal compound is modified by biomineralization to form an adhesion layer on the surface of the bacterial body.
  • the biomineralization reaction includes: metal ions combine with biomacromolecules on the cell membrane of bacteria or biomacromolecules on the cell wall to provide mineralization sites, adjust pH or introduce other salts, metal ions Metal compounds are generated at the mineralization site, continuously grow and accumulate, and bind to the bacterial surface.
  • the biomineralization reaction forms binding sites for biomacromolecules and metal compounds, and the volume of the metal compounds is further increased by deposition reactions or biomineralization.
  • the coverage of the metal compound on the bacterial surface is 0.1%-99.9%.
  • the coverage of the metal compound on the bacterial surface is adjusted by adjusting the feed ratio, reaction temperature and/or reaction time.
  • the metal element of the metal compound is selected from one or more of zinc, calcium, copper, iron, manganese and magnesium; the non-metallic component is selected from carbonate, hydroxide, sulfur ion and One or more of the phosphate radicals.
  • the metal compound is selected from zinc carbonate, calcium carbonate, copper carbonate, magnesium carbonate; zinc hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, magnesium hydroxide, zinc sulfide, copper sulfide , manganese sulfide; one or more of zinc phosphate, calcium phosphate, copper phosphate, iron phosphate, magnesium phosphate and manganese phosphate.
  • the metal compound is a manganese-containing compound.
  • the manganese-containing compound is one or more of manganese hydroxide, manganese dioxide and manganese sulfide.
  • the metal compound is manganese hydroxide and/or manganese dioxide.
  • the present application provides a manganizing bacterium, which includes a bacterium body and an insoluble or slightly soluble manganese-containing compound attached to the surface of the bacterium body.
  • the insoluble or slightly soluble manganese-containing compound is one or more of manganese hydroxide, manganese dioxide and manganese sulfide.
  • the bacterial entity is a gram-positive bacterium or a gram-negative bacterium.
  • the bacterial entity is selected from cocci, bacilli and/or spirochetes.
  • the coccus is selected from the group consisting of Staphylococcus aureus, Micrococcus urea, Streptococcus pneumoniae, Streptococcus pneumoniae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus agalactiae, One or more of Staphylococcus, Staphylococcus albus, and Staphylococcus citrus.
  • the bacillus is selected from the group consisting of Escherichia coli, Salmonella, Yersinia pestis, Shigella hyopsia, Pasteurella multocida, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Lactobacillus bifidus, Acetobacter and Corynebacterium one or more of.
  • the spirochete is selected from one or more of Helicobacter pylori, Vibrio comma and Vibrio cholerae.
  • the bacterial body is a live bacterium or an inactivated bacterium.
  • the bacterial body is a wild-type bacterium, a genetically engineered bacterium and/or an attenuated strain.
  • the present application provides a composition comprising the modified bacterium or the manganized bacterium, and optionally a pharmaceutically acceptable carrier, the modified bacterium includes the bacterial body and the modified bacterium The insoluble or insoluble biologically acceptable metal compound on the surface of the bacteria body, and the pH value of the composition is 0-14.
  • the present application provides a modified bacterium freeze-dried powder, which includes the modified bacterium and additives.
  • the additives include lyoprotectants and/or excipients.
  • the lyoprotectant is selected from one or more of carbohydrates/polyols, polymers, anhydrous solvents, surfactants, amino acids, salts and amines.
  • the excipient is selected from binders, fillers, disintegrants, lubricants, wine, vinegar, concoctions, etc., ointment bases, cream bases, preservatives, antioxidants, One or more of flavoring agents, aromatic agents, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators and coloring agents.
  • the mass fraction of additives in the modified bacterial freeze-dried powder is 0.1-99%.
  • the mass fraction here is defined as the mass fraction of the lyoprotectant in the lyophilized product.
  • the lyoprotectant is selected from the group consisting of sucrose, mannose, ⁇ -D-mannopyranose, trehalose, inosose, raffinose, inulin, dextran, maltodextrin, maltopolysaccharide, octasulfate One or more of sucrose, heparin and 2-hydroxypropyl- ⁇ -cyclodextrin.
  • the mass fraction of the lyoprotectant in the lyophilized bacterial powder is 0.1-99%.
  • the mass fraction here is defined as the mass fraction of the lyoprotectant in the lyophilized product.
  • the present application provides a freeze-dried powder of manganizing bacteria, which includes the manganizing bacteria and additives.
  • the additives include lyoprotectants and/or excipients.
  • the lyoprotectant is selected from one or more of carbohydrates/polyols, polymers, anhydrous solvents, surfactants, amino acids, salts and amines.
  • the excipient is selected from binders, fillers, disintegrants, lubricants, wine, vinegar, concoctions, etc., ointment bases, cream bases, preservatives, antioxidants, One or more of flavoring agents, aromatic agents, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators and coloring agents.
  • the mass fraction of the additive in the lyophilized bacterial powder is 0.1-99%.
  • the mass fraction here is defined as the mass fraction of the lyoprotectant in the lyophilized product.
  • the lyoprotectant is selected from the group consisting of sucrose, mannose, ⁇ -D-mannopyranose, trehalose, inosose, raffinose, inulin, dextran, maltodextrin, maltopolysaccharide, octasulfate One or more of sucrose, heparin and 2-hydroxypropyl- ⁇ -cyclodextrin.
  • the mass fraction of the lyoprotectant in the lyophilized bacterial powder is 0.1-99%.
  • the mass fraction here is defined as the mass fraction of the lyoprotectant in the lyophilized product.
  • the present application provides a method for preparing the modified bacterium or the manganized bacterium, which comprises the following steps: S1: preparing a bacterial suspension; S2: adding Soluble metal salt solution.
  • the preparation method further includes step S3: adding a soluble aqueous hydroxide solution, an aqueous carbonate solution or an aqueous phosphate solution to a pH of 8-12, or adding a soluble aqueous sulfide salt solution, and reacting The modified bacteria are produced.
  • the soluble sulfide salt solution in the preparation method includes sodium sulfide solution, potassium sulfide solution and/or ammonium sulfide solution
  • the easily soluble aqueous hydroxide solution, aqueous carbonate solution or aqueous phosphate solution in the preparation method is selected from: aqueous sodium hydroxide solution, aqueous sodium carbonate solution, and aqueous sodium phosphate solution.
  • step S2 of the preparation method according to every 1 billion bacteria, the amount of metal ions added is 0.2-13.5 mmol, and a soluble metal salt solution is added to the bacterial suspension .
  • the soluble metal salt solution in the preparation method is a permanganate solution.
  • step S2 of the preparation method includes adding a permanganate solution to the bacterial suspension, stirring for reaction, and centrifuging to obtain manganized bacteria.
  • per 1 billion bacteria 0.2-3 ⁇ mol of permanganate is added.
  • the present application provides the modified bacteria, the manganized bacteria, the modified bacterial freeze-dried powder, the manganized bacteria freeze-dried powder, or the composition in the preparation of tumor immunotherapy application in formulations.
  • the present application provides the modified bacteria, the manganizing bacteria, the modified bacterial freeze-dried powder, the manganizing bacteria freeze-dried powder, or the composition, which is used to prevent and/or treat tumors.
  • the present application also provides a method for preventing and/or treating tumors, which comprises administering an effective amount of the modified bacteria, the manganized bacteria, the modified The freeze-dried powder of bacteria, the freeze-dried powder of manganized bacteria, or the composition.
  • Figure 1 is a scanning electron microscope picture of modified inactivated bacteria attached to different metal compounds
  • Fig. 2 is the scanning electron microscope pictures of different bacteria before and after manganese dioxide modification
  • Figure 3 is a statistical chart of the relative activity of CT26 cells co-incubated with different samples for 12 hours;
  • Figure 4 is a statistical diagram of the proportion of mature cells detected by flow cytometry after co-incubation of bone marrow-derived dendritic cells with different samples;
  • Fig. 5 is a graph showing tumor growth curves of mice treated with bacteria modified with different metal compounds in a mouse tumor model.
  • Figure 6 is a statistical graph of the bioluminescent signal intensity of reporter gene expression after the reporter cells were incubated with different samples for 24 hours, after the STING pathway was activated in different groups;
  • Fig. 7 is a survival curve of mice reinoculated with the same tumor on the 60th day after the mouse subcutaneous tumor model of colon cancer was cured by manganese dioxide-modified inactivated Salmonella.
  • Fig. 8 is the XPS spectrogram of different bacteria after manganization
  • Figure 9 shows the intracellular Cy5. 5 Statistical graph of signal strength
  • Figure 10 is a statistical diagram of the content of manganese ions phagocytosed in cells after co-incubation of DC2.4 cells with manganese dioxide (MnO 2 ) or manganese dioxide manganese bacteria (MnO 2 @FS);
  • Figure 11 is a statistical diagram of the proportion of mature dendritic cells detected after co-incubating bone marrow-derived dendritic cells (BMDCs) with different samples for 12 hours;
  • BMDCs bone marrow-derived dendritic cells
  • Figure 12 is a statistical diagram of the concentration of various cytokines in the medium supernatant detected after bone marrow-derived dendritic cells (BMDCs) were co-incubated with different samples for 12 hours;
  • BMDCs bone marrow-derived dendritic cells
  • Figure 13 is a statistical diagram of the concentration of different cytokines in the culture medium after peritoneal macrophages are co-incubated with different samples;
  • Figure 14 is the western blot of interferon regulatory factor (IRF3) and phosphorylated interferon regulatory factor (p-IRF3) and the corresponding IRF3 phosphorylation ratio statistics after the reporter cells were incubated with different samples for 24 hours;
  • IRF3 interferon regulatory factor
  • p-IRF3 phosphorylated interferon regulatory factor
  • Figure 15 is a statistical diagram of downstream fluorescent signal intensity after Toll-like receptor 4 activation after Toll-like receptor 4 reporter cells were incubated with different samples for 24 hours;
  • Figure 16 is a tumor growth curve of the mouse colon cancer CT26 tumor model after receiving different adjuvants combined with inactivated bacteria;
  • Figure 17 is a graph showing the change in body weight of the mouse colon cancer CT26 tumor model after receiving different adjuvants combined with inactivated bacteria;
  • Figure 18 shows the intracellular Cy5.5 signal of CD45 positive cells in the tumor site detected by flow cytometry 24 hours after injection of Cy5.5-labeled bacteria (FS) or Cy5.5-labeled manganese dioxide manganese bacteria (MnO 2 @FS) strength chart;
  • FS Cy5.5-labeled bacteria
  • MnO 2 @FS Cy5.5-labeled manganese dioxide manganese bacteria
  • Figure 19 shows the distribution of manganese ions in major organs and tumor tissues 24 hours after intratumoral injection of different samples in mice;
  • Figure 20 is the statistical value of the oxygen content inside the tumor at different time points after injecting different samples into the tumor
  • Figure 21 is a statistical chart of the mature proportion of DC cells in the peritumoral lymph nodes after the mouse tumor model received different treatments
  • Figure 22 is a statistical chart of the percentage of immune cells infiltrating inside the tumor 24 hours after the mice received tumor treatment;
  • Figure 23 is a statistical chart of cytokine content in the tumor 24 hours after the mice received tumor treatment
  • Figure 24 is a statistical chart of the proportion of immune cells infiltrating inside the tumor on the 5th day after the mice received tumor treatment;
  • Figure 25 is a graph of tumor growth curves of mice treated with NK cell blocking antibody and normal mouse B16-OVA tumor model after treatment;
  • Fig. 26 is a graph showing tumor growth curves of mice treated with T cell blocking antibody and normal mouse CT26 tumor model after treatment;
  • Figure 27 is a graph of tumor growth curves of different tumor models (4T1 breast cancer tumor, B16-OVA melanoma, KPC pancreatic cancer) after treatment;
  • Figure 28 is a graph showing the tumor growth curve of the mouse CT26 tumor model treated with different kinds of manganese bacteria
  • Figure 29 is a graph showing the tumor growth curve of the mouse 4T1 tumor model treated with different kinds of manganese bacteria
  • Figure 30 is a tumor growth curve for the treatment of mouse tumor models with manganized bacteria prepared in different manganese sulfate/bacteria feeding ratios;
  • Figure 31 is a tumor growth curve of the New Zealand rabbit liver cancer model treated with manganized bacteria
  • Figure 32 is a graph showing the tumor growth curve after the bilateral tumor model in mice was treated with manganized bacteria
  • Figure 33 is a statistical chart of the ratio of memory T cells in peripheral blood on the 60th day after the mouse CT26 tumor model was cured by manganized bacteria;
  • Figure 34 is a graph showing the change in tumor volume of the mouse CT26 tumor model re-inoculated on the 60th day after being cured by manganizing bacteria;
  • Figure 35 is a scanning electron micrograph of manganized inactivated bacteria prepared from manganese sulfate
  • Figure 36 is a scanning electron micrograph of manganized inactivated bacteria attached to the surface of manganese sulfide
  • Figure 37 is a scanning electron micrograph of manganized inactivated bacteria prepared with potassium permanganate as a raw material
  • Fig. 38 is a scanning electron micrograph of manganized bacteria obtained at different feeding ratios.
  • modified generally refers to some modification compared with the natural state (or wild type), such as artificial modification.
  • modification can be by physical means, chemical means and/or biological means.
  • the modification may be a modification of one or more aspects.
  • the term “poorly soluble” or “poorly soluble” may be used interchangeably with “slightly soluble” or “slightly soluble”, and generally refers to a low solubility in a solvent at room temperature (20 degrees) , for example, a solubility in a solvent of about 0.01 g to 1 g/100 g or less.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • modified bacteria not only includes the modification of the surface of the bacteria itself to include biologically acceptable metal compounds, but also includes bacteria modified in other ways.
  • the bacterium may be a genetically modified bacterium.
  • the surface of the bacterial body may also contain other substances.
  • the bacteria may be an attenuated strain.
  • composition generally refers to a composition that may contain inactive ingredients in addition to active ingredients.
  • the inactive ingredients may comprise one or more (pharmaceutically acceptable) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives as appropriate preparations.
  • the acceptable ingredients of the compositions are generally nontoxic or less toxic to recipients at the dosages and concentrations employed.
  • the term "comprises” generally refers to the meanings of including, encompassing, containing or encompassing. In some cases, it also means “for” and “consisting of”.
  • treating generally refers to: (i) preventing a disease, disorder, and/or condition from occurring in a patient who may be predisposed to it but has not been diagnosed with it; (ii) inhibiting the disease , disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e. making the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subside.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • tumor generally refers to neoplastic or malignant cell growths.
  • the tumors of this application may be benign or malignant.
  • the tumors of this application may be solid or non-solid.
  • the application provides a modified bacterium and its preparation method and application.
  • This application adjusts the pH value of the solution to weak alkaline after mixing metal ions with live bacteria or inactivated bacteria (attenuated Salmonella at first), which will cause insoluble or insoluble metal ion compounds to attach on the surface of bacteria to form modified Bacteria, through intratumoral injection, the modified bacteria are injected into the mouse tumor model, which can achieve the effect of inhibiting tumor growth. After further mechanism research, it was found that the modified bacteria caused the body's tumor growth after in situ injection. Anti-tumor immune response, so as to achieve the effect of tumor therapy. When divalent metal ions are placed in alkaline conditions, hydroxides are easily formed and precipitated.
  • the surface of bacteria can provide a nucleation center for precipitates and further promote the precipitation of hydroxides.
  • hydrogen After the oxide is precipitated on the surface of the bacteria, it is transformed into a more stable oxide again.
  • an oxide adhesion layer is formed on the surface of the bacteria, and inactivated bacteria modified with metal compounds are obtained.
  • the deposition reaction described in this application that is, after some metal ion salts are mixed with bacteria, etc., the corresponding anions are introduced. Under suitable pH conditions, metal ions and anions form insoluble or insoluble metal compounds and attach to the surface of bacteria. Under stirring conditions, the target product can continuously settle on the surface of the bacteria in the liquid, and finally, the bacteria attached to the metal compound form a uniform and stable suspension.
  • this technique can also be used for other types of bacteria, including Staphylococcus aureus, Escherichia coli and Lactobacillus.
  • the modified bacteria prepared by using the above bacteria based on the same method can also activate the immune system to obtain good anti-tumor activity.
  • insoluble or insoluble metal compounds other than metal hydroxides or metal oxides can form an adhesion layer on the surface of bacteria and achieve similar immune stimulation results when the solubility is reduced.
  • metal compounds can also activate immune cells, induce a strong anti-tumor immune response after being bound to the surface of bacteria, and may produce immune memory effects, reducing cancer metastasis and recurrence The probability.
  • the modified bacterium includes the bacterium body and the insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body.
  • the bacterial body can be selected from Salmonella, Staphylococcus aureus, Escherichia coli, Lactobacillus, Salmonella attenuated strain, Staphylococcus aureus attenuated strain, Escherichia coli attenuated strain and Lactobacillus attenuated strain one or more of .
  • the bacterial body may be a live bacterium or an inactivated bacterium, for example, the bacterium body is an inactivated bacterium, and both the live bacterium and the inactivated bacterium may include attenuated bacteria.
  • the metal compound can be modified by a deposition reaction to form an adhesion layer on the surface of the bacterial body.
  • the metal element of the metal compound can be selected from one or more of zinc, calcium, copper, iron, manganese and magnesium;
  • the non-metallic component can be selected from carbonate, hydroxide, sulfur ion and One or more of the phosphate radicals.
  • the metal compound may be selected from zinc carbonate, calcium carbonate, copper carbonate, magnesium carbonate; zinc hydroxide, iron hydroxide, copper hydroxide, manganese hydroxide, magnesium hydroxide, zinc sulfide, copper sulfide, Manganese sulfide; one or more of zinc phosphate, calcium phosphate, copper phosphate, iron phosphate, magnesium phosphate and manganese phosphate.
  • the present application also provides a preparation method of the above-mentioned modified bacteria, comprising the following steps,
  • S3 adding easily soluble hydroxide aqueous solution, carbonate aqueous solution or phosphate aqueous solution to a pH of 8-12 (for example, to a pH of about 8, to a pH of about 9, to a pH of about 10, to a pH of about 11 or to a pH of about 12), or add a soluble sulfide salt solution, and react to prepare the modified bacterium.
  • a soluble aqueous hydroxide solution can be added to a pH of 8-12 (for example, to a pH of about 8, to a pH of about 9, to a pH of about 10, to a pH of about 11 or to a pH of about 12), and the reaction After centrifugation, the modified bacteria with metal hydroxide or metal oxide attached to the surface are obtained;
  • the easily soluble aqueous hydroxide solution is preferably an aqueous sodium hydroxide solution;
  • a readily soluble carbonate aqueous solution may be added to a pH of 8-12 (for example, to a pH of about 8, to a pH of about 9, to a pH of about 10, to a pH of about 11 or to a pH of about 12), and the reaction After centrifugation, the modified bacteria with metal carbonate attached to the surface are obtained;
  • the easily soluble carbonate aqueous solution is preferably an aqueous sodium carbonate solution;
  • a lyotropic phosphate aqueous solution can be added to a pH of 8-12 (for example, to a pH of about 8, to a pH of about 9, to a pH of about 10, to a pH of about 11 or to a pH of about 12), and after the reaction centrifugation to obtain the modified bacteria with metal phosphate attached to the surface;
  • the soluble phosphate aqueous solution is preferably sodium phosphate aqueous solution;
  • a soluble sulfide salt solution can be added, and centrifuged after the reaction to obtain modified bacteria with metal sulfides attached to the surface;
  • the soluble sulfide salt solution is preferably an aqueous sodium sulfide solution;
  • 0.2-13.5 mmol of metal ions can be added in an amount of 0.2-13.5 mmol per 1 billion bacteria, and a soluble metal salt solution can be added to the bacterial suspension.
  • a soluble metal salt solution can be added to the bacterial suspension.
  • about 0.2 mmol, about 0.5 mmol, about 1.0 mmol, about 2.0 mmol, about 3.0 mmol, about 4.0 mmol, about 5.0 mmol, about 6.0 mmol, about 7.0 mmol, about 8.0 mmol can be added per 1 billion bacteria.
  • Mmol, about 9.0mmol, about 10.0mmol, about 11.0mmol, about 12.0mmol, about 13.0mmol, or about 13.5mmol metal ion addition amount add soluble metal salt solution in the bacterial suspension.
  • the present application also provides a modified bacterium lyophilized powder, including a lyoprotectant and the above-mentioned modified bacterium.
  • the lyoprotectant may be selected from one of sucrose, trehalose, inosose, raffinose, inulin, dextran, maltodextrin, maltopolysaccharide and 2-hydroxypropyl- ⁇ cyclodextrin one or more species.
  • the mass/volume fraction of the lyoprotectant in the sample suspension is 0.1-20%, such as 1-5%.
  • the mass/volume fraction of the lyoprotectant in the sample suspension is 0.1-20%, such as 1-5%.
  • the mass/volume fraction of the lyoprotectant in the sample suspension is 0.1-20%, such as 1-5%.
  • the present application also provides the application of the above-mentioned modified bacteria or the freeze-dried powder of the above-mentioned modified bacteria in the preparation of tumor treatment preparations.
  • a modified bacterium which includes a bacterium body and an insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body, and the metal compound is modified on the surface of the bacterium body through a deposition reaction.
  • the surface of the bacterial body forms an adherent layer.
  • a modified bacterium which includes a bacterium body and an insoluble or insoluble biologically acceptable metal compound modified on the surface of the bacterium body.
  • the content on 8 bacteria ranged from about 0.1 ⁇ g to 600 ⁇ g.
  • the content of the metal compound per 1* 10 bacteria is about 0.1 ⁇ g, about 1 ⁇ g, about 5 ⁇ g, about 10 ⁇ g, about 20 ⁇ g, about 30 ⁇ g, about 40 ⁇ g, about 50 ⁇ g, about 60 ⁇ g, about 70 ⁇ g, about 80 ⁇ g, about 90 ⁇ g, about 100 ⁇ g, about 150 ⁇ g, about 200 ⁇ g, about 250 ⁇ g, about 300 ⁇ g, about 350 ⁇ g, about 400 ⁇ g, about 450 ⁇ g, about 500 ⁇ g, about 550 ⁇ g, or about 600 ⁇ g.
  • a composition which comprises the modified bacterium or the manganized bacterium, and optionally a pharmaceutically acceptable carrier, the modified bacterium includes the bacterial body and the modified The insoluble or insoluble biologically acceptable metal compound on the surface of the bacteria body, and the pH value of the composition is 0-14.
  • the composition has a pH of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
  • a method for preparing the modified bacteria or the manganized bacteria is also provided, which includes the following steps: S1: preparing a bacterial suspension; S2: adding Soluble metal salt solution.
  • the application mixes manganese ions with live bacteria or inactivated bacteria, and finds that by adjusting the pH value of the solution to weak alkaline, it will cause the growth of micron or nanoscale manganese compounds on the surface of bacteria, and this process is defined as microbial growth.
  • Manganizing process and the combination of microbial body and manganese compound obtained in this process is defined as manganizing bacteria.
  • manganizing bacteria For example, when divalent manganese ions are placed in alkaline conditions, manganese hydroxide is easily formed and precipitated, and bacteria are negatively charged in this solution condition, and the surface of bacteria is a typical solid-liquid interface, which can provide The nucleation center, especially some microstructures on the surface of bacteria, can further promote the precipitation of manganese hydroxide.
  • manganese hydroxide When manganese hydroxide precipitates on the surface of bacteria, it will be transformed into more stable manganese dioxide again under the action of dissolved oxygen. The continuous precipitation and transformation of bacteria will form a manganese dioxide adhesion layer on the surface of bacteria, and manganized bacteria will be obtained.
  • the phagocytic efficiency and stimulation efficiency brought about by the structural integration of manganized bacteria are significantly improved, allowing immune cells to phagocytize bacteria and manganese dioxide at the same time, so that "dioxide Manganese manganese bacteria" had a significantly enhanced immunostimulatory effect than "a mixture of manganese dioxide and inactivated bacteria".
  • Further studies have shown that after manganese bacteria are injected into the tumor, they first activate innate immunity, recruit natural killer cells (natural killer cells, NK cells), macrophages, etc. to the tumor site, and kill some tumor cells through non-specific immune responses.
