Bacteria that secrete interleukin 10

Abstract

The present invention relates to a fusion polypeptide comprising an IL10 polypeptide, which can be prokaryotically expressed and secreted in bacterial cells. The present invention also relates to a modified bacterium having anti-tumor activity in tumors and the ability to prokaryotically express and secrete an IL10 polypeptide, wherein the bacterium comprises an essential gene expression cassette controlled by an obligate hypoxia-inducible promoter, and the bacterium is deficient in at least one gene, or its functional expression product, involved in or regulating an endogenous antioxidant stress response pathway. The present invention also relates to a pharmaceutical composition comprising the modified bacterium and its anti-tumor use.

Description

[Technical Field]

[0001] The present invention relates to the field of genetic engineering. In particular, the present invention relates to a bacterium having antitumor activity that has been genetically modified to secrete interleukin-10 in situ, a pharmaceutical composition containing the modified bacterium, and its antitumor use.

[0002] [background] Cancer has become one of the most widespread diseases threatening human life and health. However, existing treatments (radiotherapy, chemotherapy, surgery, and targeted drugs) all have shortcomings, and there is an urgent need to develop novel treatment approaches. Conventional bacterial tumor therapy, typified by Coli's toxin, has a 150-year history but is known for its unstable efficacy and safety issues. Although the development of genetic engineering technology has made remarkable progress in reducing the virulence of bacterial strains, a series of clinical trials have demonstrated that attenuated engineered bacteria for human tumor therapy are safe but have limited efficacy, i.e., it is difficult to balance safety and therapeutic efficacy, and therefore cannot meet the need for tumor therapy. Further optimization of tumor therapy is essential.

[0003] Interleukin-10 (IL-10) is a cytokine primarily produced by Th2 cells. IL-10 can inhibit the production of other lymphokines but is also a major anti-inflammatory factor. IL-10 has certain therapeutic effects against transplant rejection, inflammation, and other conditions. Interleukin-10 was originally thought to be a B cell-derived T cell growth factor (B-TCGF), and therefore its function was thought to be to stimulate the activation of CD8+ T cells (MacNeil IA et al., The Journal of Immunology, 1990). Researchers subsequently found that interleukin-10 can also induce the expression of CD3 and CD8 molecules in thymocytes and activate the cytotoxicity of CD8+ T cells (Chen WF et al., The Journal of Immunology, 1991). In addition, interleukin-10 can enhance the proliferation of CD8+ T cells by directly stimulating T cell receptor signaling via anti-CD3 monoclonal antibodies.

[0004] When interleukin-10 was used in immunotherapy, it was initially found that interleukin-10 knockout mice were particularly susceptible to chemically induced skin cancer, and tumors developed more rapidly and metastasized earlier in the interleukin-10 knockout mice. Intratumoral CD8+ T cells, MHC molecules, and granzymes were all inhibited in tumors in interleukin-10 knockout mice. In contrast, in transgenic mice overexpressing interleukin-10, the above molecules were increased upon interleukin-10 overexpression (Mumm JB, Emmerich J, Zhang X, Chan I, Wu L, Mauze S, Blaisdell S, Basham B, Dai J, Grein J, Sheppard C, Hong K, Cutler C, Turner S, LaFace D, Kleinschek M, Judo M, Ayanoglu G, Langowski J, Gu D, Paporello B, Murphy E, Sriram V, Naravula S, Desai B, Medicherla S, Seghezzi W, McClanahan T, Cannon-Carlson S, Beebe AM, Oft M. IL-10 elicits IFNγ-dependent tumor immune surveillance. Cancer. Cell. 2011 Dec 13;20(6):781-96. doi:10.1016 / j.ccr.2011.11.003. PMID:22172723). Martin Soft et al. found that injecting pegylated interleukin-10 (interleukin-10 chemically modified to extend its half-life) into tumor-bearing mice significantly increased IFN-γ and granzyme expression in the tumor. They also found that pegylated interleukin-10 activated durable immune memory against tumors in vivo and protected mice against tumor rechallenge 8 months after initial tumor rejection.This sustained tumor immunity may be due to interleukin-10-induced phosphorylation of STAT1 and STAT3 in intratumoral CD8+ T cells, which activates a signaling pathway specific to intratumoral CD8+ T cells and promotes the secretion of the cytokine IFN-γ. (Mumm JB, Emmerich J, Zhang S, Desai B, Medicherla S, Seghezzi W, McClanahan T, Cannon-Carlson S, Beebe AM, Oft M.IL-10 elicits IFNγ-dependent tumor immune surveillance. Cancer Cell. 2011 Dec 13; 20(6): 781-96.doi:10.1016 / j.ccr.2011.11.003.PMID:22172723). However, in lymphoid-derived CD4+ or CD8+ T cells, interleukin-10 can only induce STAT3 phosphorylation in the cells, but not IFN-γ secretion. (Emmerich J, Mumm JB, Chan IH, LaFace D, Truong H, McClanahan T, Gorman DM, Oft M. IL-10 directly activates and expands tumor-resident CD8(+) T cells without de novo infiltration from secondary lymphoid organs. Cancer Res. 2012 Jul 15;72(14):3570-81. doi:10.1158 / 0008-5472. CAN-12-0721. Epub 2012 May 11. PMID:22581824)STAT1 is particularly important in the induction of IFN-γ because it induces the production of the cell-specific transcription factor T-bet, which is a transcription factor for IFN-γ-producing Th1 and Tc1 T cells (Afkarian M, Sedy JR, Yang J, Jacobson NG, Cereb N, Yang SY, Murphy TL, Murphy KM. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat Immunol. 2002 Jun;3(6):549-57. doi:10.1038 / ni794. Epub 2002 May 13, PMID:12006974). T-bet acts synergistically with the TCR-activated transcription factor NFAT to induce the production of IFN-γ and granulase in cytotoxic T cells (Glimcher LH, Townsend MJ, Sullivan BM, Lord GM. Recent developments in the transcriptional regulation of cytolytic effector cells. Nat Rev Immunol. 2004 Nov;4(11):900-11. doi:10.1038 / nri1490. PMID:15516969). Thus, PEGylated interleukin-10 increased IFN-γ, granzymes, and perforin in cytotoxic T cells only under TCR stimulation. Additionally, intratumoral IFN-γ produced by CD8+ T cells is thought to be involved in the induction of MHC class I and class II molecules, enabling antigen presentation in tumors.In human tumors, the expression of MHC molecules is highly correlated with improved patient prognosis (Walsh MD, Dent OF, Young JP, Wright CM, Barker MA, Leggett BA, Bokey L, Chapuis PH, Jass JR, Macdonald GA. HLA-DR expression is associated with better prognosis in sporadic Australian clinically mediated stage C colorectal cancers. Int J Cancer. 2009 Sep 1;125(5):1231-7. doi:10.1002 / ijc.24484. PMID:19462453.). In summary, interleukin-10 can ameliorate tumor-associated inflammatory responses and tumor immunodeficiency in tumor treatment, indicating that interleukin-10 may represent a novel approach with broad prospects for treating cancer patients.

[0005] Therefore, bacteria with antitumor activity that can secrete interleukin-10 in situ have improved effects on tumor treatment. However, even if bacteria can express IL-10, it is difficult to secrete the expressed IL-10 extracellularly. Therefore, there is a need to improve the expression method and prepare bacteria with antitumor activity that can secrete IL-10 in situ.

[0006] [Summary of the Invention] In the present invention, a fusion polypeptide is constructed by linking the amino acid sequence of mature IL-10 to a signal peptide (which directs extracellular secretion of proteins in prokaryotic cells) via a peptide linker containing 1 to 10 acidic amino acid residues. The fusion polypeptide of the present disclosure can be secreted extracellularly when expressed in prokaryotic cells.

[0007] According to the present invention, a synthetic biology genetic circuit is used to construct an obligate anaerobic bacterium that exhibits therapeutic effects against tumors after being administered to an animal or human body and can be excreted by normal tissues and organs in a short period of time, thereby reducing the toxic and side effects caused to the animal or human body by maintaining the bacterium in vivo for a long period of time, and thus providing more reliable anti-tumor ability and safety.

[0008] In a first aspect, the present invention provides a fusion polypeptide comprising a signal peptide, an interleukin-10 (IL-10) polypeptide, and a peptide linker connecting the signal peptide and the IL-10 polypeptide, wherein the peptide linker comprises 1 to 10 acidic amino acid residues, and wherein, when expressed intracellularly, the signal peptide directs the extracellular secretion of the IL-10 polypeptide.

[0009] In a second aspect, the present invention provides polynucleotides encoding the fusion polypeptides of the invention, vectors comprising the polynucleotides, and host cells comprising the polynucleotides and / or vectors.

[0010] In a third aspect, the present invention provides a method for the prokaryotic expression of an IL-10 polypeptide, comprising: i) transforming a bacterial cell with a vector of the invention; ii) culturing the transformed cells under conditions that allow expression of IL-10 by the vector; The present invention provides a method comprising:

[0011] In a fourth aspect, the present invention provides a modified bacterium, wherein the bacterium, compared to an unmodified starting strain, comprises one or more expression cassettes for expressing a polynucleotide of the invention and an essential gene, the expression of which is controlled by a strictly hypoxia inducible promoter, and wherein the bacterium is deficient in at least one gene, or its functional expression product, involved in or regulating an endogenous antioxidant stress response pathway.

[0012] The invention also provides modified bacteria, wherein the bacteria, compared to an unmodified starting strain, comprise one or more expression cassettes for expressing a polynucleotide of the invention and an essential gene whose expression is controlled by an obligate hypoxia-inducible promoter, and the bacterium is deficient in at least one gene, or functional expression product thereof, required for survival of the bacterium within macrophages.

[0013] The present invention also provides modified bacteria, wherein the bacterium comprises, compared to an unmodified starting strain, one or more expression cassettes for expressing a polynucleotide of the invention, an essential gene whose expression is controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette comprising a gene encoding a bacterial hemolysin protein controlled by a promoter active under acidic pH conditions, and wherein the bacterium is deficient in at least one gene or its functional expression product involved in or regulating an endogenous antioxidant stress response pathway.

[0014] The present invention also provides modified bacteria, wherein the bacterium, compared to an unmodified starting strain, comprises one or more expression cassettes for expressing a polynucleotide of the invention, an essential gene whose expression is controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette comprising a gene encoding a bacterial hemolysin protein controlled by a promoter active under acidic pH conditions, wherein the bacterium expresses wild-type lipopolysaccharide (LPS), and the bacterium is deficient in at least one gene or functional expression product thereof required for survival within macrophages.

[0015] The present invention also provides a modified Salmonella typhimurium bacterium, wherein the bacterium comprises one or more expression cassettes for expressing a polynucleotide of the invention, an essential gene whose expression is controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette comprising a gene encoding a hemolysin protein derived from the bacterium controlled by a promoter active under acidic pH conditions, compared to an unmodified starting strain, and wherein the bacterium is deficient in at least one gene or functional expression product thereof that is involved in or controls an endogenous antioxidant stress response pathway.

[0016] The present invention also provides a modified Salmonella typhimurium bacterium, wherein the bacterium, compared to an unmodified starting strain, comprises one or more expression cassettes for expressing a polynucleotide of the invention, an essential gene whose expression is controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette comprising a gene encoding a hemolysin protein derived from the bacterium controlled by a promoter active under acidic pH conditions, and wherein the bacterium expresses wild-type lipopolysaccharide (LPS) and is deficient in at least one gene or its functional expression product required for the bacterium to survive within macrophages.

[0017] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the modified bacterium of the present invention. In one embodiment, the pharmaceutical composition is used to treat malignant tumors.

[0018] The present invention also provides a method for treating a malignant tumor, the method comprising the step of administering to a subject suffering from a malignant tumor the modified bacterium or pharmaceutical composition of the present invention.

