Recombinant bacterial cells capable of expressing heterogeneous extracellular matrix (ECM) degrading polypeptides using tumor-inducible promoters.

Abstract

Provided herein are recombinant bacterial cells capable of expressing heterologous extracellular matrix (ECM)-degrading polypeptides that degrade extracellular matrix components. The invention also provides promoters, nucleic acids, and cells containing them. The invention also provides heterologous bacterial cells, promoters, nucleic acids, and the medical uses and methods of use of the cells provided herein.

Description

[Technical Field]

[0001] This invention is in the field of cancer treatment. [Background technology]

[0002] Immunological cancer therapies, including immune checkpoint inhibitors (ICIs), antibody therapies, and engineered cell therapies such as chimeric antigen receptor T-cell (CAR-T) therapy, have transformed the treatment landscape for many tumors and offer highly effective treatment options for cancers that were previously refractory.

[0003] Cancer immunotherapy typically requires effective infiltration of host cells and / or therapeutically administered cells into the tumor core. However, the extracellular matrix of the tumor can act as a physical barrier to effective immune cell infiltration into the tumor core, potentially contributing to the development of immune-exclusionary tumors.

[0004] Immune-exclusionary tumors are characterized by a tumor microenvironment (TME) that physically excludes immune cells from the tumor core, thereby limiting the effectiveness of cancer immunotherapy. The extracellular matrix (ECM) is a major contributor to the exclusive nature of the microenvironment in immune-exclusionary tumors, a subset of tumors known as "immunologically cold" tumors.

[0005] In a significant number of immunosuppressive tumors, high levels of extracellular matrix (ECM) components are present, presenting barriers to immunotherapy and dramatically reducing their therapeutic effects. The ECM can mediate elimination in several ways: for example, it constitutes a high-density, highly fibrous environment with high levels of interstitial pressure, creating a biophysical barrier to the invasion of immune cells and other biologics; and it directly captures immunosuppressive growth factors and signals in immune cells, directly affecting their behavior and inhibiting their migration and invasion. Among these elevated ECM components are hyaluronic acid (HA) and proteoglycans such as versican. Most solid tumor indications have been shown to have a certain percentage of immunosuppressive patients. For example, it is estimated that approximately 50% of patients with triple-negative breast cancer have tumors that are immunosuppressive. Among the indications with the highest percentage of immunosuppressive tumors are triple-negative breast cancer, colon cancer, and pancreatic cancer. These are also indications involving very abundant extracellular matrix deposition, including increased levels of proteoglycans (e.g., hyaluronic acid (HA)).

[0006] Immune-exclusionary tumors (IMEs) worldwide are estimated to affect more than 3 million new patients annually, and currently there are no appropriate therapies for patients with these tumor types, nor do they respond to immunotherapy strategies. Therefore, IMEs represent a significant challenge to the effective treatment of a large cohort of cancer patients and contribute to unnecessary cancer-related morbidity and mortality.

[0007] Several methods have been explored to disrupt the solid tumor stroma to remove barriers to invasion, making immune-excluded tumors accessible to immune cells and thus more manageable for immunotherapy. For example, some approaches involve using chemotherapeutic agents, radiotherapy, and inflammatory cytokines to enhance immunotherapy for immune-excluded tumors. However, these approaches do not address the fundamental physical constraints imposed on tumors by the extracellular matrix (ECM), and some of the non-targeting nature of these approaches can lead to undesirable side effects.

[0008] Previous studies have shown that using hyaluronidase to break down hyaluronic acid (HA) in a mouse model of cancer results in normalization of vascular coverage, reduction of hypoxia levels, and increased invasion without affecting tumor spread (Jacobetz et al., 2013, "Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer", Gut, 62(1):112-120; Clift et al., 2019, "Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy", Cancer Res., 79(16):4149-4159; each is incorporated herein by reference in its entirety). Prior attempts to transfer this to clinical practice involved modifying the enzyme through a PEGylation process to increase its time in circulation. Because hyaluronic acid (HA) is produced at a high metabolic turnover level in most healthy tissues, this led to toxicity associated with the circulating level of the enzyme. In fact, the dose administered to patients in clinical trials was up to three orders of magnitude smaller than the dose initially tested in mouse models. Reducing the dosage of the modified enzyme to mitigate toxicity led to decreased efficacy, and the Phase III trial ultimately failed (Van Cutsem et al., 2020, "Randomized Phase III Trial of Pegvorhyaluronidase Alfa With Nab-Paclitaxel Plus Gemcitabine for Patients With Hyaluronan-High Metastatic Pancreatic Adenocarcinoma", J Clin Oncol., 38(27):3185-3194; the whole is incorporated herein by reference).Subsequent data showed that the dose administered to patients was ineffective in the mouse model, demonstrating that dose reductions caused by toxicity levels likely resulted in the lack of efficacy observed in clinical trials.

