Anti-tumor bacteria to reduce neutrophil levels in tumors

Abstract

Provided is a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to neutrophil, and a cytotoxin or active fragment thereof. Also provided are modified bacteria having anti-tumor activity that express the fusion protein, the antibody or antigen-binding fragment thereof or the toxin or active fragment thereof in a hypoxic environment, such as a tumor. Also provided are pharmaceutical compositions comprising the modified bacteria and anti-tumor uses thereof.

Description

Anti-tumor bacteria that reduce neutrophil levels in tumors Technical Field

[0001] The present invention relates to modified bacteria with anti-tumor activity, a pharmaceutical composition comprising the modified bacteria and anti-tumor use thereof.

[0002] Background of the Invention

[0003] 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, and there is an urgent need to develop new treatments. Traditional tumor bacterial therapy, represented by Coley toxin, has a history of 150 years, but the effect is unstable and there are safety issues. The development of genetic engineering technology has made great progress in reducing the toxicity of strains. A series of clinical trials have confirmed the safety of attenuated engineered bacteria for the treatment of human tumors, but the efficacy is poor, and it is difficult to achieve both safety and therapeutic efficacy, which cannot meet the needs of clinical tumor treatment. Further in-depth optimization of tumor bacterial therapy is imperative.

[0004] Furthermore, the inventors discovered that neutrophils in the human body limit the application of tumor bacterial therapy. Therefore, it is necessary to optimize the strains used for tumor bacterial therapy to reduce the impact of neutrophils.

[0005] Summary of the Invention

[0006] The present invention constructs strictly anaerobic bacteria through synthetic biology gene circuits. After being applied to an animal or human body, the bacteria show a therapeutic effect on tumors and can be cleared by normal tissues and organs in a short time, thereby reducing the toxic side effects caused by the long-term retention of bacteria in the body to the animal or human body, and has more reliable anti-tumor efficacy and safety. In addition, exogenous proteins (such as antibodies against neutrophils or fusion proteins of the antibodies and cytotoxins) are expressed in the bacteria to inhibit the recruitment of neutrophils in tumors or kill neutrophils, thereby enhancing the anti-tumor efficacy of the bacteria.

[0007] In a first aspect, the present invention provides a fusion protein comprising an antibody (eg, a nanobody) or an antigen-binding fragment thereof that binds to neutrophils and a cytotoxin or an active fragment thereof.

[0008] In some embodiments, the antibody comprises a complementarity determining region (CDR) selected from the group consisting of: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO: 20, a CDR2 comprising residues 50-67 of SEQ ID NO: 20, and a CDR3 comprising residues 100-111 of SEQ ID NO: 20; and a CDR1 comprising residues 31-35 of SEQ ID NO: 21, a CDR2 comprising residues 50-64 of SEQ ID NO: 21, and a CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

[0009] 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 fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0010] 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.

[0011] In some embodiments, the fusion protein further comprises a linker and / or tag sequence.

[0012] In a second aspect, the invention provides an expression construct comprising a nucleotide sequence encoding an antibody (eg, a Nanobody) or an antigen-binding fragment thereof that binds to neutrophils, operably linked to a strictly hypoxia-inducible promoter.

[0013] In some embodiments, the antibody comprises a complementarity determining region (CDR) selected from the group consisting of: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO: 20, a CDR2 comprising residues 50-67 of SEQ ID NO: 20, and a CDR3 comprising residues 100-111 of SEQ ID NO: 20; and a CDR1 comprising residues 31-35 of SEQ ID NO: 21, a CDR2 comprising residues 50-64 of SEQ ID NO: 21, and a CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

[0014] 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.

[0015] In some embodiments, the nucleotide sequence further encodes a tag sequence.

[0016] In some embodiments, the nucleotide sequence encodes a fusion protein of the present invention.

[0017] In a third aspect, the present invention provides a modified bacterium.

[0018] In one embodiment, the modified bacteria comprise an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette comprising an essential gene of the bacteria under the control of a strictly hypoxia-inducible promoter, and the bacteria lack at least one gene required for survival in macrophages or its functional expression product, compared to an unmodified starting strain.

[0019] In one embodiment, the modified bacteria comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacteria under the control of a strict hypoxia-inducible promoter, and the bacteria lacks at least one gene involved in or regulating an endogenous anti-oxidative stress response pathway or its functional expression product, compared to an unmodified starting strain.

[0020] In one embodiment, compared to an unmodified starting strain, the modified bacteria comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacteria under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, and the bacteria lacks at least one gene involved in or regulating an endogenous anti-oxidative stress response pathway or its functional expression product.

[0021] In one embodiment, the modified bacteria comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacteria under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, compared to an unmodified starting strain, wherein the bacteria expresses wild-type lipopolysaccharide (LPS) and the bacteria lack at least one gene required for survival in macrophages or its functional expression product.

[0022] In one embodiment, the modified bacterium is a modified Salmonella typhimurium, wherein the bacterium comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette and a pH-regulatable expression cassette compared to an unmodified starting strain, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, and the bacterium lacks at least one gene involved in or regulating an endogenous anti-oxidative stress response pathway or its functional expression product.

[0023] In one embodiment, the modified bacterium is a modified Salmonella typhimurium, wherein the bacterium comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, compared to an unmodified starting strain, wherein the bacterium expresses wild-type lipopolysaccharide (LPS) and the bacterium lacks at least one gene required for survival in macrophages or its functional expression product.

[0024] In one embodiment, the present invention provides a pharmaceutical composition comprising the bacterium of any one of the preceding embodiments. In one embodiment, the pharmaceutical composition is for use in treating a malignant tumor.

[0025] In one embodiment, the present invention provides a method for treating a malignant tumor, comprising administering a pharmaceutical composition of the present invention to a subject suffering from a malignant tumor.

[0026] In one embodiment, the present invention provides use of the modified bacteria in the preparation of a medicament for treating a malignant tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: Schematic diagram of the DB-ZW1 strain construction scheme.

[0028] Figure 2: Changes in the distribution of DB-ZW1 in tumors and tissues over time.

[0029] Figure 3: Tumor inhibitory effects of DB-ZW1 on bladder cancer, melanoma, and in situ colon cancer. Figures 3A-C show the effects of DB-ZW1 on tumor volume of subcutaneous bladder cancer, in situ melanoma, and in situ colon cancer, respectively; Figure 3D shows the effect of DB-ZW1 on tumor number of in situ colon cancer.

[0030] Figure 4: Map of the pET-26b plasmid.

[0031] Figure 5: Map of plasmid pET26b-pelB-Nb127D01-HA-his.

[0032] Figure 6: Western blot analysis of Nanobodies expressed in 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) at different IPTG concentrations. P represents the lysed bacterial pellet, S represents the lysate supernatant, and LB represents the LB culture supernatant.

[0033] FIG7 : IPTG-inducible expression ( FIG7A ) and anaerobic-inducible expression ( FIG7B ) of the fusion protein were detected by western blot.

[0034] Figure 8: Neutralization of intratumoral neutrophils increases DB-ZW1 levels in MB49 bladder cancer tumors. Left: Images of tumors in mice administered DB-ZW1 alone; Right: Images of tumors in mice administered DB-ZW1 + Ly6G / Ly6c (Gr-1) antibodies.

[0035] Figure 9: Neutralization of intratumoral neutrophils enhances the therapeutic efficacy of DB-ZW1 against 4T1 breast cancer. Figure 9A shows flow cytometry results of neutralization of intratumoral neutrophils in a 4T1 breast cancer model using the Ly6G / Ly6c (Gr-1) antibody (αGR-1). Figure 9B shows the changes in tumor size in 4T1 tumors injected with the artificial antitumor bacteria ZW1 one day after αGR-1 neutralization. Figure 9C shows representative tumor images from each group one day after bacterial injection.

[0036] Figure 10: The proportion of neutrophils successfully bound by nanoantibodies in the total number of neutrophils, where No Nb represents the sample without the addition of nanoantibodies, and Control strain represents the sample with the addition of LB supernatant of the Escherichia coli BL21 strain.

[0037] Figure 11: Therapeutic effect of DB-ZW1 expressing nanobody / fusion protein in mouse tumor model.

[0038] Figure 12: Neutrophil levels in the blood and tumors of mice after treatment with DB-ZW1 expressing nanobody / fusion protein.

[0039] Figure 13: Expression of Nanobodies, toxin proteins, or Nanobodies fused to toxin proteins under IPTG induction. M represents marker, LB represents secreted protein in the culture supernatant, S represents soluble protein expressed but not secreted in the lysis supernatant, and P represents functional inclusion body protein contained in the bacterial debris pellet.

[0040] Detailed Description of the Invention

[0041] definition

[0042] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0043] As used herein, the term "hypoxia regulated essential gene expression cassette" refers to a segment of DNA capable of initiating expression of an essential gene under hypoxic conditions, which contains an essential gene controlled by a hypoxia-inducible promoter and, if necessary, may further contain other regulatory elements required for expression of the essential gene.

[0044] As used herein, an "essential gene" refers to a gene that plays a decisive role in the growth and / or survival of bacteria. If the bacteria lack this gene or its functional expression product, they will not be able to survive, divide, and / or grow normally. Typical examples of bacteria lacking essential genes or their functional expression products are auxotrophic strains, which, in the absence of specific exogenous supplements, cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments. Essential genes are typically present in a single copy on the bacterial chromosome.

[0045] As used herein, a "strictly hypoxia inducible promoter" refers to a promoter that can initiate transcription of a specific gene under anaerobic conditions but can hardly be activated and produce transcripts under aerobic conditions.

[0046] A "strictly hypoxia-inducible promoter" useful in the present invention can be identified by the following experiment:

[0047] Construct an expression strain containing the essential gene (e.g., dapA / dapE) under the control of the promoter to be tested. Streak a Luria-Bertani (LB) plate in an anaerobic incubator at 37°C for 24 hours. Under anaerobic conditions, pick a single colony and resuspend it in 20 μl of LB liquid medium. Add 20 μl of the resuspended single colony to each of the following four tubes: ABCD. Add 5 μl to each tube:

[0048] A. 2 ml LB liquid medium,

[0049] B. 2 ml LB liquid medium,

[0050] C.2ml LB liquid medium + DAP,

[0051] D. 2 ml LB liquid medium + DAP.

[0052] Incubate tubes AC at 37°C in an anaerobic incubator for 24 hours, and incubate tubes BD in an aerobic shaker at 37°C for 24 hours. If bacteria can grow in tubes ACD but not in tube B (OD600 value less than 0.05), the promoter is considered to be strictly hypoxia-inducible.

[0053] Table 1 lists exemplary strictly hypoxia-inducible promoters and pH-inducible promoters identified in the above experiments that can be used in the present invention.

[0054] Table 1

[0055] As used herein, "gene involved in or regulating the endogenous anti-oxidative stress response pathway" refers to a gene that participates in the resistance of bacteria (e.g., intracellular bacteria, such as Salmonella) to the killing effects of reactive oxygen species (ROS) in vivo or in vitro environments.

[0056] As used herein, "genes required for survival in macrophages" refer to genes involved in maintaining or enhancing the ability of intracellular bacteria to survive in macrophages. The functions of these gene products are related to resisting the killing effect of macrophages on microorganisms. Therefore, the deletion of these genes or their functional expression products can reduce the ability of bacteria (e.g., intracellular bacteria, such as Salmonella) to survive in macrophages.

[0057] In this article, "functional expression products" include RNA and protein, etc.

[0058] As used herein, "facultative anaerobic bacteria" refers to bacteria that can survive both in the presence and absence of oxygen.

[0059] As used herein, "pH regulated expression cassette" refers to a group of gene expression elements whose expression of related genes is regulated by the environmental pH.

[0060] As used herein, "a promoter that is active under acid pH conditions" refers to a promoter that can be activated under acidic pH conditions and regulates the expression of a related gene. In some embodiments, the promoter that is active under acidic pH conditions of the present invention can be activated or still has promoter activity when the pH value is lower 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. In some embodiments, the promoter that is active under acidic pH conditions also has promoter activity at neutral pH values ​​(e.g., pH 7.0-7.4).

[0061] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0062] As used herein, "polynucleotide" refers to a macromolecule composed of multiple nucleotides linked by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (e.g., E. coli) to improve polypeptide expression. Methods for codon optimization are known in the art.

[0063] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm using default parameters.

[0064] As used herein, the term "live bacteria" refers to a strain that is viable, has active nutritional metabolic characteristics, and is capable of performing its own biological functions. Live bacteria may include bacterial biomass produced during the metabolic process of the strain.

[0065] In this article, term " gram-negative bacteria " refers to after carrying out the known partial program of gram staining, does not retain the bacterium of the initial basic dye staining agent (such as, crystal violet) as.In exemplary gram staining, first by heating, cell is fixed on slide and dyed with basic dye (such as, crystal violet), and described basic dye is absorbed by gram-negative bacteria and gram-positive bacteria.Then, slide is processed with mordant (such as, Gram's iodine solution), and described mordant is incorporated into basic dye (such as, crystal violet) and is trapped in cell.Then, by cell acetone or ethanol washing, then with the second dye of different colors counterstaining (such as, safranin).Gram-positive organism retains initial purple dyeing, and gram-negative organism decolours by organic washing solvent, therefore shows counterstaining. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia species, Shigella species, Salmonella species, Campylobacter species, Neisseria species, Haemophilus species, Aeromonas species, Francisella species, Yersinia species, Klebsiella species, Bordetella species, Legionella species, Corynebacterium species, Citrobacter species, Chlamydia species, Brucella species, Pseudomonas species, Helicobacter species, and Vibrio species.

[0066] Term used herein " malignant solid tumor " refers to the abnormal mass of tissue, does not usually include cyst or liquid area. Solid tumor may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named with the cell type that forms them. The example of malignant solid tumor is sarcoma, carcinoma and lymphoma. Leukemia (blood cancer) does not form malignant solid tumor (according to the definition of NIH National Cancer Institute) usually. Malignant solid tumor includes but is not limited to the abnormal cell mass that may be derived from different tissue types, described different tissue types are such as liver, colon, colorectal, skin, breast, pancreas, cervix uteri, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung etc., therefore malignant solid tumor includes malignant solid liver tumor, colon tumor, colorectal tumor, skin tumor, breast tumor, pancreatic tumor, cervix tumor, uterine body tumor, bladder tumor, gallbladder tumor, kidney tumor, laryngeal tumor, lip tumor, oral cavity tumor, esophageal tumor, ovarian tumor, prostate tumor, stomach tumor, testicular tumor, thyroid tumor or lung tumor etc.

[0067] As used herein, for therapeutic purposes, the term "subject" is preferably a subject in need of treatment for a target pathological condition, such as a tumor. For preventive purposes, the subject is preferably a subject at risk of developing or prone to developing a target pathological condition. The term "subject" includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0068] As used herein, "treatment" is a process used to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying neoplastic or cancerous cells), inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating a malignant tumor, alleviating symptoms caused by a malignant tumor, improving the quality of life of a subject suffering from a malignant tumor, reducing the dosage of other drugs required to treat a malignant tumor, delaying the progression of a malignant tumor, curing a malignant tumor, and / or prolonging the survival of a patient suffering from a malignant tumor.