[0019] The present invention provides the use of the modified bacterium of the present invention in the manufacture of a medicament for treating malignant tumors. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows Western blot results demonstrating the effect of the presence and absence of the D5 peptide linker on prokaryotic expression of IL-10. Left: Protein expression of strain BL21(DE3)-plasmid-pET22b-pelB-D5-huil10-Amp in pellets (pellet), lysate supernatants (supernatant), and concentrated LB supernatants (LB). Right: Protein expression of strain BL21(DE3)-plasmid-pET22b-pelB-huil10-Amp (control without the D5 linker peptide) in pellets (pellet), lysate supernatants (supernatant), and concentrated LB supernatants (LB). [Figure 2] Western blot results showing the effect of the presence and absence of the D5 peptide linker on prokaryotic expression of IL-10. Right: Protein expression of strain BL21(DE3)-plasmid pSC101-ptac-pelB-D5-huil10-KanR in pellets (pellet), lysate supernatants (supernatant), and concentrated LB supernatants (LB). Left: Protein expression of strain BL21(DE3)-plasmid pSC101-ptac-pelB-huil10-KanR (control without D5 linker peptide) in pellets (pellet), lysate supernatants (supernatant), and concentrated LB supernatants (LB). [Figure 3] Figure 1 shows Western blot results of fusion polypeptides containing different peptide linkers expressed by inducible expression plasmids, where each fusion polypeptide was individually detected as an insoluble expression product (INS), a bacterial soluble product (S), and a supernatant secreted product (SUP) (from left to right). [Figure 4] FIG. 1 shows an outline of the construction of the DB-ZW1 strain. [Figure 5] FIG. 1 shows the distribution of DB-ZW1 in tumors and tissues over time. [Figure 6]6A-6C show the antitumor effects of DB-ZW1 on bladder cancer, melanoma, and in situ colon cancer. In particular, Figures 6A-6C illustrate the effects of DB-ZW1 on the tumor volume of subcutaneous bladder cancer, in situ melanoma, and subcutaneous in situ colon cancer, respectively, and Figure 6D illustrates the effect of DB-ZW1 on the tumor number of in situ colon cancer. [Figure 7] Expression and secretion of human interleukin-10 in E. coli Nissle and Salmonella typhimurium DB-ZW1. The left panel is a Western blot image of the expression products (INS: insoluble expression product, S: bacterial soluble product, and SUP: supernatant secreted product), and the right panel is a quantitative analysis of the secreted expression products. [Figure 8] Figure 1 shows Western blot images of interleukin-10 expression and secretion by E. coli Nissle and Salmonella typhimurium DB-ZW1 (INS: insoluble expression product, S: intrabacterial soluble product, and SUP: supernatant secreted product). [Figure 9] FIG. 1 shows the antitumor effect of E. coli Nissle cells expressing the fusion polypeptide of the present invention. [Figure 10] FIG. 1 shows the weight change of tumor-bearing mice treated with E. coli Nissle expressing the fusion polypeptide of the present invention.

[0021] [Detailed Description of the Invention] Embodiment 1: A fusion polypeptide comprising a signal peptide, an interleukin-10 (IL-10) polypeptide, and a peptide linker connecting the signal peptide and the IL-10 polypeptide, wherein the peptide linker comprises 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues, and wherein, when expressed intracellularly, the signal peptide directs the extracellular secretion of the IL-10 polypeptide.

[0022] Embodiment 2: The peptide linker is i) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 acidic amino acid residues (e.g., D and / or E); ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral amino acid residues (e.g., N); and / or iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 basic amino acid residues (e.g., K) 2. The fusion polypeptide of embodiment 1, comprising:

[0023] Embodiment 3: The peptide linker is consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glutamic acid residues; consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aspartic acid residues; or 3. The fusion polypeptide of embodiment 1 or 2, consisting of a total of 2, 3, 4, 5, 6, 7, 8, 9, or 10 glutamic acid and aspartic acid residues.

[0024] Embodiment 4: The fusion polypeptide of any one of embodiments 1 to 3, wherein the peptide linker comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) D segment consisting of at least one (e.g., 1, 2, 3, 4, or 5) aspartic acid residue, and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) E segment consisting of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) glutamic acid residue, and the D and E segments are arranged in an alternating configuration.

[0025] Embodiment 5: The fusion polypeptide of embodiment 4, wherein the peptide linker comprises one D segment and one E segment.

[0026] Embodiment 6: The peptide linker is D1E1, D2E1, D1E2, D3E1, D2E2, D1E3, D4E1, D3E2, D2E3, D1E4, D5E1, D4E2, D3E3, D2E4, D1E5, D6E1, D5E2, D4E3, D3E4, D2E5, D1E6, D7E1, D6E2, D5E3, D4E4, D3E5, D2E6, D1E7, D8E1, D7E2, D6E3, D5E4, D4E5, D3E6, D2E7, D1E8, D9E1, D8E1, D7E2, D6E3, D5E4, D4E5, D3E6, D2E7, or D1E8 E1D1, E2D1, E1D2, E3D1, E2D2, E1D3, E4D1, E3D2, E2D3, E1D4, E5D1, E4D2, E3D3, E2D4, E1D5, E6D1, E5D2, E4D3, E3D4, E2D5, E1D6, E7D1, E6D2, E5D3, E4D4, E3D5, E2D6, E1D7, E8D1, E7D2, E6D3, E5D4, E4D5, E3D6, E2D7, E1D8, E9D1, E8D1, E7D2, E6D3, E5D4, E4D5, E3D6, E2D7, or E1D8 6. The fusion polypeptide of embodiment 5, comprising the amino acid sequence of:

[0027] Embodiment 7: The fusion polypeptide of embodiment 1 or 2, wherein the peptide linker comprises a total of 2, 4, 5, 6, 7, 8, 9, or 10 acidic and neutral amino acid residues.

[0028] Embodiment 8: The fusion polypeptide of embodiment 1, 2, or 7, wherein the peptide linker consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0029] Embodiment 9: The fusion polypeptide of embodiment 1, 2, 7, or 8, wherein the peptide linker comprises at least one (e.g., 1, 2, 3, 4, or 5) X segment consisting of at least one (e.g., 1, 3, 4, 5, 6, 7, 8, or 9) acidic amino acid residue, and at least one (e.g., 1, 2, 3, 4, or 5) Z segment consisting of at least one (e.g., 1, 2, 4, 5, 6, 7, 8, or 9) neutral amino acid residue, wherein the X and Z segments are arranged in an alternating configuration.

[0030] Embodiment 10: A fusion polypeptide according to any one of embodiments 7 to 9, wherein the acidic amino acid residue is D and the neutral amino acid residue is N.

[0031] Embodiment 11: The fusion polypeptide of embodiment 10, wherein the peptide linker comprises one X segment and one Z segment.

[0032] Embodiment 12: The peptide linker is D1N1, D2N1, D1N2, D3N1, D2N2, D1N3, D4N1, D3N2, D2N3, D1N4, D5N1, D4N2, D3N3, D2N4, D1N5, D6N1, D5N2, D4N3, D3N4, D2N5, D1N6, D7N1, D6N2, D5N3, D4N4, D3N5, D2N6, D1N7, D8N1, D7N2, D6N3, D5N4, D4N5, D3N6, D2N7, D1N8, D9N1, D8N1, D7N2, D6N3, D5N4, D4N5, D3N6, D2N7, or D1N8 N1D1, N2D1, N1D2, N3D1, N2D2, N1D3, N4D1, N3D2, N2D3, N1D4, N5D1, N4D2, N3D3, N2D4, N1D5, N6D1, N5D2, N4D3, N3D4, N2D5, N1D6, N7D1, N6D2, N5D3, N4D4, N3D5, N2D6, N1D7, N8D1, N7D2, N6D3, N5D4, N4D5, N3D6, N2D7, N1D8, N9D1, N8D1, N7D2, N6D3, N5D4, N4D5, N3D6, N2D7, or N1D8 12. The fusion polypeptide of embodiment 11, comprising the amino acid sequence:

[0033] Embodiment 13: The fusion polypeptide of any one of embodiments 1 to 12, wherein the IL-10 polypeptide is a human IL-10 polypeptide or a variant thereof.

[0034] Embodiment 14: The fusion polypeptide of any one of embodiments 1 to 13, wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15, or an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 12, 13, 14, or 15, or an amino acid sequence that contains 1 to 20, 1 to 15, 1 to 10, or 1 to 5, such as 1, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., or more amino acid residue substitutions, insertions, deletions, and / or additions, preferably conservative substitutions, compared to SEQ ID NO: 12, 13, 14, or 15.

[0035] Embodiment 15: The signal peptide is i) signal peptides derived from T1SS secretion systems, such as HlyA; ii) signal peptides from T2SS secretion systems, such as pelB, ompA, ompF, ompC, ompT, PhoA, PhoE, and LPP; and iii) SicP-S ptP-dependent signal peptides derived from the T3SS secretion system 15. The fusion polypeptide according to any one of embodiments 1 to 14, wherein the signal peptide is selected from the group consisting of: e.g., the signal peptide is a pelB signal peptide, and in particular the signal peptide comprises the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 16, or an amino acid sequence which comprises 1 to 10, or 1 to 5, such as 1, 2, 3, 4, 5, 8, 9, or 10, amino acid residue substitutions, insertions, deletions, and / or additions, preferably conservative substitutions, compared to SEQ ID NO: 16.

[0036] Embodiment 16: The fusion polypeptide according to any one of embodiments 1 to 15, comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, or an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, or an amino acid sequence which comprises 1 to 20, 1 to 15, 1 to 10, or 1 to 5, such as 1, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., or more amino acid residue substitutions, insertions, deletions, and / or additions, preferably conservative substitutions, compared to SEQ ID NO: 17, 18, 19, or 20.

[0037] Embodiment 17: A polynucleotide encoding a fusion polypeptide according to any one of embodiments 1 to 16.

[0038] Embodiment 18: An expression cassette comprising the polynucleotide of embodiment 17, operably linked to a promoter.

[0039] Embodiment 19: An expression vector comprising the polynucleotide of embodiment 17 or the expression cassette of embodiment 18.

[0040] Embodiment 20: A host cell comprising the polynucleotide of embodiment 17 or the expression vector of embodiment 18, such as Escherichia coli (e.g., Nissle and BL21(DE3) strains), Salmonella typhimurium (e.g., SL7207 strain), and Bacillus subtilis.

[0041] Embodiment 21: i) transforming a bacterial cell with the expression vector of embodiment 19; ii) culturing the transformed cells under conditions that allow expression of the vector, preferably secretion of IL-10; 1. A method for the prokaryotic expression of an IL-10 polypeptide, comprising:

[0042] Embodiment 22: The method of embodiment 21, wherein the bacterial cell is a bacterial cell selected from the group consisting of Escherichia coli (e.g., Nissle strain and BL21(DE3) strain, etc.), Salmonella typhimurium (e.g., SL7207 strain, etc.), and Bacillus subtilis.

[0043] Embodiment A Embodiment A1: The bacterium has, compared to the unmodified starting strain: i) a hypoxia-regulated essential gene expression cassette comprising the expression cassette of embodiment 18 and an essential gene of said bacterium, said essential gene being controlled by an obligate hypoxia-inducible promoter; or ii) An expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter. wherein the bacterium is defective in at least one gene or functional expression product thereof that is involved in or controls an endogenous antioxidant stress response pathway.

[0044] Embodiment A2: The modified bacterium of embodiment A1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0045] Embodiment A3: The modified bacterium of embodiment A1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0046] Embodiment A4: The modified bacterium of any one of the preceding embodiments A, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the culture medium.

[0047] Embodiment A5: The modified bacterium of embodiment A4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0048] Embodiment A6: The modified bacterium of embodiment A4, wherein the essential genes are selected from dapA and dapE.

[0049] Embodiment A7: The modified bacterium of any one of the preceding embodiments A, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

[0050] Embodiment A8: The modified bacterium of any one of the preceding embodiments A, wherein the functional expression product of a gene involved in or regulating an endogenous antioxidant stress response pathway is an HtrA serine protease family related protein.

[0051] Embodiment A9: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is defective in HtrA serine protease activity.

[0052] Embodiment A10: The modified bacterium of any one of the preceding embodiments A, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0053] Embodiment A11: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is defective with respect to htrA.

[0054] Embodiment A12: The modified bacterium of any one of embodiments A1 to A11, wherein the essential gene is a gene that is naturally occurring in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0055] Embodiment A13: The modified bacterium of any one of embodiments A1 to A11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is entirely under the control of an obligate hypoxia-inducible promoter.

[0056] Embodiment A14: A modified bacterium according to embodiment A13, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 that is controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0057] Embodiment A15: A modified bacterium according to embodiment A13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or wherein the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0058] Embodiment A16: A modified bacterium according to embodiment A15, wherein an exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is present in a plasmid carried by the bacterium.

[0059] Embodiment A17: The modified bacterium of any one of the preceding embodiments A, further comprising a pH-regulated expression cassette, the pH-regulated expression cassette comprising a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions.

[0060] Embodiment A18: The modified bacterium according to embodiment A17, wherein the bacterial-derived hemolysin protein is a hemolysin protein of a Gram-negative bacterium.

[0061] Embodiment A19: The modified bacterium of embodiment A18, wherein the gene encoding a hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0062] Embodiment A20: The modified bacterium of embodiment A19, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0063] Embodiment A21: The modified bacterium of any one of embodiments A17 to A20, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0064] Embodiment A22: The modified bacterium of any one of embodiments A17 to A21, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0065] Embodiment A23: The modified bacterium of any one of embodiments A17 to A21, wherein the promoter that is active under acidic pH conditions is sseA.

[0066] Embodiment A24: The modified bacterium of any one of the preceding embodiments A, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0067] Embodiment A25: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is a bacterium of the family Enterobacteriaceae.

[0068] Embodiment A26: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is a bacterium of the genus Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, or Pantoea.

