Antitumor bacteria that reduce neutrophil levels in tumors

Abstract

The present invention provides a fusion protein comprising an antibody or its antigen-binding fragment that binds to neutrophils, and a cytotoxin or its active fragment. Also provided are modified bacteria having antitumor activity and expressing the fusion protein, antibody or its antigen-binding fragment, or toxin or its active fragment in a hypoxic environment (e.g., a tumor). Pharmaceutical compositions containing the modified bacteria and their antitumor use are also provided.

Description

[Technical Field]

[0001] This invention relates to modified bacteria having antitumor activity, pharmaceutical compositions containing the modified bacteria, and their antitumor use. [Background technology]

[0002] Cancer has become one of the most serious diseases threatening human life and health, but existing treatments (radiotherapy, chemotherapy, surgery, and targeted drugs) all have shortcomings, creating an urgent need to develop new treatment approaches. Conventional bacterial oncology, exemplified by Corey's toxin, has a 150-year history but is known for its unreliable efficacy and safety concerns. Developments in genetic engineering have made significant progress in reducing the toxicity of bacterial strains, and a series of clinical trials have demonstrated that attenuated bacteria for human oncology treatment, while safe, have limited efficacy—that is, they cannot meet the needs of clinical oncology treatment because they cannot combine safety and therapeutic efficacy. Further optimization of oncology treatment is essential.

[0003] Furthermore, the inventors found that neutrophils in the human body limit the application of bacterial therapy to tumors. Therefore, in order to reduce the effects of neutrophils, it is necessary to optimize the strains used for bacterial therapy against tumors. [Overview of the Initiative]

[0004] According to the present invention, obligate anaerobic bacteria are constructed that exhibit therapeutic effects against tumors after administration to animals or humans by means of a synthetic biological genetic circuit, and can be eliminated within a short period of time by normal tissues and organs, thereby reducing toxic effects and side effects on animals or humans caused by the long-term retention of bacteria in vivo, and providing more reliable antitumor effects and safety. Furthermore, the antitumor effect of bacteria is enhanced by expressing exogenous proteins (e.g., antibodies against neutrophils, or fusion proteins of such antibodies, and cytotoxins) in the bacteria to inhibit neutrophil recruitment or kill neutrophils in tumors.

[0005] In a first aspect, the present invention provides a fusion protein comprising an antibody (e.g., a nanobody) or an antigen-binding fragment thereof that binds to neutrophils, and a cytotoxin or an active fragment thereof.

[0006] In some embodiments, the antibody comprises a complementarity-determining region (CDR) selected from the group consisting of CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 containing residues 31-35 of SEQ ID NO: 21, CDR2 containing residues 50-64 of SEQ ID NO: 21, and CDR3 containing residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0007] In some embodiments, the cytotoxin is Pseudomonas exotoxin (PE). In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytotoxin fragment comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0008] In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0009] In some embodiments, the fusion protein further comprises a linker and / or tag sequence.

[0010] In a second aspect, the present invention provides an expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter encoding an antibody (e.g., a nanobody) or an antigen-binding fragment thereof that binds to neutrophils.

[0011] In some embodiments, the antibody comprises a complementarity-determining region (CDR) selected from the group consisting of CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 containing residues 31-35 of SEQ ID NO: 21, CDR2 containing residues 50-64 of SEQ ID NO: 21, and CDR3 containing residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0012] In some embodiments, the nucleotide sequence further encodes a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0013] In some embodiments, the nucleotide sequence further encodes the tag sequence.

[0014] In some embodiments, the nucleotide sequence encodes the fusion protein of the present invention.

[0015] In a third aspect, the present invention provides a modified bacterium.

[0016] In one embodiment, compared to an unmodified starting strain, the bacteria contain an expression construct of the present invention and a hypoxia-regulating essential gene expression cassette containing the bacterial essential genes under the control of a second strictly hypoxia-inducible promoter, and the bacteria lack at least one gene or its functional expression product required for survival in macrophages.

[0017] In one embodiment, compared to an unmodified starting strain, the bacterium comprises a hypoxia-regulating essential gene expression cassette containing the expression construct of the present invention and the bacterium's essential genes under the control of a second strictly hypoxia-inducible promoter, and the bacterium lacks at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or a functional expression product thereof.

[0018] In one embodiment, compared to an unmodified starting strain, the bacterium comprises an expression construct of the present invention, a hypoxia-regulating essential gene expression cassette containing the bacterium's essential genes under the control of a second strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions, and the bacterium lacks at least one gene or its functional expression product that is involved in or modulates the endogenous antioxidant stress response pathway.

[0019] In one embodiment, compared to an unmodified starting strain, the bacterium comprises an expression construct of the present invention, a hypoxia-regulating essential gene expression cassette containing the bacterium's essential genes under the control of a second strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial-derived hemolysin protein under the control of a promoter that is active under acidic pH conditions, and the bacterium expresses wild-type lipopolysaccharide (LPS) and lacks at least one gene or its functional expression product required for survival in macrophages.

[0020] In one embodiment, compared to an unmodified starting strain, the bacterium is a modified Salmonella typhimurium containing the expression construct of the present invention, a hypoxia-regulating essential gene expression cassette containing the bacterium's essential genes under the control of a second strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions, and the bacterium lacks at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or a functional expression product thereof.

[0021] In one embodiment, the modified bacterium is a modified Salmonella typhimurium, which includes the expression construct of the present invention, a hypoxia-regulating essential gene expression cassette containing the bacterium's essential genes under the control of a second strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions, and the modified Salmonella typhimurium expresses wild-type lipopolysaccharide (LPS) and lacks at least one gene or functional expression product required for survival in macrophages.

[0022] In one embodiment, the present invention provides a pharmaceutical composition comprising a bacterium described in any one of the prior embodiments. In one embodiment, the pharmaceutical composition is used for the treatment of a malignant tumor.

[0023] In one embodiment, the present invention provides a method for treating a malignant tumor, comprising the step of administering a pharmaceutical composition of the present invention to a subject having a malignant tumor.

[0024] In one embodiment, the present invention provides the use of modified bacteria in the preparation of pharmaceuticals for treating malignant tumors. [Brief explanation of the drawing]

[0025] [Figure 1] This is a schematic diagram of the construction scheme for DB-ZW1 strain. [Figure 2]This shows the changes in the distribution of DB-ZW1 in tumors and tissues over time. [Figure 3] The inhibitory effects of DB-ZW1 on bladder cancer, melanoma, and orthotopic colon cancer are shown. Figures 3A to 3C illustrate the effect of DB-ZW1 on tumor volume in subcutaneous bladder cancer, orthotopic melanoma, and subcutaneous orthotopic colon cancer, respectively, while Figure 3D illustrates the effect of DB-ZW1 on tumor number in orthotopic colon cancer. [Figure 4] A map of plasmid pET-26b is shown. [Figure 5] A map of plasmid pET26b-pelB-Nb127D01-HA-his is shown. [Figure 6] Western blotting of nanobody expression induced by IPTG at different concentrations shows the detection of strains BL21(DE3)-pET26b-pelB-Nb127D01-HA-his (Figure 6A), BL21(DE3)-pET26b-pelB-Nb163E3-HA-his (Figure 6B), and BL21(DE3)-pET26b-pelB-Nb97A9-HA-his (Figure 6C). P represents lysed bacterial precipitate, S represents the supernatant of the lysate, and LB represents the supernatant of LB medium. [Figure 7] Western blotting reveals the detection of IPTG-induced expression (Figure 7A) and anaerobic-induced expression (Figure 7B) of the fusion protein. [Figure 8] This study shows that DB-ZW1 levels in MB49 bladder cancer tumors are elevated due to the neutralization of intratumoral neutrophils. Left panel: Intratumoral images of mice administered DB-ZW1 alone; Right panel: Intratumoral images of mice administered DB-ZW1 + Ly6G / Ly6c(Gr-1) antibody. [Figure 9]This study demonstrates that the therapeutic effect of DB-ZW1 on 4T1 breast cancer is enhanced by the neutralization of neutrophils within the tumor. Figure 9A shows flow cytometry results of neutrophil neutralization in tumors of a 4T1 breast cancer model using Ly6G / Ly6c(Gr-1) antibody (αGR-1), Figure 9B shows the change in size of 4T1 tumors injected with the artificial antitumor bacterium ZW1 one day after neutralization with αGR-1 antibody, and Figure 9C shows typical images of tumors from each group one day after bacterial injection. [Figure 10] This shows the percentage of neutrophils to which nanobodies are effectively bound, out of the total number of neutrophils. Here, "Nb-less" represents a sample without added nanobodies, and "control strain" represents a sample to which the LB supernatant of Escherichia coli (E. coli) BL21 culture was added. [Figure 11] This study demonstrates the therapeutic effect of nanobody / fusion protein expression DB-ZW1 in a mouse tumor model. [Figure 12] This shows the blood neutrophil content and tumor neutrophil content of mice after treatment with nanobody / fusion protein expression DB-ZW1. [Figure 13] This shows the expression of nanobodies, toxin proteins, or nanobodies fused to toxin proteins under IPTG induction. M represents a marker, LB represents a secreted protein in the culture medium supernatant, S represents a soluble protein in the dissolved supernatant that is expressed but not secreted, and P represents a functional inclusion protein in the bacterial debris precipitate. [Modes for carrying out the invention]

[0026] definition In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Similarly, the terms and laboratory procedures used herein in relation to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are terms and routine steps widely used in the corresponding fields. At the same time, to further the understanding of this invention, definitions and explanations of the relevant terms are provided below.

[0027] As used herein, “hypoxia-regulated essential gene expression cassette” means a sequence of DNA capable of promoting the expression of an essential gene under hypoxic conditions, the sequence of DNA containing the essential gene under the control of a hypoxia-inducible promoter, and may further contain other regulatory elements necessary for the expression of the essential gene, if necessary.

[0028] As used herein, “essential genes” refer to genes that are critical to bacterial growth and / or survival. In the absence of essential genes or their functional expression products, bacteria cannot survive, divide, and / or grow normally. A typical example of a deletion of an essential gene or its functional expression product is a nutrient-deficient strain that cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments in the absence of certain exogenous supplements. Essential genes usually appear as a single copy on the bacterial chromosome.

[0029] As used herein, a "strictly hypoxia-inducible promoter" refers to a promoter that can initiate transcription of a specific gene under anaerobic conditions, but is virtually incapable of being activated and producing a transcript under aerobic conditions.

[0030] A "strictly hypoxia-inducible promoter" useful in the present invention can be identified by the following experiment.

[0031] Construct an expression strain containing the essential gene (e.g., dapA / dapE) under the control of the promoter to be evaluated, streak it on a Luria Bertani (abbreviated as LB) plate in an anaerobic incubator, and culture anaerobically at 37°C for 24 hours. Under anaerobic conditions, pick a single clone and resuspend it in 20 μL of LB broth. Add the 20 μL resuspension of the single clone evenly to the liquids in the following four tubes (A, B, C, and D, 5 μL per tube): A. 2 ml of LB broth, B. 2 ml of LB broth, C. 2ml LB broth + DAP, D. 2ml of LB broth + DAP.

[0032] Tubes A and C were incubated in an anaerobic incubator at 37°C for 24 hours, and tubes B and D were incubated in an aerobic shaker at 37°C for 24 hours. If bacteria can grow in tubes A, C, and D, but not in tube B (detected OD600 value is less than 0.05), the promoter is considered a strictly hypoxia-inducible promoter.

[0033] Table 1 shows exemplary strictly hypoxia-inducible promoters that can be used in the present invention, as identified by the above experiments.

[0034] [Table 1]

[0035] As used herein, “genes involved in or modulating the endogenous antioxidant stress response pathway” refers to genes involved in the resistance of bacteria (e.g., intracellular bacteria such as Salmonella) to reactive oxygen species (ROS) in an in vivo or in vitro environment.

[0036] As used herein, “genes required for survival in macrophages” refers to genes involved in maintaining or improving the survival capacity of intracellular bacteria within macrophages. Since the function of the products of these genes is related to resistance to the killing effect of macrophages on microorganisms, deletion of these genes or their functional expression products may reduce the survival capacity of bacteria (e.g., intracellular bacteria such as Salmonella) within macrophages.

[0037] The term "functional expression product" as used in this specification includes RNA, proteins, and the like.

[0038] As used herein, "facultative anaerobic bacteria" refers to bacteria that can survive under both aerobic and anaerobic conditions.

[0039] As used herein, "pH-regulating expression cassette" refers to a group of gene expression elements that regulate the expression of the corresponding gene according to the environmental pH.

[0040] As used herein, “promoter active under acidic pH conditions” refers to a promoter that can be activated under acidic pH conditions and can regulate the expression of the corresponding gene. In some embodiments, the promoter of the present invention that is active under acidic pH conditions can be activated or still have promoter activity at pH values ​​below 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or 5.5. In some embodiments, the promoter that is active under acidic pH conditions also has promoter activity at neutral pH (e.g., pH 7.0 to 7.4).

[0041] In this invention, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding native amino acids, as well as to native amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may include, but are not limited to, modified forms, glycosylation, lipid ligation, sulfated forms, γ-carboxylation of glutamic acid residues, and ADP-ribosylation.

[0042] As used herein, "polynucleotide" refers to a macromolecule formed by the linkage of multiple nucleotides via phosphodiester bonds, where nucleotides include ribonucleotides and deoxyribonucleotides. The polynucleotide sequences of the present invention can be codon-optimized for different host cells (e.g., Escherichia coli) to improve polypeptide expression. Methods for codon optimization are known in the art.

[0043] Sequence identity between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between those two sequences. Methods for evaluating the level of sequence identity between polypeptide sequences or polynucleotide sequences are publicly known in the art. Sequence identity can be evaluated using various known sequence analysis software. For example, sequence identity can be evaluated using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be evaluated using the Needleman-Wunsch algorithm with default parameters.

[0044] As used herein, the term "live bacteria" refers to a strain that is viable, characterized by active nutrient metabolism, and capable of performing its own biological functions. Live bacteria may include bacterial biomass produced in the metabolic processes of the strain.

[0045] As used herein, the term "Gram-negative bacteria" refers to bacteria that do not retain the initial basic dye stain (e.g., crystal violet) that is part of the procedure known as Gram staining. In an exemplary Gram stain, cells are first fixed to a slide by heat and stained with a basic dye (e.g., crystal violet) that is taken up by both Gram-negative and Gram-positive bacteria. The slide is then treated with a mordant (e.g., Gram iodine solution) that binds to the basic dye (e.g., crystal violet) and traps it in the cells. The cells are then washed with acetone or alcohol and counterstained with a second dye of a different color (e.g., safranin). Gram-positive organisms retain the initial violet stain, while Gram-negative organisms exhibit counterstaining because they are decolorized by the washing organic solvent. Exemplary Gram-negative bacteria include Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., and Citrobacter. This includes, but is not limited to, *Chlamydia* spp., *Brucella* spp., *Pseudomonas* spp., *Helicobacter* spp., and *Vibrio* spp.

[0046] As used herein, the term "malignant solid tumor" refers to an abnormal mass of tissue, which usually does not include cysts or fluid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of malignant solid tumors are named after the type of cells that form the tumor. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancer) does not usually form malignant solid tumors (as defined by the NIH National Cancer Institute). Malignant solid tumors include, but are not limited to, clusters of abnormal cells that may originate from different histological types, such as the liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid gland, or lung. Therefore, malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, mammary gland tumors, pancreatic tumors, cervical tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral cavity tumors, esophageal tumors, ovarian tumors, prostate tumors, gastric tumors, testicular tumors, thyroid tumors, or lung tumors.

[0047] For the purposes of the treatments described herein, the term “subject” preferably refers to a subject requiring treatment for a target condition, such as a tumor. For preventive purposes, the subject preferably refers to a subject at risk of developing or susceptible to developing a target condition. The term “subject” includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and genetically modified non-human animals. In some preferred embodiments, the subject is human.

[0048] As used herein, “treatment” refers to an approach for obtaining useful or desired clinical outcomes. For the purposes of the present invention, useful or desired clinical outcomes include, but are not limited to, one or more of the following: reduction (or destruction) of the proliferation of neoplastic or cancerous cells, inhibition of metastasis of neoplastic cells, reduction or decrease in tumor size, remission of malignant tumor, relief of symptoms resulting from malignant tumor, improvement of the quality of life of a person with malignant tumor, reduction of the dosage of other medications required to treat malignant tumor, delay in the progression of malignant tumor, cure of malignant tumor, and / or extension of survival of a patient with malignant tumor.

[0049] As used herein, “effective amount” or “effective dose” of a bacterium, pharmaceutical, or pharmaceutical composition is an amount sufficient to produce one or more of the useful or desired outcomes. With respect to prophylactic use, useful or desired outcomes include the elimination or reduction of risk, the attenuation of severity, or the delay of the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, complications of the disease, and pathological intermediate phenotypes that appear during the onset of the disease. With respect to therapeutic use, useful or desired outcomes include, for example, the reduction of one or more symptoms of a disease such as a tumor, the reduction of the dose of other medications required to treat the disease, the improvement of the effects of other medications, and / or the delay of cancer progression in a patient. For example, “effective amount” preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80% compared to an untreated subject. The ability of a compound to inhibit tumor growth can be evaluated in animal model systems that predict its effects in human tumors. Alternatively, the inhibitory properties of a composition can be evaluated by investigating the compound's inhibitory ability in vitro using assays known to skilled practitioners of such inhibition. A therapeutically effective dose of a therapeutic compound may reduce tumor size or otherwise improve symptoms in a subject. Those skilled in the art may determine such a dose based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0050] As used herein, “pharmaceutically acceptable carriers” include, but are not limited to, any material that, when combined with the active ingredient, allows the active ingredient to maintain its biological activity and is non-reactive with the target immune system, disintegrants, adhesives, fillers, buffers, tension agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion). Depending on the route of administration, the bacteria of the present invention may be coated with materials to protect the compound from the action of acids and other natural conditions that can inactivate the bacteria. pharmaceutically acceptable carriers include saline, PBS buffer, sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of solutions or dispersions for sterile injection. The use of such media and agents with respect to pharmaceutically active substances is known in the art. Any conventional media or agent may be used in the pharmaceutical compositions of the present invention unless it is incompatible with the active compound.

[0051] As used herein, the term "cytotoxin" primarily refers to protein toxins that are toxic to cells, such as exotoxins and endotoxins expressed by bacteria.

[0052] Fusion protein The present invention provides a fusion protein comprising an antibody (e.g., a nanobody) that binds to neutrophils or an antigen-binding fragment thereof, and a cytotoxin or an active fragment thereof.

[0053] Examples of antibodies (nanobodies) that bind to neutrophils include, but are not limited to, antibodies that bind to CXCR2 (e.g., nanobodies of SEQ ID NOs. 19, 20, and 21, or antibodies containing their CDRs), antibodies that bind to G-CSF-R (e.g., nanobodies of SEQ ID NOs. 61 and 62, or antibodies containing their CDRs), antibodies that bind to EGFR (e.g., nanobodies of SEQ ID NOs. 63, or antibodies containing their CDRs), antibodies that bind to CXCR4 (e.g., nanobodies of SEQ ID NOs. 64 and 65, or antibodies containing their CDRs), antibodies that bind to IL6R (e.g., nanobodies of SEQ ID NOs. 66-70, or antibodies containing their CDRs), and antibodies that bind to human TNFR1 (e.g., nanobodies of SEQ ID NOs. 71-78, or antibodies containing their CDRs).

[0054] Examples of cytotoxins include, but are not limited to, cholix toxin (e.g., SEQ ID NO: 79), cholera toxin (e.g., SEQ ID NO: 81), heat-sensitive enterotoxins (e.g., subunit A including SEQ ID NO: 82 and subunit B including SEQ ID NO: 83), diphtheria toxin (DT, e.g., SEQ ID NO: 84), Shiga toxin (e.g., chain A including SEQ ID NO: 86 and chain B including SEQ ID NO: 87), anthrax toxin (e.g., anthrax toxin edema factor of SEQ ID NO: 88, anthrax toxin lethal factor of SEQ ID NO: 89, and anthrax toxin protective antigen of SEQ ID NO: 90), lysine (e.g., lysine A chain of SEQ ID NO: 91), and pseudomonas exotoxin (PE, e.g., pseudomonas exotoxin A of SEQ ID NO: 25). The active fragments of these toxins are also included in the present invention and are known in the art, for example, the CET40 fragment of Corix toxin (domains II and III of Corix toxin, SEQ ID NO: 80), the DT390 fragment of diphtheria toxin (SEQ ID NO: 85), and the PE38 fragment and PE[LR] of Pseudomonas exotoxin (SEQ ID NOs: 26 and 27).