[0069] As used herein, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes that appear during the course of disease. For therapeutic uses, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in the patient. For example, an "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%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Such an amount can be determined by one skilled in the art 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.

[0070] As used herein, "pharmaceutically acceptable carrier" includes any material that allows the ingredient to retain biological activity when combined with the active ingredient and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For example, depending on the route of administration, the bacteria of the present invention can be wrapped in a material to protect the bacteria from the effects of acids and other natural conditions that can inactivate the bacteria. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the temporary preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any incompatibility with the active compound, may be in the pharmaceutical composition of the present invention.

[0071] As used herein, the term "cytotoxin" primarily relates to protein toxins that are toxic to cells, such as bacterially expressed exotoxins and endotoxins.

[0072] Fusion protein

[0073] The present invention provides a fusion protein comprising an antibody (eg, nanobody) or an antigen-binding fragment thereof that binds to neutrophils and a cytotoxin or an active fragment thereof.

[0074] Examples of such antibodies (Nanobodies) that bind to neutrophils include, but are not limited to, antibodies that bind to CXCR2 (such as Nanobodies of SEQ ID NOs: 19, 20 and 21, or antibodies comprising their CDRs), antibodies that bind to G-CSF-R (such as Nanobodies of SEQ ID NOs: 61 and 62, or antibodies comprising their CDRs), antibodies that bind to EGFR (such as Nanobodies of SEQ ID NO: 63, or antibodies comprising their CDRs), antibodies that bind to CXCR4 (such as Nanobodies of SEQ ID NOs: 64 and 65, or antibodies comprising their CDRs), antibodies that bind to IL6R (such as Nanobodies of SEQ ID NOs: 66-70, or antibodies comprising their CDRs), antibodies that bind to human TNFR1 (such as Nanobodies of SEQ ID NOs: 71-78, or antibodies comprising their CDRs).

[0075] Examples of such 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 enterotoxin (e.g., comprising subunit A of SEQ ID NO: 82 and subunit B of SEQ ID NO: 83), diphtheria toxin (DT, e.g., SEQ ID NO: 84), Shiga toxin (e.g., comprising the A chain of SEQ ID NO: 86 and the B chain of 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), ricin toxin (e.g., ricin A chain of SEQ ID NO: 91), and Pseudomonas exotoxin (PE, e.g., Pseudomonas exotoxin A of SEQ ID NO: 25). Active fragments of these toxins are also encompassed by the present invention and are known in the art, such as fragment CET40 of Cholix toxin (domains II and III of Cholix toxin, SEQ ID NO: 80), fragment DT390 of diphtheria toxin (SEQ ID NO: 85), and fragments PE38 and PE[LR] of Pseudomonas exotoxin (SEQ ID NOs: 26 and 27).

[0076] In some embodiments, the antibody is an antibody that binds to CXCR2. In some embodiments, the antibody comprises a complementary determining region (CDR) selected from the group consisting of: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO: 20, a CDR2 comprising residues 50-67 of SEQ ID NO: 20, and a CDR3 comprising residues 100-111 of SEQ ID NO: 20; a CDR1 comprising residues 31-35 of SEQ ID NO: 21, a CDR2 comprising residues 50-64 of SEQ ID NO: 21, and a CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

[0077] 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 a Pseudomonas exotoxin (PE). In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0078] 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.

[0079] In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker is a flexible linker, such as a GS linker, such as (G n S) m , wherein m and n are independently integers of 1 to 4. In some embodiments, the fusion protein further comprises a tag. In some embodiments, the tag is a His tag (such as 6×His) or an HA tag.

[0080] 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 fused directly 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.

[0081] In some embodiments, the fusion protein comprises SEQ ID NO: 19 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 19 and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 20 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 20 and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 21 and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 21 and SEQ ID NO: 27 from the N-terminus to the C-terminus.

[0082] In some embodiments, the fusion protein comprises 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 comprises 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 comprises 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 comprises SEQ ID NO: 28, SEQ ID NO: 20, and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 28, SEQ ID NO: 21, and SEQ ID NO: 26 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 28, SEQ ID NO: 21, and SEQ ID NO: 27 from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises 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 comprises SEQ ID NO: 29, SEQ ID NO: 19, and SEQ ID NO: 27 from N-terminus to C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 29, SEQ ID NO: 20, and SEQ ID NO: 26 from N-terminus to C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 29, SEQ ID NO: 20, and SEQ ID NO: 27 from N-terminus to C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 29, SEQ ID NO: 21, and SEQ ID NO: 26 from N-terminus to C-terminus. In some embodiments, the fusion protein comprises SEQ ID NO: 29, SEQ ID NO: 21, and SEQ ID NO: 27 from N-terminus to C-terminus.

[0083] Polynucleotides and expression constructs

[0084] The present invention provides a polynucleotide encoding the fusion protein of the present invention.

[0085] The present invention also provides an expression construct comprising a nucleotide sequence encoding the fusion protein of the present invention operably linked to a strictly hypoxia-inducible promoter.

[0086] In some embodiments, the strict hypoxia-inducible promoter is selected from pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA and Ptet-Fnr. In some embodiments, the strict hypoxia-inducible promoter comprises a nucleotide sequence selected from SEQ ID NO: 9-18.

[0087] The present invention also provides an expression construct comprising a nucleotide sequence encoding an antibody (eg, a Nanobody) or an antigen-binding fragment thereof that binds to neutrophils, operably linked to a strictly hypoxia-inducible promoter.

[0088] Examples of such antibodies (Nanobodies) that bind to neutrophils include, but are not limited to, antibodies that bind to CXCR2 (such as Nanobodies of SEQ ID NOs: 19, 20 and 21, or antibodies comprising their CDRs), antibodies that bind to G-CSF-R (such as Nanobodies of SEQ ID NOs: 61 and 62, or antibodies comprising their CDRs), antibodies that bind to EGFR (such as Nanobodies of SEQ ID NO: 63, or antibodies comprising their CDRs), antibodies that bind to CXCR4 (such as Nanobodies of SEQ ID NOs: 64 and 65, or antibodies comprising their CDRs), antibodies that bind to IL6R (such as Nanobodies of SEQ ID NOs: 66-70, or antibodies comprising their CDRs), antibodies that bind to human TNFR1 (such as Nanobodies of SEQ ID NOs: 71-78, or antibodies comprising their CDRs).

[0089] In some embodiments, the antibody is an antibody that binds to CXCR2. In some embodiments, the antibody comprises a complementary determining region (CDR) selected from the group consisting of: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO: 20, a CDR2 comprising residues 50-67 of SEQ ID NO: 20, and a CDR3 comprising residues 100-111 of SEQ ID NO: 20; a CDR1 comprising residues 31-35 of SEQ ID NO: 21, a CDR2 comprising residues 50-64 of SEQ ID NO: 21, and a CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21.

[0090] 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.

[0091] In some embodiments, the nucleotide sequence further encodes a tag sequence. In some embodiments, the tag is a His tag (such as 6×His) or an HA tag.

[0092] The present invention also provides an expression construct comprising a nucleotide sequence encoding a cytotoxin or an active fragment thereof operably linked to a strictly hypoxia-inducible promoter.

[0093] 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 capable of killing tumor cells.

[0094] Examples of such 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 enterotoxin (e.g., comprising subunit A of SEQ ID NO: 82 and subunit B of SEQ ID NO: 83), diphtheria toxin (DT, e.g., SEQ ID NO: 84), Shiga toxin (e.g., comprising the A chain of SEQ ID NO: 86 and the B chain of 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), ricin toxin (e.g., ricin A chain of SEQ ID NO: 91), or Pseudomonas exotoxin (PE, e.g., Pseudomonas exotoxin A of SEQ ID NO: 25). Active fragments of these toxins are also encompassed by the present invention and are known in the art, such as fragment CET40 of Cholix toxin (domains II and III of Cholix toxin, SEQ ID NO: 80), fragment DT390 of diphtheria toxin (SEQ ID NO: 85), and fragments PE38 and PE[LR] of Pseudomonas exotoxin (SEQ ID NOs: 26 and 27).

[0095] 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 fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0096] 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.

[0097] In some embodiments, the nucleotide sequence further encodes a tag sequence. In some embodiments, the tag is a His tag (such as 6×His) or an HA tag.

[0098] Modified bacteria

[0099] The present invention provides a modified bacterium, wherein the bacterium comprises an expression construct of the present invention and a hypoxia-regulatable essential gene expression cassette compared to an unmodified starting strain. In one embodiment, the essential gene expression cassette comprises an essential gene of the bacterium controlled by a strict hypoxia-inducible promoter. In one embodiment, the bacterium lacks at least one gene or its functional expression product that participates in or regulates an endogenous antioxidant stress response pathway. In one embodiment, the bacterium lacks at least one gene or its functional expression product that is required for survival in macrophages. In one embodiment, the bacterium expresses wild-type lipopolysaccharide (LPS).

[0100] In one embodiment, the strict hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA and Ptet-Fnr. Preferably, the strict hypoxia-inducible promoter is ssbp1 promoter.

[0101] In one embodiment, the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacteria. In one embodiment, when cultured under aerobic conditions, the growth of the bacteria is dependent on the addition of DAP or its analogues 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, the dapE comprises a nucleotide sequence having ≥81%, preferably ≥82%, more preferably ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or most preferably ≥99% sequence identity to SEQ ID NO: 57. Preferably, the nucleotide sequence of dapE comprises SEQ ID NO: 57. Preferably, the nucleotide sequence of dapE consists of SEQ ID NO:57.

[0102] In one embodiment, the gene involved in or regulating the endogenous anti-oxidative stress response pathway is a gene of the HtrA serine protease family. In one embodiment, the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is a protein associated with the HtrA serine protease family. In one embodiment, the bacterium lacks HtrA serine protease activity. In one embodiment, the gene involved in or regulating the endogenous anti-oxidative stress response pathway is htrA. In one embodiment, the bacterium lacks htrA. Preferably, the htrA comprises a nucleotide sequence having ≥81%, preferably ≥82%, more preferably ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or most preferably ≥99% sequence identity to SEQ ID NO:58. Preferably, the nucleotide sequence of htrA comprises SEQ ID NO:58. Preferably, the nucleotide sequence of htrA consists of SEQ ID NO: 58.

[0103] In one embodiment, the essential gene is a gene naturally present in the bacterial chromosome. In one embodiment, the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter. Thus, the expression of the essential gene in the bacteria is completely controlled by the strict hypoxia-inducible promoter.

[0104] In one embodiment, the essential gene expression cassette is exogenous. In one embodiment, the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated. Thus, the expression of the essential gene in the bacteria is completely controlled by the strict hypoxia inducible promoter. In one embodiment, the exogenous essential gene expression cassette is integrated into the chromosome of the bacteria. In one embodiment, the exogenous essential gene expression cassette is located outside the chromosome of the bacteria. In one embodiment, the exogenous essential gene expression cassette is present in a plasmid carried by the bacteria.

[0105] In one embodiment, the modified bacteria further comprises a pH regulating expression cassette. In one embodiment, the modified bacteria comprises a hypoxia regulating essential gene expression cassette and a pH regulating expression cassette compared to the unmodified starting strain. In one embodiment, the pH regulating expression cassette comprises a gene for a hemolysin protein encoding bacteria of a promoter that is controlled by 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 at a pH value in the range of about pH 4.0 to 7.0. In some embodiments, the promoter that is active under acidic pH conditions is active at a pH of pH 6.0 ± 1. In one embodiment, the promoter that is active under acidic pH conditions is active at a pH value lower 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 pH value ranges. In one embodiment, the promoter active under acidic pH conditions is selected from sseA, ssrA, ssaB, ssaG, ssaM and ssaR. In one embodiment, the promoter active under acidic pH conditions is sseA. In one embodiment, the bacterial-derived 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, the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae or Escherichia coli. Preferably, the hlyA comprises a nucleotide sequence having ≥81%, preferably ≥82%, more preferably ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or most preferably ≥99% sequence identity to SEQ ID NO: 59. Preferably, the nucleotide sequence of the hlyA comprises SEQ ID NO: 59. Preferably, the nucleotide sequence of the hlyA consists of SEQ ID NO: 59.

[0106] In one embodiment, the unmodified starting bacterial strain is a facultative anaerobic bacterium. In one embodiment, the bacterium is Enterobacteriaceae (Enterobacteriaceae) bacterium. Preferably, the bacterium is Escherichia (Escherichia), Salmonella (Salmonella), Shigella (Shigella), Klebsiella (Klebsiella), Yersinia (Yersinia), Citrobacter (Citrobacter), Enterobacter (Enterobacter), Serratia (Serratia), Proteus (Proteus), Morganella (Morganella), Providencia (Providencia), Hafnia (Hafnia), Pantoea (Pantoea) bacterium. Preferably, the bacteria are selected from Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis (Y.pseudotuberculosis), Yersinia aldouae, Yersinia bercov-ieri, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensii, Yersinia mollaretti, Yersinia rohdei, Yersinia ruckeri, Citrobacter freundii, Citrobacter kaseri, Citrobacter braakii, E.aerogenes, E.cloacae, E.gergoviac, E.sakazakii, E.tavlorac, E.aminigenus), E. intermedius, E. asburiac, E. cancerogenus, E. dissolvens, E. nimipressualis, S. marcescens, S. entomophila, S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S. odorifera, S. plymuthica, S. proteamaculans, Serratia rubidaea, Serratia ureilytica, P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri, P. hauseri, M. morganii, P. alcalifaciens, P. rustigianii, P. stuartii, P. rettgeri, P. heimbochae, H. alvei, and P. agglomerans. Preferably, the bacterium is Salmonella typhimurium. Preferably, the starting strain is Salmonella typhimurium SL7207.

[0107] In one embodiment, when administered to a subject with a tumor, the bacteria can survive and proliferate in the tumor tissue but are rapidly cleared from normal tissue. In one embodiment, when administered to a subject with a malignant tumor, the bacteria can induce an anti-tumor specific immune response. In one embodiment, when administered to a subject with a malignant tumor, the bacteria can induce anti-tumor immune memory.

[0108] In one embodiment, the bacteria does not express wild-type flagellin. In one embodiment, the modified bacteria lacks the fliC gene.

[0109] In one embodiment, the bacteria survive in macrophages at a level of about 50% to about 30% of the unmodified starting strain's survival level. In one embodiment, the bacteria survive in macrophages at a level of about 30% to about 10% of the unmodified starting strain's survival level. Preferably, the bacteria survive in macrophages at a level of about 10% to about 1% of the unmodified starting strain's survival level.

[0110] Pharmaceutical composition

[0111] The present invention provides a pharmaceutical composition comprising an effective amount of the modified bacteria of the present invention. In one embodiment, the modified bacteria are live bacteria.

[0112] In one embodiment, the pharmaceutical composition is used to treat a malignant tumor. In one embodiment, the pharmaceutical composition is used to induce an anti-tumor specific immune response in a subject suffering from a malignant tumor. In one embodiment, the pharmaceutical composition is used to induce anti-tumor immune memory in a subject suffering from 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 has become resistant to or failed previous anti-tumor therapy.

[0113] 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 mucosa. In one embodiment, 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 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, liver cancer, pancreatic cancer, bile duct cancer, colon cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal tumor, thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer and osteosarcoma.