[0069] Embodiment A27: The bacterium is Escherichia coli (such as, for example, the Nissle strain and the BL21(DE3) strain), Enterobacter blattae (E. blattae), Enterobacter fergusonii (E. fergusonii), Enterobacter hermannii (E. hermannii), Enterobacter vulneris (E. vulneris), Salmonella enterica (S. enterica), Salmonella bongori (S. bongori), Salmonella typhi (S. typhi), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Shigella dysenteriae (S. dysenteriae), Shigella flexneri (S. flexneri), Shigella boydii (S. boydii), Shigella sonnei (S. sonnei), Klebsiella pneumoniae (K. peumoniae), Klebsiella oxytoca (K. oxytoca), Yersinia pestis (Y. pestis), Yersinia enterocolitica (Y. enterocolitica), Yersinia pseudotuberculosis (Y. pseudotuberculosis), Yersinia aldouae (Y. aldouae), Yersinia bercovieri (Y.bercovieri), Yersinia frederiksenii (Y. frederiksenii), Yersinia intermedia (Y. intermedia), Yersinia kristersenii (Y. kristersenii), Yersinia mollaretti (Y. mollaretti), Yersinia rohdei (Y. rohdei), Yersinia ruckeri (Y. ruckeri), Citrobacter freundii (C. freundii), Citrobacter koseri (C. kaseri), Citrobacter braakii (C. braakii), Enterobacter aerogenes (E. aerogenes), Enterobacter cloacae (E. cloacae), Enterobacter gergoviae (E. gergoviae), Enterobacter sakazakii (E. sakazakii), Enterobacter taylorae (E. tavlorae), Enterobacter amnigenus (E. aminigenus), Enterobacter intermedius (E. intermedius), Enterobacter asburiae (E. asburiac), Enterobacter cancerogenus (E. cancerogenus), Enterobacter dissolvens (E.dissolvens), Enterobacter nimipressuralis (E. nimipressuralis), Serratia marcescens (S. marcescens), Serratia entomophila (S. entomophila), Serratia ficaria (S. ficaria), Serratia fonticola (S. fonticola), Serratia grimesii (S. grimesii), Serratia liquefaciens (S. liquefaciens), Serratia odorifera (S. odorifera), Serratia plymuthica (S. plymuthica), Serratia proteamaculans (S. proteamaculans), Serratia rubidaea (S. rubidaea), Serratia ureilytica (S. ureilytica), Proteus mirabilis (P. mirabilis), Proteus vulgaris (P. vulgaris), Proteus myxofaciens (P. myxofaciens), Proteus penneri (P. penneri), Proteus hauseri (P. hauseri), Morganella morganii (M. morganii), Providencia alcalifaciens (P.The modified bacterium of any one of the previous embodiments is selected from the group consisting of P. alcalifaciens, Providencia rustigianii (P. rustigianii), Providencia stuartii (P. stuartii), Providencia rettgeri (P. rettgeri), Providencia heimbachae (P. heimbochae), Hafnia alvei (H. alvei), and Pantoea agglomerans (P. agglomerans).

[0070] Embodiment A28: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is a Salmonella typhimurium bacterium.

[0071] Embodiment A29: The modified bacterium of embodiment A28, wherein the starting strain is Salmonella typhimurium SL7207.

[0072] Embodiment A30: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0073] Embodiment A31: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue and is rapidly eliminated in normal tissue.

[0074] Embodiment A32: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0075] Embodiment A33: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0076] Embodiment A34: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0077] Embodiment A35: The modified bacterium of any one of the preceding embodiments A, which does not express wild-type flagellin.

[0078] Embodiment A36: The modified bacterium of any one of the preceding embodiments A, wherein the bacterium is defective in the fliC gene.

[0079] Embodiment A37: The modified bacterium of any one of the preceding embodiments A, wherein the level of survival of the bacteria within macrophages is in the range of about 50% to about 30% of the survival level of the unmodified starting strain.

[0080] Embodiment A38: The modified bacterium of any one of embodiments A1 to A36, wherein the level of survival of the bacterium within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0081] Embodiment A39: The modified bacterium of any one of embodiments A1 to A36, wherein the level of survival of the bacterium within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0082] Embodiment B Embodiment B1: The bacterium has, compared to the unmodified starting strain: i) the expression cassette of embodiment 18, a hypoxia-regulated essential gene expression cassette comprising an essential gene of said bacterium controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette; or ii) An expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette. wherein the pH-regulated expression cassette comprises a gene encoding a bacterial hemolysin protein controlled by a promoter active under acidic pH conditions, and the bacterium is deficient in at least one gene or its functional expression product involved in or controlling an endogenous antioxidant stress response pathway.

[0083] Embodiment B2: The modified bacterium of embodiment B1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0084] Embodiment B3: The modified bacterium of embodiment B1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0085] Embodiment B4: The modified bacterium of any one of the preceding embodiments B, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the culture medium.

[0086] Embodiment B5: The modified bacterium of embodiment B4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0087] Embodiment B6: The modified bacterium of embodiment B4, wherein the essential genes are selected from dapA and dapE.

[0088] Embodiment B7: The modified bacterium of any one of the preceding embodiments B, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

[0089] Embodiment B8: The modified bacterium of any one of the preceding embodiments B, wherein the functional expression product of a gene involved in or regulating an endogenous antioxidant stress response pathway is a protein related to the HtrA serine protease family.

[0090] Embodiment B9: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium is defective in the activity of HtrA serine protease.

[0091] Embodiment B10: The modified bacterium of any one of the preceding embodiments B, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0092] Embodiment B11: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium is defective with respect to htrA.

[0093] Embodiment B12: The modified bacterium of any one of embodiments B1 to B11, wherein the essential gene is a gene that is naturally occurring in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0094] Embodiment B13: The modified bacterium of any one of embodiments B1 to B11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is entirely under the control of an obligate hypoxia-inducible promoter.

[0095] Embodiment B14: A modified bacterium according to embodiment B13, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 that is controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0096] Embodiment B15: A modified bacterium according to embodiment B13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or wherein the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0097] Embodiment B16: A modified bacterium according to embodiment B15, wherein an exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is present in a plasmid carried by the bacterium.

[0098] Embodiment B17: The modified bacterium of any one of the preceding embodiments B, wherein the bacterially derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0099] Embodiment B18: The modified bacterium of embodiment B17, wherein the gene encoding the hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0100] Embodiment B19: The modified bacterium of embodiment B18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0101] Embodiment B20: The modified bacterium of any one of the preceding embodiments B, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0102] Embodiment B21: The modified bacterium of any one of the preceding embodiments B, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0103] Embodiment B22: The modified bacterium of any one of the preceding embodiments B, wherein the promoter that is active under acidic pH conditions is sseA.

[0104] Embodiment B23: The modified bacterium of any one of the preceding embodiments B, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0105] Embodiment B24: The modified bacterium of any one of the preceding Embodiments B, wherein the bacterium is a bacterium of the family Enterobacteriaceae.

[0106] Embodiment B25: The modified bacterium of any one of embodiments B, wherein the bacterium is a bacterium of the genera Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0107] Embodiment B26: The bacterium is selected from the group consisting of Escherichia coli (such as, for example, Nissle and BL21(DE3) strains), Enterobacter blatae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter vulneris, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella zonei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia aldovae, Yersinia bercovieri, Yersinia fredericsenii, Yersinia intermedia, Yersinia christelsenii, Yersinia molarettii, Yersinia rhodei, Yersinia ruckeri, Citrobacter freundei, Citrobacter koseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter tylorolae, En Serratia amnigenus, Enterobacter intermedius, Enterobacter asbriae, Enterobacter cancelogenus, Enterobacter dissolvens, Enterobacter nimipressuralis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia liquefaciens, Serratia odorifera, Serratia primutica, Serratia proteamaculans, Serratia rubidaea, Serratia The modified bacterium of any one of the preceding Embodiment B is selected from the group consisting of Proteus ureilichia, Proteus mirabilis, Proteus vulgaris, Proteus mixofaciens, Proteus pennelli, Proteus hauseri, Morganella morganii, Providencia alcalifaciens, Providencia rustigianii, Providencia stuarchii, Providencia rettgeri, Providencia heimbaccae, Hafnia alvei, and Pantoea agglomerans.

[0108] Embodiment B27: The modified bacterium of any one of the preceding Embodiments B, wherein the bacterium is a Salmonella typhimurium bacterium.

[0109] Embodiment B28: The modified bacterium according to embodiment B27, wherein the starting strain is Salmonella typhimurium SL7207.

[0110] Embodiment B29: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0111] Embodiment B30: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue and is rapidly eliminated in normal tissue.

[0112] Embodiment B31: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0113] Embodiment B32: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0114] Embodiment B33: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0115] Embodiment B34: The modified bacterium of any one of the preceding embodiments B, which does not express wild-type flagellin.

[0116] Embodiment B35: The modified bacterium of any one of the preceding embodiments B, wherein the bacterium is defective in the fliC gene.

[0117] Embodiment B36: The modified bacterium of any one of the preceding embodiments B, wherein the level of survival of the bacteria within macrophages ranges from about 50% to about 30% of the survival level of the unmodified starting strain.

[0118] Embodiment B37: The modified bacterium of any one of embodiments B1 to B35, wherein the level of survival of the bacterium within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0119] Embodiment B38: The modified bacterium of any one of embodiments B1 to B35, wherein the level of survival of the bacterium within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0120] Embodiment C Embodiment C1: The bacterium has, compared to the unmodified starting strain: i) a hypoxia-regulated essential gene expression cassette comprising the expression cassette of embodiment 18 and an essential gene of said bacterium, said essential gene being controlled by an obligate hypoxia-inducible promoter; or ii) an expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter; 1. A modified Salmonella typhimurium bacterium comprising: a modified Salmonella typhimurium bacterium, wherein the bacterium is deficient in at least one gene, or functional expression product thereof, that is involved in or controls an endogenous antioxidant stress response pathway.

[0121] Embodiment C2: The modified bacterium of embodiment C1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0122] Embodiment C3: The modified bacterium of embodiment C1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0123] Embodiment C4: The modified bacterium of any one of the preceding Embodiments C, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the medium.

[0124] Embodiment C5: The modified bacterium of embodiment C4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0125] Embodiment C6: The modified bacterium of embodiment C4, wherein the essential genes are selected from dapA and dapE.

[0126] Embodiment C7: The modified bacterium of any one of the preceding embodiments C, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

[0127] Embodiment C8: The modified bacterium of any one of the preceding embodiments C, wherein the functional expression product of a gene involved in or regulating an endogenous antioxidant stress response pathway is a protein related to the HtrA serine protease family.

[0128] Embodiment C9: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium is defective in the activity of HtrA serine protease.

[0129] Embodiment C10: The modified bacterium of any one of the preceding embodiments C, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0130] Embodiment C11: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium is defective with respect to htrA.

[0131] Embodiment C12: The modified bacterium of any one of embodiments C1 to C11, wherein the essential gene is a gene that is naturally occurring in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0132] Embodiment C13: The modified bacterium of any one of embodiments C1 to C11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is entirely under the control of an obligate hypoxia-inducible promoter.

[0133] Embodiment C14: A modified bacterium according to embodiment C13, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0134] Embodiment C15: A modified bacterium according to embodiment C13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0135] Embodiment C16: A modified bacterium according to embodiment C15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein the expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located in a plasmid carried by the bacterium.

[0136] 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.

[0137] Embodiment C18: The modified bacterium of embodiment C17, wherein the gene encoding the hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0138] Embodiment C19: The modified bacterium of embodiment C18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0139] Embodiment C20: The modified bacterium of any one of the preceding embodiments C, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0140] Embodiment C21: The modified bacterium of any one of the preceding embodiments C, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0141] Embodiment C22: The modified bacterium of any one of the preceding embodiments C, wherein the promoter that is active under acidic pH conditions is sseA.

[0142] Embodiment C23: The modified bacterium of any one of the preceding embodiments C, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0143] Embodiment C24: The modified bacterium of any one of the preceding embodiments C, wherein the starting strain is Salmonella typhimurium SL7207.

[0144] Embodiment C25: The modified bacterium of any one of embodiments C1 to C24, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0145] Embodiment C26: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue but is rapidly eliminated in normal tissue.

[0146] Embodiment C27: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0147] Embodiment C28: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0148] Embodiment C29: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0149] Embodiment C30: The modified bacterium of any one of the preceding embodiments C, which does not express wild-type flagellin.

[0150] Embodiment C31: The modified bacterium of any one of the preceding embodiments C, wherein the bacterium is defective in the fliC gene.

[0151] Embodiment C32: The modified bacterium of any one of the preceding embodiments C, wherein the level of survival of the bacteria within macrophages ranges from about 50% to about 30% of the survival level of the unmodified starting strain.

[0152] Embodiment C33: The modified bacterium of any one of embodiments C1 to C31, wherein the level of survival of the bacteria within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0153] Embodiment C34: The modified bacterium of any one of embodiments C1 to C31, wherein the level of survival of the bacteria within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0154] Embodiment D Embodiment D1: The bacterium has, compared to the unmodified starting strain: i) a hypoxia-regulated essential gene expression cassette comprising the expression cassette of embodiment 18 and an essential gene of said bacterium, said essential gene being controlled by an obligate hypoxia-inducible promoter; or ii) An expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter. wherein the bacterium is deficient in at least one gene or functional expression product thereof required for survival within macrophages.