[0009] Therefore, there is a need to develop novel, safe, and effective therapeutic agents that degrade the extracellular matrix (ECM) components of immune-exclusionary tumors without affecting the stroma of healthy tissue.

[0010] Brief Description of the Invention The inventors have developed recombinant cells capable of degrading components of the extracellular matrix (ECM) by expressing and selectively secreting extracellular matrix (ECM)-degrading polypeptides. Surprisingly, the inventors have found that these cells are capable of degrading the extracellular matrix (ECM), including tumor extracellular matrix (ECM).

[0011] The inventors have also, remarkably, developed a pH and hypoxia-inducible promoter that enables the expression of a promoter-operatably linked gene within a specific pH range and hypoxia conditions. The pH range and hypoxia conditions in which the pH and hypoxia-inducible promoter is active are the same as those present in the tumor microenvironment. Thus, the inventors have remarkably developed an improved promoter, or in other words, a tumor-inducible promoter, that can be activated in the tumor microenvironment. By operatably linking the pH and hypoxia-inducible promoter to an extracellular matrix (ECM)-degrading polypeptide, the present invention enables the expression of an extracellular matrix (ECM)-degrading polypeptide, and the degradation of the extracellular matrix (ECM) by the extracellular matrix (ECM)-degrading polypeptide can be controlled in response to pH and hypoxia.

[0012] The inventors have further surprisingly found that when administered to a subject, the recombinant cells home to tumor tissue and specifically accumulate in tumor tissue. In in vitro experiments, the inventors have found that the recombinant cells infiltrate tumor tissue and further increase the killing of tumor cells in the tissue by chimeric antigen receptor T cells (CAR-T cells).

[0013] Therefore, the inventors have developed recombinant cells that specifically localize to the extracellular matrix (ECM) of a tumor and are capable of degrading it when administered to a subject, and that promote the improvement of tumor infiltration by therapeutic agents. Therefore, the inventors have surprisingly developed recombinant cells that can make immune-excluded tumors accessible to therapeutic agents.

[0014] The inventors have also developed medical methods, medical uses, and treatments related to the recombinant cells.

Mode for Carrying Out the Invention

[0015] The present invention is as described in the claims.

[0016] Recombinant bacterial cells In a first aspect, the present invention provides a recombinant bacterial cell capable of expressing a heterologous extracellular matrix (ECM) degrading polypeptide that degrades an extracellular matrix component. "Capable of expressing" means that the bacterial cell can express a specific polypeptide, for example, an extracellular matrix (ECM) degrading polypeptide, under certain conditions, for example, by transcribing messenger RNA (mRNA) from a nucleotide sequence encoding the polypeptide and / or translating the mRNA into the polypeptide. Thus, in a related aspect, the present invention provides a recombinant bacterial cell that expresses a heterologous extracellular matrix (ECM) degrading polypeptide that degrades an extracellular matrix component.

[0017] The heterologous ECM degrading polypeptide can be any ECM degrading polypeptide. In some embodiments, the extracellular matrix degrading polypeptide is a) Hyaluronidase, such as microbial hyaluronidase or mammalian hyaluronidase, For example, here, the microbial hyaluronidase is bacterial hyaluronidase, b) chondroitin ABC lyase, optionally microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase, For example, here, the microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase, c) neuraminidase, such as microbial neuraminidase or mammalian neuraminidase, For example, here, the microbial neuraminidase is bacterial neuraminidase, and / or d) PNGase, such as microbial PNGase or mammalian PNGase, For example, here, the microbial PNGase is bacterial PNGase, is.

[0018] Various means for producing recombinant bacterial cells capable of expressing specific polypeptides, such as extracellular matrix (ECM) degrading polypeptides, are known to those skilled in the art. For example, cells can be transformed or transfected with a nucleic acid containing a nucleotide sequence encoding a heterologous extracellular matrix degrading (ECM) polypeptide.

[0019] Thus, in one embodiment, the cell comprises a nucleic acid comprising a nucleotide sequence encoding a heterologous extracellular matrix degrading (ECM) polypeptide. The nucleic acid can be any nucleic acid. Thus, in some embodiments, the nucleic acid is DNA. Suitable forms of DNA are known in the art and are discussed herein. In some embodiments, the nucleic acid is RNA. Suitable forms of RNA are known in the art and include mRNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), self-replicating RNA (srRNA), RNA virus vectors, and the like.