[0055] In some embodiments, the antibody is an antibody that binds to CXCR2. In some embodiments, the antibody includes a complementation-determining region (CDR) selected from the group consisting of: CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; CDR1 containing residues 31-35 of SEQ ID NO: 21, CDR2 containing residues 50-64 of SEQ ID NO: 21, and CDR3 containing residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody includes an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0056] In some embodiments, the cytotoxin is a protein toxin. In some embodiments, the cytotoxin is a toxin capable of killing neutrophils. In some embodiments, the cytotoxin is Pseudomonas exotoxin (PE). In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, a fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0057] In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0058] In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker is a flexible linker, for example, (G n S) m The GS linker is such that (wherein m and n are independently integers from 1 to 4). In some embodiments, the fusion protein further includes a tag. In some embodiments, the tag is a His tag (e.g., 6×His) or an HA tag.

[0059] In some embodiments, the cytotoxin or its active fragment is fused to the C-terminus of the antibody or its antigen-binding fragment. In some embodiments, the cytotoxin or its active fragment is directly fused to the antibody or its antigen-binding fragment. In some embodiments, the cytotoxin or its active fragment is fused to the antibody or its antigen-binding fragment via a linker.

[0060] In some embodiments, the fusion protein includes SEQ ID NO: 19 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 19 and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 20 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 20 and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 21 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 21 and SEQ ID NO: 27 from the N-terminus to the C-terminus.

[0061] In some embodiments, the fusion protein includes SEQ ID NO: 28, SEQ ID NO: 19, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 28, SEQ ID NO: 19, and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 28, SEQ ID NO: 20, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 28, SEQ ID NO: 217 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 19, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 19, and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 20, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 20, and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 21, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes SEQ ID NO: 29, SEQ ID NO: 21, and SEQ ID NO: 27 from the N-terminus to the C-terminus. Polynucleotides and expression constructs The present invention provides a polynucleotide encoding the fusion protein of the present invention.

[0062] The present invention also provides an expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, encoding the fusion protein of the present invention.

[0063] In one embodiment, the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr. In some embodiments, the strictly hypoxia-inducible promoter includes a nucleotide sequence selected from SEQ ID NOs: 9-18.

[0064] The present invention also provides an expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, encoding an antibody or antigen-binding fragment thereof that binds to neutrophils.

[0065] Examples of antibodies (nanobodies) that bind to neutrophils include, but are not limited to, antibodies that bind to CXCR2 (e.g., nanobodies of SEQ ID NOs. 19, 20, and 21, or antibodies containing their CDRs), antibodies that bind to G-CSF-R (e.g., nanobodies of SEQ ID NOs. 61 and 62, or antibodies containing their CDRs), antibodies that bind to EGFR (e.g., nanobodies of SEQ ID NOs. 63, or antibodies containing their CDRs), antibodies that bind to CXCR4 (e.g., nanobodies of SEQ ID NOs. 64 and 65, or antibodies containing their CDRs), antibodies that bind to IL6R (e.g., nanobodies of SEQ ID NOs. 66-70, or antibodies containing their CDRs), and antibodies that bind to human TNFR1 (e.g., nanobodies of SEQ ID NOs. 71-78, or antibodies containing their CDRs).

[0066] In some embodiments, the antibody is an antibody that binds to CXCR2. In some embodiments, the antibody includes a complementation-determining region (CDR) selected from the group consisting of: CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; CDR1 containing residues 31-35 of SEQ ID NO: 21, CDR2 containing residues 50-64 of SEQ ID NO: 21, and CDR3 containing residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody includes an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0067] In some embodiments, the nucleotide sequence further encodes a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0068] In some embodiments, the nucleotide sequence further encodes a tag sequence. In some embodiments, the tag is a His tag (e.g., 6×His) or an HA tag.

[0069] The present invention also provides an expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter encoding a cytotoxin or an active fragment thereof.

[0070] In some embodiments, the cytotoxin is a protein toxin. In some embodiments, the cytotoxin is a toxin that can kill neutrophils. In some embodiments, the cytotoxin can kill tumor cells.

[0071] Examples of cytotoxins include, but are not limited to, Corix toxin (e.g., SEQ ID NO: 79), cholera toxin (e.g., SEQ ID NO: 81), thermosensitive enterotoxins (e.g., subunit A including SEQ ID NO: 82 and subunit B including SEQ ID NO: 83), diphtheria toxin (DT, e.g., SEQ ID NO: 84), Shiga toxin (e.g., chain A including SEQ ID NO: 86 and chain B including SEQ ID NO: 87), anthrax toxin (e.g., anthrax toxin edema factor of SEQ ID NO: 88, anthrax toxin lethal factor of SEQ ID NO: 89, and anthrax toxin protective antigen of SEQ ID NO: 90), lysine (e.g., lysine chain A of SEQ ID NO: 91), or Pseudomonas exotoxin (PE, e.g., Pseudomonas exotoxin A of SEQ ID NO: 25). The active fragments of these toxins are also included in the present invention and are known in the art, for example, the CET40 fragment of Corix toxin (domains II and III of Corix toxin, SEQ ID NO: 80), the DT390 fragment of diphtheria toxin (SEQ ID NO: 85), and the PE38 fragment and PE[LR] of Pseudomonas exotoxin (SEQ ID NOs: 26 and 27).

[0072] In some embodiments, the cytotoxin is Pseudomonas exotoxin (PE). In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytotoxin fragment comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0073] In some embodiments, the nucleotide sequence further encodes a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0074] In some embodiments, the nucleotide sequence further encodes a tag sequence. In some embodiments, the tag is a His tag (e.g., 6×His) or an HA tag.

[0075] Modified bacteria The present invention provides a modified bacterium comprising the expression construct of the present invention and a hypoxia-regulating essential gene expression cassette, compared to an unmodified starting strain. In one embodiment, the essential gene expression cassette contains the bacterium's essential genes under the control of a strictly hypoxia-inducible promoter. In one embodiment, the bacterium lacks at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or a functional expression product thereof. In one embodiment, the bacterium lacks at least one gene required for survival in macrophages, or a functional expression product thereof. In one embodiment, the bacterium expresses wild-type lipopolysaccharide (LPS).

[0076] In one embodiment, the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr. Preferably, the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0077] In one embodiment, the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) in bacteria. In one embodiment, when cultured under aerobic conditions, bacterial growth relies on additional DAP or analogues added to the culture medium. In one embodiment, the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof. Preferably, the essential gene is selected from dapA and dapE. Preferably, dapE contains a nucleotide sequence having at least 81%, preferably at least 82%, more preferably at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% sequence identity with respect to SEQ ID NO: 57. Preferably, the nucleotide sequence of dapE includes SEQ ID NO: 57. Preferably, the nucleotide sequence of dapE consists of SEQ ID NO: 57.

[0078] In one embodiment, the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family gene. In one embodiment, the functional expression product of the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family-related protein. In one embodiment, the bacterium lacks the activity of HtrA serine protease. In one embodiment, the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA. In one embodiment, the bacterium lacks htrA. Preferably, htrA contains a nucleotide sequence having at least 81%, preferably at least 82%, more preferably at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% sequence identity with respect to SEQ ID NO: 58. Preferably, the nucleotide sequence of htrA contains SEQ ID NO: 58. Preferably, the nucleotide sequence of htrA consists of SEQ ID NO: 58.

[0079] In one embodiment, the essential gene is a gene naturally present on the bacterial chromosome. In another embodiment, the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter. As a result, the expression of the essential gene in bacteria is completely under the control of the strictly hypoxia-inducible promoter.

[0080] In one embodiment, the essential gene expression cassette is exogenous. In one embodiment, essential genes naturally present on the bacterial chromosome are deleted or functionally inactivated. As a result, the expression of essential genes in bacteria is entirely under the control of a strictly hypoxia-inducible promoter. In one embodiment, the exogenous essential gene expression cassette is integrated into the bacterial chromosome. In one embodiment, the exogenous essential gene expression cassette is located outside the bacterial chromosome. In one embodiment, the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0081] In one embodiment, the modified bacterium further comprises a pH-regulating expression cassette. In one embodiment, compared to the unmodified starting strain, the modified bacterium comprises a hypoxia-regulating essential gene expression cassette and a pH-regulating expression cassette. In one embodiment, the pH-regulating expression cassette contains a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions. According to some embodiments of the present invention, the promoter that is active under acidic pH conditions is active in a pH range of approximately pH 4.0 to 7.0. In some embodiments, the promoter that is active under acidic pH conditions is also active at pH 6.0 ± 1. In some embodiments, the promoter that is active under acidic pH conditions is active at pH values ​​less than 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6 or 5.5, or between any two of these values. In one embodiment, the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR. In one embodiment, the promoter active under acidic pH conditions is sseA. In one embodiment, the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein. In one embodiment, the gene encoding the gram-negative bacterial hemolysin protein is hlyA or hlyE. In one embodiment, hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli. Preferably, hlyA contains a nucleotide sequence having at least 81%, preferably at least 82%, more preferably at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% sequence identity with respect to SEQ ID NO: 59. Preferably, the nucleotide sequence of hlyA contains SEQ ID NO: 59.Preferably, the nucleotide sequence of hlyA consists of SEQ ID NO: 59.

[0082] In one embodiment, the unmodified starting strain is a facultative anaerobic bacterium. In another embodiment, the bacterium is from the family Enterobacteriaceae. Preferably, the bacterium is from the genera Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea. Preferably, the bacteria include Escherichia coli, Enterobacter blattae (E. blattae), Enterobacter fergusonii (E. fergusonii), Enterobacter hermannii (E. hermannii), Enterobacter vulneris (E. vulneris), Salmonella enterica (S. enterica), Salmonella bongori (S. bongori), Salmonella typhi (S. typhi), Salmonella choleraesuis (S. choleraesuis), and Salmonella typhi (Salmonella Shigella typhimurium (S. typhimurium), Shigella dysenteriae (S. dysenteriae), Shigella flexneri (S. flexneri), Shigella boydii (S. boydii), Shigella sonnei (S. sonnei), Klebsiella pneumoniae (K.peumoniae), Klebsiella oxytoca (K. oxytoca), Yersinia pestis (Y. pestis), Yersinia enterocolitica (Y. enterocolitica), Mycobacterium pseudotuberculosis (Y. pseudotuberculosis), Yersinia aldouae (Y. aldouae), Yersinia bercovieri (Y. bercovieri), Yersinia frederiksenii (Y. frederiksenii), Yersinia intermedia (Y. intermedia), Yersinia kristersenii (Y. Yersinia kristersenii, Yersinia mollaretti (Y.mollaretti), Yersinia rohdei (Y.rohdei), Yersinia ruckeri (Y.ruckeri), Citrobacter freundii (C.freundii), Citrobacter koseri (C.kaseri), Citrobacter braakii (C.braakii), Enterobacter aerogenes (E.aerogenes), Enterobacter cloacae (E.cloacae), Enterobacter Enterobacter gergoviae (E. gergoviac), Enterobacter sakazakii (E. sakazakii), Enterobacter taylorae (E.Enterobacter amnigenus (E. aminigenus), Enterobacter intermedius (E. intermedius), Enterobacter asburiae (E. asburiac), Enterobacter cancerogenus (E. cancerogenus), Enterobacter dissolvens (E. dissolvens), Enterobacter nimipressuralis (E. nimipressuralis), Serratia marcescens (S. marcescens), Serratia entomophila (S. entomophila), Serratia ficaria ficaria (S. ficaria), Serratia fonticola (S. fonticola), Serratia grimesii (S. grimesii), Serratia liquefaciens (S. liquefaciens), Serratia odorifera (S. odorifera), Serratia plymuthica (S. plymuthica), Serratia proteamaculans (S. proteamaculans), Serratia rubidaea (S. rubidaea), Serratia ureilytica (S. ureilytica), Proteus mirabilis (Proteus mirabilis (P. mirabilis), Proteus vulgaris (P. vulgaris), Proteus myxofaciens (P.myxofaciens), Proteus penneri (P. penneri), Proteus hauseri (P. hauseri), Morganella morganii (M. morganii), Providencia alcalifaciens (P. alcalifaciens), Providencia rustigianii (P. rustigianii), Providencia stuartii (P. stuartii), Providencia rettgeri (P. rettgeri), Providencia heimbachae (P. heimbochae), Hafnia albeyi The bacterium is selected from the group consisting of *H. alvei* (H. alvei) and *Pantoea agglomerans* (P. agglomerans). Preferably, the bacterium is *Typhimurium typhimurense*. Preferably, the starting strain is *Typhimurium typhimurense* SL7207.

[0083] In one embodiment, when administered to a subject with a tumor, the bacteria can survive and proliferate in tumor tissue but are rapidly eliminated in normal tissue. In one embodiment, when administered to a subject with a malignant tumor, the bacteria can induce an antitumor-specific immune response. In one embodiment, when administered to a subject with a malignant tumor, the bacteria can induce antitumor immunological memory.

[0084] In one embodiment, the bacteria do not express wild-type flagellin. In another embodiment, the modified bacteria lack the fliC gene.

[0085] In one embodiment, the survival level of bacteria in macrophages is about 50% to about 30% of the survival level of the unmodified starting strain. In another embodiment, the survival level of bacteria in macrophages is about 30% to about 10% of the survival level of the unmodified starting strain. Preferably, the survival level of bacteria in macrophages is about 10% to about 1% of the survival level of the unmodified starting strain.

[0086] Pharmaceutical composition The present invention provides a pharmaceutical composition containing an effective amount of the modified bacteria of the present invention. In one embodiment, the modified bacteria are live bacteria.

[0087] In one embodiment, the pharmaceutical composition is used to treat a malignant tumor. In one embodiment, the pharmaceutical composition is used to induce an antitumor-specific immune response in a subject with a malignant tumor. In one embodiment, the pharmaceutical composition is used to induce antitumor immunological memory in a subject with a malignant tumor. In one embodiment, the pharmaceutical composition is used to prevent or treat metastasis or recurrence of a malignant tumor. In one embodiment, the pharmaceutical composition is used to treat a malignant tumor that is resistant to or has failed to respond to prior antitumor treatment.

[0088] In one embodiment, the malignant tumor is a malignant tumor from the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin and mucous membranes. In some embodiments, the malignant tumor is a sarcoma or cancer. In one embodiment, the malignant tumor is a solid tumor. In one embodiment, the malignant tumor is selected from the group consisting of glioma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, oral cancer, tongue cancer, laryngeal cancer, head and neck cancer, melanoma, bronchial cancer, lung cancer, pleural cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, bile duct cancer, colorectal cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal cancer (suprarenoma), thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer, and osteosarcoma.

[0089] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Treatment method and use The present invention provides a method for treating a malignant tumor, comprising the step of administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to a subject having a malignant tumor.

[0090] The present invention provides a method for inducing an antitumor-specific immune response in a subject having a malignant tumor, comprising the step of administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to the subject having a malignant tumor.

[0091] The present invention provides a method for inducing antitumor immunological memory in a subject having a malignant tumor, comprising the step of administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to the subject having a malignant tumor.

[0092] The present invention provides a method for preventing or treating metastasis or recurrence of a malignant tumor, comprising the step of administering an effective amount of the modified bacteria or pharmaceutical composition of the present invention to a subject having a malignant tumor.

[0093] The present invention provides a method for preventing or treating metastasis or recurrence of a malignant tumor, comprising the step of administering an effective amount of the modified bacteria or pharmaceutical composition of the present invention to a subject having a malignant tumor or being at high risk of metastasis or recurrence of a malignant tumor.

[0094] The present invention provides a method for treating malignant tumors that are resistant to or have failed to undergo prior antitumor treatment, comprising the step of administering an effective amount of the modified bacteria or pharmaceutical composition of the present invention to a subject having a malignant tumor that is resistant to or has failed to undergo prior antitumor treatment.

[0095] The present invention provides for the use of the modified bacteria in the manufacture of pharmaceuticals for the treatment of malignant tumors.

[0096] The present invention provides the use of a modified bacterium in the manufacture of a pharmaceutical product for inducing an antitumor-specific immune response in subjects with malignant tumors.

[0097] The present invention provides the use of a modified bacterium in the manufacture of a pharmaceutical product for inducing antitumor immunological memory in subjects with malignant tumors.

[0098] The present invention provides the use of the modified bacteria of the present invention in the manufacture of a pharmaceutical product for preventing or treating the metastasis or recurrence of malignant tumors.

[0099] The present invention provides the use of the modified bacteria of the present invention in the manufacture of pharmaceuticals for treating malignant tumors that are resistant to or have failed to receive prior antitumor therapies.

[0100] In one embodiment, the modified bacteria are viable bacteria.

[0101] In one embodiment, the malignant tumor is a malignant tumor of the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin and mucous membranes. In some embodiments, the malignant tumor is a sarcoma or cancer. In one embodiment, the malignant tumor is a solid tumor. In one embodiment, the malignant tumor is selected from the group consisting of glioma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, oral cancer, tongue cancer, laryngeal cancer, head and neck cancer, melanoma, bronchial cancer, lung cancer, pleural cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, bile duct cancer, colorectal cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal cancer, thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer, and osteosarcoma.

[0102] In one embodiment, the bacteria, pharmaceutical, or pharmaceutical composition of the present invention is administered intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally, transcerebrally, through the gastrointestinal tract, on the body surface, through the oral mucosa, through the nose, through the rectum, or through the vaginal route. In one embodiment, the bacteria, pharmaceutical, or pharmaceutical composition of the present invention may be formulated for administration intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally, transcerebrally, through the gastrointestinal tract, on the body surface, through the oral mucosa, through the nose, through the rectum, or through the vaginal route.

[0103] In one embodiment, the dosage form of the pharmaceutical or pharmaceutical composition of the present invention may be, for example, a solution, emulsion, lyophilized preparation or suspension; for oral administration, the dosage form may be a tablet or capsule; for nasal administration, the dosage form may be a powder, infusion or aerosol; for topical administration, the dosage form may be an aqueous solution, suspension, ointment, cream or gel; and for rectal or vaginal administration, the dosage form may be a suppository, enema, or delivered as part of an endoscopic or colonoscopy procedure. In one embodiment, a pharmaceutical or pharmaceutical composition may be formulated in the form of a solution, emulsion, lyophilized preparation or suspension; for oral administration, it may be formulated in the form of a tablet or capsule; for intranasal dosage form, it may be formulated in the form of a powder, nasal spray or aerosol; for topical administration, it may be formulated in the form of an aqueous solution, suspension, ointment, cream or gel; and for rectal or vaginal administration, it may be formulated as a suppository, enema, or delivered as part of an endoscopic or colonoscopy procedure. For methods known in the art for preparing dosage forms, see, for example, Remington's Pharmaceutical Sciences (20th edition), edited by A. Gennaro, 2000, Mack Publishing Company, Inc. Easton, PA.

[0104] In some embodiments, the bacteria, pharmaceuticals, or pharmaceutical compositions of the present invention may be administered at various intervals, including, but not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, and including a range between any two of the listed values, for example, 1 minute to 10 minutes, 1 minute to 30 minutes, 1 minute ~1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 10 minutes to 18 hours, 10 minutes to 1 day, 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 4 hours, 30 minutes to 12 hours, 30 minutes to 18 hours, 30 minutes to 1 day, 30 minutes to 2 days, 1 This includes 1 hour to 2 hours, 1 hour to 4 hours, 1 hour to 12 hours, 1 hour to 18 hours, 1 hour to 1 day, 4 hours to 12 hours, 4 hours to 18 hours, 4 hours to 1 day, 1 hour to 2 days, 1 hour to 3 days, 1 hour to 4 days, 1 hour to 5 days, 1 hour to 7 days, 1 hour to 10 days, 2 hours to 3 days, 2 hours to 4 days, 2 hours to 5 days, 2 hours to 7 days, 2 hours to 10 days, or 5 to 10 days. In some embodiments, bacteria, pharmaceuticals, or pharmaceutical compositions are administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 15 weeks, once every 20 weeks, once every 25 weeks, or once every 26 weeks.In some embodiments, the bacteria, pharmaceuticals, or pharmaceutical compositions of the present invention may be formulated to be administered at various intervals, including, but not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, and including a range between any two of the listed values, for example, 1 minute to 10 minutes, 1 minute to 30 minutes. Minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 10 minutes to 18 hours, 10 minutes to 1 day, 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 4 hours, 30 minutes to 12 hours, 30 minutes to 18 hours, 30 minutes to 1 day, 30 minutes to 2 days , including 1-2 hours, 1-4 hours, 1-12 hours, 1-18 hours, 1 hour-1 day, 4-12 hours, 4-18 hours, 4 hours-1 day, 1 hour-2 days, 1 hour-3 days, 1 hour-4 days, 1 hour-5 days, 1 hour-7 days, 1 hour-10 days, 2 hours-3 days, 2 hours-4 days, 2 hours-5 days, 2 hours-7 days, 2 hours-10 days, or 5-10 days. In some embodiments, bacteria, pharmaceuticals, or pharmaceutical compositions are formulated to be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks. The administration plan may be based on the pharmacokinetic decay pattern that practitioners predict will be achieved. Advances in treatment can be monitored through routine techniques and assays. The administration plan may change over time.