[0155] Embodiment D2: The modified bacterium of embodiment D1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0156] Embodiment D3: The modified bacterium of embodiment D1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0157] Embodiment D4: The modified bacterium of any one of the preceding embodiments D, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the medium.

[0158] Embodiment D5: The modified bacterium of embodiment D4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0159] Embodiment D6: The modified bacterium of embodiment D4, wherein the essential genes are selected from dapA and dapE.

[0160] Embodiment D7: The modified bacterium of any one of the preceding embodiments D, wherein the gene required for survival in macrophages is an STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0161] Embodiment D8: The modified bacterium of any one of the preceding embodiments D, wherein the functional expression product of the gene required for survival within macrophages is a protein related to the HtrA serine protease family.

[0162] Embodiment D9: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is defective in the activity of HtrA serine protease.

[0163] Embodiment D10: The modified bacterium of any one of the preceding embodiments D, wherein the gene required for survival within macrophages is htrA.

[0164] Embodiment D11: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is defective with respect to htrA.

[0165] Embodiment D12: The modified bacterium of any one of embodiments D1 to D11, wherein the essential gene is a gene that is naturally present in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0166] Embodiment D13: The modified bacterium of any one of embodiments D1 to D11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is placed entirely under the control of an obligate hypoxia-inducible promoter.

[0167] Embodiment D14: A modified bacterium according to embodiment D13, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0168] Embodiment D15: A modified bacterium according to embodiment D13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0169] Embodiment D16: A modified bacterium according to embodiment D15, wherein an exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is present in a plasmid carried by the bacterium.

[0170] Embodiment D17: The modified bacterium of any one of the preceding embodiments D, further comprising a pH-regulated expression cassette, wherein the pH-regulated expression cassette comprises a gene encoding a bacterial hemolysin protein under the control of a promoter that is active under acidic pH conditions.

[0171] Embodiment D18: The modified bacterium according to embodiment D17, wherein the hemolysin protein of bacterial origin is a hemolysin protein of a Gram-negative bacterium.

[0172] Embodiment D19: The modified bacterium of embodiment D18, wherein the gene encoding a hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0173] Embodiment D20: The modified bacterium of embodiment D19, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0174] Embodiment D21: The modified bacterium of any one of embodiments D17 to D20, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0175] Embodiment D22: The modified bacterium of any one of embodiments D17 to D21, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0176] Embodiment D23: The modified bacterium of any one of embodiments D17 to D21, wherein the promoter that is active under acidic pH conditions is sseA.

[0177] Embodiment D24: The modified bacterium of any one of the preceding embodiments D, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0178] Embodiment D25: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is a bacterium of the family Enterobacteriaceae.

[0179] Embodiment D26: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is a bacterium of the genera Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0180] Embodiment D27: The bacterium is selected from the group consisting of Escherichia coli (such as, for example, Nissle and BL21(DE3) strains), Enterobacter blatae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter vulneris, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella zonei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia aldovae, Yersinia bercovieri, Yersinia fredericsenii, Yersinia intermedia, Yersinia christelsenii, Yersinia molarettii, Yersinia rhodei, Yersinia ruckeri, Citrobacter freundei, Citrobacter koseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter tylorolae, En Serratia amnigenus, Enterobacter intermedius, Enterobacter asbriae, Enterobacter cancelogenus, Enterobacter dissolvens, Enterobacter nimipressuralis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia liquefaciens, Serratia odorifera, Serratia primutica, Serratia proteamaculans, Serratia rubidaea, Serratia The modified bacterium of any one of the preceding embodiments is selected from the group consisting of Proteus ureilistica, Proteus mirabilis, Proteus vulgaris, Proteus mixofaciens, Proteus pennelli, Proteus hauseri, Morganella morganii, Providencia alcalifaciens, Providencia rustigianii, Providencia stuarchii, Providencia rettgeri, Providencia heimbaccae, Hafnia alvei, and Pantoea agglomerans.

[0181] Embodiment D28: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is a Salmonella typhimurium bacterium.

[0182] Embodiment D29: The modified bacterium of embodiment D28, wherein the starting strain is Salmonella typhimurium SL7207.

[0183] Embodiment D30: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium expresses wild-type lipopolysaccharide (LPS).

[0184] Embodiment D31: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue and is rapidly eliminated in normal tissue.

[0185] Embodiment D32: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0186] Embodiment D33: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0187] Embodiment D34: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0188] Embodiment D35: The modified bacterium of any one of the preceding embodiments D, which does not express wild-type flagellin.

[0189] Embodiment D36: The modified bacterium of any one of the preceding embodiments D, wherein the bacterium is defective in the fliC gene.

[0190] Embodiment D37: The modified bacterium of any one of the preceding embodiments D, wherein the level of survival of the bacterium within macrophages ranges from about 50% to about 30% of the survival level of the unmodified starting strain.

[0191] Embodiment D38: The modified bacterium of any one of embodiments D1 to D36, wherein the survival level of the bacterium within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0192] Embodiment D39: The modified bacterium of any one of embodiments D1 to D36, wherein the level of survival of the bacterium within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0193] Embodiment E Embodiment E1: The bacterium has, compared to the unmodified starting strain: i) the expression cassette of embodiment 18, a hypoxia-regulated essential gene expression cassette comprising an essential gene of said bacterium controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette; or ii) An expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette. wherein the pH-regulated expression cassette comprises a gene encoding a bacterial hemolysin protein controlled by a promoter active under acidic pH conditions, wherein the bacterium expresses wild-type lipopolysaccharide (LPS), and wherein the bacterium is deficient in at least one gene or functional expression product thereof required for survival within macrophages.

[0194] Embodiment E2: The modified bacterium of embodiment E1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0195] Embodiment E3: The modified bacterium of embodiment E1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0196] Embodiment E4: The modified bacterium of any one of the preceding embodiments E, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the medium.

[0197] Embodiment E5: The modified bacterium of embodiment E4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0198] Embodiment E6: The modified bacterium of embodiment E4, wherein the essential genes are selected from dapA and dapE.

[0199] Embodiment E7: The modified bacterium of any one of the preceding embodiments E, wherein the gene required for survival in macrophages is an STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0200] Embodiment E8: The modified bacterium of any one of the preceding embodiments E, wherein the functional expression product of the gene required for survival in macrophages is a protein related to the HtrA serine protease family.

[0201] Embodiment E9: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium is defective in the activity of HtrA serine protease.

[0202] Embodiment E10: The modified bacterium of any one of the preceding embodiments E, wherein the gene required for survival in macrophages is htrA.

[0203] Embodiment E11: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium is defective with respect to htrA.

[0204] Embodiment E12: The modified bacterium of any one of embodiments E1 to E11, wherein the essential gene is a gene that is naturally occurring in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0205] Embodiment E13: The modified bacterium of any one of embodiments E1 to E11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is entirely under the control of an obligate hypoxia-inducible promoter.

[0206] Embodiment E14: The modified bacterium of embodiment E13, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette of embodiment 18 or an expression cassette comprising the polynucleotide of embodiment 17 controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0207] Embodiment E15: The modified bacterium of embodiment E13, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or wherein the expression cassette of embodiment 18 or the expression cassette comprising the polynucleotide of embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0208] Embodiment E16: The modified bacterium according to embodiment E15, wherein the exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is present in a plasmid carried by the bacterium.

[0209] Embodiment E17: The modified bacterium of any one of the preceding embodiments E, wherein the bacterially derived hemolysin protein is a Gram-negative bacterial hemolysin protein.

[0210] Embodiment E18: The modified bacterium of embodiment E17, wherein the gene encoding a hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0211] Embodiment E19: The modified bacterium of embodiment E18, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0212] Embodiment E20: The modified bacterium of any one of the preceding embodiments E, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0213] Embodiment E21: The modified bacterium of any one of embodiments E, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0214] Embodiment E22: The modified bacterium of any one of embodiments E, wherein the promoter that is active under acidic pH conditions is sseA.

[0215] Embodiment E23: The modified bacterium of any one of embodiments E, wherein the unmodified starting strain is a facultative anaerobic bacterium.

[0216] Embodiment E24: The modified bacterium of any one of embodiments E, wherein the bacterium is a bacterium of the family Enterobacteriaceae.

[0217] Embodiment E25: The modified bacterium of any one of embodiments E, wherein the bacterium is a bacterium of the genera Escherichia, Salmonella, Shigella, Klebsiella, Yersinia, Citrobacter, Enterobacter, Serratia, Proteus, Morganella, Providencia, Hafnia, and Pantoea.

[0218] Embodiment E26: The bacterium is selected from the group consisting of Escherichia coli (such as, for example, Nissle and BL21(DE3) strains), Enterobacter blatae, Enterobacter fergusonii, Enterobacter hermannii, Enterobacter vulneris, Salmonella enterica, Salmonella bongori, Salmonella typhi, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella zonei, Klebsiella pneumoniae, Klebsiella oxytoca, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia aldovae, Yersinia bercovieri, Yersinia fredericsenii, Yersinia intermedia, Yersinia christelsenii, Yersinia molarettii, Yersinia rhodei, Yersinia ruckeri, Citrobacter freundei, Citrobacter koseri, Citrobacter braachii, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter tylorolae, En Serratia amnigenus, Enterobacter intermedius, Enterobacter asbriae, Enterobacter cancelogenus, Enterobacter dissolvens, Enterobacter nimipressuralis, Serratia marcescens, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia liquefaciens, Serratia odorifera, Serratia primutica, Serratia proteamaculans, Serratia rubidaea, Serratia ureilistica, Proteus mirabilis, Proteus vulgaris, Proteus mixofaciens, Proteus pennelli, Proteus hauseri, Morganella morganii, Providencia alcalifaciens, Providencia rustigianii, Providencia stuarchii, Providencia rettgeri, Providencia heimbaccae, Hafnia alvei, and Pantoea agglomerans.

[0219] Embodiment E27: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium is a Salmonella typhimurium bacterium.

[0220] Embodiment E28: The modified bacterium of embodiment E27, wherein the starting strain is Salmonella typhimurium SL7207.

[0221] Embodiment E29: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue and is rapidly eliminated in normal tissue.

[0222] Embodiment E30: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0223] Embodiment E31: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0224] Embodiment E32: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0225] Embodiment E33: The modified bacterium of any one of the preceding embodiments E, which does not express wild-type flagellin.

[0226] Embodiment E34: The modified bacterium of any one of the preceding embodiments E, wherein the bacterium is defective in the fliC gene.

[0227] Embodiment E35: The modified bacterium of any one of the preceding embodiments E, wherein the level of survival of the bacteria within macrophages ranges from about 50% to about 30% of the survival level of the unmodified starting strain.

[0228] Embodiment E36: ​​The modified bacterium of any one of embodiments E1 to E34, wherein the level of survival of the bacterium within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0229] Embodiment E37: The modified bacterium of any one of embodiments E1 to E34, wherein the level of survival of the bacterium within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0230] Embodiment F Embodiment F1: The bacterium has, compared to the unmodified starting strain: i) the expression cassette of embodiment 18, a hypoxia-regulated essential gene expression cassette comprising an essential gene of said bacterium controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette; or ii) an expression cassette comprising the polynucleotide of embodiment 17 and / or an essential gene controlled by an obligate hypoxia-inducible promoter, and a pH-regulated expression cassette; a pH-regulated expression cassette comprising a gene encoding a bacterial hemolysin protein controlled by a promoter active under acidic pH conditions, wherein the bacterium expresses wild-type lipopolysaccharide (LPS), and the bacterium is deficient in at least one gene or its functional expression product required for survival within macrophages.

[0231] Embodiment F2: The modified bacterium of embodiment F1, wherein the obligate hypoxia-inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.

[0232] Embodiment F3: The modified bacterium of embodiment F1, wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

[0233] Embodiment F4: The modified bacterium of any one of the preceding embodiments F, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and wherein, when cultured under aerobic conditions, growth of the bacterium is dependent on additional DAP or an analog thereof being added to the culture medium.

[0234] Embodiment F5: The modified bacterium of embodiment F4, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

[0235] Embodiment F6: The modified bacterium of embodiment F4, wherein the essential genes are selected from dapA and dapE.

[0236] Embodiment F7: The modified bacterium of any one of the preceding embodiments F, wherein the gene required for survival in macrophages is an STM3120, STM3119, slyA, sifA, SPI-2, phoP, or htrA gene.

[0237] Embodiment F8: The modified bacterium of any one of the preceding embodiments F, wherein the gene required for survival within macrophages is a gene involved in or regulating an endogenous antioxidant stress response pathway.