[0114] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0115] Treatment methods and uses

[0116] The present invention provides a method for treating malignant tumors, comprising administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to a subject suffering from a malignant tumor.

[0117] The present invention provides a method for inducing an anti-tumor specific immune response in a subject having a malignant tumor, comprising 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.

[0118] The present invention provides a method for inducing anti-tumor immune memory in a subject having a malignant tumor, comprising administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to the subject having the malignant tumor.

[0119] The present invention provides a method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to a subject suffering from a malignant tumor.

[0120] The present invention provides a method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to a subject suffering from metastasis or recurrence of a malignant tumor or a subject at high risk of metastasis or recurrence of a malignant tumor.

[0121] The present invention provides a method for treating a malignant tumor that has become resistant to or failed to be treated with previous anti-tumor therapy, comprising administering an effective amount of the modified bacteria of the present invention or the pharmaceutical composition of the present invention to a subject suffering from a malignant tumor that has become resistant to or failed to be treated with previous anti-tumor therapy.

[0122] The present invention provides use of the modified bacteria of the present invention in preparing a drug for treating malignant tumors.

[0123] The present invention provides use of the modified bacteria of the present invention in the preparation of a medicament for inducing an anti-tumor specific immune response in a subject suffering from a malignant tumor.

[0124] The present invention provides use of the modified bacteria of the present invention in the preparation of a medicament for inducing anti-tumor immune memory in a subject suffering from a malignant tumor.

[0125] The present invention provides use of the modified bacteria of the present invention in preparing a medicament for preventing or treating metastasis or recurrence of malignant tumors.

[0126] The present invention provides use of the modified bacteria of the present invention in the preparation of a medicament for treating malignant tumors that have become resistant to or failed to be treated with previous anti-tumor therapies.

[0127] In one embodiment, the modified bacteria are live bacteria.

[0128] 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 mucosa. In one embodiment, 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 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, liver cancer, pancreatic cancer, bile duct cancer, colon cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal tumor, thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer and osteosarcoma.

[0129] In one embodiment, the bacteria, drugs or pharmaceutical compositions of the present invention are administered by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration. In one embodiment, the bacteria, drugs or pharmaceutical compositions of the present invention can be formulated into a form for administration by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0130] In one embodiment, the dosage form of the drug or pharmaceutical composition of the present invention can be, for example, in the form of a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form can be in the form of a tablet or capsule; for intranasal dosage form, the dosage form can be in the form of a powder, nasal drops or aerosol; for topical administration, the dosage form can be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form can be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure. In one embodiment, the drug or pharmaceutical composition is formulated as a solution, emulsion, freeze-dried preparation or suspension; for oral administration, it can be formulated as a tablet or capsule; for intranasal dosage form, it can be formulated as a powder, nasal drops or aerosol; for topical administration, it can be formulated as an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, it can be formulated as a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure. Methods well known in the art for preparing dosage forms are described, for example, in "Remington's Pharmaceutical Sciences" (20th ed.), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA.

[0131] In some embodiments, the bacteria, drugs, or pharmaceutical compositions of the invention can be administered, for example, at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, e.g., 1 minute to 10 minutes, 1 minute to 30 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 In some embodiments, the present invention provides the following types of time intervals: 1 hour - 30 minutes, 10 minutes - 1 hour, 10 minutes - 2 hours, 10 minutes - 4 hours, 10 minutes - 12 hours, 10 minutes - 18 hours, 10 minutes - 1 day, 30 minutes - 1 hour, 30 minutes - 2 hours, 30 minutes - 4 hours, 30 minutes - 12 hours, 30 minutes - 18 hours, 30 minutes - 1 day, 30 minutes - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days or 5 days - 10 days. In some embodiments, the bacteria, drug, or pharmaceutical composition is 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.In some embodiments, the bacteria, drug, or pharmaceutical composition is formulated to be administered, for example, at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, e.g., 1 minute to 10 minutes, 1 minute to 30 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, 1 minute to 2 0 min - 30 min, 10 min - 1 hour, 10 min - 2 hours, 10 min - 4 hours, 10 min - 12 hours, 10 min - 18 hours, 10 min - 1 day, 30 min - 1 hour, 30 min - 2 hours, 30 min - 4 hours, 30 min - 12 hours, 30 min - 18 hours, 30 min - 1 day, 30 min - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days, or 5 days - 10 days. In some embodiments, the bacteria, drug or pharmaceutical composition is 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 dosage regimen may depend on the pattern of pharmacokinetic decay that the practitioner expects to achieve. The progress of the therapy can be monitored by conventional techniques and assays. The dosage regimen can vary over time.

[0132] In some embodiments, the effective amount of bacteria in the medicament or pharmaceutical composition comprises at least about 10 4 colony forming units (cfu), for example at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 cfu, including any ranges between listed values, such as 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[[ID=5​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0133] In some embodiments, the volume of an effective dose of the bacterium, medicament, or pharmaceutical composition of the invention is less than or equal to 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. In some embodiments, the volume of an effective dose of the bacterium, medicament, 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. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml, about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. In some embodiments, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 200 ml, about 190 ml, about 180 ml, about 170 ml, about 160 ml, about 150 ml, about 140 ml, about 130 ml, about 120 ml, about 110 ml, about 100 ml, about 90 ml, about 80 ml, about 70 ml, about 60 ml, about 50 ml, about 40 ml or about 30 ml, as well as ranges between any of the listed values, for example, 100 ml-110 ml, 100 ml-120 ml, 90 ml-120 ml, 90 ml-130 ml, etc. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 205 ml, about 195 ml, about 185 ml, about 175 ml, about 165 ml, about 155 ml, about 145 ml, about 135 ml, about 125 ml, about 115 ml, about 105 ml, about 95 ml, about 85 ml, about 75 ml, about 65 ml, about 55 ml, about 45 ml, about 35 ml or about 25 ml, and ranges between any of the listed values, such as 105 ml-115 ml, 105 ml-125 ml, 95 ml-125 ml, 95 ml-135 ml, etc.Alternatively, the volume of the effective dose of the bacterium, drug 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, alternatively 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 the effective dose of the bacterium, drug or pharmaceutical composition is less than or equal to about 2.0 ml.

[0134] The concentration of the bacteria of the present invention in, for example, a medicament or pharmaceutical composition may be 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, including any ranges between the listed values, e.g. 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, 10 4 -10 12 cfu / ml, 10 4 -10 13 cfu / ml, 10 5 -10 8 cfu / ml, 10 5 -10 9 cfu / ml, 105 -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 cfu / ml. The concentration of the bacteria of the present invention in, for example, a medicament or pharmaceutical composition may be 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 13cfu / g, including ranges between any of the listed values, such as 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 [[ID=3||]]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 11 cfu / g, 10 6 [[ID=9||]]-10 12 cfu / g, 10 6 -10 13 cfu / g, :0 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 cfu / g.

[0135] The dosage ranges mentioned herein are merely exemplary and do not limit the dosage ranges that can be selected by a physician. The amount of the active ingredient (e.g., the bacteria of the present invention) in the pharmaceutical compositions of the present invention can vary according to factors such as the individual's disease state, age, sex, and weight, including the presence or absence of a malignancy, the type of malignancy being treated, the severity of the malignancy, the activity or viability of the bacteria, drug, or pharmaceutical composition, the route of administration, the duration of treatment, the drugs used in combination with the bacteria, drug, or pharmaceutical composition (if any), the diet and general health of the subject, and similar factors well known in the art. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, a single dose can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0136] Bacteria, medicine or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as growing the microorganisms in a fermentation tank, followed by centrifugal concentration and washing, filtering or dialysis, conventional granulation, mixing, dissolving, encapsulating, lyophilizing or emulsifying processes and other methods. Bacteria, medicine or pharmaceutical compositions of the present invention can be produced in various forms, including particles, precipitations or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Preparations can optionally contain stabilizers, pH adjusting agents, surfactants, bioavailability regulators and combinations thereof.

[0137] In one embodiment, the bacterium of the present invention, medicine or pharmaceutical composition can be used alone, or in the presence of a carrier with other compounds or composition combination administration. In one embodiment, the bacterium, medicine or pharmaceutical composition can be used in combination with other malignant tumor therapies (including but not limited to, radiotherapy, chemotherapy and surgical operation). The bacterium, medicine or pharmaceutical composition can be used as the adjuvant in therapy in such a case. Therefore, another aspect of the embodiment relates to a kind of bacterium, medicine or pharmaceutical composition, which is used for being used as an adjuvant in a malignant tumor therapy selected from radiotherapy and chemotherapy.

[0138] Example Implementation

[0139] Embodiment 1: A fusion protein comprising an antibody (eg, nanobody) or an antigen-binding fragment thereof that binds to neutrophils and a cytotoxin or an active fragment thereof.

[0140] Embodiment 2: The fusion protein of embodiment 1, wherein the antibody comprises a complementarity determining region (CDR) selected from the group consisting of:

[0141] a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19;

[0142] a CDR1 comprising residues 31-35 of SEQ ID NO:20, a CDR2 comprising residues 50-67 of SEQ ID NO:20, and a CDR3 comprising residues 100-111 of SEQ ID NO:20; and

[0143] A CDR1 comprising residues 31-35 of SEQ ID NO:21, a CDR2 comprising residues 50-64 of SEQ ID NO:21, and a CDR3 comprising residues 98-115 of SEQ ID NO:21.

[0144] Embodiment 3: The fusion protein of embodiment 1 or 2, wherein the antibody comprises an amino acid sequence selected from SEQ ID NO: 19, 20 and 21.

[0145] Embodiment 4: The fusion protein of any one of Embodiments 1-3, wherein the cytotoxin is a protein toxin capable of killing neutrophils.

[0146] Embodiment 5: The fusion protein of any one of embodiments 1-4, wherein the cytotoxin is Pseudomonas exotoxin (PE).

[0147] Embodiment 6: The fusion protein of any one of embodiments 1-5, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25.

[0148] Embodiment 7: The fusion protein of any one of Embodiments 1-6, wherein the active fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0149] Embodiment 8: The fusion protein of any one of Embodiments 1-7, wherein the fusion protein further comprises a signal peptide, for example, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0150] Embodiment 9: The fusion protein of any one of embodiments 1-8, wherein the fusion protein further comprises a linker, such as a flexible linker, for example a GS linker, such as (G n S) m , wherein m and n are independently integers of 1-4.

[0151] Embodiment 10: The fusion protein of any one of embodiments 1-9, wherein the fusion protein further comprises a tag, such as a His tag (such as 6×His) or an HA tag.

[0152] Embodiment 11: The fusion protein of any one of Embodiments 1-10, wherein the cytotoxin or active fragment thereof is fused to the C-terminus of the antibody or antigen-binding fragment thereof.

[0153] Embodiment 12: The fusion protein of any one of Embodiments 1-11, wherein the cytotoxin or active fragment thereof is fused to the antibody or antigen-binding fragment thereof directly or through a linker.

[0154] Embodiment 13: The fusion protein of any one of embodiments 1-12, wherein the fusion protein comprises from N-terminus to C-terminus:

[0155] SEQ ID NO: 19 and SEQ ID NO: 26,

[0156] SEQ ID NO: 19 and SEQ ID NO: 27,

[0157] SEQ ID NO: 20 and SEQ ID NO: 26,

[0158] SEQ ID NO: 20 and SEQ ID NO: 27,

[0159] SEQ ID NO: 21 and SEQ ID NO: 26, or

[0160] SEQ ID NO:21 and SEQ ID NO:27.

[0161] Embodiment 14: The fusion protein of any one of embodiments 1-13, wherein the fusion protein comprises from N-terminus to C-terminus:

[0162] SEQ ID NO:28, SEQ ID NO:19 and SEQ ID NO:26,

[0163] SEQ ID NO:28, SEQ ID NO:19 and SEQ ID NO:27,

[0164] SEQ ID NO:28, SEQ ID NO:20 and SEQ ID NO:26,

[0165] SEQ ID NO:28, SEQ ID NO:20 and SEQ ID NO:27,

[0166] SEQ ID NO:28, SEQ ID NO:21 and SEQ ID NO:26,

[0167] SEQ ID NO:28, SEQ ID NO:21 and SEQ ID NO:27,

[0168] SEQ ID NO:29, SEQ ID NO:19 and SEQ ID NO:26,

[0169] SEQ ID NO:29, SEQ ID NO:19 and SEQ ID NO:27,

[0170] SEQ ID NO:29, SEQ ID NO:20 and SEQ ID NO:26,

[0171] SEQ ID NO:29, SEQ ID NO:20 and SEQ ID NO:27,

[0172] SEQ ID NO: 29, SEQ ID NO: 21 and SEQ ID NO: 26, or

[0173] SEQ ID NO:29, SEQ ID NO:21 and SEQ ID NO:27.

[0174] Embodiment 15: An isolated polynucleotide encoding the nucleotide sequence of the fusion protein of any one of embodiments 1-14.

[0175] Embodiment 16: An expression construct comprising the polynucleotide of embodiment 15 operably linked to a strictly hypoxia-inducible promoter.

[0176] Embodiment 17: The expression construct of embodiment 16, 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.

[0177] Embodiment 18: The expression construct of embodiment 16 or 17, wherein the oxygen-inducible promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-18.

[0178] Embodiment 19: An expression construct comprising a nucleotide sequence operably linked to a strictly hypoxia-inducible promoter, the nucleotide sequence encoding an antibody (eg, a nanobody) or an antigen-binding fragment thereof that binds to neutrophils, or a toxin or an active fragment thereof.

[0179] Embodiment 20: The expression construct of embodiment 19, wherein the antibody comprises a complementarity determining region (CDR) selected from the group consisting of:

[0180] a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19;

[0181] a CDR1 comprising residues 31-35 of SEQ ID NO:20, a CDR2 comprising residues 50-67 of SEQ ID NO:20, and a CDR3 comprising residues 100-111 of SEQ ID NO:20; and

[0182] a CDR1 comprising residues 31-35 of SEQ ID NO:21, a CDR2 comprising residues 50-64 of SEQ ID NO:21, and a CDR3 comprising residues 98-115 of SEQ ID NO:21; or

[0183] The toxin comprises the amino acid sequence of SEQ ID NO: 25; or

[0184] The active fragment of the toxin comprises the active fragment of SEQ ID NO: 26 or 27.

[0185] Embodiment 21: The expression construct of embodiment 19 or 20, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 20 and 21.

[0186] Embodiment 22: The expression construct of any one of embodiments 19-21, wherein the nucleotide sequence further encodes a signal peptide, for example, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0187] Embodiment 23: The expression construct of any one of embodiments 19-22, wherein the nucleotide sequence further encodes a tag, such as a His tag (such as 6×His) or an HA tag.

[0188] Embodiment 24: An expression construct comprising a nucleotide sequence encoding a cytotoxin or an active fragment thereof operably linked to a strictly hypoxia-inducible promoter.

[0189] Embodiment 25: The expression construct of embodiment 24, wherein the cytotoxin is a protein toxin capable of killing neutrophils.

[0190] Embodiment 26: The expression construct of embodiment 24 or 25, wherein the cytotoxin is Pseudomonas exotoxin (PE).