  • Tumor-associated antigens the manganese ions gradually degraded and released by manganese dioxide can stimulate the STING pathway, and the inactivation of the bacterial body can stimulate Toll-like receptors (TLR).
  • TLR Toll-like receptors
  • the simultaneous action of the two aspects can more effectively activate antigen-presenting cells such as dendrites DCs present tumor antigens to T cells and activate T cell-mediated adaptive immunity (tumor-specific immune response).
  • Tumor-specific T cells migrate to the whole body (including in distant tumors), inhibit tumor metastasis, and generate immune memory cells to inhibit tumor recurrence.
  • manganese dioxide catalyzes the decomposition of hydrogen peroxide in the tumor to generate oxygen, improves tumor hypoxia, reverses the immunosuppressive tumor microenvironment, and is also helpful to improve the effect of immunotherapy.
  • manganizing bacteria with different manganese compounds growing on the surface can be obtained by different methods; specifically, two ideas of oxidation and reduction can be used to obtain manganizing bacteria growing manganese dioxide on the surface; or by The sulfide is added to convert the soluble manganese salt into insoluble manganese sulfide, which grows on the surface of bacteria to obtain manganizing bacteria with manganese sulfide growing on the surface.
  • the manganized bacteria provided by this application can regulate the hypoxia and weakly acidic microenvironment in the tumor, and at the same time stimulate the host immune system through the STING and Toll-like receptor (TLR) pathways, and activate the innate immune response successively And acquired immune response, produce a strong and effective anti-tumor immune effect, which is suitable for various types of tumors; at the same time, because this technology can use inactivated bacteria, the manganese inactivated bacteria has extremely high safety. It is well tolerated by mice at high doses and is expected to have a very wide safety window for clinical application.
  • TLR Toll-like receptor
  • the manganized bacteria provided by this application can improve the tumor microenvironment through manganese-containing compounds, can decompose endogenous hydrogen peroxide H 2 O 2 in tumor lesions, and significantly improve the hypoxic microenvironment of tumor sites while killing Live bacteria co-stimulate the host immune system; on the other hand, weakly alkaline manganizing bacteria can also properly regulate the acidic microenvironment.
  • tumor hypoxia and slightly acidic environment are important causes of immunosuppression at the tumor site
  • the improvement of tumor hypoxia and the neutralization of slightly acidic compounds containing manganese can reverse the immunosuppressive microenvironment in the tumor, and help manganese bacterial immune stimulation
  • the recruited immune cells infiltrate deep into the tumor, thereby significantly enhancing the ability of antigen-presenting cells to present antigens, up-regulating the secretion of cytokines, and strengthening the subsequent anti-tumor immune response.
  • the manganized bacteria provided by this application have significantly improved the efficiency of phagocytosis and stimulation through structural integration, and enabled immune cells to phagocytize bacteria and manganese-containing compounds at the same time, thus making “manganized bacteria” more effective than "containing manganese”
  • a mixture of manganese compounds and inactivated bacteria” had a significantly enhanced immunostimulatory effect. Further studies have shown that after manganese bacteria are injected into the tumor, they first activate innate immunity, recruit natural killer cells (NK cells), macrophages, etc.
  • TLR Toll-like receptor
  • the combination of the two aspects may be more effective in activating antigen-presenting cells such as dendritic cells (DC) , while DC can present tumor antigens to T cells and activate T cell-mediated adaptive immunity (tumor-specific immune response).
  • DC dendritic cells
  • Tumor-specific T cells migrate to the whole body (including in distant tumors), inhibit tumor metastasis, and generate immune memory cells to inhibit tumor recurrence.
  • the oncolytic effect is far stronger than that of a single manganese-containing compound, inactivated bacteria, and a mixture of manganese-containing compounds and bacteria, and has no obvious toxic and side effects.
  • the manganizing bacteria include the bacteria body and the insoluble or slightly soluble manganese-containing compound attached to the surface of the bacteria body.
  • the bacterial body can be a gram-positive bacterium or a gram-negative bacterium.
  • the bacterial body may be one or more of Salmonella, Staphylococcus aureus, Escherichia coli and Lactobacillus.
  • the insoluble or slightly soluble manganese-containing compound may be one or more of manganese hydroxide, manganese dioxide and manganese sulfide, preferably manganese dioxide.
  • the insoluble or slightly soluble manganese-containing compound may be manganese hydroxide, manganese dioxide, manganese sulfide or the coexistence of manganese hydroxide and manganese dioxide.
  • the bacterial body may be a live bacterium or an inactivated bacterium, for example, an inactivated bacterium, and both the live bacterium and the inactivated bacterium include attenuated bacteria.
  • the present application also provides a freeze-dried powder of manganizing bacteria, including any of the above-mentioned manganizing bacteria and a freeze-drying protectant;
  • the freeze-drying protectant is sucrose, mannose, ⁇ -D-mannopyranose, trehalose, muscle At least one of sugar, raffinose, inulin, dextran, maltodextrin, maltopolysaccharide, sucrose octasulfate, heparin and 2-hydroxypropyl-beta-cyclodextrin.
  • the mass/volume fraction of the lyoprotectant in the lyophilized bacterial powder before lyophilization may be 0.1%-20%.
  • the present application also provides a preparation method of manganizing bacteria, comprising the following steps,
  • step S2' add an appropriate amount of manganese salt solution to the bacterial suspension obtained in step S1, add a soluble sulfide salt solution, react for 1-2 hours, and centrifuge to obtain manganizing bacteria coated with manganese sulfide on the surface.
  • bacteria are live bacteria or inactivated bacteria, such as inactivated bacteria.
  • bacteria can be Gram-positive bacteria or Gram-negative bacteria.
  • pH adjustment can be achieved by adding sodium oxide solution
  • soluble sulfide salt solutions include sodium sulfide solution, potassium sulfide solution, ammonium sulfide solution, and the like.
  • 0.2 ⁇ mol-13.5 mmol of manganese salt can be added.
  • a preparation method of manganizing bacteria comprising the following steps,
  • bacteria are live bacteria or inactivated bacteria, such as inactivated bacteria.
  • bacteria are Gram-positive bacteria or Gram-negative bacteria.
  • the present application also provides the application of any one of the above-mentioned manganized bacteria in the preparation of tumor immunotherapy preparations.
  • the present application also provides the application of the freeze-dried powder prepared by any one of the above-mentioned manganizing bacteria to prepare tumor immunotherapy preparations.
  • the bacterial body may be Gram-positive bacteria or Gram-negative bacteria.
  • the bacterial entity may be selected from one or more of cocci, bacilli and spirochetes.
  • the coccus may be selected from the group consisting of Staphylococcus aureus, Micrococcus urea, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus meningitidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, albino One or more of Staphylococcus and Staphylococcus citrus.
  • the bacillus can be selected from one of Escherichia coli, Salmonella, Yersinia pestis, Shigella hyopsium, Pasteurella multocida, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Lactobacillus bifidosum, Acetobacter, and Corynebacterium or more.
  • the spirochete may be selected from one or more of Helicobacter pylori, Vibrio comma and Vibrio cholerae.
  • the bacterial body can be selected from Salmonella, Staphylococcus aureus, Escherichia coli, Lactobacillus, Salmonella attenuated strain, Staphylococcus aureus attenuated strain, Escherichia coli attenuated strain and Lactobacillus attenuated strain one or more of .
  • the bacterial body may be wild-type bacteria, genetically engineered bacteria and/or attenuated strains.
  • the bacterial body may be living bacteria or inactivated bacteria.
  • the live bacteria or inactivated bacteria may comprise attenuated bacteria.
  • the metal compound for example, a manganese-containing compound
  • the metal compound can be physically bound to modify the surface of the bacterial body to form an adhesion layer.
  • the physical association may include electrostatic attraction.
  • said physical association may include partial embedding.
  • the metal compound for example, a manganese-containing compound
  • the metal compound can be chemically combined to modify the surface of the bacterial body to form an adhesion layer.
  • the chemical combination can include conjugation.
  • the chemical association may include forming a chemical bond.
  • the chemical bonding may include bonding in a cooperative manner.
  • the metal compound for example, manganese-containing compound
  • the metal compound can be modified on the surface of the bacterial body through a deposition reaction to form an adhesion layer.
  • the metal compound eg, manganese-containing compound
  • the biomineralization reaction generally refers to the combination of metal ions with biomacromolecules on the cell membrane or cell wall of bacteria to provide mineralization sites, adjust pH or introduce other salts, and the metal ions will Metal compounds are generated at the chemical sites, which continue to grow and accumulate, and bind to the surface of bacteria.
  • the binding sites of biomacromolecules and metal compounds can be formed through the biomineralization reaction, and the volume of the metal compound can be further increased by deposition reaction or biomineralization.
  • the coverage of the metal compound on the bacterial surface may be about 0.1%-99.9%. With this coverage, the bacteria are able to come into contact with the physiological environment, stimulating the immune system.
  • the coverage of the metal compound on the bacterial surface can be adjusted by adjusting the feed ratio, reaction temperature and/or reaction time.
  • the coverage of the metal compound on the bacterial surface is (bacterial surface area-the area where the modified bacteria directly contacts with the outside world)/bacterial surface area.
  • the modified lyophilized powder of bacteria may contain modified bacteria (eg, lyophilized powder of manganized bacteria), and additives.
  • the additive may include a lyoprotectant
  • the lyoprotectant may be selected from sugars/polyols, polymers, anhydrous solvents, surfactants, amino acids, salts and amines one or more.
  • the lyoprotectant may be selected from sucrose, mannose, ⁇ -D-mannopyranose, trehalose, inosose, raffinose, inulin, dextran, maltodextrin, maltopolysaccharide, sucrose octasulfate, heparin and One or more of 2-hydroxypropyl- ⁇ -cyclodextrin.
  • the mass fraction of the lyoprotectant in the modified bacterial lyophilized powder is 0.1-99%.
  • the mass fraction of the lyoprotectant is about 1%, about 2%, about 3%, about 5%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30% %, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, About 95%, about 99%.
  • the mass fraction of the lyoprotectant in the modified bacterial lyophilized powder is 0.1-20%.
  • the additive may include excipients.
  • the excipients may be selected from binders, fillers, disintegrants, lubricants, wine, vinegar, concoctions, etc., ointment bases, cream bases, preservatives, antioxidants, flavoring agents, One or more of aromatics, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators and colorants.
  • the mass fraction of additives in the modified bacterial freeze-dried powder is 0.1-99%.
  • the mass fraction of the additive is about 1%, about 2%, about 3%, about 5%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% , about 99%.
  • Example A Preparation and basic morphology characterization of bacteria modified with different metal compounds
  • Embodiment A1 the preparation of the bacterium that the surface is modified by zinc carbonate
  • Embodiment A2 the preparation of the bacterium that the surface is modified by iron hydroxide or copper hydroxide
  • Embodiment A3 the preparation of the modified bacterium of surface attachment manganese sulfide, zinc sulfide, copper sulfide
  • Examples A1-A3 were used to make scanning electron microscope samples, and the surface morphology of bacteria modified with different metal compounds was characterized by scanning electron microscopy (SEM). The results are shown in Figure 1. Among them, the pictures marked ZnCO 3 @FS, Fe(OH) 3 @FS, Cu(OH) 2 @FS, MnS@FS, ZnS@FS, CuS@FS respectively indicate that the surface has been treated with zinc carbonate, iron hydroxide, copper hydroxide , manganese sulfide, zinc sulfide, copper sulfide modified Salmonella scanning electron micrographs.
  • Embodiment A5 Preparation of modified other bacteria
  • S2 Bacterial amplification culture, inoculate 50 ⁇ L of frozen bacteria liquid into 6 mL of fresh meat extract peptone (LB) medium, place the shaking tube in a constant temperature shaker at 37°C at a shaking frequency of 220r/min After activation for 12 hours, the activated bacterial solution was diluted and spread on the LB solid medium plate, placed in a digital display incubator at 37°C for 12 hours, picked a single colony in good condition, and inoculated it into 5 mL of fresh LB broth for culture culture medium at 37°C on a constant temperature shaker overnight to obtain a bacterial solution;
  • LB fresh meat extract peptone
  • the synthesized manganized inactivated bacteria and unmanganized inactivated bacteria were prepared as scanning electron microscope (SEM) samples, and their appearance and morphology were characterized by scanning electron microscope.
  • SEM scanning electron microscope
  • the immobilized inactivated bacteria maintain the appearance characteristics of the original bacteria, the surface of the inactivated bacteria after manganization is combined with a layer of rough sediment, and the bacteria themselves maintain their original shape and size, indicating that The manganization process does not destroy the morphology of the bacteria itself, but only attaches a layer of substances on its surface.
  • XPS X-ray photoelectron spectroscopy
  • Embodiment B1 Relative cell activity of CT26 cells (mouse colon cancer cell line) after incubation with different samples
  • CT26 cells were seeded in a 96-well plate at a density of 10 cells/well, cultured overnight in a 37°C cell incubator, and when the cells were adhered to the wall and in good condition, different samples prepared in Examples A1-A3 at different concentrations were added (where Bacteria were inactivated attenuated Salmonella) co-incubated at 37°C.
  • Bacteria modified by different metal compounds the corresponding concentration of metal compounds is 1 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, in the unmodified bacteria group, the total amount of bacteria and The total amount of modified bacteria was the same.
  • Example B2 Experiments of different samples stimulating the maturation of myeloid-derived dendritic cells
  • DC cells are highly efficient antigen-presenting cells in the body. After being stimulated by certain factors or ingesting antigens, they differentiate into mature DC cells. Mature DC cells can effectively activate initial T cells, induce the generation of cytotoxic T lymphocytes, and secrete Tumor necrosis factor ⁇ (TNF- ⁇ ), etc., play a key role in the anti-tumor immune response. Therefore, samples that can effectively stimulate the maturation of DC cells can enhance the body's anti-tumor immune response.
  • TNF- ⁇ Tumor necrosis factor ⁇
  • Bone marrow-derived stem cells were extracted from the bone marrow of C57BL/6 mice, and colony-stimulating factor (GM-CSF) was added to promote the differentiation of stem cells into myeloid-derived dendritic cells (Bone Marrow-Derived Dendritic Cells, BMDCs).
  • GM-CSF colony-stimulating factor
  • Bacteria samples (the amount of bacteria in each sample is 3.6*107; the corresponding bare metal compound content is 1.5-2.7 ⁇ g , in the rest of the groups without bacteria, the amount of the same metal compound is the same as that of the metal compound modified
  • the same bacterial group for example, ZnS and ZnS@FS two groups, containing the same amount of ZnS
  • blank blank control group
  • FS unmodified inactivated bacteria
  • ZnS zinc sulfide suspension
  • ZnCO 3 zinc carbonate suspension
  • Fe(OH) 3 iron hydroxide colloid
  • CuS copper sulfide suspension
  • Cu(OH) 2 copper hydroxide suspension
  • MnS manganese sulfide suspension
  • ZnS@FS Salmonella suspension modified with zinc sulfide
  • ZnCO 3 @FS Salmonella suspension modified with zinc carbonate
  • Fe(OH) 3 @FS Salmonella suspension modified with iron hydroxide
  • CuS@FS sulfide modified Copper-modified Salmonella suspension
  • Cu(OH) 2 @FS copper hydroxide-modified Salmonella suspension
  • MnS@FS manganese sulfide-modified Salmonella suspension.
  • LPS lipopolysaccharide
  • a potent activator of immune cells including B cells, monocytes, macrophages, and other LPS-responsive cells
  • immature DC cells tend to mature under the stimulation of such immunogenic stimuli
  • Embodiment C animal experiments
  • Example C1 Therapeutic Experiment of Bacteria Modified by Different Metal Compounds on Mouse Colon Cancer Tumor Model
  • Colon cancer tumor cells were inoculated on the back of BALB/c mice to establish a tumor model.
  • the tumor size grew to about 120 mm 3 , they were randomly divided into groups with 6 mice in each group. Mice in different groups were treated with bacteria modified with different metal compounds .
  • a single treatment was performed by intratumoral injection.
  • the amount of bacteria used in the control group is constant, which is 1.8*10 10 bacteria/kg body weight.
  • the dose of metal compound is 0.75mg/kg-1.35mg/kg body weight
  • the metal ion concentration is 300 ⁇ g/mL-1.08mg/ml.
  • the tumor volume change was recorded and the tumor growth curve was made. The results are shown in Figure 5.
  • the survival rate of the mice was recorded, the tumor inhibition rate was calculated, and the statistics of the survival rate and the calculation results of the tumor inhibition rate were shown in Table 1.
  • Table 1 Survival rate of mice treated with tumors modified by different metal compounds and tumor inhibition rate on day 19 after inoculation
  • the bacteria modified by most of the metal compounds in this application can achieve the effect of tumor therapy, while the bacteria modified by the hydroxide of Fe (Fe(OH) 3 @FS) did not show obvious tumor inhibitory effect , may be due to differences in the doses of different metal compounds to exert curative effects, and no effective dose of Fe(OH)3@FS was found in one experiment.
  • Example D1 Demonstrating the activation of the modified bacteria on the STING pathway at the cellular level
  • the modified bacteria described in this application have multi-channel stimulating effects.
  • This example is designed by taking the Salmonella modified with manganese compounds (manganese dioxide) as an example, and other metal ions have their corresponding immune stimulating mechanisms.
  • cells containing a reporter gene regulated by the activation of the STING pathway (STING + ) are used. After the STING pathway of the cell is stimulated and activated, the expression of the reporter gene luciferase will be activated, which can catalyze the fluorescence in the medium.
  • the bioluminescence signal is emitted by the substrate of the enzyme enzyme, and the higher the degree of activation of STING, the stronger the bioluminescence signal.
  • the cells were mixed with bacteria modified by manganese dioxide (MnO 2 @FS), manganese dioxide (MnO 2 ), Salmonella (FS), manganese chloride (Mn 2+ ), positive control (positive control, PC) After co-incubating for 24 hours, the bioluminescent signal intensity was detected after adding the luciferase substrate, and then the bioluminescent signal intensity of all groups were compared with the PBS group. Null cells are cells that do not express the STING pathway, so they cannot induce luciferase expression by activating the STING pathway. The incubation of these cells with different samples proves that the samples and reagents in the experiment cannot interfere with the bioluminescent signal or induce luciferase. Express.
  • the statistical graph of the ratio of bioluminescent signal intensity is shown in Figure 6.
  • the ratio of bioluminescent signal intensity in the cells co-incubated with the modified bacteria is higher, indicating that the STING pathway of the cells in this group is significantly activated; and
  • the ratio of bioluminescent signal intensity was close, indicating that the STING activation level was close, that is, the modified bacteria could effectively activate the STING pathway.
  • STING refers to the interferon gene stimulator, which is mainly expressed on the rough endoplasmic reticulum, mitochondria and outer membranes of human macrophages, T lymphocytes, dendritic cells, etc.
  • STING plays an important pivotal role in the innate immune response triggered by virus, bacterial and parasitic infection, the body's tumor immune process and the process of cell autophagy; through its own phosphorylation, ubiquitination and dimerization, it regulates protein synthesis and The expression of IFN plays a key role in multiple immune links of the body. STING is an important regulatory target in the body's anti-tumor immunity.
  • the proliferation of tumor cells can activate STING in antigen-presenting cells, thereby activating the adaptive immune process mediated by T cells and exerting anti-tumor effects. Therefore, the modified bacteria described in this application can stimulate the activation of the STING pathway, indicating that it has the potential for anti-tumor immunotherapy.
  • Example D2 Cell experiments prove that the modified bacteria can activate the TLR4 pathway
  • TLR4 + TLR4 positive cells
  • TLR4 + TLR4 positive cells
  • TLR4 TLR4 + , TLR4 positive cells
  • TLR4 TLR4 pathway activation
  • TLR4-negative cells do not express TLR4, so they will not be stimulated and express luciferase, and bioluminescence will not occur after the addition of luciferin substrate. This group of cells proved that all reagents or samples used in the experiment cannot affect biological The luminescent signal interferes with or induces luciferase expression.
  • the bioluminescent signal intensity ratios of three parallel samples in different groups are shown in Table 2.
  • the positive control group was co-incubated with cells at 3 ⁇ g/mL MPLA, which had a significant TLR4 pathway stimulating effect.
  • the modified bacteria MnO 2 @FS
  • the modified bacteria can significantly stimulate TLR4 compared with the blank control group (Blank), showing that the fluorescence intensity ratio is greater than 1; compared with the simple bacteria (FS), the modified bacteria have stronger fluorescence intensity , indicating that the modified bacteria can better stimulate the TLR4 pathway. It shows that the modified bacterium of the present application has the effect similar to TLR4 agonist.
  • TLR4 is a Toll-like receptor. The activation of TLR4 can promote DC to secrete related interleukins to enhance Th1 immune response, which is beneficial to anti-tumor immunotherapy.
  • Table 2 Statistical table of the ratio of bioluminescence intensity after adding luciferase substrate in different groups of cell culture medium
  • STING and TLR4 show that the modified inactivated bacteria in this application can effectively activate the natural immune system, which helps to relieve the immune suppression of the tumor microenvironment, thereby strengthening the body's anti-tumor immune response.
  • the modified bacteria in this application have the effect of multi-pathway agonists, and at the same time strengthen non-specific immune response and anti-tumor immune response, which is helpful for synergistic anti-tumor immunotherapy.
  • Blank blank control group
  • the second group F.SA: inactivated Staphylococcus aureus
  • the third group F.E: inactivated Escherichia coli
  • the fourth group F.L: Inactivated Lactobacillus
  • the fifth group MnO 2 @F.SA: inactivated Staphylococcus aureus modified by manganese dioxide;
  • Group 6 MnO 2 @FE: inactivated Escherichia coli modified with manganese dioxide;
  • the seventh group MnO 2 @FL inactivated Lactobacillus modified by manganese dioxide;
  • the eighth group MnO 2 @FS modified with manganese dioxide inactivated Salmonella.
  • Example A5 According to the preparation method of Example A5, different kinds of bacteria were prepared into modified bacteria, and the treatment experiment of mouse subcutaneous CT26 colon cancer model was carried out.
  • the tumor inhibition rate of each group was calculated on the 17th day after inoculation.
  • the tumor inhibition rate of the blank control group was 0.
  • the results of the tumor inhibition rate of other groups are shown in Table 3, and the tumor growth curve is shown in Figure 28.
  • the results showed that the modified inactivated bacteria of different species could all inhibit the tumor very well, and the tumor inhibition rate was about 5 times that of the single bacterial treatment. It shows that the different types of bacteria modified in this application can all realize the treatment of tumors, and the type of bacteria has no direct influence on the curative effect of the modified bacteria.
  • Table 3 Statistical table of tumor inhibition rate of each group on the 17th day after tumor inoculation in mice treated with bacteria modified by compounds with or without manganese
  • Example F Anti-tumor immune memory effect of the body produced after the modified bacteria treat the tumor
  • the average proportion of memory T cells in all T cells in the peripheral blood leukocytes in vivo was 83.86 %, significantly increased compared with the blank control group without any treatment (memory T cells account for 59.5% of the average content of all T cells in peripheral blood leukocytes in mice), indicating that the modified bacteria in this application can induce memory T cells. Produce, produce immune memory effect.
  • mice were reinoculated with the same tumor cells, and the tumor survival of the mice was observed.
  • the survival curve of the mice is shown in Figure 7.
  • the mice cured by the modified bacteria did not appear after reinoculation of tumor cells.
  • the obvious phenomenon of cancer recurrence can still have a longer survival period, that is to say, the modified bacteria can inhibit the recurrence of tumors, producing a vaccine-like effect.
  • the modified bacterial suspension obtained in Examples A1-A3 and Example A5 is mixed with various additives (such as lyoprotectants, excipients) and then freeze-dried to observe its state after freeze-drying and Whether it can be redispersed into bacterial suspension after adding solvent (water), wherein the observation results of manganese sulfide-modified Salmonella mixed with different proportions of lyoprotectant and freeze-dried are recorded in Table 4.