[0020] As discussed herein, the extracellular matrix (ECM) may contain proteoglycans. While we do not wish to be bound by any theory, the degradation of proteoglycan components in the extracellular matrix (ECM) contained in tumors is thought to improve access to tumors for therapeutic agents such as immunotherapies.

[0021] As discussed herein, the extracellular matrix (ECM) component that can be appropriately targeted for degradation is proteoglycans. Suitable ECM-degrading polypeptides capable of degrading proteoglycans are known in the art and include (but are not limited to) hyaluronidases, chondroitin ABC lyases, neuraminidases, and / or PNGases. Hyaluronidases, chondroitin ABC lyases, neuraminidases, and PNGases are widely distributed throughout eukaryotes and prokaryotes. Therefore, extracellular matrix (ECM)-degrading polypeptides are a) Eukaryotic hyaluronidase, eukaryotic chondroitin ABC lyase, eukaryotic neuraminidase, and / or eukaryotic PNGase, and / or b) Prokaryotic hyaluronidase, prokaryotic chondroitin ABC lyase, prokaryotic neuraminidase, and / or prokaryotic PNGase It can be selected from the following.

[0022] In some embodiments, the ECM component is a proteoglycan, and the extracellular matrix-degrading polypeptide is a) Hyaluronidase, for example, microbial hyaluronidase or mammalian hyaluronidase, For example, here, microbial hyaluronidase is bacterial hyaluronidase. b) Chondroitin ABC lyase, optionally microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase, For example, here, microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase. c) Neuraminidase, for example, microbial neuraminidase or mammalian neuraminidase, For example, here, microbial neuraminidase is bacterial neuraminidase, and / or d) PNGase, for example, microbial PNGase or mammalian PNGase, for example, where microbial PNGase is bacterial PNGase. That is the case.

[0023] Prokaryotic extracellular matrix (ECM) degrading polypeptides may be microbial extracellular matrix (ECM) degrading polypeptides. Microbial extracellular matrix (ECM) degrading polypeptides may appropriately be bacterial extracellular matrix (ECM) degrading polypeptides. Eukaryotic extracellular matrix (ECM) degrading polypeptides may appropriately be mammalian extracellular matrix (ECM) degrading polypeptides, such as human extracellular matrix (ECM) degrading polypeptides.

[0024] Therefore, in some embodiments, the extracellular matrix component is a proteoglycan. a) ECM-degrading polypeptides are hyaluronidases, optionally microbial hyaluronidases, or mammalian hyaluronidases. Selectively, microbial hyaluronidase is bacterial hyaluronidase, b) The ECM-degrading polypeptide is chondroitin ABC lyase, optionally microbial chondroitin ABC lyase, or mammalian chondroitin ABC lyase. Selectively, microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase, c) ECM-degrading polypeptides are neuraminidases, optionally microbial neuraminidases, or mammalian neuraminidases. Selectively, microbial neuraminidase is bacterial neuraminidase, and / or d) The ECM-degrading polypeptide is PNGase, optionally microbial PNGase, or mammalian PNGase. Selectively, microbial PNGase is bacterial PNGase.

[0025] As discussed herein, examples of proteoglycans that are extracellular matrix (ECM) components that can be appropriately targeted for degradation include, but are not limited to, hyaluronic acid (HA) and versican.

[0026] Hyaluronic acid (HA) is an anionic, non-sulfated glycosaminoglycan widely distributed throughout connective tissue, epithelial tissue, and nerve tissue. It is a major component of the extracellular matrix and contributes to cell proliferation and migration, as well as tumor progression. Hyaluronic acid (HA) has the following structure:

[0027] [ka] Here, n is an integer representing the number of monomers in the hyaluronic acid polymer. As used herein, the terms "hyaluronic acid," "hyaluronan," and "HA" are interchangeable.

[0028] Versican is a large extracellular matrix chondroitin sulfate proteoglycan present in various human tissues and encoded by the VCAN gene. Versican consists of a central protein core linked by multiple chondroitin sulfate chains and has five different isoforms created by differential or alternative splicing. Versican is involved in cell adhesion, migration, and proliferation, and increased versican expression is associated with tumor growth and metastasis.

[0029] As is known in the art, hyaluronic acid (HA) can be degraded by hyaluronidase. Versican can be degraded by different classes of enzymes that target different parts of the glycan chain, including sialic acid, glycosaminoglycans (GAGs), and glycosidic bonds. These include chondroitin ABC lyase, neuraminidase, and / or PNGase.