[0191] Embodiment 27: The expression construct of any one of Embodiments 24-26, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO:25.

[0192] Embodiment 28: The expression construct of any one of Embodiments 24-27, wherein the active fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0193] Embodiment 29: The expression construct of any one of Embodiments 24-28, wherein the nucleotide sequence further encodes a signal peptide, for example, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

[0194] Embodiment 30: The expression construct of any one of embodiments 24-29, wherein the nucleotide sequence further encodes a tag, such as a His tag (such as 6×His) or an HA tag.

[0195] Implementation Plan A

[0196] Embodiment A1: A modified bacterium, wherein compared to an unmodified starting strain, the bacterium comprises an expression construct of any one of embodiments 16-30 and a hypoxia-regulatable essential gene expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the bacterium lacks at least one gene involved in or regulating an endogenous antioxidant stress response pathway or its functional expression product.

[0197] Embodiment A2: The modified bacterium of 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.

[0198] Embodiment A3: The modified bacterium of Embodiment A1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0199] Embodiment A4: The modified bacterium of any one of the preceding embodiments A, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues to the culture medium.

[0200] Embodiment A5: The modified bacterium of Embodiment A4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0201] Embodiment A6. The modified bacterium of Embodiment A4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0202] Embodiment A7: The modified bacterium of any one of the preceding Embodiments A, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family gene.

[0203] Embodiment A8: The modified bacterium of any one of the preceding embodiments A, wherein the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family-related protein.

[0204] Embodiment A9: The modified bacterium of any preceding Embodiment A, wherein the bacterium lacks HtrA serine protease activity.

[0205] Embodiment A10: The modified bacterium of any preceding embodiment A, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0206] Embodiment A11. The modified bacterium of any preceding Embodiment A, wherein the bacterium is deficient in htrA.

[0207] Embodiment A12: The modified bacterium of any one of Embodiments A1-A11, wherein the essential gene is a gene naturally present in the bacterial chromosome, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0208] Embodiment A13: The modified bacterium of any one of Embodiments A1-A11, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0209] Embodiment A14: The modified bacterium of Embodiment A13, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0210] Embodiment A15: The modified bacterium of Embodiment A13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0211] Embodiment A16: The modified bacterium of Embodiment A15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0212] Embodiment A17: The modified bacterium of any one of the preceding Embodiments A, further comprising a pH-regulatable expression cassette comprising a gene encoding a bacterially derived hemolysin protein controlled by a promoter active under acidic pH conditions.

[0213] Embodiment A18: The modified bacterium of Embodiment A17, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0214] Embodiment A19: The modified bacterium of Embodiment A18, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0215] Embodiment A20. The modified bacterium of Embodiment A19, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0216] Embodiment A21: The modified bacterium of any one of Embodiments A17-A20, wherein the promoter active under acidic pH conditions is active at a pH value 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.

[0217] Embodiment A22: The modified bacterium of any one of Embodiments A17-A21, wherein the promoter active under acidic pH conditions is selected from sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0218] Embodiment A23: The modified bacterium of any one of Embodiments A17-A21, wherein the promoter active under acidic pH conditions is sseA.

[0219] Embodiment A24: The modified bacterium of any preceding Embodiment A, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0220] Embodiment A25. The modified bacterium of any preceding Embodiment A, wherein the bacterium is an Enterobacteriaceae bacterium.

[0221] Embodiment A26: The modified bacterium of any of the preceding embodiments A, wherein the bacterium is an Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea bacterium.

[0222] Embodiment A27: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is selected from Escherichia coli, E. blattae, E. fergusonii, E. hermannii, E. vulneris, S. enterica, S. bongori, S. typhi, S. choleraesuis, S. typhimurium, S. dysenteriae, S. flexneri, S. botulinum, S. Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia aldouae, Yersinia bercovi, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensii ), Yersinia mollaretti, Yersinia rohdei, Yersinia ruckeri, Citrobacter freundii, Citrobacter kaseri, Citrobacter braakii, E. aerogenes, E. cloacae, E. gergoviac, E. sakazakii, E. tavlorac, E. aminigenus, E. intermedius , E. asburiac, E. cancerogenus, E. dissolvens, E. nimipressualis, S. marcescens, S. entomophila, S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S. odorifera, S. plymuthica, S. longyan spot (S.proteamaculans), Serratia rubidaea, Serratia ureilytica, P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri, P. hauseri, M. morganii, P. alcalifaciens, P. rustigianii, P. stuartii, P. rettgeri, P. heimbochae, H. alvei, and P. agglomerans.

[0223] Embodiment A28. The modified bacterium of any preceding Embodiment A, wherein the bacterium is Salmonella typhimurium.

[0224] Embodiment A29. The modified bacterium of Embodiment A28, wherein the starting strain is Salmonella typhimurium SL7207.

[0225] Embodiment A30. The modified bacterium of any preceding Embodiment A, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0226] Embodiment A31: The modified bacterium of any preceding Embodiment A, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0227] Embodiment A32. The modified bacterium of any preceding Embodiment A, wherein the bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0228] Embodiment A33: The modified bacterium of any preceding Embodiment A, wherein said bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0229] Embodiment A34. The modified bacterium of any preceding Embodiment A, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0230] Embodiment A35: The modified bacterium of any preceding Embodiment A, which does not express wild-type flagellin.

[0231] Embodiment A36: The modified bacterium of any one of the preceding Embodiments A, which lacks the fliC gene.

[0232] Embodiment A37. The modified bacterium of any preceding Embodiment A, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0233] Embodiment A38. The modified bacterium of any one of Embodiments A1-A36, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0234] Embodiment A39. The modified bacterium of any one of Embodiments A1-A36, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0235] Implementation Plan B

[0236] Embodiment B1: A modified bacterium, wherein compared to an unmodified starting strain, the bacterium comprises the expression construct of any one of embodiments 16-30, a hypoxia-regulatable essential gene expression cassette, and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium controlled by a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein controlled by a promoter active under acidic pH conditions, and the bacterium lacks at least one gene involved in or regulating an endogenous antioxidant stress response pathway or its functional expression product.

[0237] Embodiment B2: The modified bacterium of embodiment B1, 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.

[0238] Embodiment B3: The modified bacterium of Embodiment B1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0239] Embodiment B4: The modified bacterium of any one of the preceding embodiments B, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues to the culture medium.

[0240] Embodiment B5. The modified bacterium of Embodiment B4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0241] Embodiment B6. The modified bacterium of Embodiment B4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0242] Embodiment B7: The modified bacterium of any preceding Embodiment B, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family gene.

[0243] Embodiment B8: The modified bacterium of any one of the preceding embodiments B, wherein the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family-related protein.

[0244] Embodiment B9: The modified bacterium of any preceding Embodiment B, wherein the bacterium lacks HtrA serine protease activity.

[0245] Embodiment B10: The modified bacterium of any preceding Embodiment B, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0246] Embodiment B11. The modified bacterium of any preceding Embodiment B, wherein the bacterium is deficient in htrA.

[0247] Embodiment B12: The modified bacterium of any one of Embodiments B1 to B11, wherein the essential gene is a gene naturally present in the bacterial chromosome, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0248] Embodiment B13: The modified bacterium of any one of Embodiments B1-B11, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0249] Embodiment B14: The modified bacterium of Embodiment B13, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0250] Embodiment B15. The modified bacterium of Embodiment B13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0251] Embodiment B16. The modified bacterium of Embodiment B15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0252] Embodiment B17: The modified bacterium of any preceding Embodiment B, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0253] Embodiment B18: The modified bacterium of Embodiment B17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0254] Embodiment B19. The modified bacterium of Embodiment B18, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0255] Embodiment B20. The modified bacterium of any preceding embodiment B, wherein the promoter active under acidic pH conditions is active at a pH of 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.

[0256] Embodiment B21. The modified bacterium of any preceding Embodiment B, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0257] Embodiment B22. The modified bacterium of any preceding Embodiment B, wherein the promoter active under acidic pH conditions is sseA.

[0258] Embodiment B23. The modified bacterium of any preceding Embodiment B, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0259] Embodiment B24. The modified bacterium of any preceding Embodiment B, wherein the bacterium is an Enterobacteriaceae.

[0260] Embodiment B25: The modified bacterium of any of the preceding Embodiments B, wherein the bacterium is an Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea bacterium.

[0261] Embodiment B26: The modified bacterium of any of the preceding embodiments A, wherein the bacterium is selected from the group consisting of Escherichia coli, Escherichia roach, Escherichia fergusonii, Escherichia hermannii, Escherichia wounds, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella bodni, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia auverii, Yersinia burnetii, Yersinia freundii, Yersinia intermedia, Yersinia kurzewi, Yersinia moorei, Yersinia rosea, Yersinia ruckeri, Citrobacter freudii, Citrobacter kozei, and B. Citrobacter glumene, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter japonici, Enterobacter sakazakii, Enterobacter tylosus, Enterobacter rivulosa, Enterobacter intermedia, Enterobacter agglomerans, Enterobacter carcinogenes, Enterobacter lyticus, Enterobacter superpressure, Serratia marcescens, Serratia entomophila, Serratia figi, Serratia izumi, Serratia glumae, Serratia liquefaciens, Serratia aromatica, Serratia puchengensis, Serratia longyanmao, Serratia rubrum, Serratia urealyticum, Proteus mirabilis, Proteus vulgaris, Proteus mucogenes, Proteus pannei, Proteus housiei, Morganella morganii, Providencia alcaligenes, Providencia ruegensis, Providencia stuartii, Providencia rettgeri, Providencia henryi, Hafnia alvei and Pantoea agglomerans.

[0262] Embodiment B27. The modified bacterium of any preceding Embodiment B, wherein the bacterium is Salmonella typhimurium.

[0263] Embodiment B28. The modified bacterium of Embodiment B27, wherein the starting strain is Salmonella typhimurium SL7207.

[0264] Embodiment B29. The modified bacterium of any preceding Embodiment B, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0265] Embodiment B30: The modified bacterium of any preceding Embodiment B, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0266] Embodiment B31. The modified bacterium of any preceding Embodiment B, wherein the bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0267] Embodiment B32. The modified bacterium of any preceding Embodiment B, wherein said bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0268] Embodiment B33. The modified bacterium of any preceding Embodiment B, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0269] Embodiment B34: The modified bacterium of any preceding Embodiment B, which does not express wild-type flagellin.

[0270] Embodiment B35: The modified bacterium of any preceding Embodiment B, which lacks the fliC gene.

[0271] Embodiment B36. The modified bacterium of any preceding Embodiment B, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0272] Embodiment B37. The modified bacterium of any one of Embodiments B1-B35, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0273] Embodiment B38. The modified bacterium of any one of Embodiments B1-B35, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0274] Implementation Plan C

[0275] Embodiment C1: A modified Salmonella typhimurium bacterium, wherein compared to an unmodified starting strain, the bacterium comprises the expression construct of any one of embodiments 16-30, a hypoxia-regulatable essential gene expression cassette, and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, and the bacterium lacks at least one gene involved in or regulating an endogenous antioxidant stress response pathway or its functional expression product.

[0276] Embodiment C2: The modified bacterium of embodiment C1, 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.

[0277] Embodiment C3: The modified bacterium of Embodiment C1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0278] Embodiment C4: The modified bacterium of any one of the preceding embodiments C, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues to the culture medium.

[0279] Embodiment C5. The modified bacterium of Embodiment C4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0280] Embodiment C6. The modified bacterium of Embodiment C4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0281] Embodiment C7: The modified bacterium of any one of the preceding Embodiments C, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family gene.

[0282] Embodiment C8: The modified bacterium of any one of the preceding embodiments C, wherein the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family-related protein.

[0283] Embodiment C9: The modified bacterium of any preceding Embodiment C, wherein the bacterium lacks HtrA serine protease activity.

[0284] Embodiment C10: The modified bacterium of any preceding Embodiment C, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0285] Embodiment C11. The modified bacterium of any preceding Embodiment C, wherein the bacterium is deficient in htrA.

[0286] Embodiment C12: The modified bacterium of any one of Embodiments C1 to C11, wherein the essential gene is a gene naturally present in the chromosome of the bacterium, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0287] Embodiment C13: The modified bacterium of any one of Embodiments C1-C11, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0288] Embodiment C14: The modified bacterium of Embodiment C13, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0289] Embodiment C15: The modified bacterium of Embodiment C13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0290] Embodiment C16: The modified bacterium of Embodiment C15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0291] Embodiment C17: The modified bacterium of any one of the preceding Embodiments C, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0292] Embodiment C18: The modified bacterium of Embodiment C17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0293] Embodiment C19: The modified bacterium of Embodiment C18, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0294] Embodiment C20: The modified bacterium of any one of the preceding embodiments C, wherein the promoter active under acidic pH conditions is active at a pH value 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.

[0295] Embodiment C21. The modified bacterium of any preceding Embodiment C, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0296] Embodiment C22: The modified bacterium of any preceding Embodiment C, wherein the promoter active under acidic pH conditions is sseA.

[0297] Embodiment C23. The modified bacterium of any preceding Embodiment C, wherein the unmodified starting strain is a facultative anaerobic Salmonella typhimurium.

[0298] Embodiment C24: The modified bacterium of any one of the preceding Embodiments C, wherein the starting strain is Salmonella typhimurium SL7207.

[0299] Embodiment C25. The modified bacterium of any one of Embodiments C1-C24, wherein said bacterium expresses wild-type lipopolysaccharide (LPS).

[0300] Embodiment C26: The modified bacterium of any preceding Embodiment C, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0301] Embodiment C27. The modified bacterium of any preceding Embodiment C, wherein the bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0302] Embodiment C28: The modified bacterium of any preceding Embodiment C, wherein the bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0303] Embodiment C29: The modified bacterium of any preceding Embodiment C, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0304] Embodiment C30: The modified bacterium of any preceding Embodiment C, which does not express wild-type flagellin.

[0305] Embodiment C31: The modified bacterium of any one of the preceding Embodiments C, which lacks the fliC gene.

[0306] Embodiment C32: The modified bacterium of any preceding Embodiment C, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0307] Embodiment C33: The modified bacterium of any one of Embodiments C1-C31, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0308] Embodiment C34: The modified bacterium of any one of Embodiments C1-C31, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0309] Implementation Plan D

[0310] Embodiment D1: A modified bacterium, wherein the bacterium comprises the expression construct of any one of embodiments 16-30 and a hypoxia-regulatable essential gene expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the bacterium lacks at least one gene required for survival in macrophages or its functional expression product, compared to an unmodified starting strain.

[0311] Embodiment D2: The modified bacterium of 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.

[0312] Embodiment D3: The modified bacterium of Embodiment D1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0313] Embodiment D4: The modified bacterium of any one of the preceding embodiments D, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues to the culture medium.

[0314] Embodiment D5: The modified bacterium of Embodiment D4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0315] Embodiment D6. The modified bacterium of Embodiment D4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0316] Embodiment D7: The modified bacterium of any one of the preceding Embodiments D, wherein the gene required for survival in macrophages is the STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0317] Embodiment D8: The modified bacterium of any one of the preceding Embodiments D, wherein the functional expression product of the gene required for survival in macrophages is an HtrA serine protease family-related protein.