  • additives such as lyoprotectants, excipients
  • the redispersible sample, its lyoprotectant and its ratio are defined as the available range, and the lyophilized sample with a better shape and can be redispersed into a suspension, the sample with a more regular shape after lyophilization is helpful
  • the used lyoprotectant and the corresponding ratio are set to a preferred range.
  • sucrose and ⁇ -cyclodextrin can make the modified bacteria have a better freeze-drying effect.
  • trehalose, inosose, raffinose, inulin, dextran, maltodextrin, Maltopolysaccharide can resuspend the sample after freeze-drying without changing its properties, while lactose and mannitol cannot achieve effective freeze-drying protection. All additives used in the examples were purchased commercially.
  • Example H1 Synthesis of manganized bacteria with different feed ratios
  • the input amount of inactivated bacteria is fixed, and the manganese chloride of different quality is added in the inactivated bacterial suspension, observes the stability of synthetic sample, because whole reaction is in liquid environment
  • the product is dispersed in the liquid phase, so the stability of the sample is mainly indicated by whether the product dispersibility is good. If there is precipitation or coagulation visible to the naked eye, it is considered that the product dispersibility is not good. On the contrary, if the liquid color of the product If the distribution is uniform and no precipitation occurs, the product is considered to have good dispersibility.
  • Table 5 From the perspective of product stability, when the input amount of bacteria is about 1 billion bacteria and the input amount of manganese chloride is not more than 13.5 mmol, manganized inactivated bacteria with good dispersion can be obtained.
  • Example A5 Take the manganized bacteria produced in Example A5, add different concentrations of the lyoprotectant according to the characteristics of different lyoprotectants, observe the redissolution of the manganized bacteria after lyophilization, and select the appropriate type and concentration of the lyoprotectant , and the results are shown in Table 6.
  • Table 6 When no lyoprotectant is added, the lyophilized manganized bacteria cannot be redispersed in the solvent (water). After adding an appropriate proportion of lyoprotectant, a good freeze-dry reconstitution effect can be achieved.
  • the "mass/volume fraction" in Table 6 refers to the mass/volume fraction of the lyoprotectant in the solution in the liquid preparation before lyophilization.
  • Embodiment H3 preparation of manganized living bacteria
  • S2 Bacterial amplification culture, inoculate 50 ⁇ L of frozen bacteria liquid into 6 mL of fresh meat extract peptone (LB) medium, place the shaking tube in a constant temperature shaker at 37°C at a shaking frequency of 220r/min After activation for 12 hours, the activated bacterial solution was diluted and spread on the LB solid medium plate, placed in a digital display incubator at 37°C for 12 hours, picked a single colony in good condition, and inoculated it into 5 mL of fresh LB broth for culture culture medium at 37°C on a constant temperature shaker overnight to obtain a bacterial solution;
  • LB fresh meat extract peptone
  • Embodiment H4 preparation of manganized living bacteria
  • the preparation method is the same as that of Example A5, the only difference being that when the pH value is adjusted to 8-10, the manganization reaction time is increased by 30% to 100% compared with Example A5, but in the obtained manganized bacterial sample, the manganese compound on the surface of the bacteria The particles are finer.
  • Embodiment H5 preparation of manganized living bacteria
  • the preparation method is the same as that of Example A5, the only difference is that when the pH value is adjusted to 11-13, the manganization reaction time is shortened as the pH increases, but when the pH is higher than 13, the morphology of bacteria will be destroyed, which is not conducive to Uniformity of the final product.
  • Embodiment I1 the influence of manganizing bacteria on cell viability
  • CT26 tumor cells were seeded in a 96-well plate at a density of 10 4 cells/well, and cultured overnight in a 37°C cell incubator. When the cells adhered to the wall and were in good condition, different samples of different concentrations were added for co-incubation. The samples and their concentrations are shown in Table 7. After incubation for 24 hours, the medium containing the samples was removed, the excess samples were washed away with PBS, and the relative activity of the cells was detected by the MTT assay. The results are shown in Table 7.
  • Embodiment I2 Manganization inactivates bacteria to promote the phagocytosis of DC2.4
  • the bacteria were labeled with Cy5.5 to obtain inactivated bacteria and manganized inactivated bacteria with fluorescent signals.
  • DC2.4 cells in good condition were co-incubated with inactivated bacteria or manganized inactivated bacteria with fluorescent signals for a period of time. The samples were quantified by the concentration of manganese ions. The final concentration of the samples was 2 ⁇ g/mL.
  • DC2.4 cells It can phagocytize inactivated bacteria and manganese inactivated bacteria. At different time points, the fluorescence signal intensity of DC2.4 cells was detected by flow cytometry, and the results obtained are shown in FIG. 9 .
  • the abscissa of the spectrum is the fluorescence intensity
  • the peak area is the cell distribution density
  • the statistical graph is the statistical value of the average fluorescence intensity.
  • DC2.4 cells were co-incubated with manganese dioxide or manganese inactivated bacteria (the same concentration of manganese element). After co-incubation for 12 hours, the percentage of manganese ion content in the cells to the total input manganese ion content was detected by ICP, so as to observe The changes in the content of manganese uptake by cells are shown in Figure 10. The results show that manganese dioxide can be more phagocytized by cells after growing on the surface of bacteria. At the same time, the manganese content in cells of DC2.4 co-incubated with manganized inactivated bacteria was significantly higher than that co-incubated with manganese dioxide DC2.4 cells. The above experimental results indicate that compared with manganese dioxide alone, manganized inactivated bacteria can be more effectively phagocytized by DC2.4 cells.
  • DC2.4 is a dendritic cell line derived from mouse bone marrow, which is used to study the interaction between drugs and DC cells in vitro.
  • DC cells are the most functional professional antigen-presenting cells in the body. Its function is to phagocytize, process and present antigens. The enhancement of phagocytosis helps DC cells absorb more manganese dioxide, thereby stimulating the STING pathway more strongly.
  • the maturation of DC cells occurs in the process of phagocytosis/processing of antigens, more antigen uptake can promote the maturation of dendritic cells, and mature DC cells can effectively activate naive T cells, triggering a series of antitumor immune response.
  • Example I3 Stimulated Maturation of Bone Marrow-Derived DC Cells
  • Bone marrow-derived stem cells were extracted from the bone marrow of C57BL/6 mice, and colony-stimulating factor (GM-CSF) was added to promote the differentiation of stem cells into bone marrow-derived dendritic cells (BMDCs).
  • GM-CSF colony-stimulating factor
  • the BMDCs were co-existed with different samples. Incubate, wherein the manganese ion of manganese dioxide is used for quantification, the final concentration is 2 ⁇ g/mL, and the final concentration of F.S is 0.12MCF, and the maturation ratio of BMDCs in different groups is detected after 12 hours.
  • DC cells are highly efficient antigen-presenting cells in the body. When stimulated by certain factors or ingesting antigens, they differentiate into mature DC cells.
  • Mature DC cells can effectively activate initial T cells, induce the generation of cytotoxic T lymphocytes, and secrete Interleukin 1 ⁇ (IL-1 ⁇ ), tumor necrosis factor ⁇ (TNF- ⁇ ), interleukin 6 (IL-6), etc., play a key role in the anti-tumor immune response.
  • tumor necrosis factor ⁇ TNF- ⁇
  • IL-1 ⁇ is a pro-inflammatory cytokine
  • IL-6 is a pro-inflammatory cytokine
  • IL-6 is a pro-inflammatory cytokine
  • IL-6 is mainly produced by macrophages and helper T cells, induces cell differentiation, and activates an inflammatory response. Therefore, samples that can effectively stimulate DC cell maturation can enhance the body's anti-tumor immune response.
  • the statistical results of the detected DC cell maturation ratio are shown in FIG. 11 , and correspondingly, the statistics of the concentration of cytokines secreted by DC cells in different groups are shown in FIG. 12 .
  • FIG. 11 The statistical results of the detected DC cell maturation ratio
  • FIG. 12 the statistics of the concentration of cytokines secreted by DC cells in different groups are shown in FIG. 12 .
  • Example I4 Cytokine expression after co-incubation of peritoneal macrophages with different samples
  • macrophages In the anti-tumor immune response, besides dendritic cells, macrophages also play an important role.
  • the macrophages extracted from the peritoneal cavity of C57BL/6 mice were co-incubated with different samples. The samples were quantified with manganese dioxide manganese ions, the final concentration was 2 ⁇ g/mL, and the final concentration of F.S was 0.12MCF, and the secretion of macrophages was detected. Cytokines, the results are shown in Figure 13. Judging from the secretion of interleukin 1 ⁇ , tumor necrosis factor ⁇ and interleukin 6, manganized inactivated bacteria have the strongest activation effect on macrophages, even better than that of the positive control. Effect.
  • cytokines play an important role in the anti-tumor immune response, among which tumor necrosis factor ⁇ (TNF- ⁇ ) can kill or inhibit tumor cells; IL-1 ⁇ is a pro-inflammatory cytokine, and the secretion of IL-6 indicates that the tumor necrosis factor ⁇ (TNF- ⁇ ) can kill or inhibit tumor cells; Phage cells are polarized and can recruit more immune cells to the tumor site, causing a stronger anti-tumor immune response; IL-6 is mainly produced by macrophages and helper T cells, induces cell differentiation, and activates inflammatory responses. This experiment shows that the activation of the innate immune response can also stimulate the proliferation and differentiation of cells involved in the immune response, and increase the activity of immune cells (such as NK cells). These three cytokines can promote anti-tumor immune responses, indicating that the present application The manganese inactivated bacteria contribute to the anti-tumor immune response and enhance the efficacy of tumor immunotherapy.
  • Example I5 Demonstrate the activation of manganese inactivated bacteria on the STING pathway at the cellular level
  • interferon regulatory factor 3 IRF3
  • interferon regulatory factor 3 phosphorylation p-IRF3
  • Phosphorylation at the 12C end of IRF-3 can induce the expression of IFN ⁇ / ⁇ , and the degree of phosphorylation in this experiment indicates the degree of activation of the STING pathway.
  • GAPDH is an internal reference commonly used in western blot experiments, and all cells have it. In the experiment, the internal reference strip is used to indicate that the amount of cells added in this experiment is the same.
  • Example I6 Cell experiments prove that manganese inactivated bacteria can activate the TLR4 pathway
  • TLR4 + Toll-like receptor 4 (TLR4) reporter gene
  • TLR4 + Toll-like receptor 4 positive cells
  • the positive control group is PC, specifically 3 ⁇ g/mL MPLA), in which the manganese ion of manganese dioxide is used for quantification, the final concentration is 2 ⁇ g/mL, and the final concentration for FS quantification is 0.12MCF.
  • TLR4 reporter gene after TLR4 is activated The cells express luciferase, which can react with the luciferin substrate and emit a fluorescent signal.
  • TLR4-negative cells TLR4 -
  • TLR4 - TLR4-negative cells
  • TLR4 is one of the receptors for the natural immune system to recognize pathogenic microorganisms.
  • the activation of STING and TLR4 shows that the manganese inactivated bacteria described in this application effectively activate the natural immune system , help to relieve the immunosuppression of the tumor microenvironment, thereby strengthening the body's anti-tumor immune response.
  • Example I5 the manganized inactivated bacteria described in this application have the effect of multi-path agonists, and at the same time strengthen the non-specific immune response and anti-tumor immune response, which is helpful for synergistic anti-tumor immunotherapy.
  • Embodiment J animal experiments
  • Example J1 Compared with different immune adjuvants combined with inactivated bacteria, the curative effect of manganized inactivated bacteria on tumors
  • Blank control group intratumoral injection of normal saline
  • Manganese dioxide (MnO 2 ): intratumoral injection of manganese dioxide
  • Inactivated Salmonella intratumoral injection inactivated Salmonella
  • Inactivated Salmonella and aluminum adjuvant intratumoral injection of a mixture of inactivated Salmonella and aluminum adjuvant.
  • the subcutaneous tumor model of CT26 colon cancer was inoculated on the back of the mice. When the tumor volume reached about 100 mm 3 , they were randomly divided into groups, and different samples were injected into the mouse tumors according to the groups. The dose of manganese was 1 mg/kg, and the tumor volume and body weight changes of the mice were recorded every two days, and the tumor growth curve and body weight change curve were made. The results are shown in Figure 16 and Figure 17 .
  • Figure 16 is the curve of tumor volume change, in which the injection of manganized inactivated bacteria has the best inhibitory effect on tumor growth in mice, while the injection of a mixture of inactivated bacteria and manganese dioxide or one of its components cannot achieve this effect, and The mixture has no slight effect on tumor growth inhibition, indicating that only when manganese dioxide grows on the surface of inactivated bacteria (MnO 2 @FS) can the manganized bacteria obtain the most ideal effect, that is, the modified Bacteria have good anti-tumor therapeutic effect.
  • the aluminum adjuvant is purchased commercially, the product number is 77161, and the brand is Thermo Scientific.
  • Intratumoral injection of inactivated bacteria mixed with manganese chloride can inhibit tumor growth to a certain extent, but its "therapeutic effect" is mainly caused by the direct toxicity of manganese chloride.
  • manganese chloride has caused large-scale necrosis of tumors and surrounding tissues, and the weight of mice injected with manganese chloride has decreased significantly, indicating that the strategy of mixing inactivated bacteria and manganese chloride to treat tumors is not an ideal solution .
  • Aluminum adjuvant is a widely recognized immune adjuvant. It is reported that aluminum adjuvant can effectively enhance the immune response. As a control group, intratumoral injection of inactivated bacteria and aluminum adjuvant cannot inhibit tumor grow.
  • manganese inactivated bacteria grown on the surface of manganese dioxide can effectively inhibit tumor growth by intratumoral injection under the premise of ensuring safety.
  • Example J2 Phagocytosis of samples by CD45 + cells at the tumor site
  • the bacteria were labeled with Cy5.5 to obtain inactivated bacteria and manganized inactivated bacteria with fluorescent signals.
  • Intratumoral injection of fluorescently labeled manganized inactivated bacteria and inactivated bacteria 24 hours later, the tumor tissue was collected, processed to obtain a cell suspension, and the CD45 + cells in the tumor site cells were labeled with flow cytometry antibodies to detect The fluorescent signal intensity of the phagocytosed bacteria in the CD45 + cells was counted, and the results obtained are shown in FIG. 18 .
  • the abscissa of the spectrum is the fluorescence intensity
  • the peak area is the cell distribution density
  • the statistical graph is the statistical value of the average fluorescence intensity.
  • the CD45 + cells in the lysed tumor tissue cell suspension of the mice injected with manganese inactivated bacteria had a stronger fluorescent signal. According to the statistical chart, it can be found that the CD45 + cells in the tumor site phagocytized The amount of manganized inactivated bacteria was significantly greater than that of non-manganized inactivated bacteria, indicating that the inactivated bacteria grown on the surface of manganese dioxide can be more phagocytized by white blood cells in vivo.
  • CD45 leukocyte cell marker CD45 + cells are leukocytes
  • the enhancement of leukocyte phagocytosis of manganese bacteria is conducive to the recruitment of more immune cells to the tumor site
  • the indiscriminate phagocytosis of more tumor-associated antigens is conducive to the development of immune response at the tumor site Activate and enhance anti-tumor specific immune response.
  • Example J3 Systemic distribution of samples after injection
  • Example J4 Changes in the oxygen content of the tumor site after sample injection
  • the changes in the oxygen content in the tumor microenvironment of tumor-bearing mice were detected with oxygen content detection probes 1 h and 4 h after injection of the samples.
  • Blank control group intratumoral injection of normal saline
  • Manganese dioxide (MnO 2 ): intratumoral injection of manganese dioxide
  • Inactivated Salmonella intratumoral injection inactivated Salmonella
  • Manganized inactivated Salmonella (MnO 2 @FS): intratumoral injection of manganized inactivated Salmonella.
  • the oxygen content in the untreated tumor tissue is about 0.28ppm, and it is in a hypoxic state.
  • the oxygen content of the manganese dioxide treatment group and the manganese inactivated bacteria treatment group both rose to about 0.34ppm, which significantly increased the oxygen content of the tumor site.
  • a similar trend can still be observed after 4 h. It shows that manganese bacteria can effectively improve the hypoxic state of the tumor microenvironment through the action of manganese dioxide.
  • the hypoxic microenvironment of the tumor is a typical immunosuppressive environment. Activated in this environment, thereby exerting an immunotherapeutic effect.
  • Example J5 The strategy of inactivating bacteria with manganese can effectively activate the anti-tumor immune system
  • the first group Blank, blank control group (intratumoral injection of normal saline);
  • the second group MnO 2 , manganese dioxide (intratumoral injection of manganese dioxide);
  • the third group F.S, inactivated Salmonella (inactivated Salmonella by intratumoral injection);
  • the fourth group MnO 2 @FS, manganized inactivated Salmonella (intratumoral injection of manganized inactivated Salmonella).
  • lymph node dendritic cells migrate to lymph nodes after ingesting antigens, mature gradually during migration, express co-stimulatory molecules and present antigens to T cells in lymph nodes .
  • the lymph nodes on the side of the tumor were removed 24 hours after the sample was injected, and after grinding, the proportion of mature dendritic cells in all dendritic cells was detected by flow cytometry, and the statistical results are shown in Figure 21.
  • the mice in the manganized inactivated bacteria treatment group had the highest DC maturity, which was 26.9 times that of the normal saline injection group. immune system.
  • the tumor microenvironment is an immunosuppressive microenvironment, and the immune response is not easy to be activated, so the content of cytokines is extremely low.
  • the mouse tumor tissue was removed at 24 hours After grinding, the contents of interleukin-1 ⁇ , interleukin-6, tumor necrosis factor- ⁇ and interferon- ⁇ were detected by enzyme-linked immunoassay. The results are shown in Figure 23.
  • mice injected with manganized inactivated bacteria the levels of interleukin 1 ⁇ , interleukin 6, tumor necrosis factor ⁇ and interferon ⁇ at the tumor site were significantly higher than those of other control groups, showing that manganized inactivated bacteria Superiority of live bacterial strategies for innate immune activation in the tumor microenvironment.
  • interferon beta is a sign of STING pathway activation, because manganese dioxide in the manganized bacterial sample can enter immune cells more (Example I2) and manganized inactivated bacteria can be more persistent in the tumor site than free manganese dioxide Retention (Example J3), thus having the highest ability to activate the STING pathway.
  • mice treated with manganese-inactivated bacteria also had changes in the polarization state of macrophages at day 5.
  • M1 macrophages are an immune activation type that can help antigen presentation and T cell differentiation.
  • M2 macrophages are the tumor-promoting type and are generally considered to be associated with tumor metastasis.
  • the experimental data indicated that on the fifth day, the state of macrophage polarization at the tumor site of mice in the manganese inactivated bacteria group shifted towards anti-tumor immunity. ( Figure 24)
  • the tumor microenvironment is effectively improved, and the cytokines related to immune activation are significantly up-regulated, which helps the infiltration of macrophages, NK cells and other immune cells into the tumor site, and also improves the tumor microenvironment. More dendritic cells migrate to lymph nodes after taking up antigens and presenting antigens to T cells in lymph nodes, completing the transition from innate immune system activation to specific immune system activation.
  • helper T cells migrate to the tumor site after differentiation, the ratio of helper T cells to killer T cells is significantly increased, specifically kills tumor cells, and helps macrophages to polarize to the anti-tumor M1 type, and the polarized macrophages can continue to Activate the anti-tumor function of T cells and complete the activation of specific immune responses.
  • Example J6 Tumor treatment experiment of manganized bacteria after NK cell blocking
  • NK cells natural killer cells
  • IgG was used as an antibody that did not affect the function of NK cells , used as a control for NK antibody in this example.
  • the first group blank: blank control group
  • the second group MnO 2 @FS&IgG: combined treatment group of manganized inactivated bacteria and immunoglobulin;
  • the third group MnO 2 @FS&a-NK 1.1: combined treatment group of manganized inactivated bacteria and NK 1.1 cell antibody.
  • the subcutaneous melanoma model was established by inoculating melanoma B16-OVA tumor cells on the back of the mice, and the treatment method was intratumoral injection of manganese inactivated bacteria (MnO 2 @FS).
  • NK blocking antibody ( ⁇ -NK 1.1) was injected on the 2nd, 4th, and 6th day after the drug administration, and the mice in the second group were injected with IgG one day before the start of tumor treatment and on the 2nd, 4th, and 6th day after the administration. Tumor size was monitored every two days, and tumor growth curves were made. The results are shown in Figure 25.
  • the manganese inactivated bacteria after the loss of NK cell function, the manganese inactivated bacteria hardly inhibited tumor growth, indicating that the preparation can activate the innate immune response, and after the activation of the innate immune response, the activated NK cells play an important role in tumor growth inhibition.
  • Example J7 Tumor treatment experiment of manganized bacteria after T cell blocking
  • CD4 antibody and CD8 antibody were used to block the action site on the surface of T cells, so that CD4 + CD8 + T cells lost their function.
  • another group was injected with IgG as comparison.
  • the first group blank: blank control group
  • the second group MnO 2 @FS& ⁇ -CD4: Manganization inactivated bacteria and anti-CD4 antibody combined treatment group;
  • the third group MnO 2 @FS& ⁇ -CD8: combined treatment group of manganized inactivated bacteria and anti-CD8 antibody;
  • the fourth group MnO 2 @FS& ⁇ -CD4& ⁇ -CD8: manganized inactivated bacteria and anti-CD4, anti-CD8 antibody combined treatment group;
  • the fifth group MnO 2 @FS&IgG: combined treatment group of manganized inactivated bacteria and immunoglobulin.
  • mice Establish a subcutaneous CT26 tumor model in mice.
  • the tumor size is about 100mm 3
  • mice are randomly divided into groups.
  • the mice in different groups are injected with corresponding antibodies intravenously one day before intratumoral administration and on the 3rd, 5th, and 7th day after intratumoral administration. , record the tumor growth, and make a tumor growth curve, the results are shown in Figure 26.
  • Embodiment J8 is a diagrammatic representation of Embodiment J8.
  • the mouse back 4T1 breast cancer tumor model, B16-OVA melanoma model and KPC pancreatic cancer model were respectively established, and then different samples were injected into the tumor for treatment, and the tumor growth curve was recorded.
  • the results are shown in Figure 27.
  • the results show that all three tumor models can be well inhibited by manganized inactivated bacteria, but a single component cannot inhibit tumor growth. Live bacteria are effective against a variety of solid tumors.
  • Example J9 Treatment experiment of different manganese bacteria on breast cancer tumor model
  • the first group blank: blank control group
  • the second group MnO 2 @FS: Manganization inactivation of Salmonella
  • the third group MnO 2 @F.SA: Manganization inactivated Staphylococcus aureus
  • the fourth group MnO 2 @FE: Escherichia coli inactivated by manganization;
  • the fifth group MnO 2 @FV: Manganization inactivated Salmonella VNP20009.
  • a mouse subcutaneous 4T1 breast cancer model was established, and different types of manganese inactivated bacteria were injected into the tumor, the tumor growth was recorded, and the tumor growth curve was made. The results are shown in Figure 29. It can be seen from the growth curve that all manganized bacteria can well inhibit tumor growth, which is similar to the result of Example J3, indicating that the manganized inactivated bacteria described in this application are universally applicable to different tumors.
  • Example J10 Therapeutic Effects of Manganized Bacteria Prepared by Different Feeding Ratio on Tumors
  • Example J1 manganese bacteria with different feeding ratios were prepared, the mouse CT26 subcutaneous tumor model was established, and the mice were randomly divided into groups. According to the group setting, each mouse was injected with the corresponding drug into the tumor, and the injection dose was 3.6*10 8 Manganizing bacteria, the corresponding amount of manganese dioxide were 3 ⁇ g, 16 ⁇ g, 89 ⁇ g. Afterwards, the tumor volume was measured every two days, and the tumor growth curve was made. The results are shown in FIG. 30 .
  • the manganized bacteria prepared with different feed ratios had inhibitory effects on tumor growth. It shows that the manganizing bacterium prepared by the present application has a wide range of selectivity in feed ratio, and in actual treatment, more research can be conducted to confirm the best formula ratio.