[0105] In some embodiments, the effective amount of bacteria in the pharmaceutical or pharmaceutical composition is at least about 10 4 Colony-forming units (CFUs), for example, at least about 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 contains cfu, and the range between any of the listed values, for example, 10 4 ~10 8 cfu, 10 4 ~10 9 cfu, 10 4 ~10 10 cfu, 10 4 ~10 11 cfu, 10 4 ~10 12 cfu, 10 4 ~10 12 cfu, 10 5 ~10 8 cfu, 10 5 ~10 9 cfu, 10 5 ~10 10 cfu, 10 5 ~10 11 cfu, 10 5 ~10 12 cfu, 10 5 ~10 12 cfu, 10 6 ~10 8 cfu, 10 6 ~10 9 cfu, 10 6 ~10 10 cfu, 10 6 ~10 11 cfu, 10 6 ~10 12 cfu, 10 6 ~10 12 cfu, 10 7 ~10 8 cfu, 10 7 ~10 9 cfu, 10 7 ~10 10 cfu, 10 7 ~10 11 cfu, 10 7 ~10 12 cfu, 107 ~10 12 cfu, 10 8 ~10 9 cfu, 10 8 ~10 10 cfu, 10 8 ~10 11 cfu, 10 8 ~10 12 cfu or 10 8 ~10 12 including cfu.

[0106] In some embodiments, the effective dose volume of the bacteria, pharmaceutical, or pharmaceutical composition of the present invention is about 3 mL, about 2.5 mL, about 2 mL, about 1.5 mL, about 1 mL, about 0.75 mL, about 0.5 mL, about 0.25 mL, or about 0.1 mL or less. In some embodiments, the effective dose volume of the bacteria, pharmaceutical, or pharmaceutical composition is about 20 mL, about 19 mL, about 18 mL, about 17 mL, about 16 mL, about 15 mL, about 14 mL, about 13 mL, about 12 mL, about 11 mL, about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, or about 1 mL. Alternatively, the effective dose volume of the bacteria, pharmaceutical, or pharmaceutical composition is approximately 20.5 mL, approximately 19.5 mL, approximately 18.5 mL, approximately 17.5 mL, approximately 16.5 mL, approximately 15.5 mL, approximately 14.5 mL, approximately 13.5 mL, approximately 12.5 mL, approximately 11.5 mL, approximately 10.5 mL, approximately 9.5 mL, approximately 8.5 mL, approximately 7.5 mL, approximately 6.5 mL, approximately 5.5 mL, approximately 4.5 mL, approximately 3.5 mL, approximately 2.5 mL, approximately 1.5 mL, or approximately 0.5 mL. In some embodiments, the effective dose volume of the bacteria, pharmaceutical or pharmaceutical composition is approximately 200 mL, approximately 190 mL, approximately 180 mL, approximately 170 mL, approximately 160 mL, approximately 150 mL, approximately 140 mL, approximately 130 mL, approximately 120 mL, approximately 110 mL, approximately 100 mL, approximately 90 mL, approximately 80 mL, approximately 70 mL, approximately 60 mL, approximately 50 mL, approximately 40 mL, or approximately 30 mL, as well as a range between any two of the listed values, for example, 100 mL to 110 mL, 100 mL to 120 mL, 90 mL to 120 mL, or 90 mL to 130 mL. Alternatively, the effective dose volume of the bacteria, pharmaceutical or pharmaceutical composition is approximately 205 mL, approximately 195 mL, approximately 185 mL, approximately 175 mL, approximately 165 mL, approximately 155 mL, approximately 145 mL, approximately 135 mL, approximately 125 mL, approximately 115 mL, approximately 105 mL, approximately 95 mL, approximately 85 mL, approximately 75 mL, approximately 65 mL, approximately 55 mL, approximately 45 mL, approximately 35 mL, or approximately 25 mL, as well as a range between any two of the listed values, for example, 105 mL to 115 mL, 105 mL to 125 mL, 95 mL to 125 mL, or 95 mL to 135 mL.Alternatively, the volume of an effective dose of bacteria, medicine or pharmaceutical composition is about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters or about 100 microliters, optionally about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters or about 50 microliters. In some embodiments, the volume of an effective dose of bacteria, medicine or pharmaceutical composition is about 2.0 mL or less.

[0107] In a medicine or pharmaceutical composition, for example, the concentration of the bacteria of the present invention is at least about 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 or 10 13 cfu / ml and can be in the range between any of the recited values, for example, 10 3 ~10 8 cfu / ml, 10 3 ~10 9 cfu / ml, 10 3 ~10 10 cfu / ml, 10 3 ~10 11 cfu / ml, 10 3 ~10 12 cfu / ml, 10 3 ~10 13 cfu / ml, 10 4 ~10 8 cfu / ml, 10 4 ~10 9 cfu / ml, 10 4 ~10 10 cfu / ml, 10 4 ~10 11 cfu / ml, 104 ~10 12 cfu / ml, 10 4 ~10 13 cfu / ml, 10 5 ~10 8 cfu / ml, 10 5 ~10 9 cfu / ml, 10 5 ~10 10 cfu / ml, 10 5 ~10 11 cfu / ml, 10 5 ~10 12 cfu / ml, 10 5 ~10 13 cfu / ml, 10 6 ~10 8 cfu / ml, 10 6 ~10 9 cfu / ml, 10 6 ~10 10 cfu / ml, 10 6 ~10 11 cfu / ml, 10 6 ~10 12 cfu / ml, 10 6 ~10 13 cfu / ml, 10 7 ~10 8 cfu / ml, 10 7 ~10 9 cfu / ml, 10 7 ~10 10 cfu / ml, 10 7 ~10 11 cfu / ml, 10 7 ~10 12 cfu / ml, 10 7 ~10 13 cfu / ml, 10 8 ~10 9 cfu / ml, 10 8 ~10 10 cfu / ml, 10 8 ~10 11 cfu / ml, 10 8 ~10 12 cfu / ml or 10 8 ~10 13 Contains cfu / ml. In a pharmaceutical or pharmaceutical composition, for example, the concentration of the bacteria of the present invention is at least about 10 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 cfu / g can be a range between any of the enumerated values, for example, 10 3 ~10 8 cfu / g, 10 3 ~10 9 cfu / g, 10 3 ~10 10 cfu / g, 10 3 ~10 11 cfu / g, 10 3 ~10 12 cfu / g, 10 3 ~10 13 cfu / g, 10 4 ~10 8 cfu / g, 10 4 ~10 9 cfu / g, 10 4 ~10 10 cfu / g, 10 4 ~10 11 cfu / g, 10 4 ~10 12 cfu / g, 10 4 ~10 13 cfu / g, 10 5 ~10 8 cfu / g, 10 5 ~10 9 cfu / g, 10 5 ~10 10 cfu / g, 10 5 ~10 11 cfu / g, 10 5 ~10 12 cfu / g, 10 5 ~10 13 cfu / g, 10 6 ~10 8 cfu / g, 10 6 ~10 9 cfu / g, 10 6 ~10 10 cfu / g, 10 6 ~10 11cfu / g, 10 6 ~10 12 cfu / g, 10 6 ~10 13 cfu / g, 10 7 ~10 8 cfu / g, 10 7 ~10 9 cfu / g, 10 7 ~10 10 cfu / g, 10 7 ~10 11 cfu / g, 10 7 ~10 12 cfu / g, 10 7 ~10 13 cfu / g, 10 8 ~10 9 cfu / g, 10 8 ~10 10 cfu / g, 10 8 ~10 11 cfu / g, 10 8 ~10 12 cfu / g or 10 8 ~10 13 Contains cfu / g

[0108] The dose ranges described herein are illustrative and do not limit the dose ranges that may be selected by a physician. The amount of the active compound (e.g., the bacteria of the present invention) in the pharmaceutical composition of the present invention may vary according to the presence or absence of malignant tumor; the type and severity of the malignant tumor to be treated; the activity or viability of the bacteria, pharmaceutical or pharmaceutical composition; the route of administration; the duration of treatment; any pharmaceuticals used in combination with the bacteria, pharmaceutical or pharmaceutical composition; the disease state of the individual, including the diet and general health status of the subject, as well as similar factors well known in the art. The administration plan is adjustable to produce an optimal therapeutic response. For example, a single dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation.

[0109] The bacteria, pharmaceuticals, or pharmaceutical compositions of the present invention may be produced by methods well known in the art, including, for example, microbial growth in a fermenter, followed by concentration, and washing by centrifugation, filtration, or dialysis, as well as conventional granulation, mixing, dissolution, encapsulation, freeze-drying, or emulsification. The bacteria, pharmaceuticals, or pharmaceutical compositions of the present invention may be produced in various forms, including granules; precipitates or fine particles; powders including freeze-dried powder, rotary-dried powder, spray-dried powder, and amorphous powder; injection solutions; emulsions; elixirs; suspensions, or solutions. The formulations may optionally contain stabilizers, pH adjusters, surfactants, bioavailability adjusters, and combinations thereof.

[0110] In one embodiment, the bacteria, pharmaceutical, or pharmaceutical composition of the present invention may be administered alone or in combination with other compounds or compositions in the presence of a carrier. In one embodiment, the bacteria, pharmaceutical, or pharmaceutical composition may be administered in combination with other malignant tumor therapies, including but not limited to radiotherapy, chemotherapy, and surgical procedures. The bacteria, pharmaceutical, or pharmaceutical composition may be used as an adjuvant in such therapies. Therefore, other aspects of this embodiment relate to bacteria, pharmaceuticals, or pharmaceutical compositions for use as an adjuvant in malignant tumor therapies selected from radiotherapy and chemotherapy.

[0111] Exemplary Embodiments Embodiment 1: A fusion protein comprising an antibody (e.g., a nanobody) that binds to neutrophils or an antigen-binding fragment thereof, and a cytotoxin or an active fragment thereof.

[0112] Embodiment 2: The antibody is CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 contains residues 31-35 of SEQ ID NO: 21, CDR2 contains residues 50-64 of SEQ ID NO: 21, and CDR3 contains residues 98-115 of SEQ ID NO: 21. The fusion protein according to Embodiment 1, comprising a complementation-determining region (CDR) selected from the group consisting of the following.

[0113] Embodiment 3: The fusion protein according to Embodiment 1 or 2, wherein the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0114] Embodiment 4: A fusion protein according to any one of Embodiments 1 to 3, wherein the cytotoxin is a protein toxin capable of killing neutrophils.

[0115] Embodiment 5: A fusion protein according to any one of Embodiments 1 to 4, wherein the cytotoxin is Pseudomonas exotoxin (PE).

[0116] Embodiment 6: A fusion protein according to any one of Embodiments 1 to 5, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25.

[0117] Embodiment 7: A fusion protein according to any one of Embodiments 1 to 6, wherein the active fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0118] Embodiment 8: A fusion protein according to any one of Embodiments 1 to 7, further comprising, for example, a signal peptide containing the amino acid sequence of SEQ ID NO: 28 or 29.

[0119] Embodiment 9: Linker, e.g., flexible linker, e.g., (G n S) m A fusion protein according to any one of Embodiments 1 to 8, further comprising a GS linker such as (wherein m and n are independently integers from 1 to 4).

[0120] Embodiment 10: A fusion protein according to any one of Embodiments 1 to 9, further comprising a tag, for example, a His tag (e.g., 6×His) or an HA tag.

[0121] Embodiment 11: A fusion protein according to any one of Embodiments 1 to 10, wherein a cytotoxin or an active fragment thereof is fused to the C-terminus of an antibody or an antigen-binding fragment thereof.

[0122] Embodiment 12: A fusion protein according to any one of Embodiments 1 to 11, wherein a cytotoxin or an active fragment thereof is fused to an antibody or an antigen-binding fragment thereof, either directly or via a linker.

[0123] Embodiment 13: From the N-terminus to the C-terminus, Sequence ID 19 and Sequence ID 26, Sequence ID 19 and Sequence ID 27, Sequence IDs 20 and 26, Sequence IDs 20 and 27, Sequence ID 21 and Sequence ID 26, or Sequence IDs 21 and 27 A fusion protein according to any one of Embodiments 1 to 12, including the above.

[0124] Embodiment 14: From the N-terminus to the C-terminus, Sequence ID 28, Sequence ID 19 and Sequence ID 26, Sequence ID 28, Sequence ID 19 and Sequence ID 27, Sequence ID 28, Sequence ID 20 and Sequence ID 26, Sequence ID 28, Sequence ID 20 and Sequence ID 27, Sequence ID 28, Sequence ID 21 and Sequence ID 26, Sequence ID 28, Sequence ID 21 and Sequence ID 27, Sequence ID 29, Sequence ID 19 and Sequence ID 26, Sequence ID 29, Sequence ID 19 and Sequence ID 27, Sequence ID 29, Sequence ID 20 and Sequence ID 26, Sequence ID 29, Sequence ID 20 and Sequence ID 27, Sequence ID 29, Sequence ID 21 and Sequence ID 26, or Sequence IDs 29, 21, and 27 A fusion protein according to any one of embodiments 1 to 13, including the above.

[0125] Embodiment 15: An isolated polynucleotide encoding the nucleotide sequence of the fusion protein described in any one of Embodiments 1 to 14.

[0126] Embodiment 16: An expression construct comprising the polynucleotide described in Embodiment 15, operably linked to a strictly hypoxia-inducible promoter.

[0127] Embodiment 17: The expression construct according to Embodiment 16, wherein the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0128] Embodiment 18: The expression construct according to Embodiment 16 or 17, wherein the hypoxia-inducible promoter comprises a nucleotide sequence selected from SEQ ID NOs: 9-18.

[0129] Embodiment 19: An expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, encoding an antibody (e.g., a nanobody) or an antigen-binding fragment thereof that binds to neutrophils, or a toxin or an active fragment thereof.

[0130] Embodiment 20: The antibody is CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 contains residues 31-35 of SEQ ID NO: 21, CDR2 contains residues 50-64 of SEQ ID NO: 21, and CDR3 contains residues 98-115 of SEQ ID NO: 21. It includes a complementarity determination region (CDR) selected from the group consisting of, or The toxin contains the amino acid sequence of SEQ ID NO: 25, or The expression construct according to Embodiment 19, wherein the active fragment of the toxin comprises the active fragment of SEQ ID NO: 26 or 27.

[0131] Embodiment 21: An expression construct according to Embodiment 19 or 20, wherein the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20, and 21.

[0132] Embodiment 22: An expression construct according to any one of Embodiments 19 to 21, wherein the nucleotide sequence further encodes a signal peptide comprising, for example, the amino acid sequence of SEQ ID NO: 28 or 29.

[0133] Embodiment 23: An expression construct according to any one of Embodiments 19 to 22, wherein the nucleotide sequence further encodes a tag, for example, a His tag (e.g., 6×His) or an HA tag.

[0134] Embodiment 24: An expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter encoding a cytotoxin or an active fragment thereof.

[0135] Embodiment 25: The expression construct according to Embodiment 24, wherein the cytotoxin is a protein toxin capable of killing neutrophils.

[0136] Embodiment 26: The expression construct according to Embodiment 24 or 25, wherein the cytotoxin is Pseudomonas exotoxin (PE).

[0137] Embodiment 27: An expression construct according to any one of Embodiments 24 to 26, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25.

[0138] Embodiment 28: An expression construct according to any one of Embodiments 24 to 27, wherein the active fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0139] Embodiment 29: An expression construct according to any one of Embodiments 24 to 28, wherein the nucleotide sequence further encodes a signal peptide comprising, for example, the amino acid sequence of SEQ ID NO: 28 or 29.

[0140] Embodiment 30: An expression construct according to any one of Embodiments 24 to 29, wherein the nucleotide sequence further encodes a tag, for example, a His tag (e.g., 6×His) or an HA tag.

[0141] Embodiment A Embodiment A1: A modified bacterium that, compared to an unmodified starting strain, includes an expression construct described in any one of Embodiments 16 to 30, and a hypoxia-regulating essential gene expression cassette containing essential bacterial genes under the control of a strictly hypoxia-inducible promoter, and lacks at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or its functional expression product.

[0142] Embodiment A2: The modified bacterium according to Embodiment A1, wherein the strictly hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0143] Embodiment A3: The modified bacterium according to Embodiment A1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0144] Embodiment A4: A modified bacterium according to any one of Embodiments A1 to A3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0145] Embodiment A5: The modified bacterium according to Embodiment A4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0146] Embodiment A6: The modified bacterium according to Embodiment A4, wherein the essential gene is selected from dapA and dapE.

[0147] Embodiment A7: A modified bacterium according to any one of Embodiments A1 to A6, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family gene.

[0148] Embodiment A8: A modified bacterium according to any one of Embodiments A1 to A7, wherein the functional expression product of a gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family-related protein.

[0149] Embodiment A9: A modified bacterium according to any one of Embodiments A1 to A8, which lacks the activity of HtrA serine protease.

[0150] Embodiment A10: A modified bacterium according to any one of Embodiments A1 to A9, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0151] Embodiment A11: A modified bacterium according to any one of Embodiments A1 to A10, lacking htrA.

[0152] Embodiment A12: A modified bacterium according to any one of Embodiments A1 to A11, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

[0153] Embodiment A13: A modified bacterium according to any one of Embodiments A1 to A11, wherein the essential gene expression cassette is exogenous, and essential genes naturally present in the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0154] Embodiment A14: The modified bacterium according to Embodiment A13, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0155] Embodiment A15: The modified bacterium according to Embodiment A13, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0156] Embodiment A16: A modified bacterium according to Embodiment A15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0157] Embodiment A17: A modified bacterium according to any one of Embodiments A1 to A16, further comprising a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions.

[0158] Embodiment A18: A modified bacterium according to Embodiment A17, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0159] Embodiment A19: The modified bacterium according to Embodiment A18, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0160] Embodiment A20: A modified bacterium according to Embodiment A19, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0161] Embodiment A21: A modified bacterium according to any one of Embodiments A17 to A20, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0162] Embodiment A22: A modified bacterium according to any one of Embodiments A17 to A21, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0163] Embodiment A23: A modified bacterium according to any one of Embodiments A17 to A21, wherein the promoter active under acidic pH conditions is sseA.

[0164] Embodiment A24: A modified bacterium according to any one of Embodiments A1 to A23, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0165] Embodiment A25: A modified bacterium according to any one of Embodiments A1 to A24, which is a bacterium of the Enterobacteriaceae family.

[0166] Embodiment A26: A modified bacterium according to any one of Embodiments A1 to A25, which is a bacterium of the genus Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0167] Embodiment A27: Escherichia coli, Enterobacter blattae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter brunelis, Salmonella enterica, Salmonella bongoli, Salmonella typhi, Salmonella swine fever, Salmonella typhi, Shigella shiga, Shigella flexinella, Shigella boy's, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoka, Plague bacillus, Yersinia enteritis, Mycobacterium pseudotuberculosis, Yersinia aldobe, Yersinia bercobieri, El Enterobacter frederiksenii, Yersinia intermedia, Yersinia cristensenii, Yersinia moralettii, Yersinia rhodei, Yersinia luckeri, Citrobacter froindii, Citrobacter coseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloaca, Enterobacter gergobie, Enterobacter sakazakii, Enterobacter teirorae, Enterobacter amnigenus, Enterobacter Enterobacter intermedius, Enterobacter asburia, Enterobacter canserogenus, Enterobacter disorbens, Enterobacter nimiplesularis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia licefaciens, Serratia odorifera, Serratia primucica, Serratia protea macrans, Serratia rubidaea, Serratia ureitica, A modified bacterium according to any one of Embodiments A1 to A26, selected from the group consisting of Proteus mirabilis, Proteus vulgaris, Proteus micsofaciens, Proteus penneri, Proteus haseri, Morganella morganii, Providencia alkalifaciens, Providencia rustigianii, Providencia stuartii, Providencia letgeri, Providencia heimbacchae, Hafnia albeyi, and Pantoea agglomerans.

[0168] Embodiment A28: A modified bacterium according to any one of Embodiments A1 to A27, which is Salmonella typhimurium.