[0318] Embodiment D9: The modified bacterium of any preceding Embodiment D, wherein the bacterium lacks HtrA serine protease activity.

[0319] Embodiment D10: The modified bacterium of any preceding Embodiment D, wherein the gene required for survival in macrophages is htrA.

[0320] Embodiment D11: The modified bacterium of any preceding Embodiment D, wherein the bacterium is deficient in htrA.

[0321] Embodiment D12: The modified bacterium of any one of Embodiments D1-D11, wherein the essential gene is a gene naturally present in the bacterial chromosome, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0322] Embodiment D13: The modified bacterium of any one of Embodiments D1-D11, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0323] Embodiment D14: The modified bacterium of Embodiment D13, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0324] Embodiment D15: The modified bacterium of Embodiment D13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0325] Embodiment D16: The modified bacterium of Embodiment D15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0326] Embodiment D17: The modified bacterium of any one of the preceding Embodiments D, further comprising a pH-regulatable expression cassette comprising a gene encoding a bacterially derived hemolysin protein controlled by a promoter active under acidic pH conditions.

[0327] Embodiment D18: The modified bacterium of Embodiment D17, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0328] Embodiment D19: The modified bacterium of Embodiment D18, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0329] Embodiment D20: The modified bacterium of Embodiment D19, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0330] Embodiment D21: The modified bacterium of any one of Embodiments D17-D20, wherein the promoter active under acidic pH conditions is active at a pH value 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.

[0331] Embodiment D22: The modified bacterium of any one of Embodiments D17-D21, wherein the promoter active under acidic pH conditions is selected from sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0332] Embodiment D23: The modified bacterium of any one of Embodiments D17-D21, wherein the promoter active under acidic pH conditions is sseA.

[0333] Embodiment D24: The modified bacterium of any one of the preceding Embodiments D, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0334] Embodiment D25: The modified bacterium of any one of the preceding Embodiments D, wherein the bacterium is an Enterobacteriaceae.

[0335] Embodiment D26: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is an Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea bacterium.

[0336] Embodiment D27: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is selected from Escherichia coli, Escherichia roach, Escherichia fergusonii, Escherichia hermannii, Escherichia wounds, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella bodega, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia auverii, Yersinia burnetii, Yersinia freundii, Yersinia intermedia, Yersinia kurzewi, Yersinia moorei, Yersinia rosea, Yersinia ruckeri, Citrobacter freundii, Citrobacter kozei, and Boula Citrobacter glumene, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter japonici, Enterobacter sakazakii, Enterobacter tylosus, Enterobacter rivulosa, Enterobacter intermedia, Enterobacter agglomerans, Enterobacter carcinogenes, Enterobacter lyticus, Enterobacter superpressure, Serratia marcescens, Serratia entomophila, Serratia figi, Serratia izumi, Serratia glumae, Serratia liquefaciens, Serratia aromatica, Serratia puchengensis, Serratia longyanmao, Serratia rubrum, Serratia urealyticum, Proteus mirabilis, Proteus vulgaris, Proteus mucogenes, Proteus pannei, Proteus housiei, Morganella morganii, Providencia alcaligenes, Providencia ruegensis, Providencia stuartii, Providencia rettgeri, Providencia henryi, Hafnia alvei and Pantoea agglomerans.

[0337] Embodiment D28: The modified bacterium of any preceding Embodiment D, wherein the bacterium is Salmonella typhimurium.

[0338] Embodiment D29: The modified bacterium of Embodiment D28, wherein the starting strain is Salmonella typhimurium SL7207.

[0339] Embodiment D30: The modified bacterium of any preceding Embodiment D, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0340] Embodiment D31: The modified bacterium of any one of the preceding Embodiments D, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0341] Embodiment D32: The modified bacterium of any preceding Embodiment D, wherein the bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0342] Embodiment D33: The modified bacterium of any preceding Embodiment D, wherein said bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0343] Embodiment D34: The modified bacterium of any preceding Embodiment D, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0344] Embodiment D35: The modified bacterium of any preceding Embodiment D, which does not express wild-type flagellin.

[0345] Embodiment D36: The modified bacterium of any one of the preceding Embodiments D, which lacks the fliC gene.

[0346] Embodiment D37: The modified bacterium of any one of the preceding Embodiments D, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0347] Embodiment D38. The modified bacterium of any one of Embodiments D1-D36, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0348] Embodiment D39: The modified bacterium of any one of Embodiments D1-D36, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0349] Implementation Plan E

[0350] Embodiment E1: A modified bacterium, wherein compared to an unmodified starting strain, the bacterium comprises the expression construct of any one of embodiments 16-30, a hypoxia-regulatable essential gene expression cassette and a pH-regulatable expression cassette, wherein the essential gene expression cassette comprises an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and the pH-regulatable expression cassette comprises a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, wherein the bacterium expresses wild-type lipopolysaccharide (LPS) and the bacterium lacks at least one gene required for survival in macrophages or its functional expression product.

[0351] Embodiment E2: The modified bacterium of Embodiment El, 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.

[0352] Embodiment E3: The modified bacterium of Embodiment El, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0353] Embodiment E4: The modified bacterium of any one of the preceding embodiments E, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues to the culture medium.

[0354] Embodiment E5. The modified bacterium of Embodiment E4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0355] Embodiment E6. The modified bacterium of Embodiment E4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0356] Embodiment E7: The modified bacterium of any preceding Embodiment E, wherein the gene required for survival in macrophages is the STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0357] Embodiment E8: The modified bacterium of any preceding embodiment E, wherein the functional expression product of the gene required for survival in macrophages is an HtrA serine protease family-related protein.

[0358] Embodiment E9: The modified bacterium of any preceding Embodiment E, wherein the bacterium lacks HtrA serine protease activity.

[0359] Embodiment E10: The modified bacterium of any preceding Embodiment E, wherein the gene required for survival in macrophages is htrA.

[0360] Embodiment E11: The modified bacterium of any preceding Embodiment E, wherein the bacterium is deficient in htrA.

[0361] Embodiment E12: The modified bacterium of any one of Embodiments E1 to E11, wherein the essential gene is a gene naturally present in the chromosome of the bacterium, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0362] Embodiment E13: The modified bacterium of any one of Embodiments E1-E11, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0363] Embodiment E14: The modified bacterium of Embodiment E13, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0364] Embodiment E15: The modified bacterium of Embodiment E13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0365] Embodiment E16: The modified bacterium of Embodiment E15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0366] Embodiment E17: The modified bacterium of any one of the preceding Embodiments E, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0367] Embodiment E18: The modified bacterium of Embodiment E17, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0368] Embodiment E19: The modified bacterium of Embodiment E18, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0369] Embodiment E20: The modified bacterium of any preceding embodiment E, wherein the promoter active under acidic pH conditions is active at a pH value 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.

[0370] Embodiment E21. The modified bacterium of any preceding Embodiment E, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0371] Embodiment E22. The modified bacterium of any preceding Embodiment E, wherein the promoter active under acidic pH conditions is sseA.

[0372] Embodiment E23: The modified bacterium of any preceding Embodiment E, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0373] Embodiment E24: The modified bacterium of any preceding Embodiment E, wherein the bacterium is an Enterobacteriaceae bacterium.

[0374] Embodiment E25: The modified bacterium of any one of the preceding Embodiments E, wherein the bacterium is an Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea bacterium.

[0375] Embodiment E26: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium is selected from the group consisting of Escherichia coli, Escherichia roach, Escherichia fergusonii, Escherichia hermannii, Escherichia wounds, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella bodega, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia auverii, Yersinia burnetii, Yersinia freundii, Yersinia intermedia, Yersinia kurzewi, Yersinia moorei, Yersinia rosea, Yersinia ruckeri, Citrobacter freudii, Citrobacter kozei, and B. Citrobacter glumene, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter japonici, Enterobacter sakazakii, Enterobacter tylosus, Enterobacter rivulosa, Enterobacter intermedia, Enterobacter agglomerans, Enterobacter carcinogenes, Enterobacter lyticus, Enterobacter superpressure, Serratia marcescens, Serratia entomophila, Serratia figi, Serratia izumi, Serratia glumae, Serratia liquefaciens, Serratia aromatica, Serratia puchengensis, Serratia longyanmao, Serratia rubrum, Serratia urealyticum, Proteus mirabilis, Proteus vulgaris, Proteus mucogenes, Proteus pannei, Proteus housiei, Morganella morganii, Providencia alcaligenes, Providencia ruegensis, Providencia stuartii, Providencia rettgeri, Providencia henryi, Hafnia alvei and Pantoea agglomerans.

[0376] Embodiment E27. The modified bacterium of any preceding Embodiment E, wherein the bacterium is Salmonella typhimurium.

[0377] Embodiment E28. The modified bacterium of Embodiment E27, wherein the starting strain is Salmonella typhimurium SL7207.

[0378] Embodiment E29: The modified bacterium of any preceding Embodiment E, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0379] Embodiment E30: The modified bacterium of any preceding Embodiment E, wherein said bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0380] Embodiment E31 : The modified bacterium of any preceding Embodiment E, wherein said bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0381] Embodiment E32: The modified bacterium of any preceding Embodiment E, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0382] Embodiment E33: The modified bacterium of any preceding Embodiment E, which does not express wild-type flagellin.

[0383] Embodiment E34: The modified bacterium of any preceding Embodiment E, which lacks the fliC gene.

[0384] Embodiment E35. The modified bacterium of any preceding Embodiment E, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0385] Embodiment E36. The modified bacterium of any one of Embodiments E1-E34, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0386] Embodiment E37. The modified bacterium of any one of Embodiments E1-E34, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0387] Implementation Plan F

[0388] Embodiment F1: A modified Salmonella typhimurium bacterium, wherein the bacterium comprises the expression construct of any one of embodiments 16-30, a hypoxia-regulatable essential gene expression cassette comprising an essential gene of the bacterium under the control of a strictly hypoxia-inducible promoter, and a pH-regulatable expression cassette comprising a gene encoding a bacterial-derived hemolysin protein under the control of a promoter active under acidic pH conditions, compared to an unmodified starting strain, wherein the bacterium expresses wild-type lipopolysaccharide (LPS) and the bacterium lacks at least one gene required for survival in macrophages or its functional expression product.

[0389] Embodiment F2: The modified bacterium of 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.

[0390] Embodiment F3: The modified bacterium of Embodiment F1, wherein the strictly hypoxia-inducible promoter is the ssbp1 promoter.

[0391] Embodiment F4: The modified bacterium of any one of the preceding embodiments F, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or an analog thereof to the culture medium.

[0392] Embodiment F5. The modified bacterium of Embodiment F4, wherein the essential gene is selected from the group consisting of dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0393] Embodiment F6. The modified bacterium of Embodiment F4, wherein the essential gene is selected from the group consisting of dapA and dapE.

[0394] Embodiment F7: The modified bacterium of any preceding Embodiment F, wherein the gene required for survival in macrophages is the STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0395] Embodiment F8: The modified bacterium of any preceding Embodiment F, wherein the gene required for survival in macrophages is a gene involved in or regulating an endogenous anti-oxidative stress response pathway.

[0396] Embodiment F9: The modified bacterium of Embodiment F8, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family gene.

[0397] Embodiment F10: The modified bacterium of embodiment F9, wherein the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family-related protein.

[0398] Embodiment F11. The modified bacterium of Embodiment F9, wherein said bacterium lacks HtrA serine protease activity.

[0399] Embodiment F12: The modified bacterium of Embodiment F9, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0400] Embodiment F13. The modified bacterium of Embodiment F9, wherein said bacterium is deficient in htrA.

[0401] Embodiment F14: The modified bacterium of any one of Embodiments F1 to F13, wherein the essential gene is a gene naturally present in the chromosome of the bacterium, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia-inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia-inducible promoter.

[0402] Embodiment F15: The modified bacterium of any one of Embodiments F1-F13, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is fully controlled by the strict hypoxia-inducible promoter.

[0403] Embodiment F16: The modified bacterium of Embodiment F15, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

[0404] Embodiment F17. The modified bacterium of Embodiment F15, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium.

[0405] Embodiment F18. The modified bacterium of Embodiment F17, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium.

[0406] Embodiment F19: The modified bacterium of any preceding Embodiment F, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0407] Embodiment F20: The modified bacterium of Embodiment F19, wherein the gene encoding the Gram-negative bacterial hemolysin protein is hlyA or hlyE.

[0408] Embodiment F21. The modified bacterium of Embodiment F20, wherein the hlyA or hlyE is from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0409] Embodiment F22. The modified bacterium of any preceding embodiment F, wherein the promoter active under acidic pH conditions is active at a pH of 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.

[0410] Embodiment F23. The modified bacterium of any preceding Embodiment F, wherein the promoter active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0411] Embodiment F24. The modified bacterium of any preceding Embodiment F, wherein the promoter active under acidic pH conditions is sseA.

[0412] Embodiment F25. The modified bacterium of any preceding Embodiment F, wherein the starting strain is Salmonella typhimurium SL7207.

[0413] Embodiment F26: The modified bacterium of any preceding Embodiment F, wherein, upon administration to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

[0414] Embodiment F27. The modified bacterium of any preceding Embodiment F, wherein said bacterium is capable of inhibiting the growth of a malignant tumor when administered to a subject having the malignant tumor.

[0415] Embodiment F28: The modified bacterium of any preceding Embodiment F, wherein said bacterium is capable of inducing an anti-tumor specific immune response when administered to a subject having a malignant tumor.

[0416] Embodiment F29: The modified bacterium of any preceding Embodiment F, wherein said bacterium is capable of inducing anti-tumor immune memory when administered to a subject having a malignant tumor.

[0417] Embodiment F30: The modified bacterium of any preceding Embodiment F, which does not express wild-type flagellin.

[0418] Embodiment F31: The modified bacterium of any preceding Embodiment F, which lacks the fliC gene.

[0419] Embodiment F32: The modified bacterium of any preceding Embodiment F, wherein the bacterium has a survival level in macrophages that is about 50% to about 30% of the survival level of the unmodified starting strain.

[0420] Embodiment F33: The modified bacterium of any one of Embodiments F1-F31, wherein the bacterium has a survival level in macrophages that is about 30% to about 10% of the survival level of the unmodified starting strain.

[0421] Embodiment F34: The modified bacterium of any one of Embodiments F1-F31, wherein the bacterium has a survival level in macrophages that is about 10% to about 1% of the survival level of the unmodified starting strain.

[0422] Implementation Plan K

[0423] Embodiment K1: A pharmaceutical composition comprising an effective amount of the modified bacterium of any one of the aforementioned Embodiments A, B, C, D, E, or F.

[0424] Embodiment K2: The pharmaceutical composition of Embodiment K1, which is used for treating malignant tumors.

[0425] Embodiment K3: The pharmaceutical composition of Embodiment K1, for inducing an anti-tumor specific immune response in a subject suffering from a malignant tumor.

[0426] Embodiment K4: The pharmaceutical composition of Embodiment K1, for inducing anti-tumor immune memory in a subject suffering from a malignant tumor.

[0427] Embodiment K5: The pharmaceutical composition of Embodiment K1, which is used for preventing or treating metastasis or recurrence of malignant tumors.