  • the New Zealand rabbit liver VX2 cancer model is one of the few liver cancer recurrence and metastasis models established in larger animals, which can better simulate the recurrence and metastasis of human liver cancer. Therefore, the New Zealand rabbit liver cancer model was established and the manganized inactivated bacteria described in this application were used MnO 2 @FV is used for tumor treatment, the amount of bacteria used in sample preparation is 10.5MCF, and the amount of manganese dioxide used is 160.7 ⁇ g. The results are shown in Figure 31. Compared with the group without any treatment, the tumor lesions of New Zealand rabbits treated with manganese inactivated bacteria (MnO 2 @FV) were almost completely eliminated, indicating its excellent anti-tumor properties .
  • a mouse bilateral tumor model was established, and the CT26 colon cancer subcutaneous tumor model was inoculated on the left and right sides of the back of the mouse.
  • the number of tumor cells inoculated on the left side was half that on the right side. metastases.
  • the tumor volume on the right side reached about 100 mm 3 , they were randomly divided into groups, and different samples were injected into the tumor on the right side of the mice according to the group, and injected into the tails of the two groups of mice at the immune checkpoint 1, 3, and 5 days after treatment.
  • Intravenous injection of anti-PD-1 (ie ⁇ -PD-1) antibody The volumes of the bilateral tumors of the mice were recorded every two days, and the results of the tumor growth curves were made, as shown in FIG. 32 .
  • the first group blank: normal saline was injected into the right tumor;
  • ⁇ -PD-1 three injections of immune checkpoint antibody anti-PD-1 into the tail vein;
  • the third group MnO 2 @FS: inject manganese bacteria into the right tumor;
  • the fourth group MnO 2 @FS& ⁇ -PD-1: Manganized bacteria were injected into the right tumor and the immune checkpoint antibody anti-PD-1 was injected three times into the tail vein.
  • the tumor growth curve on the treatment side showed an excellent therapeutic effect of manganized bacteria.
  • manganized bacteria (MnO 2 @FS ) treatment effect was significantly better than that of immune checkpoint treatment.
  • the tumor on the treated side of the mice was almost completely suppressed.
  • the combination of immune checkpoints can further strengthen the inhibitory effect of manganese bacteria on the growth of distal tumors, and the growth rate of distal tumors will slow down.
  • mice were inoculated with tumor cells again, and their growth was recorded.
  • the results are shown in Figure 34.
  • the results showed that the mice cured by manganizing bacteria had almost no tumor growth after re-inoculation of tumor cells, that is to say, manganese
  • antibacterial bacteria can also inhibit the recurrence of tumors, producing a vaccine-like effect.
  • Embodiment K1 the preparation of the manganized inactivated bacteria attached to manganese dioxide (with manganese sulfate as raw material)
  • S2 Bacterial amplification culture, inoculate 50 ⁇ L of frozen-preserved attenuated Salmonella bacteria into 6 mL of fresh meat extract peptone (LB) medium, place the shaking tube in a constant temperature shaker at 37°C at 220r/min The shaking frequency was activated for 12 hours, and then the activated bacterial solution was diluted and spread on the LB solid medium plate, placed in a digital display incubator at 37°C for 12 hours, and a single colony in good condition was picked and inoculated into 5 mL of fresh LB In the broth medium, placed on a constant temperature shaker at 37°C and cultivated overnight to obtain the bacterial liquid;
  • LB fresh meat extract peptone
  • S3 Take the bacterial liquid obtained in S2 into 20 mL of fresh LB liquid medium, place the shaking tube in a constant temperature shaker at 37°C and activate it for 12 hours at a shaking frequency of 220r/min, collect the bacterial cells by centrifugation, and wash with sterile normal saline.
  • the inactivated bacterial suspension can be stored at 4°C, and 50 ⁇ L of the bacterial solution is applied to solid culture After culturing, confirm that the bacteria in the bacterial liquid have been fully inactivated;
  • Embodiment K2 Preparation of manganized inactivated bacteria attached to manganese sulfide
  • S2 Bacterial amplification culture, inoculate 50 ⁇ L of frozen-preserved attenuated Salmonella bacteria into 6 mL of fresh meat extract peptone (LB) medium, place the shaking tube in a constant temperature shaker at 37°C at 220r/min The shaking frequency was activated for 12 hours, and then the activated bacterial solution was diluted and spread on the LB solid medium plate, placed in a digital display incubator at 37°C for 12 hours, and a single colony in good condition was picked and inoculated into 5 mL of fresh LB In the broth medium, placed on a constant temperature shaker at 37°C and cultivated overnight to obtain the bacterial liquid;
  • LB fresh meat extract peptone
  • S3 Take the bacterial liquid obtained in S2 into 20 mL of fresh LB liquid medium, place the shaking tube in a constant temperature shaker at 37°C and activate it for 12 hours at a shaking frequency of 220r/min, collect the bacterial cells by centrifugation, and wash with sterile normal saline.
  • Embodiment K3 Preparation of manganized inactivated bacteria by reduction method
  • SS2 Bacterial expansion culture, inoculate 50 ⁇ L of frozen-preserved attenuated Salmonella bacteria into 6 mL of fresh meat extract peptone (LB) medium, place the shaking tube in a constant temperature shaker at 37°C at 220r/min The shaking frequency was activated for 12 hours, and then the activated bacterial solution was diluted and spread on the LB solid medium plate, placed in a digital display incubator at 37°C for 12 hours, and a single colony in good condition was picked and inoculated into 5 mL of fresh LB In the broth medium, placed on a constant temperature shaker at 37°C and cultivated overnight to obtain the bacterial liquid;
  • LB fresh meat extract peptone
  • the absorbance value at 600nm wavelength is 3, and the inactivated bacterial suspension is obtained, which can be stored at 4°C. Take 50 ⁇ L of the bacterial liquid to coat the solid medium, and confirm that there are no viable bacteria in the bacterial liquid;
  • 0.2-3 ⁇ mol of manganese can be added to every 1 billion bacteria, and a stable manganized bacterial suspension can be formed.
  • Embodiment K4 is a diagrammatic representation of Embodiment K4
  • Embodiment K5 is a diagrammatic representation of Embodiment K5:
  • Bacteria growing manganese oxide on the surface with different feed ratios were prepared by the method of Example K1.
  • the raw materials were manganese sulfate and attenuated Salmonella.
  • the morphology of the finished product was observed with a scanning electron microscope, and the results are shown in FIG. 38 .
  • the manganizing bacteria obtained at different feed ratios had manganese oxide solids with a relatively uniform size attached to their surface. With the increase of manganese sulfate feed, the manganese oxide grown on the surface of the bacteria increased.
  • numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of the embodiments use the modifiers "about”, “approximately” or “substantially” in some examples. grooming. Unless otherwise stated, “about”, “approximately” or “substantially” indicates that the stated number allows for ⁇ variation. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that can vary depending upon the desired characteristics of individual embodiments.

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Abstract

一种经修饰的细菌及其制备方法和应用，所述经修饰的细菌包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物。所述的经修饰的细菌在瘤内注射后，能够激活抗肿瘤免疫反应，实现优异的抗肿瘤治疗效果，能够有效抑制肿瘤转移和复发。

Description

一种经修饰的细菌及其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种经修饰的细菌及其制备方法和应用。
背景技术
细菌治疗肿瘤由来已久，但是其发展缓慢，直到20世纪90年代，将卡介苗用于人的膀胱癌的治疗，才又将细菌疗法引入大众视野，近年来，又逐渐发展出减毒沙门氏菌、减毒李斯特菌等减毒菌，用于不同肿瘤的治疗，肿瘤的细菌疗法也是免疫治疗的一种引申疗法，借助细菌这种异源外来物质的刺激，引发免疫反应，进一步地抑制肿瘤生长。但是，目前使用的菌种大多为减毒处理后的活细菌，在临床使用中仍具有较高的风险，安全窗口较窄，灭活细菌则达不到应有的免疫刺激效果，总体来讲，目前的肿瘤细菌疗法很难在安全剂量下实现有效的治疗。
锰作为一种过渡金属元素同时也是人体微量元素之一，逐渐在一些专利中尝试探索应用于免疫刺激增强，如专利CN201610347815.6和CN201910319344.1公开了锰在STING通路中的功能，锰的沉淀物或胶体锰具有增强免疫的效果，其中在156专利中公开了二价锰具有cGAS-STING通路激活的功能，但二价锰的免疫增强作用效果不够理想且难以获得较高的二价锰浓度以增强效果；在441专利中采用二价新生沉淀锰、胶体锰来刺激免疫系统，得到了较二价锰离子更强的免疫刺激效果，随后将新生沉淀锰、胶体锰与抗原进行简单的混合，可以获得比该抗原更有效的疫苗。另一方面，专利CN201710795715.4中公开了一种二氧化锰纳米颗粒，在二氧化锰纳米颗粒表面装载寡聚核苷酸(CpG)和/或抗原，协同增效免疫刺激作用，随着二氧化锰在弱酸性环境中的降解，最终锰离子可排出体外。利用锰离子的类似佐剂的功能，能够激活STING通路，诱导一型干扰素的产生调节宿主免疫系统，激活T细胞特异性免疫应答并诱导巨噬细胞向抗肿瘤表型极化诱导病原特异性免疫反应，从而进一步诱导免疫记忆的产生。但是，上述多件专利的治疗策略中，锰离子的作用为免疫佐剂，与铝佐剂相似，必须要加入抗原才能诱导产生针对这一抗原的免疫反应，发挥的是疫苗的功能。例如，441专利中将锰离子(或其他含锰物质)与灭活细菌混合后注射，产生的主要是针对该细菌的免疫反应来实现预防细菌感染的目的；如果将锰离子(或其他含锰物质)与肿瘤细胞特异性表达的突变抗原混合后注射，产生的是针对该抗原的免疫反应，对于表达这个抗原的肿瘤细胞将产生免疫攻击作用，而对于不表达该抗原的肿瘤细胞则无效。
发明内容
本申请提供了一种经修饰的细菌及其制备方法和应用，所述经修饰的细菌具有下述优点中的一种或多种：(1)本申请提供了一种经修饰的细菌，可以为灭活细菌，其表面的金属化合物可通过分解为离子状态进行代谢，具有更好的安全性；(2)本申请提供的经修饰的细菌，其表面修饰的不溶性金属化合物具有中和肿瘤弱酸性微环境的作用，能够提高免疫细胞在肿瘤部位的活性，降低肿瘤的耐药性，提高疗效；(3)本申请提供的经修饰的细菌能够通过多通路的刺激激活免疫细胞、诱导强烈的抗肿瘤免疫反应、并产生免疫记忆效应降低癌症转移和复发概率，是一种新型免疫激动剂。
一方面，本申请提供一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物。
另一方面，本申请提供一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物通过沉积反应修饰于所述细菌本体的表面形成附着层。
另一方面，本申请提供一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物在每1*10 8个细菌上的含量为约0.1μg～600μg。
在某些实施方式中，所述细菌本体为革兰氏阳性菌或革兰氏阴性菌。
在某些实施方式中，所述细菌本体选自球菌、杆菌和螺旋菌中的一种或多种。
在某些实施方式中，所述球菌选自金黄色葡萄球菌、脲微球菌、肺炎双球菌、肺炎链球菌、脑膜炎双球菌、化脓性链球菌、肺炎链球菌、无乳链球菌、金黄色葡萄球菌、白色葡萄球菌和柠檬色葡萄球菌中的一种或多种。
在某些实施方式中，所述杆菌选自大肠杆菌、沙门氏菌、鼠疫杆菌、猪痢疾杆菌、多杀性巴氏杆菌、白喉棒状杆菌、结核分枝杆菌、双歧乳杆菌、醋酸杆菌、棒状杆菌中的一种或多种。
在某些实施方式中，所述螺旋菌选自幽门螺杆菌、逗号弧菌和霍乱弧菌中的一种或多种。
在某些实施方式中，所述细菌本体选自沙门氏菌、金黄色葡萄球菌、大肠杆菌、乳酸杆菌、沙门氏菌减毒株、金黄色葡萄球菌减毒株、大肠杆菌减毒株和乳酸杆菌减毒株中的一种或多种。
在某些实施方式中，所述细菌本体为野生型菌、基因工程菌和/或减毒株。
在某些实施方式中，所述细菌本体为活体细菌或灭活细菌。
在某些实施方式中，所述活体细菌或灭活细菌包含减毒细菌。
在某些实施方式中，所述金属化合物通过物理结合或化学结合的方式修饰于所述细菌本体的表面形成附着层。
在某些实施方式中，所述物理结合包括静电吸附和/或部分嵌入。
在某些实施方式中，所述化学结合包括偶联、生成化学键作用和/或以配合形式结合。
在某些实施方式中，所述金属化合物通过沉积反应修饰于所述细菌本体的表面形成附着层。
在某些实施方式中，所述金属化合物通过生物矿化反应修饰于所述细菌本体的表面形成附着层。
在某些实施方式中，所述生物矿化反应包括：金属离子与细菌的细胞膜上的生物大分子或细胞壁上的生物大分子结合，提供矿化位点，调节pH或引入其他盐，金属离子在矿化位点生成金属化合物，不断生长积累，结合在细菌表面。