[0169] Embodiment A29: A modified bacterium according to Embodiment A28, wherein the starting strain is Salmonella typhimurium SL7207.

[0170] Embodiment A30: A modified bacterium according to any one of Embodiments A1 to A29, which expresses wild-type lipopolysaccharide (LPS).

[0171] Embodiment A31: A modified bacterium according to any one of Embodiments A1 to A30, which, when administered to a subject with a tumor, can survive and proliferate in tumor tissue and is rapidly eliminated in normal tissue.

[0172] Embodiment A32: A modified bacterium according to any one of Embodiments A1 to A31, which, when administered to a subject having a malignant tumor, can inhibit the growth of the malignant tumor.

[0173] Embodiment A33: A modified bacterium according to any one of Embodiments A1 to A32, which can induce an antitumor-specific immune response when administered to a subject with a malignant tumor.

[0174] Embodiment A34: A modified bacterium according to any one of Embodiments A1 to A33, which can induce antitumor immunological memory when administered to a subject with a malignant tumor.

[0175] Embodiment A35: A modified bacterium according to any one of Embodiments A1 to A34, which does not express wild-type flagellin.

[0176] Embodiment A36: A modified bacterium according to any one of Embodiments A1 to A35, which lacks the fliC gene.

[0177] Embodiment A37: A modified bacterium according to any one of Embodiments A1 to A36, wherein the survival level of the bacteria in a macrophage is approximately 50% to approximately 30% of the survival level of the unmodified starting strain.

[0178] Embodiment A38: A modified bacterium according to any one of Embodiments A1 to A36, wherein the survival level of the bacteria in a macrophage is approximately 30% to approximately 10% of the survival level of the unmodified starting strain.

[0179] Embodiment A39: A modified bacterium according to any one of Embodiments A1 to A36, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0180] Embodiment B Embodiment B1: A modified bacterium that, compared to an unmodified starting strain, comprises an expression construct described in any one of Embodiments 16 to 30, a hypoxia-regulating essential gene expression cassette containing an essential bacterial gene under the control of a second strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions, and lacks at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or its functional expression product.

[0181] Embodiment B2: The modified bacterium according to Embodiment B1, wherein the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0182] Embodiment B3: The modified bacterium according to Embodiment B1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0183] Embodiment B4: A modified bacterium according to any one of Embodiments B1 to B3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0184] Embodiment B5: The modified bacterium according to Embodiment B4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0185] Embodiment B6: The modified bacterium according to Embodiment B4, wherein the essential gene is selected from dapA and dapE.

[0186] Embodiment B7: The modified bacterium according to any one of Embodiments B1 to B6, wherein the gene involved in the endogenous antioxidant stress response pathway or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

[0187] Embodiment B8: The modified bacterium according to any one of Embodiments B1 to B7, wherein the functional expression product of the gene involved in the endogenous antioxidant stress response pathway or regulating the endogenous antioxidant stress response pathway is a protein related to the HtrA serine protease family.

[0188] Embodiment B9: The modified bacterium according to any one of Embodiments B1 to B8, which lacks the activity of HtrA serine protease.

[0189] Embodiment B10: The modified bacterium according to any one of Embodiments B1 to B9, wherein the gene involved in the endogenous antioxidant stress response pathway or regulating the endogenous antioxidant stress response pathway is htrA.

[0190] Embodiment B11: The modified bacterium according to any one of Embodiments B1 to B10, which lacks htrA.

[0191] Embodiment B12: The modified bacterium according to any one of Embodiments B1 to B11, wherein the essential gene is a gene naturally present in the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strict hypoxic-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strict hypoxic-inducible promoter.

[0192] Embodiment B13: A modified bacterium according to any one of Embodiments B1 to B11, wherein the essential gene expression cassette is exogenous, and essential genes naturally present in the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0193] Embodiment B14: The modified bacterium according to Embodiment B13, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0194] Embodiment B15: A modified bacterium according to Embodiment B13, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0195] Embodiment B16: A modified bacterium according to Embodiment B15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0196] Embodiment B17: A modified bacterium according to any one of Embodiments B1 to B16, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0197] Embodiment B18: The modified bacterium according to Embodiment B17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0198] Embodiment B19: A modified bacterium according to Embodiment B18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0199] Embodiment B20: A modified bacterium according to any one of Embodiments B1 to B19, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0200] Embodiment B21: A modified bacterium according to any one of Embodiments B1 to B20, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0201] Embodiment B22: A modified bacterium according to any one of Embodiments B1 to B21, wherein the promoter active under acidic pH conditions is sseA.

[0202] Embodiment B23: A modified bacterium according to any one of Embodiments B1 to B22, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0203] Embodiment B24: A modified bacterium according to any one of Embodiments B1 to B23, which is a bacterium of the Enterobacteriaceae family.

[0204] Embodiment B25: A modified bacterium according to any one of Embodiments B1 to B24, which is a bacterium of the genus Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0205] Embodiment B26: Escherichia coli, Enterobacter blattae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter brunelis, Salmonella enterica, Salmonella bongoli, Salmonella typhi, Salmonella swine fever, Salmonella typhi, Shigella shiga, Shigella flexinella, Shigella boy's, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoka, Plague bacillus, Yersinia enteritis, Mycobacterium pseudotuberculosis, Yersinia aldobe, Yersinia bercobieri, El Enterobacter frederiksenii, Yersinia intermedia, Yersinia cristensenii, Yersinia moralettii, Yersinia rhodei, Yersinia luckeri, Citrobacter froindii, Citrobacter coseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloaca, Enterobacter gergobie, Enterobacter sakazakii, Enterobacter teirorae, Enterobacter amnigenus, Enterobacter Enterobacter intermedius, Enterobacter asburia, Enterobacter canserogenus, Enterobacter disorbens, Enterobacter nimiplesularis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia licefaciens, Serratia odorifera, Serratia primucica, Serratia protea macrans, Serratia rubidaea, Serratia ureitica, A modified bacterium according to any one of Embodiments B1 to B25, selected from the group consisting of Proteus mirabilis, Proteus vulgaris, Proteus micsofaciens, Proteus penneri, Proteus haseri, Morganella morganii, Providencia alkalifaciens, Providencia rustigianii, Providencia stuartii, Providencia letgeri, Providencia heimbacchae, Hafnia albeyi, and Pantoea agglomerans.

[0206] Embodiment B27: A modified bacterium according to any one of Embodiments B1 to B26, which is Salmonella typhimurium.

[0207] Embodiment B28: The modified bacterium according to Embodiment B27, wherein the starting strain is Salmonella typhimurium SL7207.

[0208] Embodiment B29: The modified bacterium according to any one of Embodiments B1 to B28, which expresses wild-type lipopolysaccharide (LPS).

[0209] Embodiment B30: The modified bacterium according to any one of Embodiments B1 to B29, which can survive and proliferate in tumor tissue and is rapidly eliminated in normal tissue when administered to a subject having a tumor.

[0210] Embodiment B31: The modified bacterium according to any one of Embodiments B1 to B30, which can inhibit the growth of a malignant tumor when administered to a subject having a malignant tumor.

[0211] Embodiment B32: The modified bacterium according to any one of Embodiments B1 to B31, which can induce an antitumor-specific immune response when administered to a subject having a malignant tumor.

[0212] Embodiment B33: The modified bacterium according to any one of Embodiments B1 to B32, which can induce antitumor immune memory when administered to a subject having a malignant tumor.

[0213] Embodiment B34: The modified bacterium according to any one of Embodiments B1 to B33, which does not express wild-type flagellin.

[0214] Embodiment B35: The modified bacterium according to any one of Embodiments B1 to B34, which is deficient in the fliC gene.

[0215] Embodiment B36: The modified bacterium according to any one of Embodiments B1 to B35, wherein the survival level of the bacterium in macrophages is about 50% to about 30% of the survival level of the unmodified starting strain.

[0216] Embodiment B37: The modified bacterium according to any one of Embodiments B1 to B35, wherein the survival level of the bacterium in macrophages is about 30% to about 10% of the survival level of the unmodified starting strain.

[0217] Embodiment B38: A modified bacterium according to any one of Embodiments B1 to B35, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0218] Embodiment C Embodiment C1: A modified Salmonella typhimurium, compared to an unmodified starting strain, comprising the expression construct described in any one of Embodiments 16 to 30, a hypoxia-regulating essential gene expression cassette containing a bacterial essential gene under the control of a strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions, and lacking at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or its functional expression product.

[0219] Embodiment C2: The modified bacterium according to Embodiment C1, wherein the strictly hypoxia-inducible promoter of Embodiment C2 is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0220] Embodiment C3: The modified bacterium according to Embodiment C1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0221] Embodiment C4: A modified bacterium according to any one of Embodiments C1 to C3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0222] Embodiment C5: The modified bacterium according to Embodiment C4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0223] Embodiment C6: The modified bacterium according to Embodiment C4, wherein the essential gene is selected from dapA and dapE.

[0224] Embodiment C7: A modified bacterium according to any one of Embodiments C1 to C6, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family gene.

[0225] Embodiment C8: A modified bacterium according to any one of Embodiments C1 to C7, wherein the functional expression product of a gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family-related protein.

[0226] Embodiment C9: A modified bacterium according to any one of Embodiments C1 to C8, lacking the activity of HtrA serine protease.

[0227] Embodiment C10: A modified bacterium according to any one of Embodiments C1 to C9, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0228] Embodiment C11: A modified bacterium according to any one of Embodiments C1 to C10, lacking htrA.

[0229] Embodiment C12: A modified bacterium according to any one of Embodiments C1 to C11, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

[0230] Embodiment C13: A modified bacterium according to any one of Embodiments C1 to C11, wherein the essential gene expression cassette is exogenous, and essential genes naturally present on the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0231] Embodiment C14: A modified bacterium according to Embodiment C13, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0232] Embodiment C15: A modified bacterium according to Embodiment C13, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0233] Embodiment C16: A modified bacterium according to Embodiment C15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0234] Embodiment C17: A modified bacterium according to any one of embodiments C1 to C16, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0235] Embodiment C18: A modified bacterium according to Embodiment C17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0236] Embodiment C19: A modified bacterium according to Embodiment C18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0237] Embodiment C20: A modified bacterium according to any one of Embodiments C1 to C19, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0238] Embodiment C21: A modified bacterium according to any one of Embodiments C1 to C20, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0239] Embodiment C22: A modified bacterium according to any one of Embodiments C1 to C21, wherein the promoter active under acidic pH conditions is sseA.

[0240] Embodiment C23: A modified bacterium according to any one of Embodiments C1 to C22, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0241] Embodiment C24: A modified bacterium according to any one of Embodiments C1 to C23, wherein the starting strain is Salmonella typhimurium SL7207.

[0242] Embodiment C25: A modified bacterium according to any one of Embodiments C1 to C24, which expresses wild-type lipopolysaccharide (LPS).

[0243] Embodiment C26: A modified bacterium according to any one of Embodiments C1 to C25, which, when administered to a subject with a tumor, can survive and proliferate in tumor tissue but is rapidly eliminated in normal tissue.

[0244] Embodiment C27: A modified bacterium according to any one of Embodiments C1 to C26, which, when administered to a subject having a malignant tumor, can inhibit the growth of the malignant tumor.

[0245] Embodiment C28: A modified bacterium according to any one of Embodiments C1 to C27, which can induce an antitumor-specific immune response when administered to a subject with a malignant tumor.

[0246] Embodiment C29: A modified bacterium according to any one of Embodiments C1 to C28, which can induce antitumor immunological memory when administered to a subject with a malignant tumor.

[0247] Embodiment C30: A modified bacterium according to any one of Embodiments C1 to C29, which does not express wild-type flagellin.

[0248] Embodiment C31: A modified bacterium according to any one of Embodiments C1 to C30, which lacks the fliC gene.

[0249] Embodiment C32: A modified bacterium according to any one of Embodiments C1 to C31, wherein the survival level of the bacteria in a macrophage is approximately 50% to approximately 30% of the survival level of the unmodified starting strain.

[0250] Embodiment C33: A modified bacterium according to any one of Embodiments C1 to C31, wherein the survival level of the bacteria in a macrophage is approximately 30% to approximately 10% of the survival level of the unmodified starting strain.

[0251] Embodiment C34: A modified bacterium according to any one of Embodiments C1 to C31, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0252] Embodiment D Embodiment D1: A modified bacterium that, compared to an unmodified starting strain, contains an expression construct described in any one of Embodiments 16 to 30, a hypoxia-regulating essential gene expression cassette containing essential bacterial genes under the control of a strictly hypoxia-inducible promoter, and lacks at least one gene or its functional expression product required for survival in macrophages.

[0253] Embodiment D2: The modified bacterium according to Embodiment D1, wherein the strictly hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0254] Embodiment D3: The modified bacterium according to Embodiment D1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0255] Embodiment D4: A modified bacterium according to any one of Embodiments D1 to D3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0256] Embodiment D5: The modified bacterium according to Embodiment D4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0257] Embodiment D6: The modified bacterium according to Embodiment D4, wherein the essential gene is selected from dapA and dapE.

[0258] Embodiment D7: A modified bacterium according to any one of Embodiments D1 to D6, wherein the gene required for survival in macrophages is the gene STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA.

[0259] Embodiment D8: A modified bacterium according to any one of Embodiments D1 to D7, wherein the functional expression product of a gene required for survival in a macrophage is an HtrA serine protease family-related protein.

[0260] Embodiment D9: A modified bacterium according to any one of Embodiments D1 to D8, lacking the activity of HtrA serine protease.

[0261] Embodiment D10: A modified bacterium according to any one of Embodiments D1 to D9, wherein the gene required for survival in a macrophage is htrA.

[0262] Embodiment D11: A modified bacterium according to any one of Embodiments D1 to D10, lacking htrA.

[0263] Embodiment D12: A modified bacterium according to any one of Embodiments D1 to D11, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

[0264] Embodiment D13: A modified bacterium according to any one of Embodiments D1 to D11, wherein the essential gene expression cassette is exogenous, and essential genes naturally present in the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0265] Embodiment D14: A modified bacterium according to Embodiment D13, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0266] Embodiment D15: A modified bacterium according to Embodiment D13, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0267] Embodiment D16: A modified bacterium according to Embodiment D15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0268] Embodiment D17: A modified bacterium according to any one of Embodiments D1 to D16, further comprising a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions.

[0269] Embodiment D18: The modified bacterium according to Embodiment D17, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0270] Embodiment D19: The modified bacterium according to Embodiment D18, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0271] Embodiment D20: A modified bacterium according to Embodiment D19, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0272] Embodiment D21: A modified bacterium according to any one of Embodiments D17 to D20, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0273] Embodiment D22: A modified bacterium according to any one of Embodiments D17 to D21, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0274] Embodiment D23: A modified bacterium according to any one of Embodiments D17 to D21, wherein the promoter active under acidic pH conditions is sseA.

[0275] Embodiment D24: A modified bacterium according to any one of Embodiments D1 to D23, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0276] Embodiment D25: A modified bacterium according to any one of Embodiments D1 to D24, which is a bacterium of the Enterobacteriaceae family.

[0277] Embodiment D26: A modified bacterium according to any one of Embodiments D1 to D25, which is a bacterium of the genus Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0278] Embodiment D27: Escherichia coli, Enterobacter blattae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter brunelis, Salmonella enterica, Salmonella bongoli, Salmonella typhi, Salmonella swine fever, Salmonella typhi, Shigella shiga, Shigella flexinella, Shigella boy's, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoka, Plague bacillus, Yersinia enteritis, Mycobacterium pseudotuberculosis, Yersinia aldobe, Yersinia bercobieri, El Enterobacter frederiksenii, Yersinia intermedia, Yersinia cristensenii, Yersinia moralettii, Yersinia rhodei, Yersinia luckeri, Citrobacter froindii, Citrobacter coseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloaca, Enterobacter gergobie, Enterobacter sakazakii, Enterobacter teirorae, Enterobacter amnigenus, Enterobacter Enterobacter intermedius, Enterobacter asburia, Enterobacter canserogenus, Enterobacter disorbens, Enterobacter nimiplesularis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia licefaciens, Serratia odorifera, Serratia primucica, Serratia protea macrans, Serratia rubidaea, Serratia ureitica, A modified bacterium according to any one of embodiments D1 to D26, selected from the group consisting of Proteus mirabilis, Proteus vulgaris, Proteus micsofaciens, Proteus penneri, Proteus haseri, Morganella morganii, Providencia alkalifaciens, Providencia rustigianii, Providencia stuartii, Providencia letgeri, Providencia heimbacchae, Hafnia albeyi, and Pantoea agglomerans.

[0279] Embodiment D28: A modified bacterium according to any one of Embodiments D1 to D27, which is Salmonella typhimurium.

[0280] Embodiment D29: A modified bacterium according to Embodiment D28, wherein the starting strain is Salmonella typhimurium SL7207.

[0281] Embodiment D30: A modified bacterium according to any one of Embodiments D1 to D29, which expresses wild-type lipopolysaccharide (LPS).

[0282] Embodiment D31: A modified bacterium according to any one of Embodiments D1 to D30, which, when administered to a subject having a tumor, can survive and proliferate in tumor tissue and is rapidly eliminated in normal tissue.

[0283] Embodiment D32: A modified bacterium according to any one of Embodiments D1 to D31, which, when administered to a subject having a malignant tumor, can inhibit the growth of the malignant tumor.

[0284] Embodiment D33: A modified bacterium according to any one of Embodiments D1 to D32, which can induce an antitumor-specific immune response when administered to a subject having a malignant tumor.

[0285] Embodiment D34: A modified bacterium according to any one of Embodiments D1 to D33, which can induce antitumor immunological memory when administered to a subject having a malignant tumor.

[0286] Embodiment D35: A modified bacterium according to any one of Embodiments D1 to D34, which does not express wild-type flagellin.

[0287] Embodiment D36: A modified bacterium according to any one of Embodiments D1 to D35, which lacks the fliC gene.

[0288] Embodiment D37: A modified bacterium according to any one of Embodiments D1 to D36, wherein the viability level of the bacteria in the macrophage is approximately 50% to approximately 30% of the viability level of the unmodified starting strain.

[0289] Embodiment D38: A modified bacterium according to any one of Embodiments D1 to D36, wherein the survival level of the bacteria in a macrophage is approximately 30% to approximately 10% of the survival level of the unmodified starting strain.

[0290] Embodiment D39: A modified bacterium according to any one of Embodiments D1 to D36, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0291] Embodiment E Embodiment E1: A modified bacterium that, compared to an unmodified starting strain, comprises the expression construct described in any one of Embodiments 16 to 30, a hypoxia-regulating essential gene expression cassette containing an essential bacterial gene under the control of a strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial-derived hemolysin protein under the control of a promoter that is active under acidic pH conditions, and expresses wild-type lipopolysaccharide (LPS) and lacks at least one gene or its functional expression product required for survival in macrophages.

[0292] Embodiment E2: The modified bacterium according to Embodiment E1, wherein the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0293] Embodiment E3: The modified bacterium according to Embodiment E1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0294] Embodiment E4: A modified bacterium according to any one of Embodiments E1 to E3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0295] Embodiment E5: The modified bacterium according to Embodiment E4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0296] Embodiment E6: The modified bacterium according to Embodiment E4, wherein the essential gene is selected from dapA and dapE.

[0297] Embodiment E7: A modified bacterium according to any one of Embodiments E1 to E6, wherein the gene required for survival in macrophages is the gene STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA.

[0298] Embodiment E8: A modified bacterium according to any one of Embodiments E1 to E7, wherein the functional expression product of a gene required for survival in a macrophage is an HtrA serine protease family-related protein.

[0299] Embodiment E9: A modified bacterium according to any one of Embodiments E1 to E8, lacking the activity of HtrA serine protease.

[0300] Embodiment E10: A modified bacterium according to any one of Embodiments E1 to E9, wherein the gene required for survival in a macrophage is htrA.

[0301] Embodiment E11: A modified bacterium according to any one of Embodiments E1 to E10, lacking htrA.

[0302] Embodiment E12: A modified bacterium according to any one of Embodiments E1 to E11, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

[0303] Embodiment E13: A modified bacterium according to any one of Embodiments E1 to E11, wherein the essential gene expression cassette is exogenous, and essential genes naturally present in the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0304] Embodiment E14: A modified bacterium according to Embodiment E13, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0305] Embodiment E15: A modified bacterium according to Embodiment E13, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0306] Embodiment E16: A modified bacterium according to Embodiment E15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0307] Embodiment E17: A modified bacterium according to any one of Embodiments E1 to E16, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0308] Embodiment E18: The modified bacterium according to Embodiment E17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0309] Embodiment E19: A modified bacterium according to Embodiment E18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0310] Embodiment E20: A modified bacterium according to any one of Embodiments E1 to E19, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0311] Embodiment E21: A modified bacterium according to any one of Embodiments E1 to E20, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0312] Embodiment E22: A modified bacterium according to any one of Embodiments E1 to E21, wherein the promoter active under acidic pH conditions is sseA.