[0238] Embodiment F9: The modified bacterium of embodiment F8, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

[0239] Embodiment F10: The modified bacterium of embodiment F9, wherein the functional expression product of the gene involved in or regulating the endogenous antioxidant stress response pathway is a protein related to the HtrA serine protease family.

[0240] Embodiment F11: The modified bacterium of embodiment F9, wherein the bacterium is defective in the activity of the HtrA serine protease.

[0241] Embodiment F12: The modified bacterium of embodiment F9, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is htrA.

[0242] Embodiment F13: The modified bacterium of embodiment F9, wherein the bacterium is defective with respect to htrA.

[0243] Embodiment F14: The modified bacterium of any one of embodiments F1 to F13, wherein the essential gene is a gene that is naturally occurring in the bacterial chromosome and the native promoter of the essential gene is functionally replaced with an obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

[0244] Embodiment F15: The modified bacterium according to any one of embodiments F1 to F13, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, whereby expression of the essential gene in the bacterium is entirely under the control of an obligate hypoxia-inducible promoter.

[0245] Embodiment F16: A modified bacterium according to embodiment F15, wherein an exogenous essential gene expression cassette is integrated into the chromosome of the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 that is controlled by an obligate hypoxia-inducible promoter is integrated into the chromosome of the bacterium.

[0246] Embodiment F17: A modified bacterium according to embodiment F15, wherein the exogenous essential gene expression cassette is located outside the chromosome of the bacterium, or wherein the expression cassette according to embodiment 18 or the expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is located outside the chromosome of the bacterium.

[0247] Embodiment F18: A modified bacterium according to embodiment F17, wherein an exogenous essential gene expression cassette is present in a plasmid carried by the bacterium, or wherein an expression cassette according to embodiment 18 or an expression cassette comprising the polynucleotide according to embodiment 17 controlled by an obligate hypoxia-inducible promoter is present in a plasmid carried by the bacterium.

[0248] Embodiment F19: The modified bacterium of any one of the preceding embodiments F, wherein the bacterial-derived hemolysin protein is a hemolysin protein of a Gram-negative bacterium.

[0249] Embodiment F20: The modified bacterium of embodiment F19, wherein the gene encoding a hemolysin protein of Gram-negative bacteria is hlyA or hlyE.

[0250] Embodiment F21: The modified bacterium of embodiment F20, wherein hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

[0251] Embodiment F22: The modified bacterium of any one of the preceding embodiments F, wherein the promoter that is active under acidic pH conditions is active at a pH 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.

[0252] Embodiment F23: The modified bacterium of any one of the preceding embodiments F, wherein the promoter that is active under acidic pH conditions is selected from the group consisting of sseA, ssrA, ssaB, ssaG, ssaM, and ssaR.

[0253] Embodiment F24: The modified bacterium of any one of the preceding embodiments F, wherein the promoter that is active under acidic pH conditions is sseA.

[0254] Embodiment F25: The modified bacterium of any one of the preceding embodiments F, wherein the starting strain is Salmonella typhimurium SL7207.

[0255] Embodiment F26: The modified bacterium of any one of the preceding embodiments F, wherein the bacterium, when administered to a subject with a tumor, has the ability to survive and grow in tumor tissue and is rapidly eliminated in normal tissue.

[0256] Embodiment F27: The modified bacterium of any one of the preceding embodiments F, wherein the bacterium has the ability to inhibit the growth of a malignant tumor when administered to a subject with a malignant tumor.

[0257] Embodiment F28: The modified bacterium of any one of the preceding embodiments F, wherein the bacterium has the ability to induce an anti-tumor-specific immune response when administered to a subject with a malignant tumor.

[0258] Embodiment F29: The modified bacterium of any one of the preceding embodiments F, wherein the bacterium has the ability to induce anti-tumor immune memory when administered to a subject with a malignant tumor.

[0259] Embodiment F30: The modified bacterium of any one of the preceding embodiments F, which does not express wild-type flagellin.

[0260] Embodiment F31: The modified bacterium of any one of the preceding embodiments F, wherein the bacterium is defective in the fliC gene.

[0261] Embodiment F32: The modified bacterium of any one of the preceding embodiments F, wherein the level of survival of the bacteria within macrophages is in the range of about 50% to about 30% of the survival level of the unmodified starting strain.

[0262] Embodiment F33: The modified bacterium of any one of embodiments F1 to F31, wherein the level of survival of the bacterium within macrophages is in the range of about 30% to about 10% of the survival level of the unmodified starting strain.

[0263] Embodiment F34: The modified bacterium of any one of embodiments F1 to F31, wherein the level of survival of the bacterium within macrophages is in the range of about 10% to about 1% of the survival level of the unmodified starting strain.

[0264] Embodiment K Embodiment K1: A pharmaceutical composition comprising an effective amount of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F.

[0265] Embodiment K2: The pharmaceutical composition of embodiment K1 for use in treating malignant tumors.

[0266] Embodiment K3: A pharmaceutical composition according to embodiment K1 for use in eliciting an anti-tumor-specific immune response in a subject with a malignant tumor.

[0267] Embodiment K4: The pharmaceutical composition of embodiment K1 for use in inducing anti-tumor immune memory in a subject with a malignant tumor.

[0268] Embodiment K5: The pharmaceutical composition of embodiment K1 for use in preventing or treating metastasis or recurrence of a malignant tumor.

[0269] Embodiment K6: The pharmaceutical composition of embodiment K1 for use in treating malignant tumors that are resistant to or have failed previous antitumor therapies.

[0270] Embodiment K7: The pharmaceutical composition of any one of embodiments K1 to K6, wherein the modified bacterium is a live bacterium.

[0271] Embodiment L Embodiment L1: A method for treating a malignant tumor, comprising administering to a subject suffering from a malignant tumor an effective amount of a modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or a pharmaceutical composition of any one of the preceding Embodiments K.

[0272] 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 a modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or a pharmaceutical composition of any one of the preceding Embodiments K.

[0273] Embodiment L3: A method for inducing anti-tumor immune memory in a subject having a malignant tumor, comprising administering to the subject an effective amount of a modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or a pharmaceutical composition of any one of the preceding Embodiments K.

[0274] Embodiment L4: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering to a subject having a malignant tumor an effective amount of a modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or a pharmaceutical composition of any one of the preceding Embodiments K.

[0275] Embodiment L5: A method for preventing or treating metastasis or recurrence of a malignant tumor, comprising administering an effective amount of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of the preceding Embodiments K, to a subject having or at high risk of metastasis or recurrence of a malignant tumor.

[0276] Embodiment L6: A method for treating a malignant tumor that is resistant to or has failed to a previous anti-tumor therapy, comprising administering an effective amount of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F, or the pharmaceutical composition of any one of the preceding Embodiments K, to a subject having a malignant tumor that is resistant to or has failed to a previous anti-tumor therapy.

[0277] Embodiment L7: The method of embodiments L1-L6, wherein the modified bacterium is a live bacterium.

[0278] Embodiment M Embodiment M1: Use of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F in the manufacture of a medicament for treating a malignant tumor.

[0279] Embodiment M2: Use of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F in the manufacture of a medicament for inducing an anti-tumor-specific immune response in a subject having a malignant tumor.

[0280] Embodiment M3: Use of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F in the manufacture of a medicament for inducing anti-tumor immune memory in a subject with a malignant tumor.

[0281] Embodiment M4: Use of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F in the manufacture of a medicament for preventing or treating metastasis or recurrence of a malignant tumor.

[0282] Embodiment M5: Use of the modified bacterium of any one of the preceding Embodiments A, B, C, D, E, or F in the manufacture of a medicament for treating a malignant tumor that is resistant to or has failed previous anti-tumor therapy.

[0283] Embodiment M6: The use according to embodiments M1 to M5, wherein the modified bacterium is a live bacterium.

[0284] Embodiment N 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 originating from the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or mucosal skin.

[0285] 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 malignant tumor is a sarcoma or a cancer.

[0286] 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.

[0287] 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 the group consisting of glioma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, cancer of the oral cavity, cancer of the tongue, cancer of the larynx, cancer of the head and neck, melanoma, cancer of the bronchus, cancer of the lung, cancer of the pleura, cancer of the esophagus, cancer of the stomach, hepatocellular carcinoma, pancreatic cancer, cancer of the bile duct, colorectal cancer, rectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, suprarenoma, 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.

[0288] 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 bacterium, medicament, or pharmaceutical composition of the invention is administered by intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, intracerebral, gastrointestinal, body surface, oral mucosal, nasal, rectal, or vaginal route.

[0289] definition The terms "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 are artificial chemical analogs of the corresponding naturally occurring amino acid(s), as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including, but not limited to, glycosylation, lipid ligation, sulfation, gamma-carboxylation of glutamic acid residues, and ADP-ribosylation. Modifications also include modifications to the polypeptide sequence, including, but not limited to, substitution, deletion, insertion, and / or addition of one or more amino acids.

[0290] The term "amino acid" includes both naturally occurring amino acids in proteins and unnatural amino acids. For amino acids that occur naturally in proteins, conventional nomenclature (one-letter and three-letter) is adopted (see Sambrook). Amino acid 1 letter 3 letters Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp Cysteine ​​C Cys Glutamine Q Gln Glutamic Acid E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valin V Val

[0291] In the present invention, to determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into a first amino acid sequence or nucleic acid sequence to achieve optimal alignment with a second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are identical in length.

[0292] Those skilled in the art know that various computer programs are available to determine identity between two sequences.

[0293] "Percent amino acid identity" or "percent amino acid sequence identity" refers to a comparison between the amino acids of two polypeptides, where, when optimally aligned, the two polypeptides have a near-determined percentage of identical amino acids. For example, "95% amino acid identity" refers to a comparison between the amino acids of two polypeptides, where, when optimally aligned, 95% of the amino acids in the two polypeptides are identical.

[0294] The term "conservative substitution," also referred to as substitution with a "homologous" amino acid residue, refers to substitution in which an amino acid is replaced with an amino acid residue having a similar side chain, e.g., amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0295] By "hypoxia-regulated essential gene expression cassette" herein is meant a stretch of DNA capable of promoting expression of an essential gene under hypoxic conditions, containing an essential gene under the control of a hypoxia-inducible promoter and, if necessary, may further contain other regulated elements required for expression of the essential gene.

[0296] As used herein, "essential gene" refers to a gene that is critical for bacterial growth and / or survival; in the absence of such a gene or its functional expression product, the bacterium cannot survive, divide, and / or grow normally. A typical example of a deficiency in an essential gene or its functional expression product is a nutritionally deficient strain that cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo in the absence of a particular exogenous supplement. Essential genes usually occur as a single copy on the bacterial chromosome.

[0297] As used herein, an "obligate hypoxia-inducible promoter" refers to a promoter that has the ability to initiate transcription of a specific gene under anaerobic conditions, but is substantially unable to activate and produce transcripts under aerobic conditions.

[0298] An "obligate hypoxia-inducible promoter" useful in the present invention can be identified by the following experiment: An expression strain containing an essential gene (e.g., dapA / dapE) controlled by a promoter of interest is constructed, streaked onto a Luria-Bertani (abbreviated as LB) plate in an anaerobic incubator, and anaerobically cultured at 37°C for 24 hours. Under anaerobic conditions, a single clone is selected and resuspended in 20 μL of LB broth. 20 μL of the resuspension of the single clone is added equally to the liquid in the following four tubes (i.e., 5 μL is added to each of tubes A, B, C, and D): A. 2 ml of LB broth, B. 2 ml of LB broth, C. 2 ml of LB broth + DAP; D. 2 ml of LB broth + DAP. Tubes A and C are incubated in an anaerobic incubator at 37° C. for 24 hours, and tubes B and D are incubated in an aerobic shaker at 37° C. for 24 hours. If bacteria can grow in tubes A, C, and D but not in tube B (detected OD600 value less than 0.05), the promoter is considered to be an obligate hypoxia-inducible promoter.

[0299] As identified by the above experiments, exemplary obligate hypoxia-inducible promoters that can be used in the present invention are shown in Table 1.

[0300] [Table 1]

[0301] As used herein, "involved in or regulating the endogenous antioxidant stress response pathway" refers to a gene involved in the resistance of bacteria (e.g., intracellular bacteria, such as Salmonella) to killing by reactive oxygen species (ROS) in vivo or in vitro.

[0302] As used herein, "genes required for survival within macrophages" refers to genes involved in maintaining or enhancing the survival rate of intracellular bacteria within macrophages. The function of such gene products is associated with resistance to the killing effect of macrophages on microorganisms, and thus, deficiency of such genes or their functional expression products can reduce the survival rate of bacteria (e.g., intracellular bacteria such as Salmonella) within macrophages.

[0303] As used herein, "functional expression products" include RNA, proteins, and the like.

[0304] As used herein, "facultative anaerobic bacteria" refers to bacteria that can survive under both aerobic and anaerobic conditions.

[0305] As used herein, a "pH-regulated expression cassette" refers to a group of gene expression elements in which the expression of the corresponding gene(s) is regulated by environmental pH.