在某些实施方式中，所述生物矿化反应形成生物大分子和金属化合物的结合位点，利用沉积反应或生物矿化进一步增加金属化合物的体积。
在某些实施方式中，所述金属化合物在细菌表面的覆盖度为0.1％-99.9％。
在某些实施方式中，所述金属化合物在细菌表面的覆盖度通过调节投料比、反应温度和/或反应时间调整。
在某些实施方式中，所述金属化合物的金属元素选自锌、钙、铜、铁、锰和镁中的一种或多种；非金属成分选自碳酸根、氢氧根、硫离子和磷酸根中的一种或多种。
在某些实施方式中，所述金属化合物选自碳酸锌、碳酸钙、碳酸铜、碳酸镁；氢氧化锌、氢氧化铁、氢氧化铜、氢氧化锰、氢氧化镁、硫化锌、硫化铜、硫化锰；磷酸锌、磷酸钙、磷酸铜、磷酸铁、磷酸镁和磷酸锰中的一种或多种。
在某些实施方式中，所述金属化合物为含锰化合物。
在某些实施方式中，所述含锰化合物为氢氧化锰、二氧化锰和硫化锰中的一种或多种。在某些实施方式中，所述金属化合物为氢氧化锰和/或二氧化锰。
另一方面，本申请提供一种锰化细菌，其包括细菌本体和附着于所述细菌本体表面的难溶性或微溶性含锰化合物。
在某些实施方式中，所述难溶性或微溶性含锰化合物为氢氧化锰、二氧化锰和硫化锰中的一种或多种。
在某些实施方式中，所述细菌本体为革兰氏阳性菌或革兰氏阴性菌。
在某些实施方式中，所述细菌本体选自球菌、杆菌和/或螺旋菌。
在某些实施方式中，所述球菌选自金黄色葡萄球菌、脲微球菌、肺炎双球菌、肺炎链球菌、脑膜炎双球菌、化脓性链球菌、肺炎链球菌、无乳链球菌、金黄色葡萄球菌、白色葡萄球菌和柠檬色葡萄球菌中的一种或多种。
在某些实施方式中，所述杆菌选自大肠杆菌、沙门氏菌、鼠疫杆菌、猪痢疾杆菌、多杀性巴氏杆菌、白喉棒状杆菌、结核分枝杆菌、双歧乳杆菌、醋酸杆菌和棒状杆菌中的一种或多种。
在某些实施方式中，所述螺旋菌选自幽门螺杆菌、逗号弧菌和霍乱弧菌中的一种或多种。
在某些实施方式中，所述细菌本体为活体细菌或灭活细菌。
在某些实施方式中，所述细菌本体为野生型菌、基因工程菌和/或减毒株。
另一方面，本申请提供组合物，其包含所述经修饰的细菌或所述的锰化细菌，以及任选地药学上可接受的载剂，所述经修饰的细菌包括细菌本体和修饰于所述细菌本体表面的难溶性或不溶性的生物学可接受的金属化合物，且所述组合物的pH值为0-14。
另一方面，本申请提供了一种经修饰的细菌冻干粉，其包括所述经修饰的细菌，以及添加剂。
在某些实施方式中，所述添加剂包括冻干保护剂和/或赋形剂。
在某些实施方式中，所述冻干保护剂选自糖类/多元醇、聚合物、无水溶剂、表面活性剂、氨基酸、盐和胺中的一种或多种。
在某些实施方式中，所述赋形剂选自黏合剂、填充剂、崩解剂、润滑剂、酒、醋、药汁等、软膏剂基质、霜剂基质、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂中的一种或多种。
在某些实施方式中，所述经修饰的细菌冻干粉中添加剂的质量分数为0.1-99％。此处质量分数的定义为冻干保护剂在冻干后的产品中的质量分数。
在某些实施方式中，所述冻干保护剂选自蔗糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-β环糊精中的一种或多种。
在某些实施方式中，所述细菌冻干粉中冻干保护剂的质量分数为0.1-99％。此处质量分数的定义为冻干保护剂在冻干后的产品中的质量分数。
另一方面，本申请提供了一种锰化细菌冻干粉，其包括所述锰化细菌，以及添加剂。
在某些实施方式中，所述添加剂包括冻干保护剂和/或赋形剂。
在某些实施方式中，所述冻干保护剂选自糖类/多元醇、聚合物、无水溶剂、表面活性剂、氨基酸、盐和胺中的一种或多种。
在某些实施方式中，所述赋形剂选自黏合剂、填充剂、崩解剂、润滑剂、酒、醋、药汁等、软膏剂基质、霜剂基质、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂中的一种或多种。
在某些实施方式中，所述细菌冻干粉中添加剂的质量分数为0.1-99％。此处质量分数的定义为冻干保护剂在冻干后的产品中的质量分数。
在某些实施方式中，所述冻干保护剂选自蔗糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-β环糊精中的一种或多种。
在某些实施方式中，所述细菌冻干粉中冻干保护剂的质量分数为0.1-99％。此处质量分数的定义为冻干保护剂在冻干后的产品中的质量分数。
另一方面，本申请提供了一种所述经修饰的细菌或所述锰化细菌的制备方法，其包括以下步骤：S1：制备细菌混悬液；S2：向所述细菌混悬液中加入可溶性金属盐溶液。
在某些实施方式中，所述制备方法还包括步骤S3：加入易溶性的氢氧化物水溶液、碳酸盐水溶液或磷酸盐水溶液至pH为8-12，或加入易溶性的硫化盐水溶液，反应制得所述经修饰的细菌。
在某些实施方式中，所述制备方法中易溶性的硫化盐水溶液包括硫化钠溶液、硫化钾溶液和/或硫化铵溶液
在某些实施方式中，所述制备方法中易溶性的氢氧化物水溶液、碳酸盐水溶液或磷酸盐水溶液选自：氢氧化钠水溶液、碳酸钠水溶液、磷酸钠水溶液。
在某些实施方式中，在所述制备方法的步骤S2中，按照每10亿个的细菌，添加0.2-13.5mmol的金属离子的加入量，向所述细菌混悬液中加入可溶性金属盐溶液。
在某些实施方式中，所述制备方法中所述可溶性金属盐溶液为高锰酸盐溶液。
在某些实施方式中，所述制备方法的步骤S2包括向所述细菌混悬液中加入高锰酸盐溶液，搅拌反应，离心得到锰化细菌。
在某些实施方式中，所述制备方法中，在每10亿个细菌，添加0.2-3μmol的高锰酸盐。
另一方面，本申请提供了所述经修饰的细菌、所述锰化细菌、所述经修饰的细菌冻干粉、所述锰化细菌冻干粉、或所述组合物在制备肿瘤免疫治疗制剂中的应用。
另一方面，本申请提供了所述经修饰的细菌、所述锰化细菌、所述经修饰的细菌冻干粉、 所述锰化细菌冻干粉、或所述组合物，其用于预防和/或治疗肿瘤。
另一方面，本申请还提供了一种预防和/或治疗肿瘤的方法，其包括向有需要的受试者施用有效量的所述经修饰的细菌、所述锰化细菌、所述经修饰的细菌冻干粉、所述锰化细菌冻干粉、或所述组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1为附着不同金属化合物的经修饰的灭活细菌扫描电镜图片；
图2为不同细菌经二氧化锰修饰前后的扫描电镜图片；
图3为CT26细胞与不同样品共孵育12小时后的相对活性统计图；
图4为骨髓来源的树突状细胞与不同样品共孵育后流式检测成熟细胞比例统计图；
图5为经不同金属化合物修饰的细菌治疗小鼠肿瘤模型后，小鼠的肿瘤生长曲线图。
图6为报告细胞与不同样品孵育24h后，不同组别中STING通路激活后，报告基因表达的生物发光信号强度统计图；
图7为小鼠结肠癌皮下肿瘤模型被经二氧化锰修饰的灭活沙门氏菌治愈后第60天再次接种同种肿瘤，小鼠的生存曲线图。
图8为不同细菌在锰化后的XPS谱图；
图9为DC2.4细胞与Cy5.5标记的细菌(F.S)或Cy5.5标记的二氧化锰锰化细菌(MnO 2@F.S)共孵育后不同时间点，流式检测的细胞内Cy5.5信号强度统计图；
图10为DC2.4细胞与二氧化锰(MnO 2)或二氧化锰锰化细菌(MnO 2@F.S)共孵育后细胞中吞噬的锰离子的含量统计图；
图11为骨髓来源树突状细胞(BMDCs)与不同样品共孵育12h后检测到的成熟树突状细胞的比例统计图；
图12为骨髓来源树突状细胞(BMDCs)与不同样品共孵育12h后检测到的培养基上清中各种细胞因子的浓度统计图；
图13为腹腔巨噬细胞与不同样品共孵育后培养基中不同细胞因子的浓度统计图；
图14为报告细胞与不同样品孵育24h后，干扰素调节因子(IRF3)、磷酸化干扰素调节因子(p-IRF3)的免疫印迹图及对应的IRF3磷酸化比值统计图；
图15为Toll样受体4报告细胞与不同样品孵育24h后，Toll样受体4激活后下游荧光信号强度统计图；
图16为小鼠结肠癌CT26肿瘤模型接受不同佐剂联合灭活细菌治疗后的肿瘤生长曲线图；
图17为小鼠结肠癌CT26肿瘤模型接受不同佐剂联合灭活细菌治疗后的体重变化曲线图；
图18为肿瘤部位注射Cy5.5标记的细菌(F.S)或Cy5.5标记的二氧化锰锰化细菌(MnO 2@F.S)后24h，流式检测的肿瘤部位CD45阳性细胞内Cy5.5信号强度统计图；
图19为小鼠瘤内注射不同样品后24h，锰离子在主要器官及肿瘤组织中的分布情况；
图20为瘤内注射不同样品后，不同时间点肿瘤内部氧气含量的统计值；
图21为小鼠肿瘤模型接受不同治疗后瘤周淋巴结内的DC细胞成熟比例统计图；
图22为小鼠接受肿瘤治疗后24h，肿瘤内部浸润的免疫细胞百分比统计图；
图23为小鼠接受肿瘤治疗后24h，肿瘤内部细胞因子含量统计图；
图24为小鼠接受肿瘤治疗后第5天，肿瘤内部浸润的免疫细胞比例统计图；
图25为经NK细胞阻断抗体处理后的小鼠及正常小鼠B16-OVA肿瘤模型接受治疗后的肿瘤生长曲线图；
图26为经T细胞阻断抗体处理后的小鼠及正常小鼠CT26肿瘤模型接受治疗后的肿瘤生长曲线图；
图27为不同肿瘤模型(4T1乳腺癌肿瘤、B16-OVA黑色素瘤、KPC胰腺癌)经过治疗后的肿瘤生长曲线图；
图28为小鼠CT26肿瘤模型经不同种锰化细菌治疗后的肿瘤生长曲线图；
图29为小鼠4T1肿瘤模型经不同种锰化细菌治疗后的肿瘤生长曲线图；
图30为不同硫酸锰/细菌投料比例制备的锰化细菌对小鼠肿瘤模型进行治疗，肿瘤生长曲线图；
图31为新西兰兔肝癌模型经锰化细菌治疗后的肿瘤生长曲线图；
图32为小鼠双侧瘤模型接受锰化细菌治疗后的肿瘤生长曲线图；
图33为小鼠CT26肿瘤模型被锰化细菌治愈后第60天，外周血中的记忆T细胞的比例统计图；
图34为小鼠CT26肿瘤模型被锰化细菌治愈后第60天再次接种肿瘤，肿瘤体积变化曲线图；
图35为以硫酸锰为原料制备的锰化灭活细菌的扫描电镜图；
图36为表面附着硫化锰的锰化灭活细菌的扫描电镜图；
图37为以高锰酸钾为原料制备的锰化灭活细菌的扫描电镜图；
图38为不同投料比得到的锰化细菌的扫描电镜图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“经修饰的”通常指与自然状态(或野生型)相比经过某些改造，例如可以是人工改造。例如，改造可以是物理手段、化学手段和/或生物学手段的改造。例如，所述改造可以是一个方面或多个方面的改造。
在本申请中，术语“难溶性的”或“难溶的”可以与“微溶性的”或“微溶的”互换使用，通常指在室温(20度)下，溶剂中的溶解度较低，例如，在溶剂中的溶解度在约0.01g～1g/100g或更低的溶解度。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本申请中，术语“经修饰的细菌”除了包含细菌本体表面包含生物学可接受的金属化合物的修饰，还可进一步包含经其他方式方法改造的细菌。例如，所述细菌可以经基因修饰的细菌。例如，所述细菌本体表面还可包含其他物质。例如，所述细菌可以为减毒菌株。
在本申请中，术语“组合物”通常指除包含活性成分外，还可包含非活性成分的组合物。例如，所述非活性成分可包含一种或多种(药学上可接受的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下一般对接受者无毒或毒副作用较小。
在本申请中，术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“受试者”通常是指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴等。
在本申请中，术语“肿瘤”通常是指赘生性或恶性细胞生长。本申请的肿瘤可能是良性的，也可能是恶性的。本申请的肿瘤可能是实体的，也可能是非实体的。
发明详述
经修饰的细菌
本申请提供了一种经修饰的细菌及其制备方法和应用。本申请将金属离子与活细菌或灭活细菌(起初使用减毒沙门氏菌)混合后调节溶液pH值到弱碱性，会导致不溶或难溶的金属离子化合物在细菌的表面附着，形成经修饰的细菌，通过瘤内注射的方式，将经修饰的细菌注射至小鼠肿瘤模型内部，能够达到抑制肿瘤生长的效果，经过进一步的机制研究发现，经修饰的细菌经原位注射后引起了机体的抗肿瘤免疫反应，从而达到肿瘤治疗的效果。当二价金属离子置于碱性条件时，极易形成氢氧化物并析出，细菌表面作为一个典型的固液界面，可提供析出物的成核中心，进一步促使氢氧化物的析出，当氢氧化物析出在细菌表面后，再次转化为更加稳定的氧化物，随着氢氧化物的不断析出、转化，细菌表面形成氧化物附着层，得到金属化合物修饰的灭活细菌。本申请所述沉积反应，即一些金属离子盐与细菌等混合后，引入相应的阴离子，在合适的pH条件下，金属离子与阴离子形成难溶或不溶的金属化合物并在细菌表面附着，在充分搅拌的条件下，目标产物可以在液体中的细菌表面上连续沉降，最终，附着有金属化合物的细菌形成均匀稳定的混悬液。
进一步研究发现，除了减毒沙门氏菌之外，这一技术也可以用于其他类型的细菌，包括金黄色葡萄球菌，大肠杆菌和乳酸杆菌等。利用上述细菌基于同样方法制备的经修饰的细菌同样可以激活免疫系统获得不错的抗肿瘤活性。
进一步探究金属氢氧化物或金属氧化物外其他难溶或不溶性金属化合物，在溶解度降低时，是否能在细菌表面形成附着层并达到相似的免疫刺激结果。通过尝试多种金属化合物附着的灭活细菌的合成，发现部分金属化合物结合在细菌表面后，同样能够激活免疫细胞、诱 导强烈的抗肿瘤免疫反应，并可能产生免疫记忆效应，降低癌症转移和复发的概率。
在本申请中，所述经修饰的细菌，包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物。
在本申请中，所述细菌本体可选自沙门氏菌、金黄色葡萄球菌、大肠杆菌、乳酸杆菌、沙门氏菌减毒株、金黄色葡萄球菌减毒株、大肠杆菌减毒株和乳酸杆菌减毒株中的一种或多种。
在本申请中，所述细菌本体可为活体细菌或灭活细菌，例如，所述细菌本体为灭活细菌，所述活体细菌和灭活细菌均可包含减毒细菌。
在本申请中，所述金属化合物可通过沉积反应修饰于所述细菌本体的表面形成附着层。
在本申请中，所述金属化合物的金属元素可选自锌、钙、铜、铁、锰和镁中的一种或多种；非金属成分可选自碳酸根、氢氧根、硫离子和磷酸根中的一种或多种。
在本申请中，所述金属化合物可选自碳酸锌、碳酸钙、碳酸铜、碳酸镁；氢氧化锌、氢氧化铁、氢氧化铜、氢氧化锰、氢氧化镁、硫化锌、硫化铜、硫化锰；磷酸锌、磷酸钙、磷酸铜、磷酸铁、磷酸镁和磷酸锰中的一种或多种。
本申请还提供了上述经修饰的细菌的制备方法，包括以下步骤，
S1：制备细菌混悬液；
S2：向所述细菌混悬液中加入可溶性金属盐溶液；
S3：加入易溶性的氢氧化物水溶液、碳酸盐水溶液或磷酸盐水溶液至pH为8-12(例如，至pH为约8，至pH为约9，至pH为约10，至pH为约11或至pH为约12)，或加入易溶性的硫化盐水溶液，反应制得所述经修饰的细菌。
具体的，所述步骤S2中，
可加入易溶性的氢氧化物水溶液至pH为8-12(例如，至pH为约8，至pH为约9，至pH为约10，至pH为约11或至pH为约12)，反应后离心得到表面附着金属氢氧化物或金属氧化物的经修饰的细菌；易溶性的氢氧化物水溶液优选为氢氧化钠水溶液；
可加入易溶性的碳酸盐水溶液至pH为8-12(例如，至pH为约8，至pH为约9，至pH为约10，至pH为约11或至pH为约12)，反应后离心得到表面附着金属碳酸盐的经修饰的细菌；易溶性的碳酸盐水溶液优选为碳酸钠水溶液；
可加入易溶性的磷酸盐水溶液至pH为8-12(例如，至pH为约8，至pH为约9，至pH为约10，至pH为约11或至pH为约12)，反应后离心得到表面附着金属磷酸盐的经修饰的细菌；易溶性的磷酸盐水溶液优选为磷酸钠水溶液；
可加入易溶性的硫化盐水溶液，反应后离心得到表面附着金属硫化物的经修饰的细菌；易溶性的硫化盐水溶液优选为硫化钠水溶液；
进一步的，所述步骤S2中，可按照每10亿个的细菌，添加0.2-13.5mmol的金属离子的加入量，向所述细菌混悬液中加入可溶性金属盐溶液。例如，可按照每10亿个的细菌，添加约0.2mmol、约0.5mmol、约1.0mmol、约2.0mmol、约3.0mmol、约4.0mmol、约5.0mmol、约6.0mmol、约7.0mmol、约8.0mmol、约9.0mmol、约10.0mmol、约11.0mmol、约12.0mmol、约13.0mmol、或约13.5mmol的金属离子的加入量，向所述细菌混悬液中加入可溶性金属盐溶液。
本申请还提供了一种经修饰的细菌冻干粉，包括冻干保护剂和上述经修饰的细菌。
在本申请中，所述冻干保护剂可选自蔗糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖和2-羟丙基-β环糊精中的一种或多种。
进一步的，所述细菌冻干粉中在进行冻干处理前，冻干保护剂在样品混悬液中的质量/体积分数为0.1-20％，例如为1-5％。例如，为约0.1％，约1％，约2％，约3％，约4％，5％，约6％，约7％，约8％，约9％，约10％，约11％，约12％，约13％，约14％，约15％，约16％，约17％，约18％，约19％，约20％的质量/体积分数。
本申请还提供了上述经修饰的细菌或上述经修饰的细菌冻干粉，在制备肿瘤治疗制剂中的应用。
在本申请中，还提供了一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物通过沉积反应修饰于所述细菌本体的表面形成附着层。
在本申请中，还提供了一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物在每1*10 8个细菌上的含量为约0.1μg～600μg。例如，所述金属化合物在每1*10 8个细菌上的含量为约0.1μg、约1μg、约5μg、约10μg、约20μg、约30μg、约40μg、约50μg、约60μg、约70μg、约80μg、约90μg、约100μg、约150μg、约200μg、约250μg、约300μg、约350μg、约400μg、约450μg、约500μg、约550μg、或约600μg。
在本申请中，还提供了组合物，其包含所述经修饰的细菌或所述的锰化细菌，以及任选地药学上可接受的载剂，所述经修饰的细菌包括细菌本体和修饰于所述细菌本体表面的难溶性或不溶性的生物学可接受的金属化合物，且所述组合物的pH值为0-14。例如，所述组合物的pH值为约1，2，3，4，5，6，7，8，9，10，11，12，13或14。在本申请中，还提供了 一种所述经修饰的细菌或所述锰化细菌的制备方法，其包括以下步骤：S1：制备细菌混悬液；S2：向所述细菌混悬液中加入可溶性金属盐溶液。
锰化细菌
作为其中一个举例，本申请将锰离子与活细菌或灭活细菌混合，发现通过调节溶液pH值到弱碱性会导致在细菌表面生长微米或纳米级锰的化合物，将该过程定义为微生物的锰化过程，并将该过程中获得的微生物本体与锰的化合物的组合定义为锰化细菌。例如，当二价锰离子置于碱性条件时，极易形成氢氧化锰并析出，而细菌在此溶液条件下带有负电荷，细菌表面作为一个典型的固液界面，可为析出物提供成核中心，特别是细菌表面一些微结构可进一步促使氢氧化锰析出，当氢氧化锰析出在细菌表面后，在溶解氧的作用下再次转化为更加稳定的二氧化锰，随着氢氧化锰的不断析出、转化，细菌表面形成二氧化锰附着层，得到锰化细菌。
将表面生长了微米或纳米二氧化锰的锰化细菌注射到各种不同模型的实体肿瘤内部，可以发现，注射了锰化细菌的肿瘤生长会受到极为有效的抑制，部分肿瘤甚至完全消融；更令人意外的是，这种表面生长二氧化锰的生物锰化灭活细菌的疗效远远强于单独的二氧化锰、灭活细菌、以及二氧化锰和细菌的混合物，而且没有明显的毒副作用。与背景技术中CN201910319344.1、CN201710795715.4相比，其显著特点是在本申请的体系中没有引入肿瘤相关抗原，产生的抗肿瘤反应也不受肿瘤抗原类别的限制，对各种类型的肿瘤均有效。
基于上述发现，进一步研究锰化细菌抑制肿瘤的机制：通过实验分析发现，这种表面负载二氧化锰的细菌能够被体内免疫细胞同时一体吞噬，进入免疫细胞的锰离子会刺激免疫细胞分泌相关细胞因子，招募更多的免疫细胞到肿瘤部位，并促进更多的锰化细菌被一体吞噬，进一步加强免疫刺激效率，形成一个“正反馈”的过程。与灭活细菌和二氧化锰简单混合体系相比，这种锰化细菌结构一体化带来的吞噬效率和刺激效率的显著提升，使得免疫细胞可以同时吞噬细菌和二氧化锰，以致“二氧化锰锰化细菌”比“二氧化锰和灭活细菌的混合物”具有显著增强的免疫刺激效果。进一步研究显示，锰化细菌注射到肿瘤内部后，首先激活天然免疫，招募天然杀伤细胞(natural killer cell，NK细胞)、巨噬细胞等到肿瘤部位，通过非特异免疫反应杀灭部分肿瘤细胞，暴露肿瘤相关抗原；随后由二氧化锰逐渐降解缓释的锰离子可以刺激STING通路，灭活细菌本体则可以刺激Toll样受体(TLR)，两方面同时作用可以更有效激活抗原呈递细胞如树突状细胞(DC)，而DC则将肿瘤抗原呈递给T细胞，激活T细胞介导的获得性免疫(肿瘤特异性免疫反应)。肿瘤特异性T细胞迁移到全身(包括远端肿瘤内)，抑制肿瘤转移，并产生免疫记忆细胞，抑制肿瘤复发。与此同时，二氧化锰催化肿 瘤内过氧化氢分解产生氧气，改善肿瘤乏氧，逆转免疫抑制的肿瘤微环境，对提升免疫治疗效果也有帮助。
进一步研究发现，除了减毒沙门氏菌之外，这一技术也可以用于其他类型的细菌，包括金黄色葡萄球菌，大肠杆菌和乳酸杆菌等，均能达到相似的效果。利用上述细菌基于同样方法制备的锰化细菌同样可以通过激活免疫系统获得不错的抗肿瘤活性。此外，根据锰化合物的性质，可通过不同的方法得到表面生长不同锰化合物的锰化细菌；具体地，可以利用氧化和还原两种思路，得到表面生长二氧化锰的锰化细菌；或者可以通过加入硫化物，将可溶性锰盐转化为不溶的硫化锰，在细菌表面生长，得到表面生长硫化锰的锰化细菌。
本申请所述锰化细菌的有益效果在于：
(1)本申请提供的锰化细菌，对肿瘤内的乏氧和弱酸性微环境具有调节功能，同时通过STING和Toll样受体(TLR)的通路刺激宿主免疫系统，先后激活先天性免疫反应和获得性免疫反应，产生强有效的抗肿瘤免疫效应，适用于多种类型的肿瘤；同时，该技术因为可以采用灭活后的细菌，故该锰化灭活细菌具有极高的安全性，在高剂量下小鼠耐受性好，预期将具有非常宽的临床应用安全窗口。
(2)本申请提供的锰化细菌，可通过含锰化合物改善肿瘤微环境，能够分解肿瘤病灶中内源性的过氧化氢H 2O 2，显著改善肿瘤部位低氧微环境的同时与灭活细菌共同刺激宿主免疫系统；另一方面，弱碱性锰化细菌对酸性微环境也能够适当调节。由于肿瘤乏氧和微酸环境是导致肿瘤部位免疫抑制的重要原因，含锰化合物对于肿瘤乏氧的改善和微酸的中和可以逆转肿瘤内免疫抑制的微环境，帮助锰化细菌免疫刺激后所募集的免疫细胞向肿瘤内部深处浸润，从而显著增强抗原提呈细胞呈递抗原的能力，上调细胞因子的分泌，强化后续抗肿瘤免疫反应。
(3)本申请提供的锰化细菌，通过结构一体化带来了吞噬效率和刺激效率的显著提升，并使得免疫细胞可以同时吞噬细菌和含锰化合物，从而使得“锰化细菌”比“含锰化合物和灭活细菌的混合物”具有显著增强的免疫刺激效果。进一步研究显示，锰化细菌注射到肿瘤内部后，首先激活天然免疫，招募天然杀伤细胞(NK细胞)、巨噬细胞等到肿瘤部位，通过非特异免疫反应杀灭部分肿瘤细胞，暴露肿瘤相关抗原；随后从含锰化合物缓释的锰离子可以刺激STING通路，灭活细菌本身则可以刺激Toll样受体(TLR)通路，两个方面结合可能更有效激活抗原呈递细胞如树突状细胞(DC)，而DC则可将肿瘤抗原呈递给T细胞，激活T细胞介导的获得性免疫(肿瘤特异性免疫反应)。肿瘤特异性T细胞迁移到全身(包括远端肿瘤内)，抑制肿瘤转移，并产生免疫记忆细胞，抑制肿瘤复发。溶瘤效果远远强于单独的含 锰化合物、灭活细菌、以及含锰化合物和细菌的混合物，而且没有明显的毒副作用。