[0030] As used herein, the terms “degrading,” “degraded,” or “degrade” relate to a process of breaking down a polymer into its constituent subunits, and may include breaking down a polymer chain of a first length into two polymer chains of a second shorter length, and / or breaking down a polymer chain into its constituent monomers or fragments thereof.

[0031] Therefore, in some embodiments, a) The ECM component is hyaluronic acid (HA), and the ECM-degrading polypeptide is hyaluronidase, optionally microbial hyaluronidase, or mammalian hyaluronidase. Selectively, microbial hyaluronidase is bacterial hyaluronidase. b) The ECM component is versican, and the ECM-degrading polypeptide is chondroitin ABC lyase, which is optionally microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase. Selectively, microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase. c) The ECM component is versican, the ECM-degrading polypeptide is neuraminidase, optionally microbial neuraminidase or mammalian neuraminidase, optionally microbial neuraminidase is bacterial neuraminidase, and / or d) The ECM component is versican, and the ECM-degrading polypeptide is PNGase, optionally microbial PNGase or mammalian PNGase. Selectively, microbial PNGase is bacterial PNGase.

[0032] As discussed herein, extracellular matrix (ECM) degrading polypeptides may be any suitable extracellular matrix (ECM) degrading polypeptide, e.g., any suitable hyaluronidase, chondroitin ABC lyase, neuraminidase, and / or PNGase. In the context of degrading extracellular matrix (ECM) components in tumors, it is understood that extracellular matrix (ECM) degrading polypeptides (e.g., hyaluronidase, chondroitin ABC lyase, neuraminidase, and / or PNGase) are "suitable" if they are capable of degrading or degrading extracellular matrix (ECM) components in the tumor environment or microenvironment. As is known in the art, the tumor environment or microenvironment may be hypoxic, anaerobic, anoxic, or microaerophilic. For example, the tumor environment or microenvironment may have an oxygen concentration of less than 2%, e.g., 0-2% and / or 1-2% (McKeown, 2014, "Defining normoxia, physioxia and hypoxia in tumors-implications for treatment response", Br.J.Radiol., 87(1035):20130676). The tumor environment or microenvironment may also be acidic, for example, having a pH of 6.2±0.25 (Chien et al., 2022, "Enhancing the tropism of bacteria via genetically programmed biosensors", Nat.Biomed.Eng., 6:94-104; its contents are incorporated herein by reference in their entirety).Methods for determining whether extracellular matrix (ECM)-degrading polypeptides are capable of degrading the extracellular matrix (ECM) under hypoxic, anaerobic, anoxic, microaerophilic, and / or acidic conditions are known in the art and can be found, for example, in Oueslati et al. 2014, "CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form," Carbohydrate Polymers, 112:102-108 (the whole of which is incorporated herein by reference).

[0033] In some embodiments, heterogeneous extracellular matrix (ECM) degrading polypeptides are a) Hyaluronidase containing or consisting of the amino acid sequence of SEQ ID NO: 1, chondroitin ABC lyase containing or consisting of the amino acid sequence of SEQ ID NO: 3 or 47, neuraminidase containing or consisting of the amino acid sequence of SEQ ID NO: 5, or PNGase containing or consisting of the amino acid sequence of SEQ ID NO: 7, or containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 47, and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 48, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 / 8, or SEQ ID NO: 48.

[0034] In some embodiments, the heterogeneous extracellular matrix (ECM) degrading polypeptide is a bacterial hyaluronidase, and optionally, Heterogeneous extracellular matrix (ECM) degrading polypeptides are a) The amino acid sequence of SEQ ID NO: 1, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2.

[0035] The term “identical to” is known to those skilled in the art and relates to the percentage of identity between two or more nucleic acid sequences or two or more amino acid sequences. The terms “identical to” and “sequence identity” are interchangeable, i.e., if a first sequence is X% “identical” to a second sequence (where X is an integer from 0 to 100), then the first sequence shares X% “sequence identity” with the second sequence.

[0036] To determine the identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length.

[0037] The percentage of identity between two sequences can also be determined using mathematical algorithms. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.USA87:2264-2268 (which is incorporated herein by reference in its entirety), which has been modified as shown in Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA90:5873-5877 (which is incorporated herein by reference in its entirety). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J.Mol.Biol.215:403 (which is incorporated herein by reference in its entirety). To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402 (the entire text is incorporated herein by reference). Alternatively, PSI BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another example of a mathematical algorithm for sequence comparison is the algorithm in Myers and Miller, 1988, CABIOS 4:1117 (the entire text is incorporated herein by reference). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.Another example of a mathematical algorithm for comparing two sequences is the algorithm by Thompson et al., 1994, Nucleic Acids Res. 22(22):4673-80 (the entire algorithm is incorporated herein by reference).