[0306] As used herein, a "promoter active under acidic pH conditions" refers to a promoter that is activated under acidic pH conditions and can control the expression of a corresponding gene. In some embodiments, a promoter active under acidic pH conditions of the present invention can be activated or still have promoter activity at a pH 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, a promoter active under acidic pH conditions also has promoter activity at a neutral pH (e.g., pH 7.0 to 7.4).

[0307] [Example] Example 1: Construction of an IPTG-inducible plasmid expressing human interleukin-10 protein 1.1. The target fragments were amplified by PCR using the plasmids pET22b-pelB-Amp (SEQ ID NO: 1) and pSC101-huil10-Amp (SEQ ID NO: 2) as templates, respectively. Fragment 1 (pET22b-pelB-Amp) was amplified from pET22b-pelB-Amp using the following primer sequences: Forward primer CACCACCACCACCACCACTGAGATCCG (SEQ ID NO: 35); and Reverse primer ATCATCATCATCATCGGCCATCGCCGGCTGGGC (SEQ ID NO: 36). Fragment 2 (D5-linker-huil10) was amplified from pSC101-huil10-Amp using the following primer sequences: Forward primer TGGCCGATGATGATGATGATAGCCCGGGTCAGGGTACGCA (SEQ ID NO: 37); and Reverse primer TCAGTGGGTGGTGGTGGTGATTACGGATTTTCATGGTCATATATGC (SEQ ID NO: 38). Control fragment 3 (huil10) was amplified from pSC101-huil10-Amp using the following primer sequences: Forward primer TGGCCAGCCCGGGTCAGGGTACGCA (SEQ ID NO: 39); and Reverse primer TCAGTGGGTGGTGGTGGTGATTACGGATTTTCATGGTCATATATGC (SEQ ID NO: 40)

[0308] PCR amplification was carried out using PrimeSTAR Max DNA polymerase (Takara, #R054A) in the following system.

[0309] [Table 2]

[0310] PCR conditions were as follows: denaturation (98°C for 10 seconds), annealing (55°C for 10 seconds), extension (72°C for 5 seconds / kb), a total of 30 cycles of denaturation-annealing-extension.

[0311] Fragment 1 and Fragment 2, and Fragment 1 and Fragment 3 were recombined using the ClonExpress II One Step Cloning Kit (Vazyme, #C112) according to the manufacturer's instructions and chemically transformed into clone competent cells (DH5α competent E. coli strain, Vazyme, #C502). The recombinant plasmids were designated as plasmid-pET22b-pelB-D5-hil10-Amp (SEQ ID NO: 3) and plasmid-pET22b-pelB-hil10-Amp (SEQ ID NO: 4).

[0312] The gene sequence of interleukin-10 fused to the prokaryotic signal peptide pelB and the N-terminal polar negatively charged aspartic acid Asp linker peptide is shown in the Appendix (SEQ ID NO: 5), and the linker peptide is shown in bold.

[0313] 1.3. Clones of competent cells DH5α carrying the recombinant plasmids (plasmid-pET22b-pelB-D5-hil10-Amp and plasmid-pET22b-pelB-hil10-Amp) were identified by PCR using the following identifying primers: A forward identification primer 1F having the sequence CTCGACGCTCTCCCTTATG (SEQ ID NO: 41), and Reverse identification primer 1R having the sequence CTGACATGGCTTTATAGATACCT (SEQ ID NO: 42).

[0314] Positive clones were cultured in LB broth containing penicillin (Amp, 100 mg / L) and the plasmids were extracted (Plasmid Mini Kit I OMEGA, #D6943-03).

[0315] Example 2: Construction of E. coli strain BL21(DE3) expressing human interleukin-10 protein by IPTG induction To express the recombinant protein, the recombinant plasmid was introduced into the BL21(DE3) strain by chemical transformation (BL21(DE3) Competent E. coli Strain, Vazyme, #C504-03). Specifically, competent cells were thawed on ice, and 100 ng of the recombinant plasmid was mixed with the competent cells and placed on ice for 30 minutes. After a 45-second heat shock in a 42°C water bath, the cells were immediately placed on ice for 3 minutes. 900 μL of LB medium (antibiotic-free) was added, and the bacteria were incubated at 37°C for 1 hour in a shaker (200–250 rpm). After centrifugation at 5,000 rpm for 5 minutes, 900 μL of the supernatant was discarded. The bacteria were resuspended in the remaining medium and gently spread onto LB plates containing penicillin (Amp, 100 mg / L) using a sterile spreader. The plates were inverted in an incubator at 37°C overnight.

[0316] The construction of the BL21(DE3)-plasmid-pET22b-pelB-D5-huil10-Amp strain and the BL21(DE3)-plasmid-pET22b-pelB-huil10-Amp strain was confirmed by PCR and electrophoresis. The identification primers used were 1F / 1R from 1.3.

[0317] Example 3: Expression and secretion of interleukin-10 by strain BL21(DE3)-plasmid-pET22b-pelB-D5-huil10-Amp 3.1. The cryopreserved strain (the strain constructed in Example 2) was removed from -80°C, placed on ice until thawed, and isolated by streaking on an LB plate containing penicillin (Amp, 100 mg / L); the plate was placed in a 37°C incubator overnight. The next day, a single colony that grew vigorously and individually was selected and inoculated into a sterile culture tube containing LB medium (4 mL) containing penicillin (Amp, 100 mg / L) and incubated at 37°C. When the culture had an OD of 0.4-0.5, IPTG was added to a final concentration of 0.1 mM and induced overnight in a shaker at 16°C (induction at 200-250 rpm for 14-16 hours), and the bacterial culture was removed and stored on ice.

[0318] The culture was centrifuged at 4,200 rpm for 10 minutes at 4°C, and the bacterial pellet and LB medium supernatant were collected separately. The LB medium supernatant was placed in an ultrafiltration tube (Amicon Ultra, #UFC901096) and centrifuged at 2,500 rpm for 2 hours at 4°C, and the concentrated LB medium supernatant (containing secreted IL-10) was collected.

[0319] 3.3. Lysis solution (Qiagen, Qproteome Bacterial Protein Prep Kit, #37900, prepared with reference to the laboratory manual "Qproteome Bacterial Protein Preparation Handbook", www.qiagen.com) was added to the bacterial pellet, the mixture was placed on ice for 30 minutes, and then centrifuged at 14,000 rpm at 4°C for 30 minutes, and the bacterial debris pellet and lysate supernatant were collected separately. The bacterial debris pellet contains functional proteins in inclusion bodies, and the lysate supernatant contains soluble proteins that were expressed but not secreted.

[0320] 3.4. Characterization of Expressed and Secreted Interleukin-10 5x SDS loading buffer (Beyotime, #P0015) was added to the collected samples (concentrated LB medium supernatant, bacterial debris precipitate, and lysate supernatant) and then incubated in a metal bath at 100°C for 5 minutes. SDS-PAGE was then performed at 80V for 20 minutes. After the samples migrated to the interface between the resolving gel and stacking gel, the voltage was increased to 120V until the dye completely escaped from the gel.

[0321] 3.4.2. A Pyxis corollary PVDF membrane and transfer device were used for membrane transfer according to the manufacturer's instructions. After membrane transfer, the membrane was washed successively with TBS buffer and TBST buffer; the PVDF membrane was blocked with 5% skim milk (dissolved in TBST buffer) at room temperature for 1 hour; and the primary antibody (6%) diluted 1:10,000 in 5% skim milk was blocked. * The PVDF membrane was incubated overnight at 4°C with his-tag monoclonal antibody (Proteintech, #66005-1-lg). The overnight-incubated PVDF membrane was washed several times with TBST buffer to remove the antibody on the membrane surface, and then incubated for 1 hour at room temperature with a secondary antibody (Affinipure goat anti-rabbit IgG (H+L) conjugated with HRP, Proteintech, #SA00001-2-100UL) diluted 1:5000 in 5% skim milk. After incubation, the PVDF membrane was washed several times with TBST buffer to remove unbound antibody on the membrane surface, and finally, the membrane was immersed in TBS. Prepared developer solutions A and B (Thermo Fisher Scientific, SuperSignal™ West Pico PLUS Chemiluminescent Substrate) were mixed in a 1:1 ratio, the film was placed in a tray, the mixed solution was poured into the tray, and then exposed for 5 minutes and imaged using a Jena multifunction imager.

[0322] As shown in Figure 1, when the D5 peptide linker is present in the fusion polypeptide, E. coli can express and secrete IL-10 upon IPTG induction, whereas when the D5 peptide linker is not present, E. coli can express IL-10 upon IPTG induction but cannot secrete the expressed IL-10 extracellularly.

[0323] Example 4: Construction of a plasmid for constitutive expression of human interleukin-10 protein 4.1. The desired fragments were amplified by PCR using the plasmids (plasmid-pET22b-pelB-D5-huil10-Amp (SEQ ID NO: 3) and plasmid-pSC101-ptac-sfGFP-KanR (SEQ ID NO: 6)) as templates, respectively. Fragment 4 (pSC101-ptac-KanR) was amplified from pSC101-ptac-sfGFP-KanR (SEQ ID NO: 6) using the following primer sequences: Forward primer CTGCTAACAAAGCCCGAAAG (SEQ ID NO: 43); and Reverse primer CTTTCCTGTGTGAATTATTTCTAGAGG (SEQ ID NO: 44). Fragment 5 (pelB-D5linker-huil10) was amplified from the plasmid pET22b-pelB-D5-huil10-Amp (SEQ ID NO: 3) using the following primer sequences, with an annealing temperature of 55°C: Forward primer AAATAATTCACACAGGAAAGtataCACATCATGAAATACCTGCTGCCG (SEQ ID NO: 45); and Reverse primer CTTTCGGGCTTTGTTAGCAGCCTTTCGGGCTTTGTTAGCA (SEQ ID NO: 46). Fragment 6 (pelB-huil10) was amplified from the plasmid pET22b-pelB-huil10-Amp (SEQ ID NO: 4) using the following primer sequences, with an annealing temperature of 55°C: Forward primer AAATAATTCACACAGGAAAGtataCACATCATGAAATACCTGCTGCCG (SEQ ID NO: 45); and Reverse primer CTTTCGGGCTTTGTTAGCAGCCTTTCGGGCTTTGTTAGCA (SEQ ID NO: 46).

[0324] 4.2. Fragment 4 and Fragment 5, and Fragment 4 and Fragment 6 were recombined using ClonExpress II One Step Cloning Kit (Vazyme, #C112), respectively, and chemically transformed into clone competent cells (DH5α competent E. coli strain, Vazyme, #C502), and the recombinant plasmids were designated as plasmid-pSC101-ptac-pelB-D5-huil10-KanR (SEQ ID NO: 7) and plasmid-pSC101-ptac-pelB-huil10-KanR (SEQ ID NO: 8).

[0325] 4.3. Competent DH5α cells after cloning carrying the recombinant plasmids (plasmid-pSC101-ptac-pelB-D5-huil10-KanR and plasmid-pSC101-ptac-pelB-huil10-KanR) were identified by PCR using the following identification primers: Forward identification primer 2F having the sequence CCGTCTTACTGTCAAGAGGAC (SEQ ID NO: 47), Reverse identification primer 2R having the sequence CTGACATGGCTTTATAGATACCT (SEQ ID NO: 48).

[0326] Positive clones were cultured in LB broth containing kanamycin (50 mg / L) and the plasmids were extracted (Plasmid Mini Kit I OMEGA, #D6943-03).

[0327] Example 5: Construction of E. coli strain BL21(DE3) for constitutive expression of human interleukin-10 protein To express the recombinant proteins, the plasmids (plasmid-pSC101-ptac-pelB-D5-huil10-KanR and plasmid-pSC101-ptac-pelB-huil10-KanR) were chemically transformed into strain BL21(DE3) using a method similar to that in Example 2, except for the use of LB plates containing 50 mg / L kanamycin (Sangon Biotech (Shanghai) Co., Ltd., B528419, see product instructions for chemical transformation, https: / / store.sangon.com / product).

[0328] The construction of the BL21(DE3)-plasmid-pSC101-ptac-pelB-D5-huil10-KanR strain and the BL21(DE3)-plasmid-pSC101-ptac-pelB-huil10-KanR strain was confirmed by PCR and electrophoresis using the above-mentioned identification primers 2F / 2R.

[0329] Example 6: Expression and secretion of interleukin-10 by strain BL21(DE3)-plasmid-pSC101-ptac-pelB-D5-huil10-KanR 6.1. The cryopreserved strain (the strain constructed in Example 5) was removed from -80°C, placed on ice until thawed, and isolated by streaking onto an LB plate containing 50 mg / L kanamycin; the plate was placed in a 37°C incubator for overnight cultivation. The next day, a vigorous single colony was selected and inoculated into a sterile culture tube containing LB broth (4 mL) containing 50 mg / L kanamycin, cultivated overnight, and the bacterial solution was harvested and stored on ice.