[0428] Embodiment K6: The pharmaceutical composition of Embodiment K1, which is used for treating malignant tumors that are resistant to or have failed to respond to previous anti-tumor therapies.

[0429] Embodiment K7: The pharmaceutical composition of Embodiments K1-K6, wherein the modified bacterium is a live bacterium.

[0430] Implementation Plan L

[0431] Embodiment L1: A method for treating a malignant tumor, comprising administering an effective amount of the modified bacteria of any one of the aforementioned embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of the embodiments G to a subject suffering from a malignant tumor.

[0432] Embodiment L2: A method for inducing an anti-tumor specific immune response in a subject having a malignant tumor, comprising administering to the subject an effective amount of the modified bacteria of any one of the aforementioned embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of the embodiments G.

[0433] Embodiment L3: A method of inducing anti-tumor immune memory in a subject suffering from a malignant tumor, comprising administering to the subject an effective amount of the modified bacteria of any of the foregoing embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any of the embodiments G.

[0434] Embodiment L4: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering to a subject suffering from a malignant tumor an effective amount of the modified bacteria of any one of the aforementioned embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of the embodiments G.

[0435] Embodiment L5: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering an effective amount of the modified bacteria of any of the foregoing embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any of embodiment G to a subject suffering from metastasis or recurrence of a malignant tumor or a subject at high risk of metastasis or recurrence of a malignant tumor.

[0436] Embodiment L6: A method for treating a malignant tumor that has become resistant to or has failed treatment with previous anti-tumor therapy, comprising administering an effective amount of the modified bacteria of any one of Embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of Embodiments G to a subject suffering from a malignant tumor that has become resistant to or has failed treatment with previous anti-tumor therapy.

[0437] Embodiment L7. The method of Embodiments L1-L6, wherein the modified bacterium is a live bacterium.

[0438] Implementation Plan M

[0439] Embodiment M1: Use of the modified bacterium of any one of the aforementioned embodiments A, B, C, D, E, or F in the preparation of a medicament for treating a malignant tumor.

[0440] Embodiment M2: Use of the modified bacterium of any one of the foregoing embodiments A, B, C, D, E, or F in the preparation of a medicament for inducing an anti-tumor specific immune response in a subject suffering from a malignant tumor.

[0441] Embodiment M3: Use of the modified bacteria of any of the foregoing embodiments A, B, C, D, E, or F in the preparation of a medicament for inducing anti-tumor immune memory in a subject suffering from a malignant tumor.

[0442] Embodiment M4: Use of the modified bacterium of any one of the aforementioned embodiments A, B, C, D, E, or F in the preparation of a medicament for preventing or treating metastasis or recurrence of a malignant tumor.

[0443] Embodiment M5: Use of the modified bacterium of any one of the foregoing embodiments A, B, C, D, E, or F in the preparation of a medicament for treating a malignant tumor that is resistant to or has failed previous anti-tumor therapy.

[0444] Embodiment M6: The use of Embodiments M1-M5, wherein the modified bacterium is a live bacterium.

[0445] Implementation Plan N

[0446] Embodiment N1: The pharmaceutical composition of any one of Embodiments K, the method of any one of Embodiments L, or the use of any one of Embodiments M, wherein 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 mucosa.

[0447] Embodiment N2: The pharmaceutical composition of any one of Embodiments K, the method of any one of Embodiments L, or the use of any one of Embodiments M, wherein the malignancy is a sarcoma or a carcinoma.

[0448] Embodiment N3: The pharmaceutical composition of any one of Embodiments K, the method of any one of Embodiments L, or the use of any one of Embodiments M, wherein the malignant tumor is a solid tumor.

[0449] Embodiment N4: The pharmaceutical composition of any one of Embodiments K, the method of any one of Embodiments L, or the use of any one of Embodiments M, wherein the malignant tumor is selected from 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, liver cancer, pancreatic cancer, bile duct cancer, colon cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, adrenal tumor, thyroid cancer, parathyroid cancer, pituitary tumor, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, bone cancer, and osteosarcoma.

[0450] Embodiment N5: The pharmaceutical composition of any one of Embodiments K, the method of any one of Embodiments L, or the use of any one of Embodiments M, wherein the bacteria, drug, or pharmaceutical composition is administered by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration. Example

[0451] The following examples are provided 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.

[0452] Example 1: Construction of variant strains of Salmonella typhimurium SL7207

[0453] 1.1 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 target fragments were amplified by PCR. For PsseA-hlyA-loxp-KnaR-loxp, the following primer sequences were used: forward primer (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6); for Pssbp1-dapE-Cm, the following primer sequences were used: 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).

[0454] 1.2 The pSim6 plasmid containing λ phage Red recombinase (BioVector NTCC Inc.: 3574840) was introduced into the facultative anaerobic Salmonella SL7207 strain (NCBI: ASM1320710v1) to prepare the SL7207 strain containing the pSim6 plasmid and electroporated competent cells SL7207 (pSim6).

[0455] 1.3 The target fragment 1 was introduced into SL7207 (pSim6) competent cells via electroporation (E = 18 kV / cm). The dapE gene (which regulates the synthesis of diaminopimelane, essential for cell wall synthesis) in the SL7207 genome was replaced with the target fragment 1 using λ-red homologous recombination technology. Specifically, the target strain containing the pSim6 plasmid was cultured at 30°C and the recombinase expression conditions were 42°C for 15 minutes. Homologous arms containing 50 bp upstream and downstream of the target gene site were added to the PCR primers. Under the action of the homologous recombinase, the homologous recombination process was completed, resulting in the SL7207 (ΔdapE::PsseA-hlyA-KnaR) strain.

[0456] 1.4 The Cre plasmid containing the P1 phage Cre recombinase (Gene Bridges: A112) was introduced into the SL7207 (ΔdapE::PsseA-hlyA-KnaR) strain to remove kanamycin resistance by recombination, thereby obtaining the SL7207 (ΔdapE::PsseA-hlyA) strain.

[0457] 1.5 Introduce the pSim6 plasmid into the SL7207 (ΔdapE::PsseA-hlyA) strain; prepare SL7207 (ΔdapE::PsseA-hlyA) (pSim6) electroporation competent cells.

[0458] 1.6 Target fragment 2 was introduced into SL7207(ΔdapE::PsseA-hlyA)(pSim6) competent cells by electroporation (E=18 kV / cm). Lambda-red homologous recombination was used to replace the htrA gene (encoding a serine protease) in the SL7207(ΔdapE::PsseA-hlyA) genome with target fragment 2, generating the DB-ZW1 strain, an SL7207 strain lacking the htrA gene and possessing the hlyA gene under the control of PsseA and the dapE gene under the strict hypoxia-inducible promoter Pssbp1 (ΔdapE::PsseA-hlyA; ΔhtrA::Pssbp1-dapE-Cm).

[0459] The strain construction scheme is shown in Figure 1.

[0460] Example 2: DB-ZW1 has tumor inhibitory effects on various tumor models

[0461] 2.1 Establishment of tumor model

[0462] C57BL / 6 mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 MB49 mouse bladder cancer cells (Merck SCC148) / B16 melanoma cells (ATCC:CRL-6475) were used to establish a mouse bladder cancer / melanoma subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100 mm 3 The experiment was conducted around 4:00 p.m. An orthotopic colorectal cancer model was induced using DSS / AOM: a single intraperitoneal injection of 10 mg / kg AOM was administered. One week later, mice were fed 2.5% DSS-containing water for 7 days, followed by normal drinking water for 14 days. This constituted one DSS treatment cycle. Three additional cycles of DSS treatment were performed to establish a mouse model with colorectal cancer.

[0463] 2.2 Distribution changes of DB-ZW1 in tumors and different organs

[0464] 1×10 7 CFU, 125 μL of DB-ZW1 was injected into MB49 bladder cancer-bearing mice via the tail vein, with 3 mice per group. Approximately 90% of DB-ZW1 was observed to reside in the tumor 1 day after injection (dpi). Over the next 2 days, the density of DB-ZW1 in the tumor increased further by 100-fold (reaching 10 8 CFU / g) and remained at a high level for the next two weeks. In contrast, the density of DB-ZW1 in normal organs steadily decreased to <10 CFU / g within two weeks. 2 CFU / g (Figure 2A).

[0465] To further determine whether DB-ZW1 preferentially proliferates in tumors, 1×10 7 The number of DB-ZW1 cells in different organs was measured within 24 hours after injection of 125 μL of DB-ZW1 bacteria (CFU). Three mice were included in each group (Figure 2B). The results showed that DB-ZW1 rapidly spread throughout the mouse body within 30 minutes after injection, and the vast majority of DB-ZW1 cells (about 99.9%) were distributed in the liver, spleen, and blood. However, within 4 hours after injection, their number decreased significantly, and the total number of DB-ZW1 cells decreased by about 90%. Subsequently, DB-ZW1 cells in the tumor were detected at a rate of 0.7 h. -1 The growth rate of DB-ZW1 cells was exponential, reaching saturation within 3 days. In contrast, the number of DB-ZW1 cells steadily decreased in normal organs. This unique tumor growth characteristic of DB-ZW1 cells is key to the validity of the following research results.

[0466] 2.3 Antitumor effect of DB-ZW1 on bladder cancer, melanoma, and colon cancer

[0467] DB-ZW1 was used to treat mice with subcutaneous bladder cancer, orthotopic melanoma, and subcutaneous and orthotopic colon cancer. 1×10 7 CFU, 125 μL of DB-ZW1 was injected into tumor-bearing mice via the tail vein, with five mice per group. As shown in Figures 3A-C, DB-ZW1 significantly reduced tumor volume. Furthermore, treatment of mice with orthotopic colon cancer with DB-ZW1 significantly reduced the number of tumors in the colon (Figure 3D).

[0468] Example 3: Construction of vectors and strains for expressing nanobodies / fusion proteins binding to CXCR2

[0469] 3.1 Construction of Nanobody Inducible Expression Vectors and Bacteria

[0470] A DNA molecule encoding a fusion of three polypeptides "signal peptide + nanobody + tag" was synthesized (Qingke Bio) and cloned into the pET26b plasmid (see Figure 4), wherein the nanobody was selected from Nb127D01, Nb163E3 and Nb97A9 (SEQ ID NOs: 19, 20 and 21), the signal peptide was the signal peptide derived from the pectate lyase of Erwinia carotovora (pelB signal peptide, SEQ ID NO: 28), the tag was an HA-His tag, and the above segments were directly connected. 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).

[0471] In addition, the coding sequence of the pelB signal peptide was replaced with the coding sequence of the psp signal peptide (SEQ ID NO: 29) derived from Paenibacillus sp. EC003 to construct plasmids pET26b-psp-Nb127D01-HA-his, pET26b-psp-Nb163E3-HA-his, and pET26b-psp-Nb97A9-HA-his (SEQ ID NOs: 34-36).

[0472] The above plasmids were transformed into competent cells of Escherichia coli strain BL21 (DE3) (purchased from Novezan) by chemical transformation to obtain strains 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 that induced the expression of nanoantibodies. Specifically, add approximately 100 ng of plasmid to the competent culture, incubate on ice for 30 minutes, place in a 42°C water bath for 45 seconds, and return to ice for 2-3 minutes. Then, add 900 μl of LB medium, incubate at 37°C for 1 hour, and screen for positive clones on LB plates containing 50 mg / L kanamycin.

[0473] 3.2 Construction of Nanobody Anaerobic Expression Vector

[0474] To construct the nanobody anaerobic expression vector, the oxygen-sensitive promoter FNRsp (SEQ ID NO: 10) was used to drive the expression of the six fusion polypeptides in Example 3.1.

[0475] Specifically, primers Nb frag F1 / R1 and Nb frag F2 / R2 were designed, respectively, and the coding sequence fragment was 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. PCR was performed using primer linearize REV / FOR and plasmid pSC101-pFNRsp (SEQ ID NO: 33) as a template to obtain a linear backbone fragment.

[0476] Primer information

[0477] PCR amplification was performed using PrimeSTAR Max DNA Polymerase (Takara #R054A). The PCR system (50 μL total volume) contained 25 μL of PrimeSTAR Max Premix, 0.2-0.3 μM of each primer, and 200 ng of template. PCR reaction conditions were: denaturation at 98°C for 10 seconds, annealing at 55°C for 10 seconds, and extension at 72°C for 5 seconds / kb. 30 cycles of denaturation-annealing-extension were performed.

[0478] The backbone fragment and the coding sequence fragment were homologously recombined using the ClonExpressⅡOne Step Cloning Kit (Vazyme#C112) to construct plasmids pSC101-pFNRsp-pelB-Nb127D01-HA-his, pSC101-pFNRsp-pelB-Nb163E3-HA-his, pSC101-pFNRsp-pelB-Nb97A9-HA-his, pSC101-pFNRsp-psp-Nb127D01-HA-his, pSC101-pFNRsp-psp-Nb163E3-HA-his and pSC101-pFNRsp-psp-Nb97A9-HA-his for anaerobic expression of nanobodies.

[0479] 3.3 Construction of Nanobody-PE Fusion Protein Inducible Expression Vector and Bacteria

[0480] The nucleotide fragments encoding the Pseudomonas exotoxin fragments PE38 (SEQ ID NO: 26, 55 kDa) and PE[LR] (SEQ ID NO: 27, 43 kDa) were inserted into the plasmids pET26b-pelB-Nb127D01-HA-his, pET26b-pelB-Nb163E3-HA-his, and pET26b-pelB-Nb97A9-HA-his constructed in 3.1 (at the 3' end of the Nanobody coding sequence) to obtain the plasmid pET26b-psp-Nb127D01-PE3 for inducible expression of the Nanobody-PE fusion protein. 8-HA-his, pET26b-psp-Nb163E3-PE38-HA-his, pET26b-psp-Nb97A9-PE38-HA-his, pET26b-psp-Nb127 D01-PE[LR]-HA-his, pET26b-psp-Nb163E3-PE[LR]-HA-his, pET26b-psp-Nb97A9-PE[LR]-HA-his (SEQ ID NO:43-48). The constructed plasmids were transformed into competent cells of Escherichia coli strain BL21 (DE3) by chemical transformation (see Example 3.1) to obtain strains BL21 (DE3)-pET26b-psp-Nb127D01-PE38-HA-his, BL21 (DE3)-pET26b-psp-Nb163E3-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, BL21(DE3)-pET26b-psp-Nb97A9-PE[LR]-HA-his.

[0481] The strains BL21(DE3)-pET26b-psp-PE38-HA-his and BL21(DE3)-pET26b-psp-PE[LR]-HA-his expressing PE38 and PE[LR] were constructed in a similar manner.

[0482] 3.4 Construction of anaerobic expression vector for nanobody-PE fusion protein

[0483] 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 with primers Fragment.FOR / REV (SEQ ID NOs: 55 and 56) to obtain the coding sequence fragment (see Example 3.2 for PCR system and conditions). The coding sequence fragment was homologously recombined with the backbone fragment prepared in Example 3.2 by the method described 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).