[0313] Embodiment E23: A modified bacterium according to any one of Embodiments E1 to E22, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0314] Embodiment E24: A modified bacterium according to any one of Embodiments E1 to E23, which is a bacterium of the Enterobacteriaceae family.

[0315] Embodiment E25: A modified bacterium according to any one of Embodiments E1 to E24, which is a bacterium of the genus Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0316] Embodiment E26: Escherichia coli, Enterobacter blattae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter brunelis, Salmonella enterica, Salmonella bongoli, Salmonella typhi, Salmonella swine fever, Salmonella typhi, Shigella shiga, Shigella flexinella, Shigella boy's, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoka, Plague bacillus, Yersinia enteritis, Mycobacterium pseudotuberculosis, Yersinia aldobe, Yersinia bercobieri, El Enterobacter frederiksenii, Yersinia intermedia, Yersinia cristensenii, Yersinia moralettii, Yersinia rhodei, Yersinia luckeri, Citrobacter froindii, Citrobacter coseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloaca, Enterobacter gergobie, Enterobacter sakazakii, Enterobacter teirorae, Enterobacter amnigenus, Enterobacter Enterobacter intermedius, Enterobacter asburia, Enterobacter canserogenus, Enterobacter disorbens, Enterobacter nimiplesularis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia licefaciens, Serratia odorifera, Serratia primucica, Serratia protea macrans, Serratia rubidaea, Serratia ureitica, A modified bacterium according to any one of Embodiments E1 to E25, selected from the group consisting of Proteus mirabilis, Proteus vulgaris, Proteus micsofaciens, Proteus penneri, Proteus haseri, Morganella morganii, Providencia alkalifaciens, Providencia rustigianii, Providencia stuartii, Providencia letgeri, Providencia heimbacchae, Hafnia albeyi, and Pantoea agglomerans.

[0317] Embodiment E27: A modified bacterium according to any one of Embodiments E1 to E26, which is Salmonella typhimurium.

[0318] Embodiment E28: A modified bacterium according to Embodiment E27, wherein the starting strain is Salmonella typhimurium SL7207.

[0319] Embodiment E29: A modified bacterium according to any one of Embodiments E1 to E28, which, when administered to a subject with a tumor, can survive and proliferate in tumor tissue and is rapidly eliminated in normal tissue.

[0320] Embodiment E30: A modified bacterium according to any one of Embodiments E1 to E29, which, when administered to a subject having a malignant tumor, can inhibit the growth of the malignant tumor.

[0321] Embodiment E31: A modified bacterium according to any one of Embodiments E1 to E30, which can induce an antitumor-specific immune response when administered to a subject having a malignant tumor.

[0322] Embodiment E32: A modified bacterium according to any one of Embodiments E1 to E31, which can induce antitumor immunological memory when administered to a subject having a malignant tumor.

[0323] Embodiment E33: A modified bacterium according to any one of Embodiments E1 to E32, which does not express wild-type flagellin.

[0324] Embodiment E34: A modified bacterium according to any one of Embodiments E1 to E33, which lacks the fliC gene.

[0325] Embodiment E35: A modified bacterium according to any one of Embodiments E1 to E34, wherein the survival level of the bacteria in a macrophage is approximately 50% to approximately 30% of the survival level of the unmodified starting strain.

[0326] Embodiment E36: ​​A modified bacterium according to any one of Embodiments E1 to E34, wherein the survival level of the bacteria in a macrophage is approximately 30% to approximately 10% of the survival level of the unmodified starting strain.

[0327] Embodiment E37: A modified bacterium according to any one of Embodiments E1 to E34, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0328] Embodiment F Embodiment F1: A modified Salmonella typhimurium, compared to an unmodified starting strain, comprising the expression construct described in any one of Embodiments 16 to 30, a hypoxia-regulating essential gene expression cassette containing an essential bacterial gene under the control of a strictly hypoxia-inducible promoter, and a pH-regulating expression cassette containing a gene encoding a bacterial-derived hemolysin protein under the control of a promoter that is active under acidic pH conditions, and expressing wild-type lipopolysaccharide (LPS), and lacking at least one gene or functional expression product required for survival in macrophages.

[0329] Embodiment F2: The modified bacterium according to Embodiment F1, wherein the strictly hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0330] Embodiment F3: The modified bacterium according to Embodiment F1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0331] Embodiment F4: A modified bacterium according to any one of Embodiments F1 to F3, wherein the expression product of an essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or its analogues added to the culture medium.

[0332] Embodiment F5: The modified bacterium according to Embodiment F4, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0333] Embodiment F6: The modified bacterium according to Embodiment F4, wherein the essential gene is selected from dapA and dapE.

[0334] Embodiment F7: A modified bacterium according to any one of Embodiments F1 to F6, wherein the gene required for survival in macrophages is the gene STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA.

[0335] Embodiment F8: A modified bacterium according to any one of Embodiments F1 to F7, wherein the gene required for survival in macrophages is a gene involved in or regulating the endogenous antioxidant stress response pathway.

[0336] Embodiment F9: The modified bacterium according to Embodiment F8, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family gene.

[0337] Embodiment F10: The modified bacterium according to Embodiment F9, wherein the functional expression product of a gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family-related protein.

[0338] Embodiment F11: A modified bacterium according to Embodiment F9, lacking the activity of HtrA serine protease.

[0339] Embodiment F12: A modified bacterium according to Embodiment F9, wherein the gene htrA is involved in or modulates the endogenous antioxidant stress response pathway.

[0340] Embodiment F13: A modified bacterium according to Embodiment F9, lacking htrA.

[0341] Embodiment F14: A modified bacterium according to any one of Embodiments F1 to F13, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by a strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

[0342] Embodiment F15: A modified bacterium according to any one of Embodiments F1 to F13, wherein the essential gene expression cassette is exogenous, and essential genes naturally present in the bacterial chromosome are deleted or functionally inactivated, so that the expression of essential genes in the bacterium is completely under the control of a strictly hypoxia-inducible promoter.

[0343] Embodiment F16: A modified bacterium according to Embodiment F15, wherein an exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

[0344] Embodiment F17: A modified bacterium according to Embodiment F15, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

[0345] Embodiment F18: A modified bacterium according to Embodiment F17, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0346] Embodiment F19: A modified bacterium according to any one of Embodiments F1 to F18, wherein the bacterial hemolysin protein is a gram-negative bacterial hemolysin protein.

[0347] Embodiment F20: The modified bacterium according to Embodiment F19, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0348] Embodiment F21: A modified bacterium according to Embodiment F20, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0349] Embodiment F22: A modified bacterium according to any one of Embodiments F1 to F21, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.5.

[0350] Embodiment F23: A modified bacterium according to any one of Embodiments F1 to F22, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0351] Embodiment F24: A modified bacterium according to any one of Embodiments F1 to F23, wherein the promoter active under acidic pH conditions is sseA.

[0352] Embodiment F25: A modified bacterium according to any one of Embodiments F1 to F24, wherein the starting strain is Salmonella typhimurium SL7207.

[0353] Embodiment F26: A modified bacterium according to any one of Embodiments F1 to F25, which, when administered to a subject with a tumor, can survive and proliferate in tumor tissue and is rapidly eliminated in normal tissue.

[0354] Embodiment F27: A modified bacterium according to any one of Embodiments F1 to F26, which, when administered to a subject having a malignant tumor, can inhibit the growth of the malignant tumor.

[0355] Embodiment F28: A modified bacterium according to any one of Embodiments F1 to F27, which can induce an antitumor-specific immune response when administered to a subject with a malignant tumor.

[0356] Embodiment F29: A modified bacterium according to any one of Embodiments F1 to F28, which can induce antitumor immunological memory when administered to a subject with a malignant tumor.

[0357] Embodiment F30: A modified bacterium according to any one of Embodiments F1 to F29, which does not express wild-type flagellin.

[0358] Embodiment F31: A modified bacterium according to any one of Embodiments F1 to F30, which lacks the fliC gene.

[0359] Embodiment F32: A modified bacterium according to any one of Embodiments F1 to F31, wherein the survival level of the bacteria in a macrophage is approximately 50% to approximately 30% of the survival level of the unmodified starting strain.

[0360] Embodiment F33: A modified bacterium according to any one of Embodiments F1 to F31, wherein the survival level of the bacteria in a macrophage is approximately 30% to approximately 10% of the survival level of the unmodified starting strain.

[0361] Embodiment F34: A modified bacterium according to any one of Embodiments F1 to F31, wherein the survival level of the bacteria in the macrophage is approximately 10% to approximately 1% of the survival level of the unmodified starting strain.

[0362] Embodiment K Embodiment K1: A pharmaceutical composition comprising an effective amount of the modified bacteria described in any one of the preceding embodiments A, B, C, D, E, or F.

[0363] Embodiment K2: The pharmaceutical composition according to Embodiment K1, for use in the treatment of malignant tumors.

[0364] Embodiment K3: The pharmaceutical composition according to Embodiment K1, for use in inducing an antitumor-specific immune response in subjects with malignant tumors.

[0365] Embodiment K4: The pharmaceutical composition according to Embodiment K1, for use in inducing antitumor immunological memory in subjects having malignant tumors.

[0366] Embodiment K5: The pharmaceutical composition according to Embodiment K1, for use in the prevention or treatment of metastasis or recurrence of malignant tumors.

[0367] Embodiment K6: The pharmaceutical composition according to Embodiment K1, for use in treating malignant tumors that are resistant to or have failed to receive prior antitumor treatment.

[0368] Embodiment K7: The pharmaceutical composition according to any one of Embodiments K1 to K6, wherein the modified bacteria are live bacteria.

[0369] Embodiment L Embodiment L1: A method for treating a malignant tumor, comprising the step of administering to a subject having a malignant tumor an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of embodiments G.

[0370] Embodiment L2: A method for inducing a specific immune response against a tumor in a subject having a malignant tumor, comprising the step of administering to the subject having a malignant tumor an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of embodiments G.

[0371] Embodiment L3: A method for inducing immunological memory against a tumor in a subject having a malignant tumor, comprising the step of administering to the subject having a malignant tumor an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of embodiments G.

[0372] Embodiment L4: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising the step of administering to a subject having a malignant tumor an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of embodiments G.

[0373] Embodiment L5: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising the step of administering an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of the preceding embodiments G, to a subject having a malignant tumor or at high risk of metastasis or recurrence of a malignant tumor.

[0374] Embodiment L6: A method for treating a malignant tumor that is resistant to or has failed to a prior antitumor treatment, comprising the step of administering an effective amount of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F, or a pharmaceutical composition described in any one of the preceding embodiments G, to a subject having a malignant tumor that is resistant to or has failed to a prior antitumor treatment.

[0375] Embodiment L7: The method according to any one of Embodiments L1 to L6, wherein the modified bacteria are viable bacteria.

[0376] Embodiment M Embodiment M1: Use of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F in the preparation of a pharmaceutical for treating malignant tumors.

[0377] Embodiment M2: Use of a modified bacterium according to any one of the preceding embodiments A, B, C, D, E, or F in the preparation of a pharmaceutical for inducing a specific immune response against a tumor in a subject having a malignant tumor.

[0378] Embodiment M3: Use of a modified bacterium according to any one of the preceding embodiments A, B, C, D, E, or F in the preparation of a pharmaceutical for inducing immunological memory against a tumor in a subject having a malignant tumor.

[0379] Embodiment M4: Use of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F in the preparation of a pharmaceutical for preventing or treating metastasis or recurrence of malignant tumors.

[0380] Embodiment M5: Use of a modified bacterium described in any one of the preceding embodiments A, B, C, D, E, or F in the preparation of a pharmaceutical for treating malignant tumors that are resistant to or have failed to receive prior antitumor treatment.

[0381] Embodiment M6: The use according to any one of Embodiments M1 to M5, wherein the modified bacteria are viable bacteria.

[0382] Embodiment N Embodiment N1: The pharmaceutical composition according to any one of Embodiments K1 to K7, the method according to any one of Embodiments L1 to L7, or the use according to any one of Embodiments M1 to M6, wherein the malignant tumor is a malignant tumor from the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin mucosa.

[0383] Embodiment N2: The pharmaceutical composition according to any one of Embodiments K1 to K7, the method according to any one of Embodiments L1 to L7, or the use according to any one of Embodiments M1 to M6, wherein the malignant tumor is a sarcoma or cancer.

[0384] Embodiment N3: The pharmaceutical composition according to any one of Embodiments K1 to K7, the method according to any one of Embodiments L1 to L7, or the use according to any one of Embodiments M1 to M6, wherein the malignant tumor is a solid tumor.

[0385] Embodiment N4: A pharmaceutical composition according to any one of Embodiments K1 to K7, a method according to any one of Embodiments L1 to L7, or a use according to any one of Embodiments M1 to M6, wherein the malignant tumor is selected from the group consisting of glioma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, oral cancer, tongue cancer, laryngeal cancer, head and neck cancer, melanoma, bronchial cancer, lung cancer, pleural cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, bile duct cancer, colorectal cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal cancer, thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer, and osteosarcoma.

[0386] Embodiment N5: The use of the bacterium, pharmaceutical, or pharmaceutical composition of the present invention, administered intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally, transcerebrally, gastrointestinally, on the body surface, on the oral mucosa, transnasally, transrectally, or transvaginally, as described in any one of Embodiments K1 to K7, the method described in any one of Embodiments L1 to L7, or any one of Embodiments M1 to M6. [Examples]

[0387] The following examples are used to illustrate certain specific features and / or embodiments of the present invention. These examples should not be construed as limiting the present invention to the specific features or embodiments described.

[0388] Example 1: Construction of a variant strain of Salmonella typhimurium SL7207 1.1 Target fragments were obtained by PCR amplification using the PsseA-hlyA-loxp-KnaR-loxp plasmid (SEQ ID NO: 3) and the Pssbp1-dapE-Cm plasmid (SEQ ID NO: 4) as templates. The following primer sequences were used: for PsseA-hlyA-loxp-KnaR-loxp: forward primer (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6); for Pssbp1-dapE-Cm: forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8). The annealing temperature was 55°C. Target fragment 1 was PsseA-hlyA-loxp-KnaR-loxp (SEQ ID NO: 1), and target fragment 2 was Pssbp1-dapE-Cm (SEQ ID NO: 2).

[0389] 1.2 The pSim6 plasmid containing lambdaphage red recombinase (BioVector NTCC Inc.: 3574840) was introduced into facultative anaerobic Salmonella SL7207 strain (NCBI: ASM1320710v1) to prepare electrotransformation-competent cells SL7207 (pSim6) containing the pSim6 plasmid.

[0390] 1.3 Target fragment 1 was introduced into SL7207 (pSim6) competent cells by electrotransformation (E=18kV / cm). Using λ-red homologous recombination, the dapE gene (which regulates the synthesis of diaminopimelic acid, essential for cell wall synthesis) in the SL7207 genome was replaced with the desired fragment 1. Specifically, the target strain containing the pSim6 plasmid was cultured at 30°C, recombinase expression was performed at 42°C for 15 minutes, and the homologous recombination process was completed by adding homologous arms containing 50 bp away from each upstream and downstream of the target gene locus to PCR primers under the effect of homologous recombinase, thereby obtaining the SL7207 strain (ΔdapE::PsseA-hlyA-KnaR).

[0391] 1.4 To achieve recombination for the removal of kanamycin resistance, a Cre plasmid containing P1 phage Cre recombinase (Gene Bridges: A112) was introduced into the SL7207 strain (ΔdapE::PsseA-hlyA-KnaR) to obtain the SL7207 strain (ΔdapE::PsseA-hlyA).

[0392] 1.5 The pSim6 plasmid was introduced into the SL7207(ΔdapE::PsseA-hlyA) strain to prepare SL7207(ΔdapE::PsseA-hlyA)(pSim6) electrotransformation competent cells.

[0393] 1.6 Target fragment 2 was introduced into competent SL7207 (ΔdapE::PsseA-hlyA)(pSim6) cells by electrotransformation (E=18kV / cm). Using λ-red homologous recombination, the htrA gene (encoding a serine protease) in the SL7207 genome (ΔdapE::PsseA-hlyA) was replaced with the desired fragment 2 to obtain the htrA gene-deficient SL7207 strain (ΔdapE::PsseA-hlyA;ΔhtrA::Pssbp1-dapE-Cm)DB-ZW1 strain, which contains the hlyA gene under the control of PsseA and the dapE gene under the control of the strictly hypoxia-inducible promoter Pssbp1.

[0394] The stock construction scheme is shown in Figure 1.

[0395] Example 2: DB-ZW1 exhibits tumor inhibitory effects against various tumor models. 2.1 Construction of a tumor model C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing approximately 18g, kept in an SPF environment) were fed 1 × 10⁶ 6A mouse subcutaneous tumor model of bladder cancer / melanoma was established by subcutaneous inoculation of 100 MB49 mouse bladder cancer cells / B16 melanoma cells (ATCC:CRL-6475). 14-18 days after inoculation, the tumor volume was approximately 100 mm². 3 The experiment was conducted at that time. An in situ colon cancer model was induced by DSS / AOM. Specifically, one cycle of DSS treatment was defined as one week after a single intraperitoneal injection of 10 mg / kg of AOM, followed by 7 days of water containing 2.5% DSS, and then 14 days of regular drinking water. Three more cycles of DSS treatment were repeated to obtain a mouse model with colon cancer.

[0396] 2.2 Changes in DB-ZW1 distribution in tumors and different organs In MB49 bladder cancer-bearing mice, 1 × 10 7 CFU of DB-ZW1 was injected via the tail vein in a volume of 125 μL, with 3 mice in each group. Approximately 90% of DB-ZW1 in the mice was observed to be present in the tumors on day 1 after injection (1 dpi). The density of DB-ZW1 in the tumors increased 100-fold (10) within the following 2 days. 8 The levels (up to CFU / g) further increased and remained high over the following two weeks. In contrast, the density of DB-ZW1 in normal organs remained <10 over the two weeks. 2 The levels continued to decrease to CFU / g (Figure 2A).

[0397] To further determine whether DB-ZW1 preferentially proliferated in the tumor, 125 μL of 1 × 10⁶ oz was administered to MB49 bladder cancer-bearing mice. 7 The number of DB-ZW1 bacteria distributed in different organs was determined 24 hours after injection of CFU via the tail vein; each group consisted of 3 mice (Figure 2B). The results showed that DB-ZW1 spread rapidly within 30 minutes of injection, and the majority of DB-ZW1 cells (approximately 99.9%) were distributed in the liver, spleen, and blood. The number of DB-ZW1 cells decreased significantly within 4 hours of injection, and the total number of DB-ZW1 cells decreased by approximately 90%. Subsequently, DB-ZW1 in tumors decreased approximately 0.7 hours. -1It proliferated exponentially at a growth rate and reached saturation within 3 days. In contrast, the number of DB-ZW1 cells continued to decrease in normal organs. This specific tumor growth profile of DB-ZW1 provided a clue to validate the following findings.

[0398] 2.3 Tumor inhibitory effects of DB-ZW1 on bladder cancer, melanoma, and colon cancer Mice with subcutaneous bladder cancer, orthotopic melanoma, and subcutaneous orthotopic colon cancer were treated with DB-ZW1, and 1 × 10⁻¹⁶ mice were treated with DB-ZW1. 7 CFU-based DB-ZW1 was injected into tumor-bearing mice via the tail vein in a volume of 125 μL, with 5 mice per group. As shown in Figures 3A-3C, DB-ZW1 significantly reduced tumor size. Furthermore, treatment of mice with orthotopic colon cancer using DB-ZW1 also significantly reduced the number of tumors in the colon of the mice (Figure 3D).

[0399] Example 3: Construction of vectors and strains for nanobody / fusion protein expression that bind to CXCR2 3.1 Vectors and bacterial construction for inducible expression of nanobodies A DNA molecule encoding a fusion of three polypeptides, "signal peptide + nanobody + tag," was synthesized (by Beijing Tsingke Biotech Co., Ltd.) and cloned into the pET26b plasmid (see Figure 4). In this plasmid, the nanobody was selected from the group consisting of Nb127D01, Nb163E3, and Nb97A9 (SEQ ID NOs. 19, 20, and 21), the signal peptide was a signal peptide derived from Erwinia crotovora pectin lyase (pelB signal peptide, SEQ ID NO. 28), and the tag was an HA-His tag. The aforementioned segments were directly linked. The obtained plasmids were named pET26b-pelB-Nb127D01-HA-his (see Figure 5), pET26b-pelB-Nb163E3-HA-his, and pET26b-pelB-Nb97A9-HA-his (SEQ ID NOs. 30, 31, and 32), respectively.