[0330] The culture was centrifuged at 4,200 rpm for 10 minutes, and the bacterial pellet and LB medium supernatant were collected separately. The LB medium supernatant was placed in an ultrafiltration tube (Amicon Ultra, #UFC901096) and centrifuged at 2,500 rpm at 4°C for 2 hours, and the concentrated LB medium supernatant (containing secreted IL-10) was collected.

[0331] Lysis solution (Qiagen, #37900) was added to the bacterial pellet, the mixture was placed on ice for 30 minutes, then centrifuged at 14,000 rpm at 4°C for 30 minutes, and the bacterial debris pellet and lysate supernatant were collected separately. The bacterial debris pellet contains functional proteins within inclusion bodies, and the lysate supernatant contains expressed but non-secreted soluble proteins.

[0332] 6.4. Western Blot Analysis of Expressed and Secreted IL-10 IL-10 in each sample was analyzed by Western blot as described in Example 3.4. As shown in Figure 2, when the D5 peptide linker is present in the fusion polypeptide, E. coli can express and secrete IL-10 from the ptac constitutive promoter, whereas when the D5 peptide linker is not present, the IL-10 expressed by E. coli cannot be secreted extracellularly.

[0333] Example 7: Effect of peptide linkers on IL-10 secretion The purpose of this example was to determine the effect of different peptide linkers on the expression and secretion of IL-10 in and from bacteria.

[0334] To achieve this goal, the present inventors constructed peptide linkers as shown in Table 2 based on the type of amino acid, basic and polar uncharged amino acids, and the number of amino acids. Peptide linkers and their coding nucleotide sequences

[0335] [Table 3]

[0336] The nucleotide sequences encoding the peptide linkers in Table 2 were inserted between the nucleotide sequence encoding the signal peptide and the nucleotide sequence encoding huIL10 in the plasmid (plasmid-pET22b-pelB-huil10-Amp (SEQ ID NO: 4)) to obtain plasmids encoding different pelB + peptide linker + huil10 fusion polypeptides (plasmid-pET22b-pelB-peptide linker-huil10-Amp), where plasmid-pET22b-pelB-D5-huil10-Amp was prepared as described in Example 1.

[0337] The prepared plasmids were transformed into E. coli, and the expression of the fusion polypeptides was detected as described in Examples 2 and 3. As shown in Figure 3, the fusion polypeptides containing the peptide linkers D4 or D5 could be expressed from E. coli and secreted into the culture supernatant, while the fusion polypeptides containing other peptide linkers could be expressed from E. coli, but their secretion into the culture supernatant from E. coli was not detected.

[0338] Example 8 The objective of this example was to construct a plasmid for constitutive high-level expression and secretion of human / mouse interleukin-10 and to test the secretion of a fusion polypeptide containing the peptide linker D5 (pelB-D5-huil10) using different bacterial chassis and plasmids with different copy numbers.

[0339] 8.1. Construction of a Plasmid for Constitutive High-Level Expression and Secretion of Human Interleukin-10 The pelB-D5-huil10 DNA fragment was amplified by PCR using the plasmid (plasmid-pET22b-pelB-D5-huil10-Amp (SEQ ID NO: 3)) as a template, and the linear fragment was amplified by PCR using the plasmid (pUC-ptac-bba0032-KanR (SEQ ID NO: 50)) as a template. pelB-D5-huil10 was amplified using the following primers, using an annealing temperature of 55°C: Forward primer AAATAATTCACACAGGAAAGtataCACATCATGAAATACCTGCTGCCG (SEQ ID NO: 45); and Reverse primer CTTTCGGGCTTTGTTAGCAGCCTTTCGGGCTTTGTTAGCA (SEQ ID NO: 46). pUC-ptac-bba0032-KanR was amplified using the following primers, using an annealing temperature of 55°C: Forward primer CTTTCCTGTGTGAattatttctagagg (SEQ ID NO: 44); and Reverse primer cacgcttagcaataactag (SEQ ID NO: 49).

[0340] The fragments were recombined using the ClonExpress II One Step Cloning Kit (Vazyme, #C112) and chemically transformed into clonal competent cells (DH5α competent E. coli strain, Vazyme, #C502), respectively, and the recombinant plasmid was designated as plasmid-pUC-ptac-pelB-D5-huil10-KanR (SEQ ID NO: 51). Transformed clones were identified using the following primers: iden-bb-F:ctgattctgtggACAAGAGG; and iden-huil10-R:CAGGATCCTGATTTTCTGCC.

[0341] 8.2. Construction of a Plasmid for Constitutive High-Level Expression and Secretion of Mouse Interleukin-10 A plasmid containing the nucleotide sequence encoding mouse interleukin-10 (plasmid-pET22b-pelB-D5-mil10-Amp) was synthesized as a template, and pelB-D5-mil10 was amplified as described above. The amplified pelB-D5-mil10 was then recombined with the pUC-ptac-bba0032-KanR plasmid backbone via the Vazyme One-Step Cloning Kit to obtain a recombinant plasmid for constitutive expression of mouse interleukin-10, designated as plasmid-pUC-ptac-pelB-D5-mil10-KanR (SEQ ID NO: 52).

[0342] The primers used to amplify pelB-D5-mil10 were identical to those used to construct the plasmids for the above human interleukins (annealing temperature 55°C): Forward primer AAATAATTCACACAGGAAAGtataCACATCATGAAATACCTGCTGCCG (SEQ ID NO: 45); and Reverse primer CTTTCGGGCTTTGTTAGCAGCCTTTCGGGCTTTGTTAGCA (SEQ ID NO: 46). The forward primer for amplifying pUC-ptac-bba0032-KanR was CTTTCCTGTGTGAatatttctagg (SEQ ID NO: 44); the reverse primer was cacgctagcaataactag (SEQ ID NO: 49) (annealing temperature 55° C.).

[0343] The fragments were recombined using the ClonExpress II One Step Cloning Kit (Vazyme, #C112) and chemically transformed into cloning competent cells (DH5α competent E. coli strain, Vazyme, #C502), and the recombinant plasmid was designated as plasmid-pUC-ptac-pelB-D5-mil10-KanR (SEQ ID NO: 52). Transformed clones were identified using the following primers: iden-bb-F:ctgattctgtggACAAGAGG; and iden-huil10-R:CAGGATCCTGATTTTCTGCC.

[0344] Example 9: Construction of variant strains of Salmonella typhimurium SL7207 9.1. The desired fragment was obtained by PCR amplification using the PsseA-hlyA-loxp-KnaR-loxp plasmid (SEQ ID NO: 9) and the Pssbp1-dapE-Cm plasmid (SEQ ID NO: 10) as templates. For PsseA-hlyA-loxp-KnaR-loxp, the following primer sequences were used: forward primer TTCACAGAAAAGTGTTGCCCCCTTCCATGGCGGAAGGGGGACAAAGGTGAATTTGTCCTACTCAGGAGAGAGCGTTCA (SEQ ID NO: 11) and reverse primer TCTGACGTACACAGCAATTTTGCGTTACCTGTTAATCGAGATTGAAACACCGAAGAAAGGCCCACCCG (SEQ ID NO: 12). For Pssbp1-dapE-Cm, the following primer sequences were used: forward primer TTGGTTTCAGTGAATCCCGTTATCAGCAGTTTTTTGATGAGGTGTAGTCTATTTGTCCTACTCAGGAGAGCG (SEQ ID NO: 13) and reverse primer CAATATTTTGCCAGCCAGTCCATGCTTATTTCCTCTTACCGGAACGCTCACGAAGAAAGGCCCACCCG (SEQ ID NO: 14), with an annealing temperature of 55°C. Desired fragment 1 was PsseA-hlyA-loxp-KnaR-loxp (SEQ ID NO: 1), and desired fragment 2 was Pssbp1-dapE-Cm (SEQ ID NO: 2).

[0345] 9.2. The pSim6 plasmid (BioVector NTCC Inc.: 3574840) containing λ phage Red recombinase was introduced into the facultative anaerobic Salmonella strain SL7207 (NCBI: ASM1320710v1) to prepare electrocompetent cells of the SL7207 strain containing the pSim6 plasmid, SL7207(pSim6).

[0346] 9.3. The desired fragment 1 was introduced into SL7207 (pSim6) competent cells by electroporation (E = 18 KV / cm). The λ-red homologous recombination method was used to replace the dapE gene (which controls the synthesis of diaminopimelic acid, essential for cell wall synthesis) in the SL7207 genome with the desired fragment 1. Specifically, the target strain containing the pSim6 plasmid was cultured at 30°C; recombinase expression was carried out at 42°C for 15 minutes; and the homologous recombination process was completed by adding 50 bp-containing homologous arms to PCR primers under the influence of the homologous recombinase, away from the upstream and downstream of the target locus, respectively, to obtain strain SL7207 (ΔdapE::PsseA-hlyA-KnaR).

[0347] The Cre plasmid (Gene Bridges: A112) containing the 9.4.P1 phage Cre recombinase was introduced into the recombinant strain SL7207 (ΔdapE::PsseA-hlyA-KnaR) to remove the kanamycin resistance, thereby obtaining the strain SL7207 (ΔdapE::PsseA-hlyA).

[0348] 9.5. The pSim6 plasmid was introduced into strain SL7207(ΔdapE::PsseA-hlyA) to prepare SL7207(ΔdapE::PsseA-hlyA)(pSim6) electrocompetent cells.

[0349] Desired fragment 2 was introduced into electrocompetent SL7207 (ΔdapE::PsseA-hlyA) (pSim6) cells by electroporation (E = 18 kV / cm). Using λ-red homologous recombination, the htrA gene (encoding a serine protease) in the SL7207 genome (ΔdapE::PsseA-hlyA) was replaced with desired fragment 2 to obtain strain DB-ZW1, which lacked the htrA gene and contained the hlyA gene under the control of PsseA and the dapE gene under the control of the obligate hypoxia-inducible promoter Pssbp1 (ΔdapE::PsseA-hlyA; ΔhtrA::Pssbp1-dapE-Cm). The strain construction scheme is shown in Figure 4.

[0350] Example 10: Antitumor effects of DB-ZW1 on multiple tumor models 10.1. Tumor model construction C57BL / 6 mice (weighing approximately 18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in an SPF environment) were inoculated with 1 × 10 6 A subcutaneous tumor model for mouse bladder cancer / melanoma was established by subcutaneous inoculation of MB49 mouse bladder cancer cells (Merck, SCC148) / B16 melanoma cells (ATCC: CRL-6475). On days 14-18 after inoculation, tumor volumes reached approximately 100 mm. 3 The experiment was carried out at 100 s. The in situ colon cancer model was induced by DSS / AOM: one week after a single intraperitoneal injection of 10 mg / kg AOM, the mice were fed with water containing 2.5% DSS for 7 days, followed by normal drinking water for 14 days, which constituted one cycle of DSS treatment. The DSS treatment was repeated for three more cycles to obtain a mouse model bearing colorectal cancer.

[0351] 10.2. Changes in the distribution of DB-ZW1 in tumors and different organs 1×10 7CFU of DB-ZW1 (125 μL) was injected into MB49 bladder tumor-bearing mice (3 mice / group) via the tail vein. Approximately 90% of DB-ZW1 in the mice was observed to be present in the tumor 1 day post-injection (1 dpi). The intratumoral DB-ZW1 concentration increased 100-fold (10 8 CFU / g) and remained at a high level for the next 2 weeks. In contrast, DB-ZW1 concentrations in normal tissues increased to 10 2 There was a steady decline to less than CFU / g (Fig. 5A).

[0352] To further clarify whether DB-ZW1 preferentially grows within tumors, 1 × 10 7 CFU of DB-ZW1 bacteria (125 μL volume) were injected into MB49 bladder tumor-bearing mice via the tail vein, and the number of bacteria distributed in different organs was determined 24 h later (Figure 7B). Results showed that DB-ZW1 rapidly spread within the mice within 30 min after injection, with most DB-ZW1 cells (approximately 99.9%) distributed in the liver, spleen, and blood. The number of DB-ZW1 cells significantly decreased within 4 h after injection, and the total number of DB-ZW1 cells decreased by approximately 90%. Subsequently, DB-ZW1 cells in the tumors grew exponentially at a growth rate of approximately 0.7 h / h and reached saturation within 3 days. In contrast, the number of DB-ZW1 cells in all normal organs steadily decreased. This particular tumor growth profile of DB-ZW1 was important in ensuring the validity of the findings described below.

[0353] 10.3. Antitumor effects of DB-ZW1 against bladder cancer, melanoma, and colon cancer DB-ZW1 was used to treat subcutaneous bladder cancer, in situ melanoma, and subcutaneous and in situ colon cancer mice, and 1×10 7CFU of DB-ZW1 (volume: 125 μL) were injected into tumor-bearing mice via the tail vein. As shown in Figures 8A-8C, DB-ZW1 significantly reduced tumor volume. In addition, treating mice with in situ colon cancer with DB-ZW1 significantly reduced the number of tumors in the mouse colon (Figure 6D).