在本申请中，所述锰化细菌，包括细菌本体和附着于所述细菌本体表面的难溶性或微溶性含锰化合物。
进一步的，所述细菌本体可为革兰氏阳性菌或革兰氏阴性菌。
具体地，所述细菌本体可为沙门氏菌、金黄色葡萄球菌、大肠杆菌和乳酸杆菌中的一种或多种。
进一步的，所述难溶性或微溶性含锰化合物可为氢氧化锰、二氧化锰和硫化锰中的一种或多种，优选为二氧化锰。
具体的，所述难溶性或微溶性含锰化合物可为氢氧化锰、二氧化锰、硫化锰或者氢氧化锰与二氧化锰二者并存。
进一步的，所述细菌本体可为活体细菌或灭活细菌，例如为灭活细菌，所述活体细菌和灭活细菌均包含减毒细菌。
本申请还提供了一种锰化细菌冻干粉，包括上述任一锰化细菌和冻干保护剂；所述冻干保护剂为蔗糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-beta-环糊精中的至少一种。
进一步地，所述冻干保护剂在进行冻干处理前的所述细菌冻干粉中的质量/体积分数可为0.1％-20％。
本申请还提供了一种锰化细菌的制备方法，包括以下步骤，
S1：制备细菌混悬液；
S2：加入锰盐溶液，调节pH为弱碱性8-12(pH为8-12，例如pH区间10-11)，反应1-2小时，离心得到表面包裹氢氧化锰和/或二氧化锰的锰化细菌。
或S2’：向步骤S1得到的细菌混悬液中加入适量锰盐溶液，加入可溶性硫化盐溶液，反应1-2小时，离心得到表面包裹硫化锰的锰化细菌。
进一步的，所述细菌为活体细菌或灭活细菌，例如为灭活细菌。
进一步的，所述细菌可为革兰氏阳性菌或革兰氏阴性菌。
具体的，pH调节可以采用加入氧化钠溶液实现，可溶性硫化盐溶液包括硫化钠溶液、硫化钾溶液、硫化铵溶液等。
进一步的，每10亿个细菌，可添加0.2μmol-13.5mmol的锰盐。例如，每10亿个的细菌，添加约0.2μmol、约0.5μmol、约1.0μmol、约2.0μmol、约3.0μmol、约4.0μmol、约5.0μmol、约6.0μmol、约7.0μmol、约8.0μmol、约9.0μmol、0.02mmol、约0.05mmol、 约0.1mmol、约0.2mmol、约0.5mmol、约1.0mmol、约2.0mmol、约3.0mmol、约4.0mmol、约5.0mmol、约6.0mmol、约7.0mmol、约8.0mmol、约9.0mmol、约10.0mmol、约11.0mmol、约12.0mmol、约13.0mmol、或约13.5mmol的锰盐。
在本申请中，还提供了一种锰化细菌的制备方法，包括以下步骤，
SS1：制备细菌混悬液；
SS2：加入高锰酸盐溶液，搅拌反应，离心得到所述锰化细菌。
进一步的，所述细菌为活体细菌或灭活细菌，例如为灭活细菌。
进一步的，所述细菌为革兰氏阳性菌或革兰氏阴性菌。
进一步的，每10亿个细菌，添加0.2-3μmol的高锰酸盐。
本申请还提供了上述任一锰化细菌在制备肿瘤免疫治疗制剂中的应用。
本申请还提供了上述任一锰化细菌制备的冻干粉再制备肿瘤免疫治疗制剂中的应用。
细菌本体
在本申请中，所述细菌本体可以为革兰氏阳性菌或革兰氏阴性菌。
在本申请中，所述细菌本体可选自选自球菌、杆菌和螺旋菌中的一种或多种。例如，所述球菌可选自金黄色葡萄球菌、脲微球菌、肺炎双球菌、肺炎链球菌、脑膜炎双球菌、化脓性链球菌、肺炎链球菌、无乳链球菌、金黄色葡萄球菌、白色葡萄球菌和柠檬色葡萄球菌中的一种或多种。例如，所述杆菌可选自大肠杆菌、沙门氏菌、鼠疫杆菌、猪痢疾杆菌、多杀性巴氏杆菌、白喉棒状杆菌、结核分枝杆菌、双歧乳杆菌、醋酸杆菌、棒状杆菌中的一种或多种。例如，所述螺旋菌可选自幽门螺杆菌、逗号弧菌和霍乱弧菌中的一种或多种。
在本申请中，所述细菌本体可选自沙门氏菌、金黄色葡萄球菌、大肠杆菌、乳酸杆菌、沙门氏菌减毒株、金黄色葡萄球菌减毒株、大肠杆菌减毒株和乳酸杆菌减毒株中的一种或多种。
在本申请中，所述细菌本体可以为野生型菌、基因工程菌和/或减毒株。
在本申请中，所述细菌本体可以为活体细菌或灭活细菌。例如，所述活体细菌或灭活细菌可包含减毒细菌。
金属化合物的附着方式
在本申请中，所述金属化合物(例如，含锰化合物)可通过物理结合的方式修饰于所述细菌本体的表面形成附着层。例如，所述物理结合可包括静电吸附。例如，所述物理结合可包括部分嵌入。
在本申请中，所述金属化合物(例如，含锰化合物)可通过化学结合的方式修饰于所述 细菌本体的表面形成附着层。例如，所述化合结合可包括偶联。例如，所述化学结合可包括生成化学键作用。例如，所述化学结合可包括以配合形式结合。
在本申请中，所述金属化合物(例如，含锰化合物)可通过沉积反应修饰于所述细菌本体的表面形成附着层。
在本申请中，所述金属化合物(例如，含锰化合物)可通过生物矿化反应修饰于所述细菌本体的表面形成附着层。如本文所用，所述生物矿化反应通常是指金属离子与细菌的细胞膜上的生物大分子或细胞壁上的生物大分子结合，提供矿化位点，调节pH或引入其他盐，金属离子在矿化位点生成金属化合物，不断生长积累，结合在细菌表面。例如，可以通过所述生物矿化反应形成生物大分子和金属化合物的结合位点，利用沉积反应或生物矿化进一步增加金属化合物的体积。
在本申请中，所述金属化合物在细菌表面的覆盖度可为约0.1％-99.9％。在该覆盖度下，细菌能够与生理环境接触，刺激免疫系统。例如，所述金属化合物在细菌表面的覆盖度通过调节投料比、反应温度和/或反应时间调整。
在本申请中，所述金属化合物在细菌表面的覆盖度为(细菌表面积-经修饰的细菌直接与外界接触的面积)/细菌表面积。
经修饰的细菌冻干粉
在本申请中，所述经修饰的细菌冻干粉(例如，锰化细菌冻干粉)可包含经修饰的细菌(例如，锰化细菌)，以及添加剂。
在本申请中，所述添加剂可包括冻干保护剂，例如，所述冻干保护剂可选自糖类/多元醇、聚合物、无水溶剂、表面活性剂、氨基酸、盐和胺中的一种或多种。例如，所述冻干保护剂可选自蔗糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-β环糊精中的一种或多种。
在本申请中，所述经修饰的细菌冻干粉中冻干保护剂的质量分数为0.1-99％。例如，所述冻干保护剂的质量分数为约1％、约2％、约3％、约5％、约8％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约99％。例如，所述经修饰的细菌冻干粉中冻干保护剂的质量分数为0.1-20％。
在本申请中，所述添加剂可包括赋形剂。例如，所述赋形剂可选自黏合剂、填充剂、崩解剂、润滑剂、酒、醋、药汁等、软膏剂基质、霜剂基质、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂中的一种或多种。
在本申请中，所述经修饰的细菌冻干粉中添加剂的质量分数为0.1-99％。例如，所述添加剂的质量分数为约1％、约2％、约3％、约5％、约8％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约99％。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
材料来源
本申请实施例中各菌株来源如下表所示：
Figure PCTCN2022097190-appb-000001
Figure PCTCN2022097190-appb-000002
本申请实施例中各细胞的来源如下表所示：
Figure PCTCN2022097190-appb-000003
实施例中使用的抑瘤率计算公式为：抑瘤率＝[(1-某处理组结束治疗时平均瘤体积一该处理组开始治疗时平均瘤体积)/(对照组结束治疗时平均瘤体积-对照组开始治疗时平均瘤体积)]×100％
实施例A：经不同金属化合物修饰的细菌的制备及基本形貌表征
实施例A1：表面经碳酸锌修饰的细菌的制备
S1：制备硫酸锌水溶液；
S2：减毒沙门氏菌(以下简称F.S)经扩增培养后，离心获得菌体并进行清洗，去除菌体中残留的培养基及其他物质，再向菌体中加入多聚甲醛进行细菌的灭活和形貌固定，离心收集固定后的灭活细菌，用无菌生理盐水清洗菌体两次，最后将灭活菌体用生理盐水重悬为浓度为24MCF的灭活细菌混悬液，检测确认菌液中无活细菌，可于低温保存备用；
S3：在灭活细菌混悬液(原始菌液浊度为24MCF，约72亿个细菌/mL，体积为1mL)中加入1mL浓度为10mM的硫酸锌溶液，以及适量生理盐水，室温下短暂搅拌，随后加入碳酸钠溶液，使得pH为8-12，继续搅拌不少于1小时，离心收集沉淀，用无菌生理盐水洗两遍，最后用无菌生理盐水重悬，低温保存，产物为ZnCO 3@F.S。
在实验中，尝试加入不同原始浓度和体积比的细菌混悬液与金属离子盐溶液，探究可形成经金属化合物修饰的细菌的投料比范围，固定反应体系中细菌的总量，通过调节金属离子盐的投料量，探究产物为较稳定混悬液的投料比例，结果发现，当细菌量为10亿个、金属离子的浓度超出13.5mmol时，无论怎样调节反应体系的pH、浓度及反应时间，产物均为不可混悬的沉淀，无法实现后续应用。参与反应的金属离子盐的含量较低时，不影响产物的重新混悬，在实验中，尝试的最低投料比为，每10亿个细菌与0.2μmol的金属离子反应。
实施例A2：表面经氢氧化铁或氢氧化铜修饰的细菌的制备
如实施例A1中S1-S2的方法制备得到铁的可溶性离子盐溶液和灭活细菌混悬液；区别在于：
S3：在灭活细菌混悬液(原始菌液浊度为24MCF，约72亿个细菌/mL，此处加样体积为1mL)中加入10mM的氯化铁或硫酸铜溶液，以及适量生理盐水，室温下短暂搅拌，随后加入氢氧化钠溶液或氨水调节pH至8-12，继续搅拌不少于1小时，离心收集沉淀，用无菌生理盐水洗两遍，最后用生理盐水重悬，低温保存，产物为Fe(OH) 3@F.S、Cu(OH) 2@F.S。
实施例A3：表面附着硫化锰、硫化锌、硫化铜的经修饰的细菌的制备
S1：制备氯化锰、硫酸铜、硫酸锌水溶液，浓度为10mM；
S2：将减毒沙门氏菌进行扩增培养后，清洗获得不含培养基的菌体，不使用甲醛处理细菌，直接使用生理盐水重悬得到活菌的细菌混悬液；
S3：在细菌混悬液(原始菌液浊度为24MCF，约72亿个细菌/mL，此处加样体积为1 mL)中分别加入10mM的氯化锰、硫酸铜、硫酸锌溶液，以及适量生理盐水，室温搅拌，随后加入硫化钠溶液继续搅拌，离心收集沉淀，用无菌生理盐水洗两遍，最后用生理盐水重悬，分别得到MnS@F.S、CuS@F.S、ZnS@F.S，低温保存。
实施例A4：经修饰的细菌的基本表征
将实施例A1-A3中的产物制作扫描电镜样品，用扫描电子显微镜(SEM)表征经不同金属化合物修饰的细菌的表面形貌，结果如图1所示。其中，图片标注ZnCO 3@F.S、Fe(OH) 3@F.S、Cu(OH) 2@F.S、MnS@F.S、ZnS@F.S、CuS@F.S分别表示表面经过碳酸锌、氢氧化铁、氢氧化铜、硫化锰、硫化锌、硫化铜修饰的沙门氏菌的扫描电镜图。结果显示，经金属化合物修饰后的细菌表面均有大量固体附着，且环境中无过多的固体物质残留，并且，根据金属化合物本身晶型的不同，细菌表面附着不同金属化合物后形貌也发生了变化，说明制备过程中，金属化合物依附于细菌表面形成覆盖层。
实施例A5：经修饰的其他细菌的制备
S1：制备浓度为10mM的氯化锰水溶液；
S2：细菌的扩增培养，将50μL的冻存菌液接种在6mL的新鲜肉膏蛋白胨(LB)培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，随后将活化后的菌液稀释涂布在LB固体培养基平板上，置于数显培养箱在37℃中培养12h，挑取状态良好的单菌落，接种至5mL新鲜LB肉汤培养基中，放置于37℃恒温摇床上培养过夜，得到菌液；
S3：取50μLS2所得菌液至20mL新鲜的LB液体培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，离心收集菌体，用无菌生理盐水重悬，再次离心后加入5mL的4％多聚甲醛固定24h，离心收集固定后的灭活细菌，用无菌生理盐水清洗菌体两次，最后将灭活菌体用5-8mL生理盐水重悬，使得最终菌液的浓度为24MCF，即菌液在600nm波长处的吸光值为3，得到灭活细菌混悬液，可于4℃保存，取50μL菌液涂布固体培养基，确认菌液中无活细菌；
S4：在灭活细菌混悬液中加入10mM的氯化锰溶液，以及适量生理盐水，搅拌15min，随后加入NaOH溶液调节溶液pH至10.0-11.0，继续搅拌1h，离心收集沉淀，用无菌生理盐水洗两遍，最后用生理盐水重悬，4℃保存。
将合成的锰化灭活细菌以及未锰化的灭活细菌分别制备扫描电镜(SEM)样品，用扫描电镜对其外观形貌进行表征，得到的电镜图如图2所示，其中F.E表示固定的灭活大肠杆菌，F.SA表示固定的灭活金黄色葡萄球菌，F.S表示固定的灭活沙门氏菌，F.L表示固定的灭活乳 酸杆菌，F.V表示固定的灭活沙门氏菌VNP20009，锰化后的细菌用MnO 2@前缀表示。从图2可以看出，固定后的灭活细菌保持了原本细菌的外观特征，锰化后的灭活细菌表面结合了一层不光滑的沉淀物，细菌本身保持原有的形态和大小，说明锰化过程不破坏细菌本身的形貌，只是在其表面附着了一层物质。
随后对细菌表面附着物质进行表征，用X射线光电子能谱(XPS)技术分析细菌表面元素及其价态，结果如图8所示，不同细菌表面的XPS图谱均具有两个特征峰，分别位于654.2eV和642.4eV左右，分别对应四价锰离子(Mn 4+)的2p1/2和2p3/2自旋轨道能级，结合反应体系中的元素种类和XPS结果，说明锰化灭活细菌表面为二氧化锰而非锰元素的其他形式。
实施例B：细胞实验
实施例B1：CT26细胞(小鼠结肠癌细胞系)与不同样品孵育后的细胞相对活性
将CT26细胞以10 4个/孔的密度接种在96孔板中，37℃细胞培养箱培养过夜，待细胞贴壁且状态良好时加入不同浓度的实施例A1-A3制备得到的不同样品(其中细菌为灭活的减毒沙门氏菌)于37℃进行共孵育。经不同的金属化合物修饰的细菌，其对应的金属化合物浓度为1μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL，不经修饰的细菌组别中，细菌的总量与经修饰的细菌的总量相同。孵育12小时后，移除含有样品的培养基，用PBS洗去多余样品，用MTT检测法检测细胞相对活性，结果如图3所示(每个浓度中的数据从左到右依次为：F.S，MnS@F.S，CuS@F.S，Cu(OH) 2@F.S，ZnS@F.S，Fe(OH) 3@F.S，ZnCO 3@F.S)。在低浓度下，经修饰的细菌没有表现出较高的细胞毒性，与经修饰的细菌共孵育后的细胞仍具有80％以上的活性，随着样品浓度的增加，经修饰的细菌逐渐表现出对细胞相对活性的影响，当浓度达到40μg/mL时，细胞活性仍大于50％。说明经修饰的细菌的安全性与使用浓度相关，在一定浓度范围内具有较高的安全性。
实施例B2：不同样品刺激髓源树突状细胞成熟的实验
DC细胞是机体内高效的抗原呈递细胞，在受到某些因素刺激或摄取抗原后分化为成熟的DC细胞，成熟的DC细胞能够有效激活初始T细胞，诱导细胞毒性T淋巴细胞的生成，并分泌肿瘤坏死因子α(TNF-α)等，在抗肿瘤免疫反应中起到关键作用。因此，能够有效刺激DC细胞成熟的样品可以增强机体的抗肿瘤免疫反应。
从C57BL/6小鼠骨髓中提取骨髓来源的干细胞，加入集落刺激因子(GM-CSF)促进干细胞分化为髓源树突状细胞(Bone Marrow-Derived Dendritic Cells,BMDCs)，将BMDCs与不同经修饰的细菌样品(每份样品中细菌的用量为3.6*10 7个；对应的裸金属化合物含量为1.5-2.7μg，其余不含细菌的组别中，同种金属化合物的用量与该金属化合物修饰的细菌组别中相 同，例如ZnS和ZnS@F.S两组中，含有等量的ZnS)共孵育，检测12小时后不同组别中BMDCs的成熟比率。
BMDCs成熟比例统计结果见图4所示。其中各实验组代表不同物质加入细胞培养基中与BMDCs共孵育，具体物质如下：
blank：空白对照组；
F.S：不经修饰的灭活细菌；ZnS：硫化锌混混悬液；ZnCO 3：碳酸锌混混悬液；Fe(OH) 3：氢氧化铁胶体；CuS：硫化铜混混悬液；Cu(OH) 2：氢氧化铜混混悬液；MnS：硫化锰混混悬液；
ZnS@F.S：硫化锌修饰的沙门氏菌混悬液；ZnCO 3@F.S：碳酸锌修饰的沙门氏菌混悬液；Fe(OH) 3@F.S：氢氧化铁修饰的沙门氏菌混悬液；CuS@F.S：硫化铜修饰的沙门氏菌混悬液；Cu(OH) 2@F.S：氢氧化铜修饰的沙门氏菌混悬液；MnS@F.S：硫化锰修饰的沙门氏菌混悬液。
从图4中可以看出，经修饰的细菌刺激BMDCs的成熟比例均在40％以上，而单纯的金属化合物与BMDCs共孵育后，细胞成熟的比例不超过20％。综合DC细胞成熟比例统计情况，可以得出经修饰的细菌能够更好地刺激BMDCs的成熟，大部分组别的结果与空白对照组(blank)具有显著性差异，个别实验组甚至得到了优于阳性对照组(2μg/mL脂多糖，LPS)的结果，说明本申请经修饰的细菌具有非常优异的刺激DC细胞成熟的能力。LPS是脂多糖，是免疫细胞(包括B细胞，单核细胞，巨噬细胞和其他LPS反应性细胞)的强活化剂，未成熟的DC细胞在这类免疫原性刺激物的刺激下趋向成熟，是体外DC细胞成熟实验中常用的阳性对照物质。
实施例C：动物实验
实施例C1：经不同金属化合物修饰的细菌对小鼠结肠癌肿瘤模型的治疗实验
在BALB/c小鼠背部接种结肠癌肿瘤细胞建立肿瘤模型，待肿瘤大小长至120mm 3左右时，随机进行分组，每组6只，不同组别小鼠接受经不同金属化合物修饰的细菌的治疗。通过瘤内注射的方式进行单次治疗。其中，控制组别间细菌用量一定，均为1.8*10 10个细菌/kg体重，根据每种金属化合物和细菌的结合效率的差异，金属化合物剂量在0.75mg/kg-1.35mg/kg体重，金属离子浓度为300μg/mL-1.08mg/ml。期间记录肿瘤体积变化并制作肿瘤生长曲线，结果如图5所示，同时记录小鼠的存活率、计算抑瘤率，存活率统计情况及抑瘤率计算结果如表1所示。
表1：经不同金属化合物修饰的细菌治疗肿瘤后的小鼠生存率和接种后第19天的抑瘤率
Figure PCTCN2022097190-appb-000004
Figure PCTCN2022097190-appb-000005
图5及表1中的结果显示，与单独的灭活细菌治疗相比，经过不同金属化合物修饰的细菌治疗小鼠肿瘤，其抑瘤率明显提高，说明经不同金属化合物修饰的细菌相较于单独的细菌治疗，具有更好的治疗效果。经某些金属化合物修饰的细菌能够抑制肿瘤的生长，接受附着不同金属化合物的细菌的治疗后，小鼠肿瘤体积增加明显变缓，且生存率明显增加，最高可达66.7％存活，并且抑瘤率基本都达到60％以上。综上，证明使用经本申请中大多数金属化合物修饰的细菌均能够实现肿瘤治疗的效果，而Fe的氢氧化物修饰的细菌(Fe(OH) 3@F.S)未表现出明显的肿瘤抑制效果，可能是因为不同金属化合物发挥疗效的剂量有差异，一次实验中未找到Fe(OH)3@F.S的有效剂量。
实施例D：经修饰的细菌的免疫刺激机制研究
实施例D1：细胞水平上证明经修饰的细菌对STING通路的激活作用
本申请所述的经修饰的细菌具有多通路刺激效果，以锰的化合物(二氧化锰)修饰的沙门氏菌为例设计了本实施例，其他金属离子有其对应的免疫刺激机制。本实施例中使用了含有受STING通路活化调控的报告基因的细胞(STING +)，该细胞的STING通路受到刺激被激活后，会激活报告基因荧光素酶的表达，可催化培养基中的荧光素酶底物发出生物发光信号，STING激活程度越高，生物发光信号越强。将该细胞分别与经二氧化锰修饰的细菌(MnO 2@F.S)、二氧化锰(MnO 2)、沙门氏菌(F.S)、氯化锰(Mn 2+)、阳性对照组(positive control，PC)共孵育，24h后，加入荧光素酶底物后检测生物发光信号强度，再将所有组别的生物发光信号强度分别与PBS组做比值。Null细胞为不表达STING通路的细胞，因此无法通过激活STING通路诱导荧光素酶表达，借助该细胞与不同样品的孵育证明实验中的样品 及试剂本身无法对生物发光信号造成干扰或诱导荧光素酶表达。生物发光信号强度比值统计图如图6所示，与对照组相比，与经修饰的细菌共孵育的细胞中，生物发光信号强度比值更高，说明该组细胞的STING通路被显著激活；与阳性对照组(positive control，PC)相比，生物发光信号强度比值接近，说明其STING激活水平接近，即经修饰的细菌能够有效激活STING通路。
STING(stimulator of interferon genes)是指干扰素基因刺激因子，主要表达于人的巨噬细胞、T淋巴细胞、树突状细胞等的粗面内质网、线粒体及微粒体的外膜上。STING在病毒、细菌及寄生虫感染触发的天然免疫反应、机体的肿瘤免疫过程以及细胞自噬过程中发挥重要的枢纽作用；通过自身的磷酸化、泛素化和二聚化修饰调节蛋白质合成和IFN表达，在机体的多个免疫环节中发挥关键作用。STING是机体抗肿瘤免疫中重要的调节靶点，肿瘤细胞增殖能使抗原提呈细胞中的STING活化，从而激活T细胞介导的适应性免疫过程，发挥抗肿瘤作用。因此，本申请所述的经修饰的细菌能够刺激STING通路激活，说明其具有用于抗肿瘤免疫治疗的潜力。
实施例D2：细胞实验证明经修饰的细菌能够激活TLR4通路
本实施例中使用含有受TLR4通路活化调控的报告基因的细胞(TLR4 +，TLR4阳性细胞)，其TLR4受到刺激被激活后，会激活报告基因荧光素酶的表达，可以催化荧光素酶底物，产生生物发光信号。将该细胞与不同样品共孵育(n＝3)。孵育结束，加入荧光素酶底物后检测生物发光信号强度，再将所有组别的生物发光信号强度分别与PBS组的做比值，可用于判断TLR4的激活程度，比值越高说明TLR4激活程度越高。而TLR 4阴性细胞不表达TLR4，因此不会受到刺激并表达荧光素酶，加入荧光素底物后则不会发生生物发光，用该组细胞证明了实验中使用的所有试剂或样品无法对生物发光信号造成干扰或诱导荧光素酶表达。
不同组别三个平行样的生物发光信号强度比值如表2所示，阳性对照组为3μg/mL的MPLA与细胞共孵育，具有明显的TLR4通路刺激效果。其中经修饰的细菌(MnO 2@F.S)与空白对照组(Blank)相比能够显著刺激TLR4，表现为荧光强度比值大于1；相较于单纯细菌(F.S)，经修饰的细菌荧光强度更强，说明经修饰的细菌能够更好的刺激TLR4通路。说明本申请经修饰的细菌具有类似TLR4激动剂的作用。
TLR4是一种Toll样受体，TLR4的活化能促进DC分泌相关白细胞介素从而增强Th1型免疫应答，有利于抗肿瘤免疫治疗。
表2：不同组别细胞培养基中加入荧光素酶底物后的生物发光强度比值统计表
Figure PCTCN2022097190-appb-000006
Figure PCTCN2022097190-appb-000007
STING和TLR4的激活说明本申请中经修饰的灭活细菌有效激活了天然免疫系统，有助于解除肿瘤微环境的免疫抑制，从而强化机体抗肿瘤免疫反应。综合实施例D1和实施例D2，本申请中经修饰的细菌具有多通路激动剂的效果，同时强化非特异性免疫反应和抗肿瘤免疫反应，有助于增效抗肿瘤免疫治疗。
实施例E：不同细菌经金属化合物修饰后对结肠癌肿瘤模型的治疗实验
分组：
第一组：Blank：空白对照组；
第二组：F.SA：灭活金黄色葡萄球菌；
第三组：F.E：灭活大肠杆菌；
第四组：F.L：灭活乳酸杆菌；
第五组：MnO 2@F.SA：经二氧化锰修饰的灭活金黄色葡萄球菌；
第六组：MnO 2@F.E：经二氧化锰修饰的灭活大肠杆菌；
第七组：MnO 2@F.L经二氧化锰修饰的灭活乳酸杆菌；
第八组：MnO 2@F.S经二氧化锰修饰的灭活沙门氏菌。