[0400] Furthermore, by substituting the coding sequence of the pelB signal peptide with the coding sequence of the psp signal peptide derived from Paenibacillus sp. EC003 (SEQ ID NO: 29), plasmids pET26b-psp-Nb127D01-HA-his, pET26b-psp-Nb163E3-HA-his, and pET26b-psp-Nb97A9-HA-his (SEQ ID NOs: 34-36) were constructed.

[0401] The aforementioned plasmids were transformed into competent cells of E. coli strain BL21(DE3) (purchased from Novidin) by chemical transformation to obtain strains for inducible expression of nanobodies: BL21(DE3)-pET26b-pelB-Nb127D01-HA-his, BL21(DE3)-pET26b-pelB-Nb163E3-HA-his, BL21(DE3)-pET26b-pelB-Nb97A9-HA-his, BL21(DE3)-pET26b-psp-Nb127D01-HA-his, BL21(DE3)-pET26b-psp-Nb163E3-HA-his, and BL21(DE3)-pET26b-psp-Nb97A9-HA-his. In detail, approximately 100 ng of plasmid was added to competent cells, which were then left to stand on ice for 30 minutes, followed by 45 seconds in a 42°C water bath, and then returned to ice for 2-3 minutes. Next, 900 μl of LB medium was added, and the cells were incubated at 37°C for 1 hour. Positive clones were then screened on LB plates containing 50 mg / L kanamycin.

[0402] 3.2 Construction of vectors for anaerobic expression of nanobodies To construct a vector for anaerobic expression of nanobodies, the oxygen-sensitive promoter FNRsp (SEQ ID NO: 10) was used to drive the expression of the six fusion polypeptides in Example 3.1.

[0403] In detail, primers Nb frag F1 / R1 and Nb frag F2 / R2 were designed, and coding sequence fragments were amplified using the plasmids pET26b-pelB-Nb127D01-HA-his, pET26b-pelB-Nb163E3-HA-his, pET26b-pelB-Nb97A9-HA-his, pET26b-pelB-Nb127D01-HA-his, pET26b-pelB-Nb163E3-HA-his, and pET26b-pelB-Nb97A9-HA-his constructed in 3.1 as templates. Using primers and linearized REV / FOR, PCR was performed using plasmid pSC101-pFNRsp (SEQ ID NO: 33) as a template to obtain linear skeleton fragments.

[0404] [Table 2]

[0405] PCR amplification was performed using PrimeSTAR Max DNA polymerase (Takara, product code R054A). The PCR system (total volume 50 μL) contained 25 μL of PrimeSTAR Max Premix, 0.2-0.3 μM primers, and 200 ng of template. The PCR conditions were: denaturation: 98°C for 10 seconds, annealing: 55°C for 10 seconds, extension: 72°C for 5 seconds / kb, for a total of 30 cycles of denaturation-annealing-extension.

[0406] Using the Vazyme One-Step Cloning Kit (ClonExpress II One-Step Cloning Kit, Vazyme, No. C112), homologous recombination was performed between the skeletal fragment and the coding sequence fragment to construct the plasmids pSC101-pFRRsp-pelB-Nb127D01-HAhis, pSC101-pFRRsp-pelB-Nb163E3-HA-his, pSC101-pFRRsp-pelB-Nb97A9-HA-his, pSC101-pFRRsp-ps-Nb127D01-HA-his, pSC101-pFRRsp-ps-Nb163E3-HA-his, and pSC101-pFRRsp-ps-Nb97A9-HA-his for anaerobic expression of nanobodies. 3.3 Vectors and bacterial construction for inducible expression of nanobody-PE fusion proteins The nucleotide fragments encoding the Pseudomonas exotoxin fragments PE38 (SEQ ID NO: 26, 55kDa) and PE[LR] (SEQ ID NO: 27, 43kDa) were inserted into the plasmids pET26b-pelB-Nb127D01-HA-his, pET26b-pelB-Nb163E3-HA-his, and pET26b-pelB-Nb97A9-HA-his (the 3' end of the nanobody coding sequence) constructed in 3.1, and the plasmids were used for inducible expression of the nanobody-PE fusion protein. Lasmid pET26b-psp-Nb127D01-PE38-HA-his, pET26b-psp-Nb163E3-PE38-HA-his, pET26b-psp-Nb97A9-PE38-HA-his, pET26b-p sp-Nb127D01-PE[LR]-HA-his, pET26b-psp-Nb163E3-PE[LR]-HA-his, and pET26b-psp-Nb97A9-PE[LR]-HA-his (SEQ ID NOS: 43 to 48) were obtained. The constructed plasmids were transformed into competent cells of E. coli strain BL21(DE3) by chemical transformation (see Example 3.1), resulting in the BL21(DE3)-pET26b-psp-Nb127D01-PE38-HA-his strain, BL21(DE3)-pET26b-psp-Nb163E3-PE38-HA-his strain, and BL21(DE3)-pET 26b-psp-Nb97A9-PE38-HA-his strain, BL21(DE3)-pET26b-psp-Nb127D01-PE[LR]-HA-his strain, BL21(DE3 )-pET26b-psp-Nb163E3-PE[LR]-HA-his strain and BL21(DE3)-pET26b-psp-Nb97A9-PE[LR]-HA-his strain were obtained.

[0407] The expression strains for PE38 and PE[LR], BL21(DE3)-pET26b-psp-PE38-HA-his and BL21(DE3)-pET26b-psp-PE[LR]-HA-his, were constructed in the same manner.

[0408] 3.4 Construction of vectors for anaerobic expression of nanobody-PE fusion proteins Using the plasmids pET26b-psp-Nb127D01-PE38-HA-his, pET26b-psp-Nb163E3-PE38-HA-his, pET26b-psp-Nb97A9-PE38-HA-his, pET26b-psp-Nb127D01-PE[LR]-HA-his, pET26b-psp-Nb163E3-PE[LR]-HA-his, and pET26b-psp-Nb97A9-PE[LR]-HA-his constructed in Example 3.3 as templates, PCR amplification was performed using primers and fragments FOR / REV (SEQ ID NOs. 55 and 56) to obtain coding sequence fragments (see Example 3.2 for PCR system and conditions). By the method described in Example 3.2, coding sequence fragments were homologously recombined with the skeleton fragments prepared in Example 3.2 to obtain plasmids pSC101-pFNRsp-psp-Nb127D01-PE38-HA-his, pSC101-pFNRsp-psp-Nb163E3-PE38-HA-his, pSC101-pFNRsp-psp-Nb97A9-PE38-HA-his, pSC101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his, pSC101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his, and pSC101-pFNRsp-psp-Nb97A9-PE[LR]-HA-his (SEQ ID NOs. 49-54).

[0409] The plasmid described above was used to transform competent cells of Salmonella typhimurium ZW1 strain prepared in Example 1 by electrotransformation (see Example 1 for details on the electrotransformation method; the screening medium was LB + chloramphenicol 25 mg / L), resulting in the following strains: ZW1-psc101-pFNRsp-psp-Nb127D01-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE38-HA-his, and ZW1-psc101-pFNRsp -psp-Nb97A9-PE38-HA-his strain, ZW1-psc101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his strain, ZW1-psc101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his strain, ZW 1-psc101-pFNRsp-psp-Nb97A9-PE[LR]-HA-his strain, ZW1-psc101-pFNRsp-psp-PE38-HA-his strain, and ZW1-psc101-pFNRsp-psp-PE[LR]-HA-his strain were obtained.

[0410] The expression strains for PE38 and PE[LR], ZW1-psc101-pFNRsp-psp-PE38-HA-his and ZW1-psc101-pFNRsp-psp-PE[LR]-HA-his, were constructed in the same manner.

[0411] Example 4: Inducible expression of nanobodies and fusion proteins by bacteria To detect the inducible expression of nanobodies and fusion proteins, the following strains constructed in Examples 3.1 and 3.3 were used: BL21(DE3)-pET26b-pelB-Nb127D01-HA-his, BL21(DE3)-pET26b-pelB-Nb163E3-HA-his, BL21(DE3)-pET26b-pelB-Nb97A9-HA-his, BL21(DE3)-pET26b-psp-Nb127D01-PE38-HA-his, BL21(DE3)-pET26b-psp-Nb163 Prokaryotic expression was performed using E3-PE38-HA-his, BL21(DE3)-pET26b-psp-Nb97A9-PE38-HA-his, BL21(DE3)-pET26b-psp-Nb127D01-PE[LR]-HA-his, BL21(DE3)-pET26b-psp-Nb163E3-PE[LR]-HA-his, and BL21(DE3)-pET26b-psp-Nb97A9-PE[LR]-HA-his, and strains expressing PE[LR] and PE38 were used as controls.

[0412] In detail, bacteria were inoculated into sugar-free LB medium (containing 50 mg / L kanamycin) and cultured at 37°C and 220 rpm in a shaker until the OD600 was between 0.4 and 0.6. IPTG was added to the culture medium at different concentrations (final concentrations of 0 mM, 0.1 mM, 0.2 mM, and 0.5 mM), and then induced overnight in a shaker at 16°C and 220 rpm, after which the bacterial cultures were collected. The collected bacterial cultures were centrifuged, and the LB medium supernatant and bacterial precipitate were collected separately. The LB medium supernatant was concentrated by centrifugation using a 3 kDa ultrafiltration tube (2,500 rpm, 2-3 hours). The bacterial precipitate was dissolved using a protein extraction kit from Qiagen (Qiagen, Qproteome Bacterial Protein Prep Kit, No. 37900, see Experimental Manual Qproteome Bacterial Protein Preparation Handbook, www.qiagen.com). The dissolved material was centrifuged, and the dissolved pellet and the supernatant were collected separately.

[0413] Furthermore, the strain prepared in Example 3.4 was tested for expression under anaerobic conditions. The method was the same as described above, except that the antibiotic added to the culture medium was 25 mg / L chloramphenicol, and cultivation and induced expression were performed under anaerobic conditions.

[0414] The samples were subjected to electrophoresis on a 12.5% ​​polyacrylamide gel (80V). For membrane transfer, the Pyxis Transfer Stack Kit was used according to the manufacturer's instructions (purchased from Pyxis, catalog number SPJ-T20S). Rabbit anti-6×His antibody was used as the primary antibody (purchased from Thermo Fisher, dilution 1:10000), and goat anti-rabbit antibody was used as the secondary antibody (purchased from Thermo Fisher, dilution 1:5000). Protein expression and secretion by bacteria were detected by Western blotting (color development was performed using SuperSignal™ West Pico PLUS chemiluminescent agent purchased from Thermo Fisher).

[0415] As shown in Figures 6A to 6C, nanobody expression is positively correlated with IPTG concentration, and a distinct band of nanobody is present in the LB supernatant, indicating that the signal peptide can induce the secretion of expressed nanobody from bacterial cells.

[0416] As shown in Figure 7, a prominent band of nanobody-PE fusion protein is present in the LB supernatant during IPTG-induced and hypoxia-induced expression, indicating that the signal peptide can guide the expressed nanobody-PE fusion protein to be secreted outside the bacterial cell.

[0417] Example 5: Neutrophil neutralization in tumors increased DB-ZW1 levels in MB49 bladder cancer tumors. C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing approximately 18g, raised in an SPF environment) were fed 1 x 10 6A subcutaneous tumor model of mouse bladder cancer was established by subcutaneous inoculation of 1 MB49 mouse bladder cancer cells (Merck, SCC148). 14-18 days after inoculation (tumor volume approximately 100 mm) 3 ), mice were divided into a PBS group and a ZW1 treatment group (administered DB-ZW1 only, 1 × 10⁶ mice). 7The cfu bacteria were suspended in 125 μL of PBS and administered to mice via tail vein injection. The mice were then divided into two groups, the ZW1+αGr-1 group and the ZW1+αGr-1 group, with three mice in each group. The ZW1+αGr-1 group received 0.2 mg of InVivoMab anti-mouse Ly6G / Ly6c(Gr-1) antibody (purchased from BioXCell) via tail vein injection three days before bacterial treatment, on the day of bacterial injection, and the day after bacterial injection (2 dpi). Three days after bacterial treatment (3 dpi), the mice were euthanized, the tumors were removed and embedded, and the tumors were sequentially sectioned near the center, with the sections placed on slides. The samples were fixed with acetone for 10 minutes, air-dried, washed twice with 0.01 mol / L PBS at room temperature, and then added dropwise to the samples with goat serum (normal goat serum purchased from Jackson ImmunoResearch). The samples were then blocked at 4°C for 45 minutes. The samples were incubated with primary antibodies (antibody for labeling bacteria, pAb anti-Salmonella, purchased from Novus and diluted 200-fold; antibody for labeling neutrophils, purified anti-mouse Ly-6G antibody, purchased from Biolegend, 1:500) for 2 hours at room temperature in the dark, then washed 5 times with PBS. The samples were incubated with secondary antibodies (secondary antibody for labeling bacteria, goat anti-rabbit HL&, 1:500; secondary antibody for labeling neutrophils, donkey pAb against rat IgG, 1:1000, both purchased from Abcam) for 1 hour at room temperature in the dark, then washed 5 times with PBS. A mounting medium containing DAPI (ProLong™ Gold anti-fade medium with DAPI, purchased from Invitrogen) was added to prevent fading. Observation was performed using a fluorescence microscope after covering with a coverslip. Lipopolysaccharide O antigen and flagellin H antigen on the bacterial surface showed red fluorescence when labeled with an antibody containing the AF555 fluorescent group, the neutrophil lymphocyte membrane surface complex Ly-6G showed green fluorescence when labeled with an antibody containing the AF488 fluorescent group, and the nucleus showed blue fluorescence when stained with DAPI. As shown in Figure 8, neutrophil elimination by αGr-1 was beneficial for bacterial diffusion throughout the tumor.

[0418] Example 6: Neutrophil neutralization improved the therapeutic effect of artificial antitumor bacteria on a 4T1 breast cancer model. An orthotopic 4T1 breast cancer model was constructed by injecting a PBS suspension of 4T1 breast cancer cells (ATCC CRL-2539) into the right side of the second pair of mammary fat pads of Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF grade). Each mouse was given 10 6 Tumor cells were injected at a dose of 125 μL. The tumor volume was approximately 200 mm³ 20 days after injection. 3 The following was observed: Neutrophils were neutralized by injecting 125 μL of 1.6 mg / mL InVivoMab anti-mouse Ly6G / Ly6c(Gr-1) antibody via the tail vein. 24 hours after antibody injection, mouse tumors and blood were collected for flow cytometry analysis. 7 CFU DB-ZW1 was administered to mice via the tail vein, and mice injected with PBS, antibody alone, and DB-ZW1 alone were used as controls.

[0419] As shown in Figure 9A, more than 90% of neutrophils were eliminated from 4T1 tumors after antibody injection. As shown in Figure 9B, 4T1 tumor growth was not inhibited by injection of Ly6G / Ly6c(Gr-1) antibody alone or DB-ZW1 alone, while 4T1 tumors in mice injected with both the antibody and DB-ZW1 were significantly smaller and showed a statistically significant difference compared to the three control groups (P<0.01). As shown in Figure 9C, on day 1 after DB-ZW1 injection, tumors in the experimental mice showed redness and darkening, and significant scabbing occurred. These results indicate that a reduction in neutrophil content in tumors can improve the effect of antitumor bacteria (DB-ZW1) on tumor inhibition.

[0420] Example 7: In Vitro Test of Bacterial Nanobodies To identify the binding of nanobodies expressed by E. coli to neutrophils, primary neutrophils were isolated from the blood of C57BL / 6 mice, and the binding of the expressed nanobodies to CXCR2 on the surface of the neutrophils was detected.

[0421] In detail, blood was obtained from mice by post-orbital hematopoietic collection. Erythrocyte lysate (containing 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.2-7.4) was added to the obtained blood and incubated at room temperature for 5 minutes to completely dissolve the erythrocytes. Erythrocyte fragments were removed by filtration to obtain purified immunocellular cells. Live and dead cells were counted, and the purified immunocellular cells were incubated with Zombie Aqua (purchased from Biolegend) and TruStain FcX (anti-mouse CD16 / 32) (purchased from Biolegend, catalog number 101320) for 15 minutes at room temperature in the dark, for staining purposes to block the Fc receptor and prevent the generation of nonspecific signals. Then, purified nanobodies (purified using an AKATA protein purifier with a nickel column for his tag) were incubated with the purified immunocellular cells for 1 hour, and cells without nanobodies were used as a control. Flow cytometry was used to co-stain neutrophils for CD45 (Alexa Fluor 700 anti-mouse CD45, Biolegend), His tag (PE anti-His tag, Biolegend), and Ly6G (anti-mouse, Biolegend). The sorting purity of neutrophils and the binding of nanobodies to neutrophils were then quantified by counting using flow cytometry.

[0422] As shown in Figure 10, all three nanobodies expressed by E. coli bound to neutrophils, with Nb97A7 showing the highest binding rate at over 25%.

[0423] Example 8: Effect of nanobody and fusion protein expression DB-ZW1 on tumor inhibition in a tumor-bearing mouse model. In this example, the strains constructed in Example 3.4: ZW1-psc101-pFNRsp-psp-Nb127D01-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE38-HA-his, ZW1-psc101-pFNR sp-psp-Nb97A9-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his, ZW1-ps The MB49 subcutaneous tumor mouse model prepared in Example 5 was treated with c101-pFNRsp-psp-Nb97A9-PE[LR]-HA-his, ZW1-psc101-pFNRsp-psp-PE38-HA-his, ZW1-psc101-pFNRsp-psp-PE[LR]-HA-his, ZW1-psc101-pFnr-SP-psp-Nb127D01-CM, ZW1-psc101-pFnr-SP-psp-Nb163E3-CM, and ZW1-psc101-pFnr-SP-psp-Nb97A9-CM.

[0424] Mice injected with the aforementioned bacterial strain expressing nanobodies or fusion proteins were used as the experimental group, mice injected with the DB-ZW1 strain were used as controls, mice injected with PBS were used as blank controls, and mice injected with Gr-1 antibody after DB-ZW1 injection were used as positive controls. 1 × 10 7 CFU bacteria were suspended in 125 μL of PBS and administered to mice via tail vein. Mouse body weight was recorded, and tumor volume was measured every two days. All mice had comparable body weight, demonstrating the safety of treatment with the antitumor bacteria of the present invention.

[0425] As shown in Figure 11, ZW1-psc101-pFNRsp-psp-Nb127D01-PE38-HA-his demonstrated the best therapeutic effect, significantly improving inhibition of tumor growth compared to DB-ZW1.

[0426] On the other hand, four days after injection, blood and solid tumors were collected from the mice, and blood neutrophils and tumor neutrophils were counted by flow cytometry.

[0427] As shown in Figure 12, compared to mice injected with PBS, intratumoral neutrophils were significantly increased in mice injected with DB-ZW1, significantly decreased in positive control mice, and intratumoral neutrophil levels in mice of the other groups were comparable to those in mice injected with PBS. Furthermore, neutrophil levels in the blood of the other groups were comparable, except that neutrophil levels in the blood of the positive control group were significantly lower (almost completely eliminated). This demonstrates that the strain of the present invention can inhibit neutrophils in tumors in a site-specific manner.

[0428] Example 9: Plasmid for IPTG-induced expression of nanobodies and nanobodies-fusion toxin proteins 9.1 Construction of plasmids for IPTG-induced expression of nanobody or toxin proteins The objective of this example was to construct plasmids for the expression of nanobodies 238D2 (SEQ ID NO: 64), 238D4 (SEQ ID NO: 65), EGa1 (SEQ ID NO: 63), VHH1 (SEQ ID NO: 61), and VHH26 (SEQ ID NO: 62), as well as toxin proteins CET40 and DT390 (SEQ ID NOs: 80 and 85).

[0429] The coding sequence was synthesized (by Beijing Tsingke Biotech Co., Ltd.), and the nanobodies 238D2, ​​238D4, EGa1, and VHH1 contained the same pelB signal peptide as in Example 3.1, 6 * Using the his tag, the nanobody VHH26 and the toxin proteins CET40 and DT390 were cloned into the pET26 skeleton using the same HA-his tag as in Example 3.1.