[0354] Example 11: Construction and characterization of bacteria that express and secrete IL-10 The aim of this example was to determine the effect of constitutive expression and secretion of the fusion polypeptide of the invention in different bacteria.

[0355] The plasmids obtained in Example 8 were electrotransformed into E. coli Nissle and Salmonella typhimurium DB-ZW1 (see Example 9) to obtain the strains Nissle-pelB-D5-huIL10, Nissle-pelB-D5-mIL10, DB-ZW1-pelB-D5-huIL10, and DB-ZW1(Salmonella)-pelB-D5-mIL10.

[0356] Bacteria were grown to express the fusion polypeptides (pelB-D5-huil10 and pelB-D5-mil10) and subjected to Western blot analysis as described in Example 6. Proteins were quantified by ImageJ analysis of Western blot images.

[0357] As shown in Figure 7, the fusion polypeptide of the present invention can be expressed in E. coli Nissle and antitumor Salmonella typhimurium DB-ZW1 and can be efficiently secreted outside the bacterial cells. In particular, the secretion efficiency of the fusion polypeptide of the present invention from E. coli Nissle and Salmonella typhimurium is approximately 80% and 70%, respectively.

[0358] As shown in FIG. 8, the fusion polypeptide of the present invention can be expressed in E. coli Nissle strain and antitumor Salmonella typhimurium DB-ZW1, but can also be efficiently secreted outside the bacterial cells.

[0359] Example 12: Antitumor effect of interleukin-10-secreting bacteria The purpose of this example is to verify the antitumor effect of antitumor bacteria that express and secrete interleukin-10 through in vivo experiments.

[0360] A subcutaneous tumor model for murine MB49 bladder cancer was constructed as described in Example 10.1.

[0361] 12.1. E. coli strain Nissle-pelB-D5-IL10, E. coli strain Nissle (control), and PBS (blank control) were administered to tumor-bearing mice (n=6), respectively (1 × 10 7 CFU, 125 μL volume). Tumor growth in the mice was recorded using calipers on the day the tumor-bearing mice were injected with bacteria, and on days 0, 2, 4, 6, 8, 10, 12 and 14 after injection.

[0362] The longest diameter (a) and the maximum transverse diameter (b) of the tumor in the mouse were accurately measured using a vernier caliper. The tumor volume (V) was calculated as V = a × b 2 Calculated based on the formula (unit: mm 3 ) The tumor volume exceeded the ethical limit of 2000 mm 3 Mice were considered dead when the bacterial count exceeded 0. In addition, the body weight changes of tumor-bearing mice were weighed on days 0, 2, 4, 6, 8, 10, 12, and 14 after bacterial injection to evaluate the biological safety of the bacteria.

[0363] As shown in Figure 9, the tumor size in mice injected with Nissl-pelB-D5-IL10 was significantly reduced compared with mice injected with Nissl and PBS (P<0.005), demonstrating that the bacterially expressed and secreted interleukin-10 had biological function and therapeutic effect on tumors.

[0364] As shown in Figure 10, the body weights of mice injected with Nissl-pelB-D5-IL10 and the Nissl control gradually recovered to normal levels as the number of days after bacterial injection increased. On day 14 after bacterial injection, the body weights of the mice were not statistically different from those of the PBS group, demonstrating that the interleukin-10 expressed and secreted by the bacteria is biologically safe.

[0365] 12.2. DB-ZW1-pelB-D5-IL10, DB-ZW1 (control), and PBS (blank control) were administered to tumor-bearing mice (n=6) (1×10 7 CFU via tail vein injection, volume 125 μL). On the day the bacteria were injected into tumor-bearing mice, and on days 2, 4, 6, and 8 after injection, tumor growth in the mice was recorded using calipers and body weight was measured.

[0366] The longest diameter (a) and the maximum transverse diameter (b) of the tumor in the mouse were accurately measured using a vernier caliper. The tumor volume (V) was calculated as V = a × b 2 Calculated based on the formula (unit: mm 3 ) The tumor volume exceeded the ethical limit of 2000 mm 3 When the serotonin concentration exceeded 100kJ / mL, the mice were considered dead.

[0367] Example 13: Determination of expression by secreted bacteria of interleukin-10 in tumor-bearing mice The purpose of this example was to verify that the antitumor bacteria were successfully expressed in mice, and that the bacteria secreted interleukin-10 and produced promising antitumor effects by detecting the expression levels of interleukin-10 in the liver, spleen, and tumors of tumor-bearing mice.

[0368] A subcutaneous tumor model for murine MB49 bladder cancer was constructed as described in Example 10.1, and mice were injected with E. coli strain Nissl-pelB-D5-IL10, E. coli strain Nissl (control), and PBS (blank control) as described in Example 12.1.

[0369] Mice were dissected 3 days after injection, and the spleen, liver, and tumor tissues were weighed. RIPA lysis solution (medium) (Beyotime, #P0013C) was used to completely lyse the tissue samples on ice according to the manufacturer's instructions (see https: / / m.beyotime.com / mobilegoods.do?method=code&code=P0013C).

[0370] An interleukin-10 ELISA detection kit (Biolegend ELISA MAX™ Deluxe Set IL-10 #430604 / #431414) was used to quantitatively detect the concentration of expressed interleukin-10 in each tissue sample according to the product instructions (see https: / / www.biolegend.com / en-us / products).

[0371] Sequence Listing SEQ ID NO: 1: pET22b-pelB-Amp

[0372] SEQ ID NO: 2: pSC101-huil10-Amp

[0373] SEQ ID NO: 3: Plasmid pET22b-pelB-D5-huil10-Amp

[0374] SEQ ID NO: 4: Plasmid pET22b-pelB-huil10-Amp

[0375] [Table 4]

[0376] SEQ ID NO: 6: pSC101-ptac-sfGFP-KanR

[0377] SEQ ID NO: 7: Plasmid pSC101-ptac-pelB-D5-huil10-KanR

[0378] SEQ ID NO: 8: Plasmid pSC101-ptac-pelB-huil10-KanR

[0379] [Table 5]

[0380] [Table 6]

[0381] [Table 7]

[0382] SEQ ID NO: 12: huIL-10 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0383] SEQ ID NO: 13: huIL-10 monomer SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0384] SEQ ID NO: 14: super-huIL-10 SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0385] SEQ ID NO: 15: super-huIL-10-monomer SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0386] SEQ ID NO: 16: pelB signal peptide MKYLLPTAAAGLLLLAAQPAMA

[0387] SEQ ID NO: 17: pelB-D5-huIL-10 MKYLLPTAAAGLLLLAAQPAMADDDDD SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0388] SEQ ID NO: 18: pelB-D5-huIL-10 monomer MKYLLPTAAAGLLLLAAQPAMADDDDD SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0389] SEQ ID NO: 19: pelB-D5-super-huIL-10 MKYLLPTAAAGLLLLAAQPAMADDDDD SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0390] SEQ ID NO: 20: pelB-D5-super-huIL-10-monomer MKYLLPTAAAGLLLLAAQPAMADDDDD SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0391] SEQ ID NO: 21: pelB-D4-huIL-10 MKYLLPTAAAGLLLLAAQPAMADDDD SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0392] SEQ ID NO: 22: pelB-D4-huIL-10 monomer MKYLLPTAAAGLLLLAAQPAMADDDD SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0393] Sequence number 23: pelB-D4-super-huIL-10 MKYLLPTAAAGLLLLAAQPAMADDDD SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0394] Sequence number 24: pelB-D4-super-huIL-10-monomer MKYLLPTAAAGLLLLAAQPAMADDDD SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0395] Sequence number 50 pUC-ptac-bba0032-KanR

[0396] SEQ ID NO: 51 Plasmid-pUC-ptac-pelB-D5-huil10-KanR

[0397] SEQ ID NO: 52 Plasmid-pUC-ptac-pelB-D5-mil10-KanR

[0398] SEQ ID NO: 53: PsseA-hlyA-loxp-KnaR-loxp

[0399] SEQ ID NO: 54: Pssbp1-dapE-Cm

[0400] SEQ ID NO: 55: PsseA-hlyA-loxp-KnaR-loxp plasmid

[0401] SEQ ID NO: 56: Pssbp1-dapE-Cm plasmid

[0402] SEQ ID NO: 57: PsseA-hlyA-loxp-KnaR-loxp forward primer TTCACAGAAAAGTGTTGCCCCCTTCCATGGCGGAAGGGGGACAAAGGTGAATTTGTCCTACTCAGGAGAGCGTTCA

[0403] SEQ ID NO: 58: PsseA-hlyA-loxp-KnaR-loxp reverse primer TCTGACGTACACAGCAATTTTGCGTTACCTGTTAATCGAGATTGAAACACCGAAGAAAGGCCCACCCG

[0404] SEQ ID NO: 59: Pssbp1-dapE-Cm forward primer TTGGTTTCAGTGAATCCCGTTATCAGCAGTTTTTTGATGAGGTGTAGTCTATTTGTCCTACTCAGGAGAGCG

[0405] SEQ ID NO: 60: Pssbp1-dapE-Cm reverse primer CAATATTTTGCCAGCCAGTCCATGCTTATTTCCTTCTTACCGGAACGCTCACGAAGAAAGGCCCACCCG

Claims

1. A modified bacterium comprising a polynucleotide or expression vector encoding a fusion polypeptide, wherein the fusion polypeptide comprises a signal peptide, an interleukin-10 (IL-10) polypeptide, and a peptide linker linking the signal peptide and the IL-10 polypeptide, the peptide linker consisting of 4 or 5 aspartic acid residues, and the modified bacterium has the ability to express and secrete the IL-10 polypeptide.

2. The modified bacterium of claim 1 , wherein the IL-10 polypeptide is a human IL-10 polypeptide or a variant thereof.

3. The modified bacterium of claim 1 or 2, wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

4. The modified bacterium of any one of claims 1 to 3, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

16.

5. The modified bacterium of any one of claims 1 to 4, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24.

6. 6. The modified bacterium of any one of claims 1 to 5, wherein the bacterium further comprises one or more expression cassettes for expressing essential genes, wherein expression of the essential genes is controlled by an obligate hypoxia-inducible promoter, and wherein the bacterium is deficient in at least one gene, or a functional expression product thereof, involved in or regulating an endogenous antioxidant stress response pathway compared to an unmodified starting strain.

7. The modified bacterium of claim 6 , wherein the obligate hypoxia-inducible promoter is the ssbp1 promoter.

8. 8. The modified bacterium according to claim 6 or 7, wherein the expression product of the essential gene is involved in the synthesis of 2,6-diaminopimelic acid (DAP) in the bacterium, and when cultured under aerobic conditions, the growth of the bacterium is dependent on additional DAP or an analog thereof added to the medium.

9. 9. The modified bacterium of claim 8, wherein the essential genes are selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.

10. 9. The modified bacterium of claim 8, wherein the essential genes are selected from dapA and dapE.

11. The modified bacterium according to any one of claims 6 to 10, wherein the gene involved in or regulating the endogenous antioxidant stress response pathway is a gene of the HtrA serine protease family.

12. 12. The modified bacterium of any one of claims 6 to 11, wherein the essential gene is a gene that naturally occurs in the bacterial chromosome, and the native promoter of the essential gene functionally replaces the obligate hypoxia-inducible promoter, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

13. 12. The modified bacterium of any one of claims 6 to 11, wherein the essential gene expression cassette is exogenous and an essential gene naturally occurring in the bacterial chromosome has been deleted or functionally inactivated, thereby placing expression of the essential gene in the bacterium entirely under the control of the obligate hypoxia-inducible promoter.

14. The modified bacterium of any one of claims 6 to 13, further comprising a pH-regulated expression cassette, the pH-regulated expression cassette comprising a gene hlyA or hlyE encoding a bacterial hemolysin protein, placed under the control of a promoter that is active under acidic pH conditions.

15. 15. The modified bacterium of claim 14, wherein the hlyA or hlyE is derived from Listeria monocytogenes, Vibrio cholerae, or Escherichia coli.

16. 16. The modified bacterium of claim 14 or 15, wherein the promoter active under acidic pH conditions is sseA.

17. The modified bacterium of any one of claims 1 to 16, wherein the unmodified starting strain is a facultative anaerobic bacterium.

18. The modified bacterium of any one of claims 1 to 17, wherein the bacterium is a bacterium of the family Enterobacteriaceae.

19. The modified bacterium of any one of claims 1 to 18, wherein the bacterium is E. coli or Salmonella typhimurium.

20. 20. The modified bacterium of claim 19, wherein the starting strain is E. coli Nissle or Salmonella typhimurium SL7207.

21. A pharmaceutical composition for treating malignant tumors, comprising the modified bacterium of any one of claims 1 to 20.

22. A method for treating a malignant tumor, comprising administering an effective amount of a modified bacterium described in any one of claims 1 to 20 or a pharmaceutical composition described in claim 21 to a subject having a malignant tumor.

23. Use of the modified bacterium of any one of claims 1 to 20 or the pharmaceutical composition of claim 21 in the preparation of a medicament for treating malignant tumors.

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