根据实施例A5的制备方法将不同种细菌制备成经修饰的细菌，进行小鼠皮下CT26结肠癌模型治疗实验。计算各组在接种后第17天的抑瘤率，空白对照组抑瘤率为0，其他各组别的抑瘤率结果如表3所示，肿瘤生长曲线如图28所示。结果显示，不同种的经修饰的灭活细菌均能够对肿瘤进行很好的抑制，抑瘤率为单独细菌治疗的5倍左右。说明本申请中经修饰的不同种类细菌，均实现了对肿瘤的治疗，细菌种类对经修饰的细菌的疗效不起直接影响。
表3：有无锰的化合物修饰的细菌治疗小鼠实验中，肿瘤接种后第17天各组抑瘤率统计表
Figure PCTCN2022097190-appb-000008
Figure PCTCN2022097190-appb-000009
实施例F：经修饰的细菌治疗肿瘤后产生的机体抗肿瘤免疫记忆效果
为了验证本申请中经修饰的细菌的疫苗效果，建立小鼠结肠癌肿瘤模型，并使用二氧化锰修饰的减毒沙门氏菌(MnO 2@F.S)进行治疗，最终小鼠皮下肿瘤完全消失，实施治疗后第60天，用流式细胞仪分析小鼠外周血中记忆T细胞的比例，被经修饰的细菌治愈肿瘤的小鼠，体内记忆T细胞占外周血白细胞中所有T细胞的平均比例为83.86％，较没有经过任何处理的空白对照组(小鼠体内记忆T细胞占外周血白细胞中所有T细胞的平均含量为59.5％)显著增加，说明本申请中经修饰的细菌能够诱导记忆T细胞的产生，产生免疫记忆效应。
对已经被治愈的小鼠再次接种同种肿瘤细胞，观察小鼠肿瘤生存情况，小鼠存活曲线如图7所示，被经修饰的细菌治愈的小鼠，再次接种肿瘤细胞后，并没有出现明显的癌症复发现象，依然能够有较长的生存期，也就是说，经修饰的细菌能够抑制肿瘤的复发，产生了类似疫苗的效果。
实施例G：经修饰的细菌的冻干保护剂研究
将实施例A1-A3、实施例A5中的得到的经修饰的细菌混悬液与多种添加剂(例如冻干保护剂、赋形剂)混合后进行冻干，观察其冻干后的状态及加入溶剂(水)后是否可重新分散为细菌混悬液，其中硫化锰修饰的沙门氏菌与不同比例冻干保护剂混合后冻干的观察结果记录如表4。其中将可重新分散的样品，其冻干保护剂及比例定义为可用范围，将冻干形貌较好、并且可重新分散为混悬液的样品，冻干后形貌较为规则的样品有助于实际生产、包装和使用的便利性，所使用的冻干保护剂及相应比例设为优选范围。
其中蔗糖、β-环糊精能够使得经修饰的细菌具有较好的冻干效果，除了蔗糖、β环糊精之外，海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖均能够使得样品冻干后实现重悬，并且不发生性质改变，而乳糖、甘露醇则不能够实现有效的冻干保护。实施例中使用的所有添加剂均为商业购买获得。
表4：MnS@F.S的冻干保护条件及冻干后状态记录
Figure PCTCN2022097190-appb-000010
Figure PCTCN2022097190-appb-000011
实施例H
实施例H1：不同投料比的锰化细菌合成
按照实施例A5的合成方法，在S4步骤中，固定灭活细菌的投入量，向灭活细菌混悬液中加入不同质量的氯化锰，观察合成样品的稳定性，由于整个反应在液体环境中进行，产物分散在液相中，因此样品的稳定性主要由产物分散性是否良好表示，如有肉眼可见的沉淀、聚沉，则视为产物分散性不好，反之，如果产物的液体色泽分布均匀、没有沉淀产生，则认为产物分散性良好。结果如表5所示。从产物稳定性来看，当细菌投入量约为10亿细菌时，氯化锰投入量不大于13.5mmol时，均可以得到分散性良好的锰化灭活细菌。
表5加入NaOH后，不同细菌和氯化锰投料量对应的产物稳定性
Figure PCTCN2022097190-appb-000012
Figure PCTCN2022097190-appb-000013
实施例H2：冻干保护剂的选择
取实施例A5产物锰化细菌，根据不同种冻干保护剂的特性，加入不同浓度的冻干保护剂，观察锰化细菌冻干后的复溶情况，筛选合适的冻干保护剂种类及浓度，结果如表6所示。当不加任何冻干保护剂时，冻干后的锰化细菌不能够重新在溶剂(水)中分散，加入适当比例的冻干保护剂后，能够实现良好的冻干复溶效果。表6中所述“质量/体积分数”是指在冻干前的液体制剂中的冻干保护剂在溶液中的质量/体积分数。
表6锰化细菌MnO 2@F.S在不同冻干保护剂条件下的冻干后复溶情况
Figure PCTCN2022097190-appb-000014
Figure PCTCN2022097190-appb-000015
实施例H3：锰化活细菌制备
S1：制备浓度为10mM的氯化锰水溶液；
S2：细菌的扩增培养，将50μL的冻存菌液接种在6mL的新鲜肉膏蛋白胨(LB)培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，随后将活化后的菌液稀释涂布在LB固体培养基平板上，置于数显培养箱在37℃中培养12h，挑取状态良好的单菌落，接种至5mL新鲜LB肉汤培养基中，放置于37℃恒温摇床上培养过夜，得到菌液；
S3：取50μLS2所得菌液至20mL新鲜的LB液体培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，离心收集菌体，用无菌生理盐水重悬，再次离心后用无菌生理盐水清洗菌体两次，最后细菌用5-8mL生理盐水重悬，使得最终菌液的24MCF，菌液在600nm波长处的吸光值为3，得到细菌混悬液；
S4：在细菌混悬液中加入10mM的氯化锰溶液，以及适量生理盐水，搅拌15min，随后加入NaOH溶液调节溶液pH至10.0-11.0，继续搅拌1h，离心收集沉淀，用无菌生理盐水洗两遍，最后用生理盐水重悬，4℃保存。
实施例H4：锰化活细菌制备
制备方法与实施例A5相同，区别仅在于：当pH值调节到8-10，锰化反应时间较实施例A5增加30％～100％，但得到的锰化细菌样品中，细菌表面的锰化物颗粒更细腻。
实施例H5：锰化活细菌制备
制备方法与实施例A5相同，区别仅在于：当pH值调节到11-13，随着pH的增加，锰化反应时间缩短，但pH高于13后，会导致细菌形貌被破坏，不利于最终产物的均一性。
实施例I
实施例I1：锰化细菌对细胞活性的影响
将CT26肿瘤细胞以10 4个/孔的密度接种在96孔板中，37℃细胞培养箱培养过夜，待细胞贴壁且状态良好时加入不同浓度的不同样品进行共孵育，各组别中添加的样品及其浓度如表7所示，孵育24h后，移除含有样品的培养基，用PBS洗去多余样品，用MTT检测法检测细胞相对活性，结果如表7所示，在细胞实验所用2μg/mL二氧化锰和/或0.12MCF(麦氏单位，表示细菌浊度，1麦氏单位约为3亿个细菌)约0.36亿个灭活沙门氏菌的浓度下，所有处理组均未见明显毒性，细胞活力都接近百分之百，这显示了本申请中各样品良好的安全性。继续增加浓度至细胞实验的10倍剂量(20μg/mL二氧化锰)后，MnO 2处理组和MnO 2@F.S组开始出现少量细胞毒性，而F.S组仍没有毒性。此外，实验结果显示与MnO 2@F.S相比，MnO 2&F.S处理组在5μg/mL的浓度下便显示出极大的毒性，只有65.3％的细胞活力，这进一步证明将二氧化锰生长在细菌表面可以有效减少二氧化锰带来的细胞毒性，锰化细菌(MnO 2@F.S)整体的安全性比同等剂量的细菌与二氧化锰的混合物更好。
表7细胞相对活性的检测
Figure PCTCN2022097190-appb-000016
实施例I2：锰化灭活细菌促进DC2.4的吞噬作用
用Cy5.5对细菌进行标记，得到带有荧光信号的灭活细菌及锰化灭活细菌。将状态良好 的DC2.4细胞与带有荧光信号的灭活细菌或锰化灭活细菌共孵育一段时间，其中样品用锰离子的浓度定量，样品的终浓度为2μg/mL，DC2.4细胞会吞噬灭活细菌和锰化灭活细菌，在不同的时间点，用流式细胞仪对DC2.4细胞的荧光信号强度进行检测，得到的结果如图9所示。谱图横坐标为荧光强度，峰面积为细胞分布密度，统计图为平均荧光强度的统计值。与空白细胞相比，DC2.4细胞与荧光标记的锰化细菌共孵育后能够被流式细胞仪检出荧光信号，根据统计图，可以发现锰化灭活细菌与DC2.4细胞共孵育后，DC2.4细胞吞噬的锰化灭活细菌的量显著大于未锰化的灭活细菌，说明表面生长了二氧化锰的灭活细菌能够被更多地吞噬。
将DC2.4细胞与二氧化锰或锰化灭活细菌(相同浓度的锰元素)共同孵育，在共孵育12h后，用ICP检测细胞中锰离子含量占总投入锰离子含量的百分比，从而观察细胞摄入锰的含量变化，统计值如图10所示。结果显示，二氧化锰生长在细菌表面后能更多地被细胞吞噬，在同样的时间里，与锰化灭活细菌共孵育的DC2.4细胞内锰含量明显高于与二氧化锰共孵育的DC2.4细胞。上述实验结果说明与单独二氧化锰相比，锰化灭活细菌能够更有效地被DC2.4细胞吞噬。
DC2.4是小鼠骨髓来源的树突状细胞系，用于体外水平研究药物与DC细胞的相互作用。DC细胞是机体内功能最强的专职抗原呈递细胞，它的功能是吞噬、加工及呈递抗原，吞噬功能增强，有助于DC细胞更多的摄入二氧化锰，从而更强烈地刺激STING通路的激活；与此同时，DC细胞的成熟发生在吞噬/加工处理抗原过程中，更多的抗原摄取能够促进树突状细胞的成熟，成熟的DC细胞能够有效地激活初始T细胞，引发一系列的抗肿瘤免疫反应。
实施例I3：骨髓来源的DC细胞的刺激成熟
从C57BL/6小鼠骨髓中提取骨髓来源的干细胞，加入集落刺激因子(GM-CSF)促进干细胞分化为髓源树突状细胞(Bone Marrow-Derived Dendritic Cells,BMDC)，将BMDCs与不同样品共孵育，其中以二氧化锰的锰离子定量，终浓度为2μg/mL，F.S定量终浓度为0.12MCF，检测12h后不同组别中BMDCs的成熟比率。DC细胞是机体内高效的抗原呈递细胞，在受到某些因素刺激或摄取抗原时分化为成熟的DC细胞，成熟的DC细胞能够有效激活初始T细胞，诱导细胞毒性T淋巴细胞的生成，并分泌白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、白介素6(IL-6)等，在抗肿瘤免疫反应中起到关键作用。其中肿瘤坏死因子α(TNF-α)能够杀伤或抑制肿瘤细胞；IL-1β是一种促炎细胞因子，IL-6的分泌说明巨噬细胞被极化，能够招募更多的免疫细胞到肿瘤部位，引起更强烈的抗肿瘤免疫反应；IL-6主要由巨噬细胞，辅助性T细胞产生，诱导细胞分化，炎症反应激活，因此，能够有效刺激DC细胞成熟的样品可以增强机体的抗肿瘤免疫反应。
检测的DC细胞成熟比例统计结果见图11，相应的，不同组别的DC细胞分泌细胞因子的浓度统计见图12。综合DC成熟比例和细胞因子分泌情况，可以看到锰化灭活细菌能够更好的刺激BMDCs的成熟，其各项结果与阴性对照组具有显著性差异，甚至得到了优于阳性对照组(2μg/mL脂多糖，LPS)的结果，说明本申请所述锰化灭活细菌具有非常优异的刺激DC细胞分化的能力，能够有效提高抗肿瘤免疫反应的效率。
实施例I4：腹腔巨噬细胞与不同样品共孵育后的细胞因子表达
在抗肿瘤免疫反应中，除了树突状细胞之外，巨噬细胞也发挥着重要作用。将提取自C57BL/6小鼠腹腔的巨噬细胞和不同样品共孵育，其中样品以二氧化锰的锰离子定量，终浓度为2μg/mL，F.S定量终浓度为0.12MCF，检测巨噬细胞分泌细胞因子的情况，结果如图13所示，从白介素1β、肿瘤坏死因子α和白介素6的分泌情况来看，锰化灭活细菌对巨噬细胞的激活作用最强，甚至优于阳性对照的效果。这三种细胞因子在抗肿瘤免疫反应中具有重要作用，其中肿瘤坏死因子α(TNF-α)能够杀伤或抑制肿瘤细胞；IL-1β是一种促炎细胞因子，IL-6的分泌说明巨噬细胞被极化，能够招募更多的免疫细胞到肿瘤部位，引起更强烈的抗肿瘤免疫反应；IL-6主要由巨噬细胞，辅助性T细胞产生，诱导细胞分化，炎症反应激活。此实验中表示先天免疫反应的激活，还能够刺激参与免疫反应的细胞的增殖、分化，提高免疫细胞(例如NK细胞)的活性，这三种细胞因子能够促进抗肿瘤免疫反应，说明本申请所述锰化灭活细菌有助于抗肿瘤免疫反应，增强肿瘤免疫治疗的疗效。
实施例I5：细胞水平上证明锰化灭活细菌对STING通路的激活作用
据报道，锰离子具有激活STING通路的功能，为了证明本申请所述锰化灭活细菌表面的锰也能够实现类似STING刺激剂的功效，设计了本实施例。将人宫颈癌(HELA)细胞分别与锰化灭活细菌、二氧化锰、灭活细菌、氯化锰(阳性对照)共孵育，其中以二氧化锰的锰离子定量，终浓度为2μg/mL，F.S终浓度为0.12MCF，24h后，用western blot检测干扰素调节因子3(IRF3)和干扰素调节因子3磷酸化(p-IRF3)的水平，并统计IRF3磷酸化的比例。干扰素调节因子3(p-IRF3表示磷酸化的IRF3)。IRF-3 12C端磷酸化，会诱导IFNα/β的表达，此实验中磷酸化程度表示STING通路激活的程度。其中GAPDH是western blot实验中常用的内参，所有的细胞都有，实验中用内参条带表示此实验所加的细胞量是一样的。结果如图14所示，与对照组相比，与锰化灭活细菌共孵育的细胞，磷酸化干扰素调节因子3的比例显著提高，即这组细胞的STING通路激活程度最高；与阳性对照(positive control，PC)50μM MnCl 2相比，其STING激活水平接近，说明锰化灭活细菌能够有效激活STING通路。
实施例I6：细胞实验证明锰化灭活细菌能够激活TLR4通路
将含有Toll样受体4(TLR4)报告基因的细胞(记为TLR4 +，TLR4阳性细胞)与不同样品共孵育(实验组为MnO 2@F.S，空白对照组为PBS，一般对照组为F.S，阳性对照组为PC，具体为3μg/mL MPLA)，其中以二氧化锰的锰离子定量，终浓度为2μg/mL，F.S定量终浓度为0.12MCF，TLR4报告基因的细胞，其TLR4被激活后该细胞会表达荧光素酶，可以与荧光素底物反应，发出荧光信号，收取细胞培养基加入荧光素底物后检测荧光强度，再将所有组别与PBS组荧光强度做比值。而TLR 4阴性细胞(TLR4 -)不表达TLR4，因此不会受到刺激以表达荧光素酶，加入荧光素底物后则不会产生荧光信号，用该组细胞证明了实验中使用的所有样品对荧光素底物及荧光信号的检测没有影响。
不同组别细胞培养基中的荧光强度统计情况如图15所示，其中锰化灭活细菌能够显著刺激TLR4，其刺激TLR4的能力优于灭活的减毒沙门氏菌。说明本申请所述锰化灭活细菌具有类似TLR4激动剂的作用。
STING通路的激活能够明显增强机体抗肿瘤免疫的能力，而TLR4是天然免疫系统识别病原微生物的受体之一，STING和TLR4的激活说明本申请所述锰化灭活细菌有效激活了天然免疫系统，有助于解除肿瘤微环境的免疫抑制，从而强化机体抗肿瘤免疫反应。
综合实施例I5和实施例I6，本申请所述锰化灭活细菌具有多通路激动剂的效果，同时强化非特异性免疫反应和抗肿瘤免疫反应，有助于增效抗肿瘤免疫治疗。
实施例J：动物实验
实施例J1：与不同免疫佐剂联合灭活细菌对比，锰化灭活细菌对肿瘤的疗效
分组及治疗方式：
(1)空白对照组(Blank)：瘤内注射生理盐水；
(2)二氧化锰(MnO 2)：瘤内注射二氧化锰；
(3)灭活沙门氏菌(F.S)：瘤内注射灭活沙门氏菌；
(4)灭活沙门氏菌与二氧化锰混合物(F.S&MnO 2)：瘤内注射灭活沙门氏菌与二氧化锰的混合物；
(5)表面生长二氧化锰的灭活沙门氏菌(MnO 2@F.S)：瘤内注射锰化灭活细菌；
(6)灭活沙门氏菌与氯化锰混合物(F.S&MnCl 2)：瘤内注射灭活沙门氏菌与氯化锰的混合物；
(7)灭活沙门氏菌与铝佐剂(F.S&Al)：瘤内注射灭活沙门氏菌与铝佐剂的混合物。
在小鼠背部接种CT26结肠癌皮下肿瘤模型，待肿瘤体积达到100mm 3左右，随机进行分组，根据组别向小鼠瘤体内注射不同样品，其中，不同组别间控制锰和细菌的剂量一致， 其中锰的剂量为1mg/kg，每两天记录小鼠肿瘤体积大小及体重变化，制作肿瘤生长曲线图及体重变化曲线图，结果如图16和图17所示。图16为肿瘤体积变化曲线，其中注射锰化灭活细菌对小鼠肿瘤生长抑制效果最佳，而注射灭活细菌与二氧化锰的混合物或者其中一个组分，均不能达到这种疗效，且混合物对肿瘤的生长抑制没有丝毫作用，说明只有当二氧化锰生长在灭活细菌表面后(MnO 2@F.S)得到的锰化细菌能发挥最理想的效果，即本申请所述的经修饰的细菌具有较好的抗肿瘤治疗作用。
其中铝佐剂为商业购买，货号为77161，品牌是Thermo Scientific。
将灭活细菌与氯化锰混合后进行瘤内注射，虽然能够在一定程度上抑制肿瘤生长，但是其“治疗效果”的诱因主要为氯化锰的直接毒性，从小鼠体重变化及注射样品后小鼠照片来看，氯化锰带来了肿瘤乃至周边组织的大面积坏死，并且，注射氯化锰的小鼠体重下降明显，说明灭活细菌与氯化锰混合治疗肿瘤的策略不是理想方案。
铝佐剂是被广泛认可的一种免疫佐剂，据报道铝佐剂能够有效增强免疫反应，作为对照组之一，灭活细菌与铝佐剂混合后进行瘤内注射，并不能够抑制肿瘤生长。
综合来看，表面生长了二氧化锰的锰化灭活细菌，瘤内注射能够在保证安全性的前提下，有效地抑制肿瘤生长。
实施例J2：肿瘤部位CD45 +细胞对样品的吞噬
用Cy5.5对细菌进行标记，得到带有荧光信号的灭活细菌及锰化灭活细菌。将带有荧光标记的锰化灭活细菌和灭活细菌进行瘤内注射，24h后，取肿瘤组织，处理获得细胞混悬液，对肿瘤部位细胞中的CD45 +细胞进行流式抗体标记，检测CD45 +细胞中吞噬的细菌荧光信号强度，并进行统计，得到的结果如图18所示。谱图横坐标为荧光强度，峰面积为细胞分布密度，统计图为平均荧光强度的统计值。与对照组相比，注射锰化灭活细菌的小鼠，裂解得到的肿瘤组织细胞混悬液中的CD45 +细胞具有更强的荧光信号，根据统计图，可以发现肿瘤部位CD45 +细胞吞噬的锰化灭活细菌的量显著大于未锰化的灭活细菌，说明表面生长了二氧化锰的灭活细菌能够被活体内的白细胞更多地吞噬。CD45的白细胞的细胞标记物，CD45 +细胞即为白细胞，白细胞对锰化细菌吞噬增强有利于招募更多免疫细胞向肿瘤部位募集，无差别吞噬更多肿瘤相关抗原，有利于肿瘤部位免疫反应的激活及增强抗肿瘤特异性免疫反应。
实施例J3：样品注射后的全身分布情况
向CT26荷瘤小鼠瘤内注射锰化灭活细菌和相同剂量的二氧化锰，24h后取心、肝、脾、肺、肾以及肿瘤组织，称重后进行研磨，用ICP法定量器官中肿瘤组织以及脏器中的锰含量，并统计实验结果如图19所示。与单独注射二氧化锰相比，锰化在细菌上的二氧化锰能更多的 滞留在肿瘤部位，这将能为肿瘤微环境中的免疫细胞持续提供免疫刺激能力。而在锰代谢的重要部位——肝脏则观察到锰化在细菌上的二氧化锰处理组锰含量更低，说明锰化细菌样品不会给肝脏在短时间内造成负担，进一步反映锰化细菌样品的安全性优势。
实施例J4：样品注射后的肿瘤部位氧含量的变化
为了证明锰化灭活细菌能够有效改善肿瘤微环境的乏氧状态，在注射样品后的1h和4h用氧含量检测探针分别检测了荷瘤小鼠肿瘤微环境中氧含量的变化。
分组及治疗方式：
(1)空白对照组(Blank)：瘤内注射生理盐水；
(2)二氧化锰(MnO 2)：瘤内注射二氧化锰；
(3)灭活沙门氏菌(F.S)：瘤内注射灭活沙门氏菌；
(4)锰化灭活沙门氏菌(MnO 2@F.S)：瘤内注射锰化灭活沙门氏菌。
实验结果统计数据如图20所示，在未处理的肿瘤组织中氧气含量约为0.28ppm左右，处于乏氧状态。而在瘤内注射样品1h后，二氧化锰处理组与锰化灭活细菌处理组氧气含量均上升至0.34ppm左右，显著提高了肿瘤部位的氧气含量。4h后仍能观察到相似的趋势。说明锰化细菌可以通过二氧化锰的作用有效地改善肿瘤微环境的乏氧状态，肿瘤乏氧微环境是典型的免疫抑制环境，乏氧微环境的改善有利于后续免疫细胞浸润病灶部位，并在该环境中激活，从而发挥免疫治疗效果。
实施例J5：锰化灭活细菌策略可以有效激活抗肿瘤免疫系统
为了证明锰化细菌能够有效地激活固有免疫系统与获得性免疫系统，改善肿瘤免疫抑制微环境，并能够高效增强抗肿瘤免疫反应。在对CT26荷瘤小鼠瘤内注射不同样品24h后，取小鼠淋巴结检测树突状细胞的成熟度(图21)，取肿瘤组织检测其中细胞因子含量(图23)，在治疗24h和5天后取肿瘤组织检测其中免疫细胞浸润情况(图22与图24)。
分组及治疗方式：
第一组：Blank，空白对照组(瘤内注射生理盐水)；
第二组：MnO 2，二氧化锰(瘤内注射二氧化锰)；
第三组：F.S，灭活沙门氏菌(瘤内注射灭活沙门氏菌)；
第四组：MnO 2@F.S，锰化灭活沙门氏菌(瘤内注射锰化灭活沙门氏菌)。
淋巴结树突状细胞的成熟是免疫激活的重要一步，未成熟的树突状细胞在摄取抗原后向淋巴结迁移，在迁移的过程中逐渐成熟，表达共刺激分子并在淋巴结中向T细胞呈递抗原。在样品注射24h后取下肿瘤侧的淋巴结，研磨后用流式细胞仪检测成熟的树突状细胞在所有 树突状细胞中所占的比例，统计结果如图21所示。在四组中，锰化灭活细菌处理组的小鼠具有最高的DC成熟度，是注射生理盐水组的26.9倍，这说明了锰化灭活细菌优异的免疫刺激能力，可以高效激活小鼠的免疫系统。
肿瘤微环境是免疫抑制的微环境，免疫反应不易被激活，因此细胞因子含量极低，为了证明锰化细菌样品对免疫反应的激活以及免疫抑制的改善，在24h时，取下小鼠肿瘤组织，研磨后用酶联免疫分析法检测了其中白介素1β，白介素6，肿瘤坏死因子α以及干扰素β的含量。结果如图23所示，瘤内注射锰化灭活细菌的小鼠，肿瘤部位白介素1β，白介素6，肿瘤坏死因子α以及干扰素β的含量均显著高于其他对照组，显示出锰化灭活细菌策略对肿瘤微环境中天然免疫激活的优越性。且干扰素β是STING通路激活的标志，由于锰化细菌样品中二氧化锰能够更多的进入免疫细胞(实施例I2)且锰化灭活细菌比游离二氧化锰能够更持续的在肿瘤部位滞留(实施例J3)，因此具有最高的激活STING通路的能力。
在肿瘤治疗后的24h与5天，分别对固有免疫反应的激活(图22)与特异性免疫反应的激活(图24)进行了评估与对比。在24h与5天时取下肿瘤组织后研磨，进行流式抗体的染色，用流式细胞仪检测其中固有免疫系统——巨噬细胞与NK细胞，以及获得性免疫细胞——T细胞的浸润情况如图22所示。在24h后，肿瘤部位巨噬细胞与NK细胞比例显著上调，显示出固有免疫系统的激活，结果与图23也相符合。而特异性免疫系统的T细胞在四组中却无显著差异，这说明这一阶段主要是巨噬细胞与NK细胞在起作用。而在治疗5天后，锰化灭活细菌处理组的小鼠CD4 +T细胞(辅助性T细胞)与CD8 +T细胞(杀伤性T细胞)在肿瘤中的比例显著上调，证明该组小鼠进入特异性免疫激活阶段，而其他三组小鼠均无明显差异。(图24)
除此之外，锰化灭活细菌治疗过的小鼠在第5天时巨噬细胞极化状态也出现了变化。M1型巨噬细胞是免疫激活类型，可以帮助抗原呈递与T细胞的分化。M2型巨噬细胞则是促肿瘤类型，一般认为与肿瘤转移相关。实验数据表示在第五天时，锰化灭活细菌组的小鼠肿瘤部位巨噬细胞极化的状态朝抗肿瘤免疫的方向转变。(图24)
综上所述，锰化灭活细菌瘤内注射治疗后，肿瘤微环境被有效改善，免疫激活相关的细胞因子显著上调，帮助了巨噬细胞、NK细胞等免疫细胞对肿瘤部位的浸润，也使更多的树突状细胞摄取抗原后向淋巴结迁移并将抗原呈递给淋巴结的T细胞，完成了固有免疫系统激活并向特异性免疫系统激活的过渡。T细胞分化后迁移至肿瘤部位，辅助性T细胞与杀伤性T细胞比例显著上调，特异性杀伤肿瘤细胞，并帮助巨噬细胞向抗肿瘤M1型极化，极化的巨噬细胞又可以继续激活T细胞的抗肿瘤功能，完成特异性免疫反应的激活。
实施例J6：NK细胞阻断后的锰化细菌肿瘤治疗实验
为了证明锰化灭活细菌能够诱导先天性免疫的激活，对小鼠自然杀伤细胞(NK细胞)进行阻断，使得小鼠NK细胞功能丧失，同时，IgG作为一种不影响NK细胞功能的抗体，在本实施例中作为NK抗体的对照。
分组：
第一组：blank：空白对照组；
第二组：MnO 2@F.S&IgG：锰化灭活细菌和免疫球蛋白联合治疗组；
第三组：MnO 2@F.S&a-NK 1.1：锰化灭活细菌和NK 1.1细胞抗体联合治疗组。