[0430] The resulting recombinant plasmid was named as follows: pET26b-Kana-T7-pelB-238D2-his(sequence number 92); pET26b-Kana-T7-pelB-238D4-his(sequence number 93); pET26b-Kana-T7-pelB-EGa1-his(sequence number 94); pET26b-Kana-T7-pelB-VHH1-his(sequence number 95); pET26b-Kana-T7-pelB-VHH26-HA-his (SEQ ID NO: 96); pET26b-Kana-T7-pelB-CET40-HA-his(Sequence ID 97); pET26b-Kana-T7-pelB-DT390-HA-his (Sequence ID 98).

[0431] 9.2 Construction of a plasmid for IPTG-induced expression of a fusion protein of nanobody and toxin PE38 The objective of this example was to construct plasmids for the expression of nanobodies 238D2, ​​238D4, EGa1, VHH1, and VHH26 fused to toxin proteins.

[0432] As described in Example 3.3, the coding sequence for the toxin protein PE38 was inserted into the nanobody expression plasmid constructed in Example 9.1. The resulting recombinant plasmid was named as follows: pET26b-Kana-T7-pelB-Nb238D2-PE38-his(sequence number 99); pET26b-Kana-T7-pelB-Nb238D4-PE38-his(Sequence ID 100); pET26b-Kana-T7-pelB-NbEGa1-PE38-his(sequence number 101); pET26b-Kana-T7-pelB-VHH1-PE38-his(sequence number 102); pET26b-Kana-T7-pelB-VHH26-PE38-his (SEQ ID NO: 103).

[0433] 9.3 Construction of plasmids for IPTG-induced expression of a fusion protein of nanobody Nb127D01 and toxin CET40 or DT390 As described in Example 3.3, nucleotide sequences encoding the toxin proteins CET40 and DT390 were inserted into the nanobody Nb127D01 expression plasmid (pET26b-pelB-Nb127D01-HA-his). The resulting recombinant plasmid was named as follows: pET26b-Kana-T7-pelB-127D01-CET40-HA-his(Sequence ID 104) pET26b-Kana-T7-pelB-127D01-DT390-HA-his(Sequence ID 105).

[0434] 9.4 Construction of strains expressing nanobodies, toxin proteins, and nanobodies fused to toxin proteins under IPTG induction. Recombinant plasmids were used to transform recombinant protein-expressing strain BL21(DE3) (BL21(DE3) competent Escherichia coli strain, Vazyme, number C504-03) by chemical transformation. Specifically, competent cells were thawed on ice, 100 ng of the recombinant plasmid was mixed with the competent cells, and the mixture was left on ice for 30 minutes. After a 45-second heat shock in a 42°C water bath, the mixture was immediately placed on ice for 3 minutes. 900 μL of LB medium (without antibiotics) was added, and the mixture was shaken at 37°C for 1 hour (rotation speed 200-250 rpm), followed by centrifugation at 5,000 rpm for 5 minutes. 900 μL of supernatant was discarded, the bacteria were resuspended in the remaining amount of medium, and slightly spread on a kanamycin-containing LB plate (KanR, 100 mg / L) using a sterile spreading rod. The plate was flipped over and incubated overnight in a 37°C incubator.

[0435] The following strains were obtained that expressed nanobodies, toxin proteins, and nanobodies fused to toxin proteins under IPTG induction: BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D2-his; BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D4-his; BL21(DE3)-pET26b-Kana-T7-pelB-EGa1-his; BL21(DE3)-pET26b-Kana-T7-pelB-VHH1-his; BL21(DE3)-pET26b-Kana-T7-pelB-VHH26-his; BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D2-PE38-his; BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D4-PE38-his; BL21(DE3)-pET26b-Kana-T7-pelB-NbEGa1-PE38-his; BL21(DE3)-pET26b-Kana-T7-pelB-VHH1-PE38-his; BL21(DE3)-pET26b-Kana-T7-pelB-VHH26-PE38-his; BL21(DE3)-pET26b-Kana-T7-pelB-CET40-HA-his; BL21(DE3)-pET26b-Kana-T7-pelB-127D01-CET40-HA-his; BL21(DE3)-pET26b-Kana-T7-pelB-DT390-HA-his; BL21(DE3)-pET26b-Kana-T7-pelB-127D01-DT390-HA-his.

[0436] 9.5 Prokaryotic expression of nanobodies and nanobodies fused to toxin proteins under IPTG induction. The objective of this example was to detect the ability of the constructed strain to express heterologous proteins.

[0437] To achieve this objective, prokaryotic expression was performed using the BL21(DE3) strain constructed in Example 9.4. For details, i) The cryopreserved bacteria were restored in liquid medium, and when the culture reached OD=0.4-0.5, IPTG was added at a final concentration of 0.5 mM, and the culture was induced overnight in a 16°C shaker (induction for 14-16 hours at 200-250 rpm). The bacterial culture was then removed and placed on ice.

[0438] ii) The culture was centrifuged at 4,200 rpm at 4°C for 10 minutes, and the bacterial pellet and LB medium supernatant were collected separately. The LB medium supernatant was placed in an ultrafiltration tube (Amicon Ultra, part number UFC901096) and centrifuged at 2,500 rpm at 4°C for 2 hours, and the concentrated LB medium supernatant containing secreted proteins was collected.

[0439] iii) The lysis solution (Qiagen, Qproteome Bacterial Protein Prep Kit, No. 37900, see Experimental Manual Qproteome Bacterial Protein Preparation Handbook, www.qiagen.com) was added to the bacterial pellet and left on ice for 30 minutes. Then, it was centrifuged at 14,000 rpm at 4°C for 30 minutes, and the bacterial debris precipitate and the supernatant of the lysis solution were collected separately. The bacterial debris precipitate contained proteins in functional inclusion bodies, and the supernatant contained soluble proteins that were expressed but not secreted.

[0440] iv) 5× SDS loading buffer (Beyotime, no. P0015) was added to the collected samples (concentrated LB medium supernatant, bacterial debris precipitate, and dissolved supernatant), and a 100°C metal bath was used for 5 minutes, followed by SDS-PAGE. In other words, electrophoresis was performed at 80V for 20 minutes until the sample moved to the interface between the separation gel and the concentration gel, at which point the voltage was increased to 120V to completely separate the gel from the dye.

[0441] v) Following the instructions, membrane transfer was performed using the Pyxis® M protein transfer stack (PVDF membrane) and transfection apparatus. After membrane transfer, the membrane was washed sequentially with TBS buffer and TBST buffer, and the PVDF membrane was blocked at room temperature for 1 hour with 5% skim milk (dissolved in TBST buffer) and primary antibody (6* His (his-tagged monoclonal antibody, Proteintech, No. 66005-1-lg) was diluted in 5% skim milk at a ratio of 1:10000 and incubated overnight at 4°C. The incubated PVDF membrane was washed multiple times with TBST buffer to remove antibodies from the membrane surface. A secondary antibody (HRP conjugate affinity goat anti-rabbit IgG(H+L), Proteintech, No. SA00001-2-100UL) was diluted in 5% skim milk at a ratio of 1:5000 and incubated at room temperature for 1 hour. The incubated PVDF membrane was washed multiple times with TBST buffer to remove unbound antibodies from the membrane surface, and finally, the membrane was immersed in TBS. The prepared color-developing solutions A and B were mixed in a 1:1 ratio, the film was placed in a tray, solutions A and B were added, the film was exposed for 5 minutes, and imaging was performed using a multi-functional imaging device (Thermo Fisher, SuperSignal® West Pico PLUS chemiluminescent substrate).

[0442] As shown in Figure 13 below, all heterologous proteins were successfully expressed under IPTG induction.

[0443] Example 10: Anaerobic expression of nanobodies and nanobodies fused to toxin proteins First, plasmids and strains for anaerobic expression of nanobodies and nanobodies fused to toxin proteins were constructed.

[0444] Plasmids pET26b-Kana-T7-pelB-238D2-his, pET26b-Kana-T7-pelB-238D4-his, pET26b-Kana-T7-pelB-EGa1-his, pET26b-Kana-T7-pelB-VHH1-his, pET26b-Kana-T7-p constructed in Example 9 elB-VHH26-HA-his, pET26b-Kana-T7-pelB-Nb238D2-PE38-his, pET26b-Kana-T7-pelB-Nb238D4-PE38-his, pET26b-Kana-T7-pelB-NbEGa1-PE38-his, pET26b-Kana-T7-pelB-VH H1-PE38-his, pET26b-Kana-T7-pelB-VHH26-PE38-his, pET26b-Kana-T7-pelB-CET40-HA-his, pET26b-Kana-T7-pelB-DT390-HA-his, pET26b-Kana-T7-pelB-127D01-CET40-HA-his, and pET26b-Kana-T7-pelB-127D01-DT390-HA-his were used as templates for amplifying linear fragments encoding nanobodies, toxin proteins, or nanobodies fused to toxin proteins, while pSC101-pFNRsp was used as a template for amplifying the vector skeleton. Linear fragments of the vector skeleton were amplified by PCR. The primers used are shown in Table 5, and the PCR system and conditions were as described in Example 3.2.

[0445] [Table 3]

[0446] The plasmids obtained were as follows: pSC101-Cm-FNRSP-0033-pelB-238D2-His(Sequence ID 106); pSC101-Cm-FNRSP-0033-pelB-238D4-His(Sequence ID 107); pSC101-Cm-FNRSP-0033-pelB-EGa1-His(Sequence ID 108); pSC101-Cm-FNRSP-0033-pelB-VHH1-His(Sequence ID 109); pSC101-Cm-FNRSP-0033-pelB-VHH26-His(Sequence ID 110); pSC101-Cm-FNRSP-0033-pelB-238D2-PE38-His(Sequence ID 111); pSC101-Cm-FNRSP-0033-pelB-238D4-PE38-His(Sequence ID 112); pSC101-Cm-FNRSP-0033-pelB-EGa1-PE38-His(Sequence ID 113); pSC101-Cm-FNRSP-0033-pelB-VHH1-PE38-His(Sequence ID 114); pSC101-Cm-FNRSP-0033-pelB-VHH26-PE38-His(Sequence ID 115); pSC101-Cm-FNRSP-0033-pelB-CET40-HA-His(Sequence ID 116); pSC101-Cm-FNRSP-0033-pelB-DT390-HA-His(Sequence ID 117); pSC101-Cm-FNRSP-0033-pelB-127D01-CT390-HA-His (SEQ ID NO: 118); pSC101-Cm-FNRSP-0033-pelB-127D01-CET40-HA-His (SEQ ID NO: 119).

[0447] The plasmid described above was introduced into competent cells of the DB-ZW1 strain by electrotransformation (E=18kV / cm). The resulting strains were as follows: ZW1-pSC101-Cm-FNRSP-0033-pelB-238D2-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-238D4-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-EGa1-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH1-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH26-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-238D2-PE38-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-238D4-PE38-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-EGa1-PE38-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH1-PE38-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH26-PE38-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-CET40-HA-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-DT390-HA-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-127D01-CT390-HA-His; ZW1-pSC101-Cm-FNRSP-0033-pelB-127D01-CET40-HA-His.

[0448] The anaerobic expression of the fusion protein of the aforementioned strain, and the effect of the fusion protein on the treatment of tumors, were tested as described in Examples 4 and 8.