[0484] The above plasmids were transformed into the competent cells of the Salmonella typhimurium strain ZW1 prepared in Example 1 by electroporation (see Example 1 for the electroporation method, and the screening medium was LB + chloramphenicol 25 mg / L) to obtain the anaerobic expression nanobody 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, ZW1-psc101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his, ZW1-psc101 -pFNRsp-psp-Nb97A9-PE[LR]-HA-his, ZW1-psc101-pFNRsp-psp-PE38-HA-his, and ZW1-psc101-pFNRsp-psp-PE[LR]-HA-his.

[0485] The strains ZW1-psc101-pFNRsp-psp-PE38-HA-his and ZW1-psc101-pFNRsp-psp-PE[LR]-HA-his expressing PE38 and PE[LR] were constructed in a similar manner.

[0486] Example 4: Bacterial inducible expression of nanobodies and fusion proteins

[0487] In order to detect the inducible expression of nanobodies and fusion proteins, prokaryotic expression was performed with the following strains constructed in Examples 3.1 and 3.3: BL21 (DE3) -pET26b-pelB-Nb127D01-HA-his, BL21 (DE3) -pET26b-pelB-Nb163E3-HA-his, BL21 (DE3) -pET26b-pelB-Nb97A9-HA-his, BL21 ( 38-HA-his, BL21(DE3)-pET26b-psp-Nb127D01-PE38-HA-his, BL21(DE3)-pET26b-psp-Nb163E3-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.

[0488] Specifically, bacteria were inoculated into LB sugar-free medium (containing 50 mg / L kanamycin) and cultured in a 37°C shaker at 220 rpm until the OD600 was between 0.4 and 0.6. Different concentrations of IPTG (final concentrations of 0 mM, 0.1 mM, 0.2 mM, and 0.5 mM) were added to the culture medium, followed by overnight induction in a 16°C shaker at 220 rpm. The bacterial culture was then collected. The collected culture was centrifuged, and the LB culture supernatant and bacterial pellet were collected separately. The LB culture supernatant was concentrated by centrifugation using a 3 kDa ultrafiltration tube (2,500 rpm, 2-3 hours). The bacterial pellet was lysed using a Qiagen protein extraction kit (Qproteome Bacterial Protein Prep Kit #37900, refer to the experimental manual Qproteome Bacterial Protein Preparation Handbook, www.qiagen.com). The lysate was centrifuged, and the lysed bacterial pellet and lysate supernatant were collected separately.

[0489] In addition, the strain prepared in Example 3.4 was also tested for expression under anaerobic expression conditions. Similar to the above method, the antibiotic added to the culture medium was 25 mg / L chloramphenicol, and the culture and expression were induced under anaerobic conditions.

[0490] The samples were electrophoresed on a 12.5% ​​polyacrylamide gel (80 V) and transferred to the membrane using the Pyxis Transfer Stack kit (purchased from Pyxis, catalog number SPJ-T20S) according to the manufacturer's instructions. Rabbit anti-6×His antibody was used as the primary antibody (purchased from Thermo Fisher, dilution ratio 1:10,000) and goat anti-rabbit antibody was used as the secondary antibody (purchased from Thermo Fisher, dilution ratio 1:5,000). Bacterial protein expression and secretion were detected by Western blotting (developed with SuperSignal™ West Pico PLUS Chemiluminecent from Thermo Fisher).

[0491] As shown in Figures 6A-6C, the expression of nanobodies was positively correlated with the IPTG concentration, and there were obvious nanobody bands in the LB supernatant, indicating that the signal peptide could guide the secretion of the expressed nanobody outside the bacterial cells.

[0492] As shown in Figure 7 , when IPTG-induced and hypoxia-induced expression occurred, there were obvious bands of the Nanobody-PE fusion protein in the LB supernatant, indicating that the signal peptide could guide the expressed Nanobody-PE fusion protein to be secreted outside the bacterial cells.

[0493] Example 5: Neutralization of intratumoral neutrophils increases DB-ZW1 levels in MB49 bladder cancer tumors

[0494] C57BL / 6 mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 MB49 mouse bladder cancer cells (Merck SCC148) were used to establish a mouse bladder cancer subcutaneous tumor model. 14-18 days after inoculation (tumor volume of approximately 100 mm 3 ), the mice were divided into PBS group, ZW1 treatment group (only DB-ZW was administered, 1×10 7CFU of bacteria were suspended in 125 μL of PBS and administered to mice via tail vein injection. Three mice were included in each group (ZW1+αGr-1 group). In the ZW1+αGr-1 group, 0.2 mg of InVivoMab anti-mouse Ly6G / Ly6c (Gr-1) antibody (purchased from BioXCell) was injected into the tail vein of each mouse on day 3 before bacterial treatment, on the day of bacterial injection, and on the day after bacterial injection (2 dpi). On day 3 dpi, mice were sacrificed, tumors were removed, and embedded. Serial sections near the center of the tumor were then placed on slides. Samples were fixed with acetone for 10 minutes, air-dried, and returned to room temperature. The samples were then washed twice with 0.01 mol / L PBS, dripped with normal goat serum (purchased from Jackson ImmunoResearch), and blocked at 4°C for 45 minutes. The cells were incubated with primary antibodies (pAb anti-Salmonella antibody for labeling bacteria, purchased from Novus, diluted 200 times; Purified anti-mouse Ly-6G Antibody for labeling neutrophils, purchased from Biolegend, 1:500) in the dark at room temperature for 2 h, washed 5 times with PBS, and incubated with secondary antibodies (Goat Anti-Rabbit H&L for labeling bacteria, 1:500; Donkey pAb to Rat IgG for labeling neutrophils, 1:1000, both purchased from Abcam) in the dark at room temperature for 1 h, washed 5 times with PBS, and then covered with anti-fluorescence quenching mounting medium containing DAPI (ProLongTM Gold antifade reagent with The cells were then covered with a coverslip and observed under a fluorescence microscope. The lipopolysaccharide O antigen and flagellin H antigen on the bacterial surface were labeled with an antibody carrying the AF555 fluorescent group, resulting in red fluorescence. Ly-6G, the complex on the lymphocyte membrane surface of neutrophils, was labeled with an antibody carrying the AF488 fluorescent group, resulting in green fluorescence. The cell nuclei stained with DAPI exhibited blue fluorescence. As shown in Figure 8, the elimination of neutrophils by αGr-1 facilitated the spread of bacteria throughout the tumor area.

[0495] Example 6: Neutralizing neutrophils to enhance the therapeutic effect of artificial antitumor bacteria on the 4T1 breast cancer model

[0496] Balb / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., SPF grade) were injected with PBS suspension of 4T1 breast cancer cells (ATCC CRL-2539) into the second mammary fat pad on the right side. 6 Tumor cells / 125 μL were used to construct an orthotopic 4T1 breast cancer model. 20 days after injection, the tumor volume was approximately 200 mm 3125 μL of 1.6 mg / mL InVivoMab anti-mouse Ly6G / Ly6c (Gr-1) antibody was injected into the tail vein to neutralize neutrophils. 24 hours after the injection of the antibody, the tumors and blood of the mice were collected for flow cytometry analysis, and the mice were given 10 7 CFU DB-ZW1, and mice injected with PBS, antibodies only, and DB-ZW1 only were used as controls.

[0497] The results, as shown in Figure 9A, show that after injection of the antibody, over 90% of neutrophils in the 4T1 tumor were eliminated. As shown in Figure 9B, neither injection of the Ly6G / Ly6c (Gr-1) antibody alone nor injection of DB-ZW1 alone inhibited 4T1 tumor growth. However, 4T1 tumors in mice injected with both the antibody and DB-ZW1 significantly shrank, with significant differences compared to all three control groups (P < 0.01). As shown in Figure 9C, on the first day after DB-ZW1 injection, the tumors in the experimental group of mice turned red and dark, forming a noticeable scab. These results suggest that reducing the neutrophil content in tumors can improve the tumor-suppressing effect of the anti-tumor bacteria (DB-ZW1).

[0498] Example 7: In vitro testing of bacterially expressed Nanobodies

[0499] To identify neutrophil binding of E. coli -expressed nanobodies, primary neutrophils were isolated from the blood of C57BL / 6 mice, and the binding of the expressed nanobodies to CXCR2 on the surface of neutrophils was detected.

[0500] Specifically, blood was obtained from mice by taking blood from the eyeball, and red blood cell lysis solution (containing 150mM NH4Cl, 10mM KHCO3 and 0.1mM Na2EDTA, pH 7.2-7.4) was added to the obtained blood, incubated at room temperature for 5min to fully lyse the red blood cells, and the red blood cell fragments were filtered to remove the purified immune cells. The purified immune cells were incubated with Zombie Aqua (purchased from Biolegend) and TruStain FcX (anti-mouse CD16 / 32) (purchased from Biolegend, product number 101320) in the dark for 15min at room temperature for staining to count live and dead cells and block Fc receptors to prevent the generation of nonspecific signals. The purified nanoantibodies (purified by nickel column for his-tag using AKATA protein purifier) ​​were then incubated with the purified immune cells for 1 hour, and cells without nanoantibodies were set as controls. Co-staining for CD45 (Alexa Fluor 700 anti-mouse CD45, Biolegend), His tag (PE anti-His Tag, Biolegend) and Ly6G (anti-mouse, Biolegend) was performed by flow cytometry, and the purity of neutrophil sorting and the binding of nanoantibodies to neutrophils were determined by flow cytometry counting.

[0501] As shown in FIG10 , all three nanobodies expressed by E. coli can bind to neutrophils, among which Nb97A7 has the highest binding rate, exceeding 25%.

[0502] Example 8: Tumor Inhibitory Effect of DB-ZW1 Expressing Nanobodies and Fusion Proteins in a Tumor-Bearing Mouse Model

[0503] The strains constructed in Example 3.4 were used in this example: ZW1-psc101-pFNRsp--psp-Nb127D01-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb97A9-PE38-HA-his, ZW1-psc101-pFNRsp-psp-Nb127D01-PE[LR]-HA-his, ZW1-psc101-pFNRsp-psp-Nb163E3-PE[LR]-HA-his, W1-psc101-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, ZW1-psc101-pFnr-SP-psp-Nb97A9-CM, were used to treat the MB49 subcutaneous tumor-bearing mouse model prepared as described in Example 5.

[0504] The mice injected with the bacterial strain expressing the nanobody or fusion protein were used as the experimental group, the mice injected with the DB-ZW1 strain were used as the control group, and the mice injected with PBS were used as the blank control group. The mice injected with Gr-1 antibody after DB-ZW1 were used as the positive control group. 7 CFU of bacteria were suspended in 125 μL of PBS and injected into mice via tail vein. The body weight of mice was recorded and tumor volume was measured every two days. All mice had similar body weights, indicating that the therapy with the anti-tumor bacteria of the present invention is safe.

[0505] As shown in FIG11 , ZW1-psc101-pFNRsp--psp-Nb127D01-PE38-HA-his showed the best therapeutic effect, significantly improving tumor growth inhibition compared to DB-ZW1.

[0506] At the same time, the blood and solid tumors of the mice were collected on the 4th day after injection, and the neutrophils in the blood and tumors were counted by flow cytometry.

[0507] As shown in Figure 12, compared with mice injected with PBS, the number of neutrophils in the tumors of mice injected with DB-ZW1 increased significantly, while the number of neutrophils in the tumors of positive control mice decreased significantly. The levels of neutrophils in the tumors of mice in other groups were comparable to those of mice injected with PBS. In addition, except for the significantly lower (almost completely eliminated) neutrophil levels in the blood of the positive control group, the neutrophil levels in the blood of the other groups were comparable. This shows that the strain of the present invention can specifically inhibit neutrophils in the tumor location.

[0508] Example 9: IPTG-induced expression of nanobodies and nanobody-fused toxin protein plasmids

[0509] 9.1 Construction of plasmids expressing nanobodies or toxin proteins under IPTG induction

[0510] The purpose of this example is to construct a plasmid expressing nanoantibodies 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), and toxin proteins CET40, DT390 (SEQ ID NO: 80 and 85).

[0511] The coding sequences were synthesized (Qingke Bio) and cloned into the pET26 backbone using the same pelB signal peptide as in Example 3.1, a 6*his tag for Nanobodies 238D2, ​​238D4, EGa1 and VHH1, and the same HA-his tag as in Example 3.1 for Nanobody VHH26 and toxin proteins CET40 and DT390.

[0512] The obtained recombinant plasmids were named as follows:

[0513] pET26b-Kana-T7-pelB-238D2-his (SEQ ID NO:92);

[0514] pET26b-Kana-T7-pelB-238D4-his (SEQ ID NO:93);

[0515] pET26b-Kana-T7-pelB-EGa1-his (SEQ ID NO:94);

[0516] pET26b-Kana-T7-pelB-VHH1-his (SEQ ID NO:95);

[0517] pET26b-Kana-T7-pelB-VHH26-HA-his (SEQ ID NO:96);

[0518] pET26b-Kana-T7-pelB-CET40-HA-his (SEQ ID NO:97);

[0519] pET26b-Kana-T7-pelB-DT390-HA-his (SEQ ID NO:98).

[0520] 9.2 Construction of a plasmid expressing a fusion protein of nanobodies and toxin protein PE38 under IPTG induction

[0521] The purpose of this example is to construct plasmids expressing Nanobodies 238D2, ​​238D4, EGa1, VHH1 and VHH26 fused to toxin proteins.

[0522] As described in Example 3.3, the coding sequence of the toxin protein PE38 was inserted into the plasmid expressing the Nanobody constructed in Example 9.1. The recombinant plasmids obtained are named as follows:

[0523] pET26b-Kana-T7-pelB-Nb238D2-PE38-his (SEQ ID NO:99);

[0524] pET26b-Kana-T7-pelB-Nb238D4-PE38-his (SEQ ID NO: 100);

[0525] pET26b-Kana-T7-pelB-NbEGa1-PE38-his (SEQ ID NO: 101);

[0526] pET26b-Kana-T7-pelB-VHH1-PE38-his (SEQ ID NO: 102);

[0527] pET26b-Kana-T7-pelB-VHH26-PE38-his (SEQ ID NO: 103).

[0528] 9.3 Construction of plasmids expressing fusion proteins of nanobody Nb127D01 and toxin protein CET40 or DT390 under IPTG induction

[0529] As described in Example 3.3, the nucleotide sequences encoding the toxin proteins CET40 and DT390 were inserted into the plasmid expressing the nanobody Nb127D01 (pET26b-pelB-Nb127D01-HA-his). The obtained recombinant plasmids were named as follows:

[0530] pET26b-Kana-T7-pelB-127D01-CET40-HA-his(SEQ ID NO:104)

[0531] pET26b-Kana-T7-pelB-127D01-DT390-HA-his(SEQ ID NO:105)

[0532] 9.4 Construction of strains expressing nanobodies, toxin proteins, and nanobodies fused to toxin proteins under IPTG induction

[0533] The recombinant plasmid was chemically transformed into the recombinant protein expression strain BL21(DE3) (BL21(DE3) Competent E. coli Strain, Vazyme#C504-03). Specifically, thaw the competent cells on ice, mix 100ng of the above recombinant plasmid with the competent cells, and let it stand on ice for 30 minutes. After heat shock in a 42°C water bath for 45 seconds, immediately place it on ice for 3 minutes. Add 900μL LB medium (without antibiotics), shake at 37°C for 1 hour (speed 200-250rpm), centrifuge at 5,000rpm for 5 minutes, discard 900μL of supernatant, resuspend the cells in the remaining medium, and gently spread evenly on an LB plate containing kanamycin (KanR, 100mg / L) using a sterile spreader. Invert the plate and culture overnight in a 37°C incubator.