在小鼠背部接种黑色素瘤B16-OVA肿瘤细胞构建皮下黑色素瘤模型，治疗方法为瘤内注射锰化灭活细菌(MnO 2@F.S)，第三组小鼠在开始肿瘤治疗前1天和给药后第2、4、6天注射NK阻断抗体(α-NK 1.1)，第二组小鼠在开始肿瘤治疗前1天和给药后第2、4、6天注射IgG。每两天监测肿瘤大小，并制作肿瘤生长曲线，结果如图25所示。其中，NK细胞功能丧失后，该锰化灭活细菌几乎没有抑制肿瘤生长，说明该制剂能够激活先天免疫反应，且激活先天免疫反应后，活化的NK细胞在肿瘤生长抑制中发挥重要作用。
实施例J7：T细胞阻断后的锰化细菌肿瘤治疗实验
为了证明锰化灭活细菌能够激活特异性免疫反应，使用CD4抗体和CD8抗体，阻断T细胞表面的作用位点，使得CD4 +CD8 +T细胞丧失功能，同时，另一组中通过注射IgG作为对照。
分组：
第一组：blank：空白对照组；
第二组：MnO 2@F.S&α-CD4：锰化灭活细菌和anti-CD4抗体联合治疗组；
第三组：MnO 2@F.S&α-CD8：锰化灭活细菌和anti-CD8抗体联合治疗组；
第四组：MnO 2@F.S&α-CD4&α-CD8：锰化灭活细菌和anti-CD4、anti-CD8抗体联合治疗组；
第五组：MnO 2@F.S&IgG：锰化灭活细菌和免疫球蛋白联合治疗组。
建立小鼠皮下CT26肿瘤模型，待肿瘤大小约100mm 3时，进行随机分组，瘤内给药前1天及给药后第3、5、7天对不同组别小鼠分别通过静脉注射相应抗体，记录肿瘤生长情况，并制作肿瘤生长曲线图，结果如图26所示。从图中可以看出，同样给予锰化灭活细菌的情况下，利用针对CD4或CD8的抗体阻断小鼠体内相应的CD4 +T细胞或CD8 +T细胞的功能即可大幅降低肿瘤治疗效果，只有T细胞正常发挥作用时，才能够得到较好的疗效，说明锰化 灭活细菌起效要依赖于特异性免疫反应的激活，依靠T细胞介导的抗肿瘤免疫反应，抑制肿瘤生长。
实施例J8：
分别建立小鼠背部4T1乳腺癌肿瘤模型、B16-OVA黑色素瘤模型和KPC胰腺癌模型，然后通过瘤内注射不同样品进行治疗，记录肿瘤生长曲线，结果如图27所示。结果显示，三种肿瘤模型均能够被锰化灭活细菌很好地抑制，而单一组分并不能够抑制肿瘤生长，该实施例的结果印证了实施例J1，说明本申请所述锰化灭活细菌对多种实体瘤均有效果。
实施例J9：不同锰化细菌对乳腺癌肿瘤模型的治疗实验
第一组：blank：空白对照组；
第二组：MnO 2@F.S：锰化灭活沙门氏菌；
第三组：MnO 2@F.SA：锰化灭活金黄色葡萄球菌；
第四组：MnO 2@F.E：锰化灭活大肠杆菌；
第五组：MnO 2@F.V：锰化灭活沙门氏菌VNP20009。
建立小鼠皮下4T1乳腺癌模型，瘤内注射不同种类的锰化灭活细菌，记录肿瘤生长情况，制作肿瘤生长曲线，结果如图29所示。从生长曲线可以看出，所有锰化细菌都能够很好的抑制肿瘤生长，与实施例J3的结果相似，说明本申请所述锰化灭活细菌对不同肿瘤具有普适性。
实施例J10：不同投料比制备的锰化细菌对肿瘤的治疗效果
分组情况：
Figure PCTCN2022097190-appb-000017
根据实施例J1的方法，制备不同投料比例的锰化细菌，建立小鼠CT26皮下肿瘤模型，随机分组，根据组别设置，对各小鼠进行瘤内注射对应药物，注射剂量为3.6*10 8个锰化细菌，相应的二氧化锰用量分别为3μg、16μg、89μg。之后每两天测量一次肿瘤体积，制作肿瘤生长曲线，结果如图30所示。
从肿瘤生长曲线分析，对比空白组(第四组)肿瘤生长曲线，不同投料比制备的锰化细菌对肿瘤生长均有抑制效果。说明本申请制备的锰化细菌的投料比范围选择性较广，实际治 疗中，可通过更多的研究确认最佳的配方配比。
实施例J11：
新西兰兔肝VX2癌症模型是少数建立在较大动物的肝癌复发转移模型，能够更好地模拟人的肝癌复发转移，因此建立新西兰兔的肝癌模型，并使用本申请所述的锰化灭活细菌MnO 2@F.V进行肿瘤治疗，样品制备时细菌用量为10.5MCF，二氧化锰用量为160.7μg。结果如图31所示，与不做任何治疗处理的组别相比，锰化灭活细菌(MnO 2@F.V)治疗的新西兰兔，其肿瘤病灶几乎被完全清除，说明其优异的抗肿瘤特性。
实施例J12：
建立小鼠双边肿瘤模型，在小鼠背部左右两侧接种CT26结肠癌皮下肿瘤模型，其中左侧接种的肿瘤细胞数是右侧的一半量，右侧肿瘤模拟原发瘤，同时左侧肿瘤模拟转移瘤。待右侧肿瘤体积达到100mm 3左右，随机进行分组，根据组别向小鼠右侧治疗侧瘤体内注射不同样品，并于治疗后的1，3，5天向免疫检查点两组小鼠尾静脉注射anti-PD-1(即α-PD-1)的抗体。每两天分别记录小鼠双侧肿瘤体积大小，制作肿瘤生长曲线图结果如图32所示。
分组与治疗方式：
第一组：blank：右侧瘤内注射生理盐水；
第二组：α-PD-1：尾静脉三次注射免疫检查点抗体anti-PD-1；
第三组：MnO 2@F.S：右侧瘤内注射锰化细菌；
第四组：MnO 2@F.S&α-PD-1：右侧瘤内注射锰化细菌并尾静脉三次注射免疫检查点抗体anti-PD-1。
如图32结果显示，治疗侧的肿瘤生长曲线显示出锰化细菌优异的治疗效果，与单独注射免疫检查点抗体(α-PD-1)的组别相比，锰化细菌(MnO 2@F.S)的治疗效果明显好于免疫检查点的治疗，使用MnO 2@F.S的组别，小鼠的治疗侧肿瘤几乎被完全抑制。观察远端瘤的肿瘤生长情况则发现，联合免疫检查点后，可以进一步强化锰化细菌对远端肿瘤生长的抑制效果，远端肿瘤生长速度变缓。说明锰化细菌能够抑制原位肿瘤生长，并引起全身性抗肿瘤免疫反应，具有抑制远端肿瘤生长的效果，结合免疫检查点阻断疗法，能更有效地抑制远端肿瘤生长，具有与免疫检查点阻断疗法联合的潜力。
实施例J13：
免疫记忆效果
为了验证本申请所述锰化细菌的疫苗效果，建立小鼠CT26结肠癌肿瘤模型，并按照实施例J12所述方法进行单边肿瘤的治疗，最终小鼠皮下肿瘤完全消失，实施治疗后第60天， 用流式细胞仪分析小鼠外周血中记忆T细胞的比例，统计结果如图33所示，被锰化细菌治愈肿瘤的小鼠，体内记忆T细胞的含量显著增加，说明本申请所述锰化细菌能够诱导记忆T细胞的产生，产生免疫记忆效应。
同时对治愈的小鼠再次接种肿瘤细胞，记录其生长情况，结果如图34所示，结果显示，被锰化细菌治愈的小鼠，重新接种肿瘤细胞后几乎没有肿瘤生长，也就是说，锰化细菌治愈肿瘤的同时还能够抑制肿瘤的复发，产生了类似疫苗的效果。
实施例K
实施例K1：附着二氧化锰的锰化灭活细菌的制备(以硫酸锰、为原料)
S1：配制浓度为10mM的硫酸锰水溶液；
S2：细菌的扩增培养，将50μL的冻存减毒沙门氏菌菌液接种在6mL的新鲜肉膏蛋白胨(LB)培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，随后将活化后的菌液稀释涂布在LB固体培养基平板上，置于数显培养箱在37℃中培养12h，挑取状态良好的单菌落，接种至5mL新鲜LB肉汤培养基中，放置于37℃恒温摇床上培养过夜，得到菌液；
S3：取S2所得菌液至20mL新鲜的LB液体培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，离心收集菌体，用无菌生理盐水重悬，再次离心后加入5mL的4％多聚甲醛固定24h，离心收集固定后的灭活细菌，用无菌生理盐水清洗菌体两次，最后将灭活菌体用生理盐水重悬，使得最终菌液的OD 600＝3，即菌液在600nm波长处的吸光值为3，得到灭活细菌混悬液，灭活细菌混悬液可于4℃保存，取50μL菌液涂布固体培养基，培养后确认菌液中细菌已充分灭活；
S4：在灭活细菌混悬液中加入10mM的硫酸锰溶液，以及适量生理盐水，搅拌15min，随后加入氢氧化钠调节pH至10-11，继续搅拌1h，离心收集沉淀，用无菌生理盐水洗两遍，最后用1mL生理盐水重悬，4℃保存。
实施例K2：附着硫化锰的锰化灭活细菌的制备
S1：配制浓度为10mM的氯化锰水溶液；
S2：细菌的扩增培养，将50μL的冻存减毒沙门氏菌菌液接种在6mL的新鲜肉膏蛋白胨(LB)培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，随后将活化后的菌液稀释涂布在LB固体培养基平板上，置于数显培养箱在37℃中培养12h，挑取状态良好的单菌落，接种至5mL新鲜LB肉汤培养基中，放置于37℃恒温摇床上培养过夜，得到菌液；
S3：取S2所得菌液至20mL新鲜的LB液体培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，离心收集菌体，用无菌生理盐水重悬，再次离心后加入5mL的4％多聚甲醛固定24h，离心收集固定后的灭活细菌，用无菌生理盐水清洗菌体两次，最后将灭活菌体用生理盐水重悬，使得最终菌液的OD 600＝3，即菌液在600nm波长处的吸光值为3，得到灭活细菌混悬液，可于4℃保存，取50μL菌液涂布固体培养基，确认菌液中无活细菌；
S4：在灭活细菌混悬液中加入10mM的氯化锰溶液，以及适量生理盐水，搅拌15min，随后加入0.1M的硫化钠，继续搅拌1h，离心收集沉淀，用无菌生理盐水洗两遍，最后用1mL生理盐水重悬，4℃保存。
实施例K3：还原法制备锰化灭活细菌
SS1：制备10mM的高锰酸钾水溶液备用；
SS2：细菌的扩增培养，将50μL的冻存减毒沙门氏菌菌液接种在6mL的新鲜肉膏蛋白胨(LB)培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，随后将活化后的菌液稀释涂布在LB固体培养基平板上，置于数显培养箱在37℃中培养12h，挑取状态良好的单菌落，接种至5mL新鲜LB肉汤培养基中，放置于37℃恒温摇床上培养过夜，得到菌液；
SS3：取SS2所得菌液至20mL新鲜的LB液体培养基中，将摇菌管倾斜置于37℃恒温摇床中以220r/min的震荡频率活化12h，离心收集菌体，用无菌生理盐水重悬，再次离心后加入5mL的4％多聚甲醛固定24h，离心收集固定后的灭活细菌，将灭活菌体用生理盐水重悬，使得最终菌液的OD 600＝3，即菌液在600nm波长处的吸光值为3，得到灭活细菌混悬液，可于4℃保存，取50μL菌液涂布固体培养基，确认菌液中无活细菌；
SS4：在灭活细菌混悬液中加入10mM的高锰酸钾溶液，以及适量生理盐水，搅拌1.5h，离心收集沉淀，用无菌生理盐水洗两遍，最后用1mL生理盐水重悬，4℃保存。
根据加入高锰酸钾溶液的体积不同，每10亿个细菌中，可加入0.2-3μmol的锰，均能够形成稳定的锰化细菌混悬液。
实施例K4：
取适量实施例K1-K3的最终产物制备扫描电镜样品，用扫描电镜对其外观形貌进行表征，得到的电镜图如图35(硫酸锰为原料制备得到的MnO 2@F.S)、图36(附着硫化锰的锰化细菌MnS@F.S)、图37(还原法制备的MnO 2@F.S)所示，固定后的灭活细菌保持了原本细菌的外观特征，锰化后的细菌表面结合了一层不光滑的沉淀物，细菌本身保持原有的形态和大 小，说明附着不同金属化合物时不破坏细菌本身，只是在其表面附着了一层物质，采用不同的制备方法均可以得到多种锰化细菌。
实施例K5：
用实施例K1的方法制备不同投料比的表面生长氧化锰的细菌，原料为硫酸锰和减毒沙门氏菌，将制得的成品用扫描电镜观察其形貌，结果如图38所示。不同投料比得到的锰化细菌，其表面附着了大小较为均一的氧化锰固体，随着硫酸锰投料量的增加，细菌表面生长的氧化锰增加。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。
同理，应当注意的是，为了简化本申请披露的表述，从而帮助对一个或多个发明实施例的理解，前文对本申请实施例的描述中，有时会将多种特征归并至一个实施例、附图或对其的描述中。但是，这种披露方法并不意味着本申请对象所需要的特征比权利要求中提及的特征多。实际上，实施例的特征要少于上述披露的单个实施例的全部特征。
一些实施例中使用了描述成分、属性数量的数字，应当理解的是，此类用于实施例描述的数字，在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明，“大约”、“近似”或“大体上”表明所述数字允许有±变化。相应地，在一些实施例中，说明书和权利要求中使用的数值参数均为近似值，该近似值根据个别实施例所需特点可以发生改变。
最后，应当理解的是，本申请中所述实施例仅用以说明本申请实施例的原则。其他的变形也可能属于本申请的范围。因此，作为示例而非限制，本申请实施例的替代配置可视为与本申请的教导一致。相应地，本申请的实施例不仅限于本申请明确介绍和描述的实施例。

Claims (61)

  1. 一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物。
  2. 一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物通过沉积反应修饰于所述细菌本体的表面形成附着层。
  3. 一种经修饰的细菌，其包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，所述金属化合物在每1*10 8个细菌上的含量为约0.1μg～600μg。
  4. 根据权利要求1-3中任一项所述的经修饰的细菌，其中所述细菌本体为革兰氏阳性菌或革兰氏阴性菌。
  5. 根据权利要求1-4中任一项所述的经修饰的细菌，其中所述细菌本体选自球菌、杆菌和螺旋菌中的一种或多种。
  6. 根据权利要求5所述的经修饰的细菌，其中所述球菌选自金黄色葡萄球菌、脲微球菌、肺炎双球菌、肺炎链球菌、脑膜炎双球菌、化脓性链球菌、肺炎链球菌、无乳链球菌、金黄色葡萄球菌、白色葡萄球菌和柠檬色葡萄球菌中的一种或多种。
  7. 根据权利要求5-6中任一项所述的经修饰的细菌，其中所述杆菌选自大肠杆菌、沙门氏菌、鼠疫杆菌、猪痢疾杆菌、多杀性巴氏杆菌、白喉棒状杆菌、结核分枝杆菌、双歧乳杆菌、醋酸杆菌和棒状杆菌中的一种或多种。
  8. 根据权利要求5-7中任一项所述的经修饰的细菌，其中所述螺旋菌选自幽门螺杆菌、逗号弧菌和霍乱弧菌中的一种或多种。
  9. 根据权利要求1-8中任一项所述的经修饰的细菌，所述细菌本体选自沙门氏菌、金黄色葡萄球菌、大肠杆菌、乳酸杆菌、沙门氏菌减毒株、金黄色葡萄球菌减毒株、大肠杆菌减毒株和乳酸杆菌减毒株中的一种或多种。
  10. 根据权利要求1-9中任一项所述的经修饰的细菌，其中所述细菌本体为野生型菌、基因工程菌和/或减毒株。
  11. 根据权利要求1-10中任一项所述的经修饰的细菌，其中所述细菌本体为活体细菌或灭活细菌。
  12. 根据权利要求11所述的经修饰的细菌，其中所述活体细菌或灭活细菌包含减毒细菌。
  13. 根据权利要求1-12中任一项所述的经修饰的细菌，其中所述金属化合物通过物理结合或化学结合的方式修饰于所述细菌本体的表面形成附着层。
  14. 根据权利要求13所述的经修饰的细菌，其中所述物理结合包括静电吸附和/或部分嵌 入。
  15. 根据权利要求13所述的经修饰的细菌，其中所述化学结合包括偶联、生成化学键作用和/或以配合形式结合。
  16. 根据权利要求1-15中任一项所述的经修饰的细菌，其中所述金属化合物通过沉积反应修饰于所述细菌本体的表面形成附着层。
  17. 根据权利要求1-15中任一项所述的经修饰的细菌，其中所述金属化合物通过生物矿化反应修饰于所述细菌本体的表面形成附着层。
  18. 根据权利要求17所述的经修饰的细菌，其中所述生物矿化反应包括：金属离子与细菌的细胞膜上的生物大分子或细胞壁上的生物大分子结合，提供矿化位点，调节pH或引入其他盐，金属离子在矿化位点生成金属化合物，不断生长积累，结合在细菌表面。
  19. 根据权利要求17-18中任一项所述的经修饰的细菌，其中所述生物矿化反应形成生物大分子和金属化合物的结合位点，利用沉积反应或生物矿化进一步增加金属化合物的体积。
  20. 根据权利要求1-19中任一项所述的经修饰的细菌，其中所述金属化合物在所述细菌本体表面的覆盖度为0.1％-99.9％。
  21. 根据权利要求20所述的经修饰的细菌，其中所述金属化合物在所述细菌本体表面的覆盖度通过调节投料比、反应温度和/或反应时间调整。
  22. 根据权利要求1-21中任一项所述的经修饰的细菌，其中所述金属化合物的金属元素选自锌、钙、铜、铁、锰和镁中的一种或多种；非金属元素选自碳酸根、氢氧根、硫离子和磷酸根中的一种或多种。
  23. 根据权利要求1-22中任一项所述的经修饰的细菌，其中所述金属化合物选自碳酸锌、碳酸钙、碳酸铜、碳酸镁；氢氧化锌、氢氧化铁、氢氧化铜、氢氧化锰、氢氧化镁、硫化锌、硫化铜、硫化锰；磷酸锌、磷酸钙、磷酸铜、磷酸铁、磷酸镁和磷酸锰中的一种或多种。
  24. 根据权利要求1-23中任一项所述的经修饰的细菌，其中所述金属化合物为含锰化合物。
  25. 根据权利要求24所述的经修饰的细菌，其中所述含锰化合物为氢氧化锰、二氧化锰和硫化锰中的一种或多种。
  26. 根据权利要求1-25中任一项所述的经修饰的细菌，其中所述金属化合物为氢氧化锰和/或二氧化锰。
  27. 一种锰化细菌，其包括细菌本体和附着于所述细菌本体表面的难溶性或微溶性含锰化合物。
  28. 根据权利要求27所述的锰化细菌，其中所述难溶性或微溶性含锰化合物为氢氧化锰、二氧化锰和硫化锰中的一种或多种。
  29. 根据权利要求27-28中任一项所述的锰化细菌，所述细菌本体为革兰氏阳性菌或革兰氏阴性菌。
  30. 根据权利要求27-29中任一项所述的锰化细菌，其中所述细菌本体选自球菌、杆菌和/或螺旋菌。
  31. 根据权利要求30所述的锰化细菌，其中所述球菌选自金黄色葡萄球菌、脲微球菌、肺炎双球菌、肺炎链球菌、脑膜炎双球菌、化脓性链球菌、肺炎链球菌、无乳链球菌、金黄色葡萄球菌、白色葡萄球菌和柠檬色葡萄球菌中的一种或多种。
  32. 根据权利要求30-31中任一项所述的锰化细菌，其中所述杆菌选自大肠杆菌、沙门氏菌、鼠疫杆菌、猪痢疾杆菌、多杀性巴氏杆菌、白喉棒状杆菌、结核分枝杆菌、双歧乳杆菌、醋酸杆菌和棒状杆菌中的一种或多种。
  33. 根据权利要求30-32中任一项所述的锰化细菌，其中所述螺旋菌选自幽门螺杆菌、逗号弧菌和霍乱弧菌中的一种或多种。
  34. 根据权利要求27-33中任一项所述的锰化细菌，所述细菌本体为活体细菌或灭活细菌。
  35. 根据权利要求27-34中任一项所述的锰化细菌，其中所述细菌本体为野生型菌、基因工程菌和/或减毒株。
  36. 组合物，其包含权利要求1-26中任一项所述的经修饰的细菌或权利要求27-35中任一项所述的锰化细菌，以及任选地药学上可接受的载剂，所述经修饰的细菌包括细菌本体和修饰于所述细菌本体表面的难溶或不溶性的生物学可接受的金属化合物，且所述组合物的pH值为0-14。
  37. 一种经修饰的细菌冻干粉，其包括权利要求1-26中任一项所述的经修饰的细菌，以及添加剂。
  38. 根据权利要求37所述的经修饰的细菌冻干粉，其中所述添加剂包括冻干保护剂和/或赋形剂。
  39. 根据权利要求38所述的经修饰的细菌冻干粉，其中所述冻干保护剂选自糖类/多元醇，聚合物，无水溶剂，表面活性剂，氨基酸，盐和胺中的一种或多种。
  40. 根据权利要求38-39中任一项所述的细菌冻干粉，其中所述赋形剂选自黏合剂、填充剂、崩解剂、润滑剂、酒、醋、药汁等、软膏剂基质、霜剂基质、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂中的一种或多种。
  41. 根据权利要求38-40中任一项所述的经修饰的细菌冻干粉，其中所述冻干保护剂选自蔗 糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-β环糊精中的一种或多种。
  42. 根据权利要求38-41中任一项所述的经修饰的细菌冻干粉，其中所述细菌冻干粉中添加剂的质量分数为0.1-99％。
  43. 根据权利要求19-20中任一项所述的经修饰的细菌冻干粉，其中所述细菌冻干粉中冻干保护剂的质量分数为0.1-99％。
  44. 一种锰化细菌冻干粉，其包括权利要求27-35中任一项所述的锰化细菌，以及添加剂。
  45. 根据权利要求44所述的锰化细菌冻干粉，其中所述添加剂包括冻干保护剂和/或赋形剂。
  46. 根据权利要求45所述的锰化细菌冻干粉，其中所述冻干保护剂选自糖类/多元醇、聚合物、无水溶剂、表面活性剂、氨基酸、盐和胺中的一种或多种。
  47. 根据权利要求45-46中任一项所述的锰化细菌冻干粉，其中所述赋形剂选自黏合剂、填充剂、崩解剂、润滑剂、酒、醋、药汁等、软膏剂基质、霜剂基质、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂中的一种或多种。
  48. 根据权利要求45-47中任一项所述的锰化细菌冻干粉，其中所述冻干保护剂选自蔗糖、甘露糖、a-D-吡喃甘露糖、海藻糖、肌糖、棉白糖、菊糖、右旋糖酐、麦芽糖糊精、麦芽多糖、八硫酸蔗糖、肝素和2-羟丙基-β环糊精中的一种或多种。
  49. 根据权利要求45-48中任一项所述的锰化细菌冻干粉，其中所述细菌冻干粉中添加剂的质量分数为0.1-99％。
  50. 根据权利要求45-49中任一项所述的锰化细菌冻干粉，其中所述细菌冻干粉中冻干保护剂的质量分数为0.1-99％。
  51. 一种权利要求1-26中任一项所述的经修饰的细菌或权利要求27-35中任一项所述的锰化细菌的制备方法，其包括以下步骤：
    S1：制备细菌混悬液；
    S2：向所述细菌混悬液中加入可溶性金属盐溶液。
  52. 根据权利要求51所述的制备方法，其还包括步骤S3：加入易溶性的氢氧化物水溶液、碳酸盐水溶液或磷酸盐水溶液至pH为8-12，或加入易溶性的硫化盐水溶液，反应制得所述经修饰的细菌。
  53. 根据权利要求52所述的制备方法，其中所述易溶性的硫化盐水溶液包括硫化钠溶液、硫化钾溶液和/或硫化铵溶液。
  54. 根据权利要求52-53中任一项所述的制备方法，其中所述易溶性的氢氧化物水溶液、碳 酸盐水溶液或磷酸盐水溶液选自：氢氧化钠水溶液、碳酸钠水溶液、磷酸钠水溶液。
  55. 根据权利要求51-54中任一项所述的制备方法，在所述步骤S2中，按照每10亿个的细菌，添加0.2-13.5mmol的金属离子的加入量，向所述细菌混悬液中加入可溶性金属盐溶液。
  56. 根据权利要求51-55中任一项所述的制备方法，其中所述可溶性金属盐溶液为高锰酸盐溶液。
  57. 根据权利要求51-56中任一项所述的制备方法，其中所述步骤S2包括向所述细菌混悬液中加入高锰酸盐溶液，搅拌反应，离心得到锰化细菌。
  58. 根据权利要求51-57中任一项所述的制备方法，其中在每10亿个细菌，添加0.2-3μmol的高锰酸盐。
  59. 权利要求1-26中任一项所述的经修饰的细菌、权利要求27-35中任一项所述的锰化细菌、权利要求37-43中任一项所述的经修饰的细菌冻干粉、权利要求44-50中任一项所述的锰化细菌冻干粉、或权利要求36所述的组合物在制备肿瘤免疫治疗制剂中的应用。
  60. 权利要求1-26中任一项所述的经修饰的细菌、权利要求27-35中任一项所述的锰化细菌、权利要求37-43中任一项所述的经修饰的细菌冻干粉、权利要求44-50中任一项所述的锰化细菌冻干粉、或权利要求36所述的组合物，其用于预防和/或治疗肿瘤。
  61. 一种预防和/或治疗肿瘤的方法，其包括向有需要的受试者施用有效量的权利要求1-26中任一项所述的经修饰的细菌、权利要求27-35中任一项所述的锰化细菌、权利要求37-43中任一项所述的经修饰的细菌冻干粉、权利要求44-50中任一项所述的锰化细菌冻干粉、或权利要求36所述的组合物。
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