[0449] List of arrays Sequence ID 1: PsseA-hlyA-loxp-KnaR-loxp Sequence ID 2: Pssbp1-dapE-Cm Sequence ID 3: Plasmid PsseA-hlyA-loxp-KnaR-loxp Sequence ID 4: Plasmid Pssbp1-dapE-Cm Sequence ID 5: PsseA-hlyA-loxp-KnaR-loxp forward primer TTCACAGAAAAGTGTTGCCCCCTTCCATGGCGGAAGGGGGACAAAGGTGAATTTGTCCTACTCAGGAGAGCGTTCA Sequence ID 6: PsseA-hlyA-loxp-KnaR-loxp reverse primer TCTGACGTACACAGCAATTTTGCGTTACCTGTTAATCGAGATTGAAACACCGAAGAAAGGCCCACCCG Sequence ID 7: Pssbp1-dapE-Cm forward primer TTGGTTTCAGTGAATCCCGTTATCAGCAGTTTTTTGATGAGGTGTAGTCTATTTGTCCTACTCAGGAGAGCG Sequence ID 8: Pssbp1-dapE-Cm reverse primer CAATATTTTGCCAGCCAGTCCATGCTTATTTCCTTCTTACCGGAACGCTCACGAAGAAAGGCCCACCCG Sequence ID 9: Promoter pepTp GCAGGGGTAAAAGTGACCTGACGCAATATTTGTCTTTTTCTTGCTTCTTAATAATGTTGTCACAAAAAGTGAGGGTGACTACATGG Sequence ID 10: Promoter fnrSp CGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGCAGGTGAATGCAACGTCAAGCGAT Sequence ID 11: PromoterysgAp TCAGAAGAAGCAAAAAGACACTTTACCGAAGGGTTTAACATTTTTTCGTGATACTCATCACCATGACGCAAATGCGTTGCATAAA Sequence ID 12: Promoter ssbp1 AACCGAGGTCACAACATAGTAAAAGCGCTATTGGTAATGGTACAATCGCGCGTTTACACTTATTCAGAACGATTTTTTTCAGGAG Sequence ID 13: Promoter Hip1 GGATAAAAGTGACCTGACGCAATATTTGTCTTTTCTTGCTTAATAATGTTGTCA Sequence ID 14: Promoter BBa_I14018 TGTAAGTTTATACATAGGCGAGTACTCTGTTATGG Sequence ID 15: Promoter BBa_R1074 TTAAATTTCCTCTCGTCAGGCCGGAATAACTCCCTATAATGCGCCACCACACTGATAGTGCTAGTGTAGATCAC Sequence ID 16: Promoter Ptet-Fnr AAAATTGATCTGAATCAATATTTTGACAAAAATTGATCTGAATCAATATTTACTGAGC Sequence ID 17: Promoter Ptet-arcA GTTAATAAAATGTTATTGACAGTTAATAAAATGTTATACTGAGC Sequence ID 18: Promoter PsseA TTAGCACGTTAATTATCTATCGTGTATATG Sequence ID 19: Nb127D01 (CDR is in bold and underlined) EVQLVESGGGLVQAGESLRLSCAASGSTFD FKVMG WYRQPPGKQREGVA AIRLSGNMHYAESVKG RFAISKANAKNTVYLQMNSLRPEDTAVYYCKV NIRGQDY WGQGTQVTVSSGSG Sequence ID 20 Nb163E3 (CDR is in bold and underlined) EVQLVESGGGLVQPGGSVRLSCVASGRIFS SNAMG WFRQAPGKEREFVA AITWRSGGSAYYADSAKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA GGSSWLSFPPDY WGQGTQVTVSS Sequence ID 21 Nb97A9 (CDR is in bold and underlined) EVQLVESGGGLVQPGGSLRLSCVASGSIVR INTMG WYRQTPGKQRELVA DITSGGNINYIDAVK GRFTISRDNTKKRVYLQMNSLKPEDTAVYYCNA EIVVLVGVWTQRARTGNY WGQGTQVTVSS Sequence ID 22 Nb127D01 GAGGTTCAATTAGTGGAATCTGGCGGGGGATTGGTTCAAGCCGGTGAGTCCTTGCGTTTGAGCTGCGCCGCATCTGGGAGCACATTTGATTTCAAAGTGATGGGCTGGTATCGTCAGCCGCCCGGAAAACAGCGCGAGGGTGTAGCAGCCATCCGCTTGAGCGGAAATATGCACTAC GCCGAGTCTGTAAAAGGGCGCTTTGCTATTTCCAAAGCGAACGCTAAGAACACAGTGTATCTTCAGATGAACTCCTTGCGTCCCGAAGACACTGCCGTTTATTATTGCAAAGTAAACATCCGTGGGCAAGACTACTGGGGTCAAGGAACGCAGGTCACAGTCTCTTCTGGCTCTGGC Sequence ID 23 Nb163E3 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGCCTGGGGGCTCTGTGAGACTCTCCTGTGTAGCCTCTGGACGCATCTTCAGTAGCAATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGGCCATTACCTGGAGGAGTGGCGGTAGCGCGTACTATGCAGACTCCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCAGCTGGTGGTAGTTCCTGGTTAAGTTTTCCGCCGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA Sequence number 24 Nb97A9 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAAGCATCGTCAGAATTAATACCATGGGCTGGTACCGCCAGACTCCAGGGAAGCAGCGCGAGTTGGTCGCAGATATTACCAGTGGTGGTAACATAAACTATATAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAAGAGGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGAGATCGTTGTTCTGGTGGGAGTTTGGACCCAGCGTGCGCGGACCGGCAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA Sequence number 25 PE AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFV RAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWN QVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARS QDLDAIWRGFYIAGDPALAYGYAQDQEPDARGIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 26 PE38 PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGY HGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDL Sequence ID 27 PE[LR] RHRQPRGWEQLPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGAL LRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK SEQ ID NO: 28 pelB signal peptide MKYLLPTAAAGLLLLAAQPAMA SEQ ID NO: 29 Signal Peptide PSP MGLKMKKRSGKKAWMLLVMSLLIAVPITASA Sequence ID 30 pET26b-pelB-Nb127D01-HA-his Sequence ID 31 pET26b-pelB-Nb163E3-HA-his Sequence ID 32 pET26b-pelB-Nb97A9-HA-his Sequence ID 33 pSC101-pFNRsp Sequence ID 34 pET26b-psp-Nb127D01-HA-his Sequence ID 35 pET26b-psp-Nb163E3-HA-his Sequence ID 36 pET26b-psp-Nb97A9-HA-his Layout number 43 pET26b-psp-Nb127D01-PE38-HA-his Layout number 44 pET26b-psp-Nb163E3-PE38-HA-his Layout number 45 pET26b-psp-Nb97A9-PE38-HA-his Layout number 46 pET26b-psp-Nb127D01-PE[LR]-HA-his Accession number 47 pET26b-psp-Nb163E3-PE[LR]-HA-his Accession No. 48 pET26b-psp-Nb97A9-PE[LR]-HA-his Sequence ID 49: pSC101-pFNRsp-psp-Nb127D01-PE38-HA-his Sequence ID 50: pSC101-pFNRsp-psp-Nb163E3-PE38-HA-his Sequence ID 51: pSC101-pFNRsp-psp-Nb97A9-PE38-HA-his Sequence ID 52: pSC101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his Array number 53 pSC101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his Sequence ID 54: pSC101-pFNRsp-psp-Nb97A9-PE[LR]-HA-his Sequence ID 55 Fragment. FOR GATTACGGCGAGCGCCGAGGTGCAGCTGGTGGAG Sequence ID 56 Fragment.REV GCCAGGCTACCACCTTCAGGTGAGGAGACGGTGACCTGG Sequence ID 57: dapE (encodes succinyl diaminoheptanediate decussinylase, gene ID: 1254005) Sequence ID 58: htrA (codes serine protease, gene ID: 1251727) Sequence ID 59: hlyA (encodes listeria hemolysin, gene ID: 987033) Code sequence of the HA-his tag, sequence number 60. Tacccctatgacgtgcctgattacgccggctctggccaccaccaccaccaccac Sequence ID 61 G-CSF-R nanobody 1 (CDR is in bold and underlined) (VHH1) GGGSVQAGGSLKLSCEAS LPSTTTPYSMA WFRQAPGREREAVA TISARGGTKL YSDSVRERFTISRDNRKNTVYLKMDHLNTDDTAIYYCAA TGSILFNSRPFAESTYPF WGQGTLVTVSS Sequence ID 62 G-CSF-R nanobody 2 (CDR is in bold and underlined) (VHH26) GGGTVQAGGSLKLSCAAR GWRFSGYCVA WFRQAPGKEREAVA IIPGAGVMY TDDSVKGRFTYSRDNVKNTVSLQMNSLEPEDTATYYCAV DWAYSHSCPKNTEDYRY WGQGTQVTVSS Sequence ID 63 EGFR nanobody 1 (CDR is in bold and underlined) (EGa1) MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGLVQPGGSLRLSCAAS GRTFSSYAMG WFRQAPGKEREFVA AIRWSGGYTY YTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAA TYLSSDYSRYALPQRPLDYDY WGQGTQVTVSSAAAEQKLISEEDLNGLE Sequence ID 64 CXCR4 Nanobody 1 (CDR is in bold and underlined) (238D2) EVQLVESGGGLVQTGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS GIKSSGDSTRYAGSVKG RFTISRDNAKNMLYLQMYSLKPEDTAVYYCAK SRVSRTGLYTYDN RGQzeqVTVSS Sequence ID 65 CXCR4 Nanobody 2 (CDR is in bold and underlined) (238D4) EVQLMESGGGLVQAGGSLLRLSCAASGRTFN NYAMGWFRRAPGKEREFVA AITRSGVRSGVSAIYGDSVKD RFTISRDNAKNTLYLQMNSLKPEDTAVYTCAA SAIGSGALRRFEYDY SGQGTQVTVS Sequence ID 66 IL6R nanobody 1 (CDR is in bold and underlined) EVQLVESGGGLVQAGDSLRLSCLASGRSFK DDAMG WFRQAPGKEREFVS GIDWRGNIVDAESVKG RFTISRENAKNTLYLQMNHLKPEDTAVYYCAA SSVFSDLRYRKNPKY WGQGTQVTVSS Sequence ID 67 IL6R nanobody 2 (CDR is in bold and underlined) EVQLVESGGGLVQAGGSLRRLSCAASGSIFR INAMG WYRQAPGKQRELVA AAISGGSTNYADFVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNA LVVSHNYSDYVPFPDDY WGQGTQVTVSS Sequence ID 68 IL6R nanobody 3 (CDR is in bold and underlined) EVQLVESGGGLVQPGGSLRLSCAASGFT LDYYAIG WFRQAPGKEREGVS CISSTDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLNPEDTGVYTCAA VLTTAQAMGVPTRSYEYDY WGQGTQVTVSS Sequence ID 69 IL6R nanobody 4 (CDR is in bold and underlined) EVQLVESGGGLVEAGGSLRLSCAAAGRTLS SYSMA WFRQAPGKEREFVA IIRGNPSRTYHSDSVKG RFTASRDNAKNTVYLQMNRLEPEDTAVYYCAA DPPPFASDYSAPQSYDY WGQGTQVTVSS Sequence ID 70 IL6R nanobody 5 (CDR is in bold and underlined) EVQLVESGGGLVQAGGSLNLSCNASGDIGS INAMG WHRQAPGQQREWVA VITDTDSTIYPDSVKG RFAISRDGAKNTVHLRMSSLKPEDTAVYYCNA YQAGWGDY WGQGTQVTVSS Sequence ID 71 hTNFR1 nanobody 1 (CDR is in bold and underlined) QVQLQESGGGVVQPGGSLTLSCTRT GFTASTNAYG WYRQGPGKKCEWVSYMTIPSGRTTYADAVKGRFAMSRDKAKSTVLLQMNSLKPEDTAVYYCGD VPFSTLPAMCTNDG PWGQGTQVTVSS Sequence ID 72 hTNFR1 nanobody 2 (CDR is in bold and underlined) QVQLQESGGGVVQPGGSLTLSCTRT GFTASTNAYG WYRQGPGKKCEWVS YMTIPSGRTTYADAVKG RFAMSRDKAKSTVFLQMNSLKPEDTAVYYCGD VPFSTLPAMCTNDG PWGQGTQVTVSS Sequence ID 73 hTNFR1 nanobody 3 (CDR is in bold and underlined) QVQLQESGGGLVQAGGPLRLSCAAS WRTFSSYAMG WFRQAPGKEREFVA AISWSGGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV LHEDLYEYDY WGQGTQVTVSS Sequence ID 74 hTNFR1 nanobody 4 (CDR is in bold and underlined) QVQLQESGGGVVQPGGSLTLSCTRT GLTPSTGAYG WYRQAPGKKCELVS YITIPSGRTTYTDSVKG RFAISRDKAKNTVFLQMNSLKPEDTALYYCGD VPYSTIQAMCTDDG PWGQGTQVTVSS Sequence ID 75 hTNFR1 nanobody 5 (CDR is in bold and underlined) QVQLQESGGGVVQPGGSLTLSCTRT GLTASTNAYG WYRQAPGKKCELVS YITIPSGRTTYADSVKG RFAISRDKAKNTVFLQMNSLKPEDTALYYCGD VPYSTIQAMCTDDGP WGQGTQVTVSS Sequence ID 76 hTNFR1 nanobody 6 (CDR is in bold and underlined) QVQLQESGGGLVQSGGSLRLSCRAS GFTFSHYPMSWYRQAPGKKRELVA GIYTDGSTNYADSVKG RFTISRDNAKNTVYLQMSSLKPEDTAVYYCR RSMVSVY WGQGTQVTVSS Sequence ID 77 hTNFR1 nanobody 7 (CDR is in bold and underlined) QVQLQESGGGLVQPGGSLRLSCAAS GRTFSYYTMA WFRQAPGKEREFVA SISWSGGITYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RKEPGSAYFG SWGQGTQVTVSS Sequence ID 78 hTNFR1 nanobody 8 (CDR is in bold and underlined) QVQLQESGGGVVQPGGSLTLSCTRT GFTASTNAYG WYRQAPGKKCELVS YITIPSGRTTYADAVKG RFAISRDKAKNTVFLQMNSLKPEDTAVYYCGD VPFSTLPAMCTNDGP WGQGTQVTVSS SEQ ID NO: 79 Corix Toxin LYLTFYLEKVMKKMLLIAGATVISSMAHPTFAVEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCA YQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAA QTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK Sequence ID 80 CET40 EALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTR EGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK Sequence ID 81 Cholera toxin MVKIIFVFFIFLSSFSYANDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILSGHSTYYIYVIATAPNMFNVNDVLGAYSPHPDEQEVSALGGIPYSQIYGWYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHRA WREEPWIHHAPPGCGNAPRSSMSNTCDEKTQSLGVKFLDEYQSKVKRQIFSGYQSDIDTHNRIKDELMIKLKFGVFFTVLLSSAYAHGTPQNITDLCAESHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPSSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN Sequence ID No. 82: Heat-labile enterotoxin subunit A MKNITFIFFILLASPLYANGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSPHPYEQ EVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYRLAGFPPDHQAWREEPWIHAPQGCGNSSRTITGDTCNEETQNLSTIYLREYQSKVKRQIFSDYQSEVDIYNRIRDEL Sequence ID No. 83: Heat-labile enterotoxin subunit B APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKN Sequence ID No. 84 Diphtheria Toxin GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS Sequence ID 85 DT390 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSV GSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFL Sequence ID 86 Shiga Toxin Chain A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILM Sequence ID 87 Shiga Toxin Chain B TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR Sequence ID 88 Anthrax Toxin Edema Factor [Bacillus anthracis] MTRNKFIPNKFSIISFSVLLFAISSSQAIEVNAMNEHYTESDIKRNHKTEKNKTEKEKFKDSINNLVKTEFTNETLDKIQQTQDLLKKIPKDVLEIYSELGGEIYFTDIDLVEHKELQDLSEEEKNSMNSRGEKVPFASRFVFEKKRETPKLIINIKDYAINSEQSKEVYYEIGKGISLDIISKDKSLDPEFLNLIKSLS DDSDSSDLLFSQKFKEKLELNNKSIDINFIKENLTEFQHAFSLAFSYYFAPDHRTVLELYAPDMFEYMNKLEKGGFEKISESLKKEGVEKDRIDVLKGEKALKASGLVPEHADAFKKIARELNTYILFRPVNKLATNLIKSGVATKGLNVHGKSSDWGPVAGYIPFDQDLSKKHGQQLAVEKGNLENKKSITEHEGEIGK IPLKLDHLRIEELKENGIILKGKKEIDNGKKYYLLESNNQVYEFRISDENNEVQYKTKEGKITVLGEKFNWRNIEVMAKNVEGVLKPLTADYDLFALAPS LTEIKKQIPQKEWDKVVNTPNSLEKQKGVTNLIKYGIERKPDSTKGTLSNWQKQMLDRLNEAVKYTGYTGGDVVNHGTEQDNEEFPEKDNEIFIINPEG EFILTKNWEMTGRFIEKNITGKDYLYYFNRSYNKIAPGNKAYIEWTDPITKAKINTIPTSAEFIKNLSSIRRSSNVGVYKDSGDKDEFAKKESVKKIAGY LSDYYNSANHIFSQEKKRKISIFRGIQAYNEIENVLKSKQIAPEYKNYFQYLKERITNQVQLLLTHQKSNIEFKLLYKQLNFTENETDNFEVFQKIIDEK Sequence ID 89: Anthrax Toxin Lethal Factor [Bacillus anthracis] MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLARYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS Sequence number 90, Bacillus anthracis anthrax toxin protective antigen MKKRKVLIPLMALSTILVSSTGNLEVIQAEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG Sequence number 91, lysine chain A SNAMIFPKQYPINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRFILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQLAG NLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQESNQGASPIQLQRRNGSKFSVYDVSILIPIIALMVYRCAPPPSSQF SEQ ID NO:92 pET26b-Kana-T7-pelB-238D2-his Sequence ID 93 pET26b-Kana-T7-pelB-238D4-his Sequence ID 94 pET26b-Kana-T7-pelB-EGa1-his Sequence ID 95 pET26b-Kana-T7-pelB-VHH1-his Allocation number 96 pET26b-Kana-T7-pelB-VHH26-HA-his Sequence ID 97 pET26b-Kana-T7-pelB-CET40-HA-his Sequence ID 98 pET26b-Kana-T7-pelB-DT390-HA-his Sequence ID 99 pET26b-Kana-T7-pelB-Nb238D2-PE38-his Sequence ID 100 pET26b-Kana-T7-pelB-Nb238D4-PE38-his Sequence ID 101: pET26b-Kana-T7-pelB-NbEGa1-PE38-his Sequence ID 102: pET26b-Kana-T7-pelB-VHH1-PE38-his Layout number 103 pET26b-Kana-T7-pelB-VHH26-PE38-his Sequence ID 104 pET26b-Kana-T7-pelB-127D01-CET40-HA-his Sequence ID 105 pET26b-Kana-T7-pelB-127D01-DT390-HA-his Sequence ID 106 pSC101-Cm-FNRSP-0033-pelB-238D2-His Sequence ID 107 pSC101-Cm-FNRSP-0033-pelB-238D4-His Sequence ID 108 pSC101-Cm-FNRSP-0033-pelB-EGa1-His Sequence ID 109: pSC101-Cm-FNRSP-0033-pelB-VHH1-His Sequence ID 110 pSC101-Cm-FNRSP-0033-pelB-VHH26-His Sequence ID 111 pSC101-Cm-FNRSP-0033-pelB-238D2-PE38-His Sequence ID 112 pSC101-Cm-FNRSP-0033-pelB-238D4-PE38-His Sequence ID 113 pSC101-Cm-FNRSP-0033-pelB-EGa1-PE38-His Sequence ID 114 pSC101-Cm-FNRSP-0033-pelB-VHH1-PE38-His Sequence ID 115 pSC101-Cm-FNRSP-0033-pelB-VHH26-PE38-His Sequence ID 116 pSC101-Cm-FNRSP-0033-pelB-CET40-HA-His Sequence ID 117 pSC101-Cm-FNRSP-0033-pelB-DT390-HA-His Sequence ID 118 pSC101-Cm-FNRSP-0033-pelB-127D01-DT390-HA-His Layout number 119 pSC101-Cm-FNRSP-0033-pelB-127D01-CET40-HA-His

Claims

1. A fusion protein comprising an antibody or its antigen-binding fragment that binds to neutrophils, and a cytotoxin or its active fragment.

2. The fusion protein according to claim 1, wherein the antibody is a nanobody.

3. The aforementioned antibody CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 contains residues 31-35 of SEQ ID NO: 21, CDR2 contains residues 50-64 of SEQ ID NO: 21, and CDR3 contains residues 98-115 of SEQ ID NO:

21. The fusion protein according to claim 2, comprising a complementarity-determining region (CDR) selected from the group consisting of the following.

4. The fusion protein according to any one of claims 1 to 3, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

5. The fusion protein according to any one of claims 1 to 4, wherein the cytotoxin is Pseudomonas exotoxin (PE).

6. The fusion protein according to any one of claims 1 to 5, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO:

25.

7. The fusion protein according to any one of claims 1 to 6, wherein the fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

8. A fusion protein according to any one of claims 1 to 7, further comprising a signal peptide.

9. The fusion protein according to claim 8, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

10. A fusion protein according to any one of claims 1 to 9, further comprising a linker and / or tag sequence.

11. An expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, encoding an antibody or its antigen-binding fragment that binds to neutrophils.

12. The expression construct according to claim 11, wherein the antibody is a nanobody.

13. The aforementioned antibody CDR1 containing residues 31-35 of SEQ ID NO: 19, CDR2 containing residues 50-65 of SEQ ID NO: 19, and CDR3 containing residues 98-104 of SEQ ID NO: 19; CDR1 containing residues 31-35 of SEQ ID NO: 20, CDR2 containing residues 50-67 of SEQ ID NO: 20, and CDR3 containing residues 100-111 of SEQ ID NO: 20; and CDR1 contains residues 31-35 of SEQ ID NO: 21, CDR2 contains residues 50-64 of SEQ ID NO: 21, and CDR3 contains residues 98-115 of SEQ ID NO:

21. The expression construct according to claim 12, comprising a complementarity-determining region (CDR) selected from the group consisting of the following.

14. The expression construct according to any one of claims 11 to 13, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

15. The expression construct according to any one of claims 11 to 14, wherein the nucleotide sequence further encodes a signal peptide.

16. The expression construct according to claim 15, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

17. The expression construct according to any one of claims 11 to 16, wherein the nucleotide sequence further encodes a tag sequence.

18. An expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, encoding the fusion protein according to any one of claims 1 to 10.

19. The expression construct according to any one of claims 11 to 18, wherein the strictly hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr promoters.

20. A modified bacterium comprising the expression construct described in any one of claims 11 to 19, compared to an unmodified starting strain.

21. The modified bacterium according to claim 20, further comprising a hypoxia-regulating essential gene expression cassette containing essential bacterial genes under the control of a strictly hypoxia-inducible promoter, and lacking at least one gene involved in or regulating the endogenous antioxidant stress response pathway, or a functional expression product thereof.

22. The modified bacterium according to claim 21, wherein the strictly hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr promoters.

23. The modified bacterium according to claim 21, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

24. The modified bacterium according to any one of claims 21 to 23, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, bacterial growth depends on additional DAP or an analogue thereof added to the culture medium.

25. The modified bacterium according to claim 24, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

26. The modified bacterium according to claim 24, wherein the essential gene is selected from dapA and dapE.

27. The modified bacterium according to any one of claims 21 to 26, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family gene.

28. The modified bacterium according to any one of claims 21 to 27, wherein the functional expression product of the gene involved in or regulating the endogenous antioxidant stress response pathway is an HtrA serine protease family-related protein.

29. A modified bacterium according to any one of claims 21 to 28, which lacks the activity of HtrA serine protease.

30. The modified bacterium according to any one of claims 21 to 29, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

31. A modified bacterium according to any one of claims 21 to 30, which lacks htrA.

32. The modified bacterium according to any one of claims 21 to 31, wherein the essential gene is a gene naturally present on the bacterial chromosome, and the natural promoter of the essential gene is functionally replaced by the strictly hypoxia-inducible promoter, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

33. The modified bacterium according to any one of claims 21 to 31, wherein the essential gene expression cassette is exogenous, and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, so that the expression of the essential gene in the bacterium is completely under the control of the strictly hypoxia-inducible promoter.

34. The modified bacterium according to claim 33, wherein the exogenous essential gene expression cassette is incorporated into the bacterial chromosome.

35. The modified bacterium according to claim 33, wherein the exogenous essential gene expression cassette is located outside the bacterial chromosome.

36. The modified bacterium according to claim 35, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

37. A modified bacterium according to any one of claims 21 to 36, further comprising a pH-regulating expression cassette containing a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions.

38. The modified bacterium according to claim 37, wherein the bacterial-derived hemolysin protein is a gram-negative bacterial hemolysin protein.

39. The modified bacterium according to claim 38, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

40. The modified bacterium according to claim 39, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

41. The modified bacterium according to any one of claims 37 to 40, wherein the promoter, which is active under acidic pH conditions, is active at pH values ​​of 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or less than 5.

5.

42. The modified bacterium according to any one of claims 37 to 41, wherein the promoter, which is active under acidic pH conditions, is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

43. The modified bacterium according to any one of claims 37 to 41, wherein the promoter that is active under acidic pH conditions is sseA.

44. The modified bacterium according to any one of claims 21 to 43, wherein the unmodified starting strain is a facultative anaerobic bacterium.

45. A modified bacterium according to any one of claims 21 to 44, which is a bacterium of the family Enterobacteriaceae.

46. Escherichia sp., Salmonella sp., Shigella sp., Klebsiella sp., Yersinia sp., Citrobacter sp., Enterobacter sp., Serratia sp., Proteus sp., Morganella sp., Providencia sp., Hafnia sp., and Pantoea sp. A modified bacterium according to any one of claims 21 to 45, which is a bacterium of type sp.

47. Escherichia coli, Enterobacter blattae (E. blattae), Enterobacter fergusonii (E. fergusonii), Enterobacter hermannii (E. hermannii), Enterobacter vulneris (E. vulneris), Salmonella enterica (S. enterica), Salmonella bongoli (Salmonella bongori (S. bongori), Salmonella typhi (S. typhi), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Shigella dysenteriae (S. dysenteriae), Shigella flexneri (S. flexneri), Shigella boydii (S. boydii), Shigella sonnei Sonnei (S. sonnei), Klebsiella pneumoniae (K. peumoniae), Klebsiella oxytoca (K. oxytoca), Yersinia pestis (Y. pestos), Yersinia enterocolicica (Y. enterocolicica), Mycobacterium pseudotuberculosis (Y. pseudotuberculosis), Yersinia aldobe (Yersinia) aldouae (Y. aldouae), Yersinia bercovieri (Y. bercovieri), Yersinia frederiksenii (Y. frederiksenii), Yersinia intermedia (Yersiniaintermedia (Y. intermedia), Yersinia kristersenii (Y. kristersenii), Yersinia mollaretti (Y. mollaretti), Yersinia rohdei (Y. rohdei), Yersinia ruckeri (Y. ruckeri), Citrobacter freundii (C. freundii), Citrobacter koseri (C. kaseri), Citrobacter blaakii (Citrobacter Enterobacter braakii (C. braakii), Enterobacter aerogenes (E. aerogenes), Enterobacter cloacae (E. cloacae), Enterobacter gergoviae (E. gergoviac), Enterobacter sakazakii (E. sakazakii), Enterobacter taylorae (E. tavlorae), Enterobacter amnigenus (Enterobacter Enterobacter amnigenus (E. amnigenus), Enterobacter intermedius (E. intermedius), Enterobacter asburiae (E. asburiac), Enterobacter cancergenus (E. cancergenus), Enterobacter dissolvens (E. dissolvens), Enterobacter nimiplesularis (Enterobacter E. nimipressuralis, Serratia marcescensSerratia marcescens (S. marcescens), Serratia entomophylla (S. entomophylla), Serratia ficaria (S. ficaria), Serratia fonticola (S. fonticola), Serratia grimesii (S. grimesii), Serratia liquefaciens (S. liquefaciens), Serratia odolifera (Serratia odorifera (S. odorifera), Serratia plymuthica (S. plymuthica), Serratia proteamaclans (S. proteamaclans), Serratia rubidaea (S. rubidaea), Serratia ureilytica (S. ureilytica), Proteus mirabilis (P. mirabilis), Proteus vulgaris (Proteus vulgaris (P. vulgaris), Proteus myxofaciens (P. myxofaciens), Proteus penneri (P. penneri), Proteus hauseri (P. hauseri), Morganella morganii (M. morganii), Providencia alcalifaciens (P. alcalifaciens), Providencia raschigianii (Providencia Providencia rustigianii (P. rustigianii), Providencia stuartii (P. stuartii), Providencia retgeri (ProvidenciaA modified bacterium according to any one of claims 21 to 46, selected from the group consisting of P. rettgeri, Providencia heimbachae, Hafnia alvei, and Pantoea agglomerans.

48. A modified bacterium according to any one of claims 21 to 47, which is Salmonella typhimurium.

49. The modified bacterium according to claim 48, wherein the starting strain is Salmonella typhimurium SL7207.

50. A modified bacterium according to any one of claims 21 to 49, which expresses wild-type lipopolysaccharide (LPS).

51. A modified bacterium according to any one of claims 21 to 50, which, when administered to a subject with a tumor, can survive and proliferate in tumor tissue but is rapidly eliminated in normal tissue.

52. A modified bacterium according to any one of claims 21 to 51, which, when administered to a subject having a tumor, can express and secrete in tumor tissue an antibody or its antigen-binding fragment that binds to neutrophils, or a fusion protein according to any one of claims 1 to 10.

53. The modified bacterium according to claim 52, wherein the secreted antibody or its antigen-binding fragment or fusion protein can inhibit neutrophil recruitment or kill neutrophils.

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