[0534] The strains obtained that express nanobodies, toxin proteins, and nanobodies fused to toxin proteins under IPTG induction are as follows:

[0535] BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D2-his

[0536] BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D4-his

[0537] BL21(DE3)-pET26b-Kana-T7-pelB-EGa1-his

[0538] BL21(DE3)-pET26b-Kana-T7-pelB-VHH1-his

[0539] BL21(DE3)-pET26b-Kana-T7-pelB-VHH26-his

[0540] BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D2-PE38-his

[0541] BL21(DE3)-pET26b-Kana-T7-pelB-Nb238D4-PE38-his

[0542] BL21(DE3)-pET26b-Kana-T7-pelB-NbEGa1-PE38-his

[0543] BL21(DE3)-pET26b-Kana-T7-pelB-VHH1-PE38-his

[0544] BL21(DE3)-pET26b-Kana-T7-pelB-VHH26-PE38-his

[0545] BL21(DE3)-pET26b-Kana-T7-pelB-CET40-HA-his

[0546] BL21(DE3)-pET26b-Kana-T7-pelB-127D01-CET40-HA-his

[0547] BL21(DE3)-pET26b-Kana-T7-pelB-DT390-HA-his

[0548] BL21(DE3)-pET26b-Kana-T7-pelB-127D01-DT390-HA-his

[0549] 9.5 Prokaryotic Expression of Nanobodies and Nanobodies Fused with Toxin Proteins Under IPTG Induction

[0550] The purpose of this example is to detect the ability of the constructed strain to express heterologous proteins.

[0551] To this end, prokaryotic expression was performed using the BL21 (DE3) strain constructed in Example 9.4. Specifically:

[0552] i) Resuscitate frozen bacteria in liquid culture. When the culture reaches an OD value of 0.4-0.5, add IPTG to a final concentration of 0.5 mM and induce overnight in a shaker at 16°C (14-16 hours, 200-250 rpm). Remove the culture and place it on ice.

[0553] ii) Centrifuge the culture at 4,200 rpm at 4°C for 10 min, and collect the bacterial pellet and LB culture supernatant. Place the LB culture supernatant in an ultrafiltration tube (Amicon Ultra, #UFC901096) and centrifuge at 2,500 rpm at 4°C for 2 h. Collect the concentrated LB culture supernatant, which contains the secreted protein.

[0554] iii) Add lysis buffer (Qiagen, Qproteome Bacterial Protein Prep Kit #37900, refer to the Qproteome Bacterial Protein Preparation Handbook, www.qiagen.com) to the bacterial pellet, incubate on ice for 30 minutes, and then centrifuge at 14,000 rpm at 4°C for 30 minutes. Collect the bacterial pellet and lysis buffer supernatant. The bacterial pellet contains the functional inclusion body protein, and the lysis buffer supernatant contains the expressed, unsecreted soluble protein.

[0555] iv) 5× SDS loading buffer (Biyuntian, #P0015) was added to the collected samples (concentrated LB culture supernatant, bacterial fragment pellet, and lysate supernatant) and incubated at 100°C in a metal bath for 5 min. SDS-PAGE was then performed: electrophoresis was performed at 80 V for 20 min. After the sample was transferred to the interface of the separating gel and the stacking gel, the voltage was increased to 120 V and continued until the dye completely left the gel.

[0556] v) According to the instructions, use Pyxis transfection transfer membrane (PVDF membrane) and transfection instrument to transfer the membrane. After transfer, the membrane was washed with TBS buffer and TBST buffer in sequence; the PVDF membrane was blocked with 5% skim milk (dissolved in TBST buffer) at room temperature for 1 h; the primary antibody (6*his, his-tag monoclonal antibody, Proteintech, #66005-1-lg) was diluted at a ratio of 1:10000 in 5% skim milk and incubated overnight at 4°C; the PVDF membrane after overnight incubation was washed several times with TBST buffer to remove the antibodies on the membrane surface; the secondary antibody (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L), Proteintech, #SA00001-2-100UL) was diluted at a ratio of 1:5000 in 5% skim milk and incubated at room temperature for 1 h; the incubated PVDF membrane was washed several times with TBST buffer to remove unbound antibodies on the membrane surface, and finally the membrane was immersed in TBS. The prepared developing AB solution was mixed in a 1:1 ratio, the membrane was placed in a tray, the AB solution was poured in, and the exposure reaction was performed for 5 minutes and imaging was performed using a Jena multifunctional imager (Thermo Fisher, SuperSignal TM West Pico PLUS Chemiluminescent Substrate).

[0557] As shown in Figure 13 below, all heterologous proteins were successfully expressed under the induction of IPTG.

[0558] Example 10: Anaerobic expression of nanobodies and nanobody-fused toxin proteins

[0559] First, plasmids and strains for anaerobically expressing nanobodies and nanobody-fused toxin proteins are constructed.

[0560] Plasmids constructed according to Example 9: 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-pelB-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 na-T7-pelB-VHH1-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, pET26b-Kana-T7-pelB-127D01-DT390-HA-his were used as templates to amplify linear fragments encoding Nanobodies, toxin proteins, or Nanobodies fused to toxin proteins, and pSC101-pFNRsp was used as a template to amplify the vector backbone, and the linear fragment of the vector backbone was amplified by PCR. The primers used are shown in Table 5, and the PCR reaction system and conditions are as described in Example 3.2.

[0561] Table 5

[0562] The obtained plasmids are as follows:

[0563] pSC101-Cm-FNRSP-0033-pelB-238D2-His (SEQ ID NO: 106);

[0564] pSC101-Cm-FNRSP-0033-pelB-238D4-His (SEQ ID NO: 107);

[0565] pSC101-Cm-FNRSP-0033-pelB-EGa1-His (SEQ ID NO: 108);

[0566] pSC101-Cm-FNRSP-0033-pelB-VHH1-His (SEQ ID NO: 109);

[0567] pSC101-Cm-FNRSP-0033-pelB-VHH26-His (SEQ ID NO: 110);

[0568] pSC101-Cm-FNRSP-0033-pelB-238D2-PE38-His (SEQ ID NO: 111);

[0569] pSC101-Cm-FNRSP-0033-pelB-238D4-PE38-His (SEQ ID NO: 112);

[0570] pSC101-Cm-FNRSP-0033-pelB-EGa1-PE38-His (SEQ ID NO: 113);

[0571] pSC101-Cm-FNRSP-0033-pelB-VHH1-PE38-His (SEQ ID NO: 114);

[0572] pSC101-Cm-FNRSP-0033-pelB-VHH26-PE38-His (SEQ ID NO: 115);

[0573] pSC101-Cm-FNRSP-0033-pelB-CET40-HA-His (SEQ ID NO: 116);

[0574] pSC101-Cm-FNRSP-0033-pelB-DT390-HA-His (SEQ ID NO: 117);

[0575] pSC101-Cm-FNRSP-0033-pelB-127D01-CT390-HA-His (SEQ ID NO: 118);

[0576] pSC101-Cm-FNRSP-0033-pelB-127D01-CET40-HA-His (SEQ ID NO: 119).

[0577] The above plasmids were introduced into competent cells of strain DB-ZW1 by electroporation (E=18 kV / cm). The following strains were obtained:

[0578] ZW1-pSC101-Cm-FNRSP-0033-pelB-238D2-His;

[0579] ZW1-pSC101-Cm-FNRSP-0033-pelB-238D4-His;

[0580] ZW1-pSC101-Cm-FNRSP-0033-pelB-EGa1-His;

[0581] ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH1-His;

[0582] ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH26-His;

[0583] ZW1-pSC101-Cm-FNRSP-0033-pelB-238D2-PE38-His;

[0584] ZW1-pSC101-Cm-FNRSP-0033-pelB-238D4-PE38-His;

[0585] ZW1-pSC101-Cm-FNRSP-0033-pelB-EGa1-PE38-His;

[0586] ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH1-PE38-His;

[0587] ZW1-pSC101-Cm-FNRSP-0033-pelB-VHH26-PE38-His;

[0588] ZW1-pSC101-Cm-FNRSP-0033-pelB-CET40-HA-His;

[0589] ZW1-pSC101-Cm-FNRSP-0033-pelB-DT390-HA-His;

[0590] ZW1-pSC101-Cm-FNRSP-0033-pelB-127D01-CT390-HA-His;

[0591] ZW1-pSC101-Cm-FNRSP-0033-pelB-127D01-CET40-HA-His.

[0592] As described in Examples 4 and 8, the anaerobic expression and tumor treatment effects of the fusion protein of the above strains were tested.

[0593] Sequence Listing

Claims

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

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

3. The fusion protein of claim 2, wherein the antibody comprises a complementarity determining region (CDR) selected from the group consisting of: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO:20, a CDR2 comprising residues 50-67 of SEQ ID NO:20, and a CDR3 comprising residues 100-111 of SEQ ID NO:20; and A CDR1 comprising residues 31-35 of SEQ ID NO:21, a CDR2 comprising residues 50-64 of SEQ ID NO:21, and a CDR3 comprising residues 98-115 of SEQ ID NO:

21.

4. The fusion protein of any one of claims 1-3, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 20 and 21.

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

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

25.

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

8. The fusion protein of any one of claims 1 to 7, further comprising a signal peptide. 9 . The fusion protein of claim 8 , wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.

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

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

12. The expression construct of claim 11, wherein the antibody is a Nanobody.

13. The expression construct of claim 12, wherein the antibody comprises a complementarity determining region (CDR) selected from: a CDR1 comprising residues 31-35 of SEQ ID NO: 19, a CDR2 comprising residues 50-65 of SEQ ID NO: 19, and a CDR3 comprising residues 98-104 of SEQ ID NO: 19; a CDR1 comprising residues 31-35 of SEQ ID NO:20, a CDR2 comprising residues 50-67 of SEQ ID NO:20, and a CDR3 comprising residues 100-111 of SEQ ID NO:20; and A CDR1 comprising residues 31-35 of SEQ ID NO:21, a CDR2 comprising residues 50-64 of SEQ ID NO:21, and a CDR3 comprising residues 98-115 of SEQ ID NO:

21.

14. The expression construct of any one of claims 11-13, 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 of any one of claims 11 to 14, wherein the nucleotide sequence further encodes a signal peptide.

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

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

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

19. The expression construct of 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, wherein the bacterium comprises the expression construct of any one of claims 11 to 19 compared to an unmodified starting strain.

21. The modified bacterium of claim 20, wherein the bacterium comprises a hypoxia-regulatable essential gene expression cassette comprising an essential gene of the bacterium controlled by a strict hypoxia-inducible promoter, and the bacterium lacks at least one gene involved in or regulating an endogenous anti-oxidative stress response pathway or a functional expression product thereof, compared to an unmodified starting strain.

22. The modified bacterium of claim 21, wherein the strict 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.

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

24. The modified bacterium of any one of claims 21-23, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelate (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on the additional addition of DAP or its analogues in the culture medium.

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

26. The modified bacterium of claim 24, wherein the essential gene is selected from the group consisting of dapA and dapE.

27. The modified bacterium of any one of claims 21-26, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is a HtrA serine protease family gene.

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

29. The modified bacterium of any one of claims 21-28, wherein the bacterium lacks HtrA serine protease activity.

30. The modified bacterium of any one of claims 21-29, wherein the gene involved in or regulating an endogenous anti-oxidative stress response pathway is htrA.

31. The modified bacterium of any one of claims 21-30, wherein the bacterium lacks htrA.

32. The modified bacterium of any one of claims 21-31, wherein the essential gene is a gene naturally present in the bacterial chromosome, wherein the natural promoter of the essential gene is functionally replaced by the strict hypoxia inducible promoter, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia inducible promoter.

33. The modified bacterium of any one of claims 21-31, wherein the essential gene expression cassette is exogenous and the essential gene naturally present in the bacterial chromosome is deleted or functionally inactivated, whereby the expression of the essential gene in the bacterium is completely controlled by the strict hypoxia inducible promoter.

34. The modified bacterium of claim 33, wherein the exogenous essential gene expression cassette is integrated into the chromosome of the bacterium.

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

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

37. The modified bacterium of any one of claims 21-36, further comprising a pH-regulatory expression cassette comprising a gene encoding a bacterially derived hemolysin protein under the control of a promoter active under acidic pH conditions.

38. The modified bacterium of claim 37, wherein the bacterial-derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

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

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

41. The modified bacterium of any one of claims 37-40, wherein the promoter active under acidic pH conditions is active at a pH value 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.

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

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

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

45. The modified bacterium of any one of claims 21-44, wherein the bacterium is an Enterobacteriaceae bacterium.

46. ​​The modified bacterium of any one of claims 21-45, wherein the bacterium is an Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea bacterium.

47. The modified bacterium of any one of claims 21-46, wherein the bacterium is selected from the group consisting of Escherichia coli, E. blattae, E. fergusonii, E. hermannii, E. vulneris, S. enterica, S. bongori, S. typhi, S. choleraesuis, S. typhimurium, S. dysenteriae, S. flexneri, S. bournei, (S.boydii), Shigella sonnei (S.sonnei), Klebsiella pneumoniae (K.peumoniae), Klebsiella oxytoca (K.oxytoca), Yersinia pestis (Y.pestos), Yersinia enterocolitica (Y.ent-ocolitica), Yersinia pseudotuberculosis (Y.pseudotuberculosis), Yersinia aldouae (Y.aldouae), Yersinia bercov-ieri (Y.bercov-ieri), Yersinia frederiksenii (Y.frederiksenii), Yersinia intermedia (Y.intermedia), Yersinia kristensii (Y.kristersenii), Yersinia mollaretti, Yersinia rohdei, Yersinia ruckeri, C.freundii, C.kaseri, C.braakii, E.aerogenes, E.cloacae, E.gergoviac, E.sakazakii, E.tavlorac, E.aminigenus, E.intermedius, E. asburiac, E. cancerogenus, E. dissolvens, E. nimipressualis, S. marcescens, S. entomophila, S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S. odorifera, S. plymuthica, S.proteamaculans), S. rubidaea, S. ureilytica, P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri, P. hauseri, M. morganii, P. alcalifaciens, P. rustigianii, P. stuartii, P. rettgeri, P. heimbochae, H. alvei, and P. agglomerans.

48. The modified bacterium of any one of claims 21-47, wherein the bacterium is Salmonella typhimurium.

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

50. The modified bacterium of any one of claims 21-49, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

51. The modified bacterium of any one of claims 21-50, wherein when administered to a subject having a tumor, the bacterium is able to survive and proliferate in tumor tissue but is rapidly cleared from normal tissue.

52. The modified bacterium of any one of claims 21-51, wherein when administered to a subject having a tumor, the bacterium is capable of expressing and secreting the antibody or antigen-binding fragment thereof that binds to neutrophils, or the fusion protein of any one of claims 1-10 in tumor tissue.

53. The modified bacterium of claim 52, wherein the secreted antibody or antigen-binding fragment thereof or fusion protein is capable of inhibiting the recruitment of neutrophils or killing of neutrophils.

Images