[0038] The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted when calculating the percentage of identity.

[0039] In some embodiments, extracellular matrix (ECM) degrading polypeptides are a) Hymenoptera hyaluronidase, optionally, wasp venom hyaluronidase, hornet venom hyaluronidase (wasp venom hyaluronidase), or giant hornet venom hyaluronidase (tiger wasp venom hyaluronidase), b) Human hyaluronidase-1 (hHyal-1), human hyaluronidase-2 (hHyal-2), human PH-20 (hPH-20), or bovine testicular hyaluronidase (BTS), c) Streptomyces koganeiensis hyaluronidase, Streptomyces zooepidemic hyaluronidase, or Streptomyces pristinaespiralis hyaluronidase d) Streptococcus zooepidemicus hyaluronidase, or e) Penicillium funiculosum hyaluronidase, isn't it.

[0040] In some embodiments, extracellular matrix (ECM) degrading polypeptides are a) Influenza neuraminidase, optionally, avian influenza neuraminidase, b) Clostridium perfringens neuraminidase, optionally Nani or NanJ derived from Clostridium perfringens. c) Chinese hamster ovary (CHO) cell sialidase or Chinese hamster ovary (CHO) cell neuraminidase, d) Bacteroides fragilis neuraminidase, optionally NanH derived from Bacteroides fragilis, or e) Streptococcus pneumoniae neuraminidase isn't it.

[0041] As can be understood, for genes expressed from nucleic acids, it may be advantageous or necessary to incorporate a promoter into the nucleic acid. Therefore, in some embodiments, the nucleic acid further comprises a promoter. The term “promoter” is known to those skilled in the art and is typically a portion of a nucleic acid containing a nucleotide sequence to which a protein (e.g., sigma factor, transcription factor, and RNA polymerase) binds in order to initiate or drive the transcription of a single nucleic acid transcript (e.g., RNA or mRNA) from a nucleic acid (e.g., DNA) downstream of the promoter. In some embodiments, a nucleotide sequence encoding a heterogeneous extracellular matrix (ECM) degrading polypeptide is operably ligated to the promoter. “Operatably ligated” means that the promoter is capable of initiating or driving the transcription of a single nucleic acid transcript (e.g., RNA or mRNA) containing a nucleotide sequence encoding a heterogeneous extracellular matrix (ECM) degrading polypeptide from a nucleic acid (e.g., DNA).

[0042] The promoter may be a constitutive promoter or an inductive promoter. The terms “constitutive promoter” and “inductive promoter” are known to those skilled in the art. In some cases, it may be advantageous to control the expression of a gene encoding a heterogeneous extracellular matrix (ECM) degrading polypeptide, or the transcription of a single nucleic acid transcript (e.g., RNA or mRNA) containing a nucleotide sequence encoding a heterogeneous extracellular matrix (ECM) polypeptide from a nucleic acid (e.g., DNA). As understood, this can be achieved using an inductive promoter in which gene expression or single nucleic acid transcription is initiated or driven in response to the presence or concentration of a particular molecule, or in response to specific environmental conditions such as pH, oxygen concentration, and / or temperature. Thus, in some embodiments, the promoter is an inductive promoter.

[0043] In particular, it may be advantageous to restrict the expression of xenocellular extracellular matrix (ECM) degrading polypeptides by cells to the tumor environment or tumor microenvironment. Therefore, in some embodiments, the inducible promoter is a tumor-inducible promoter. As discussed herein, the tumor environment or tumor microenvironment may be anaerobic, anoxic, or microaerophilic, and / or may have exemplary oxygen concentrations of less than 2%, e.g., 0-2% and / or 1-2% (McKeown, 2014, "Defining normoxia, physioxia and hypoxia in tumors-implications for treatment response", Br.J.Radiol., 87(1035):20130676). The tumor environment or microenvironment may be acidic, for example, having a pH of 6.2 ± 0.25 (Chien et al., 2022, "Enhancing the tropism of bacteria via genetically programmed biosensors", Nat. Biomed. Eng., 6:94-104; the whole is incorporated herein by reference). Therefore, in order to restrict the expression of extracellular matrix (ECM) degrading polypeptides to the tumor environment or tumor microenvironment, the initiation of gene expression or transcription of a single nucleic acid transcript by an inductive promoter may appropriately occur under hypoxic, anaerobic, anoxic, or microaerophilic and / or acidic conditions; in other words, the inductive promoter (e.g., a tumor-inducible promoter) may appropriately be a hypoxia-inducible promoter and / or a pH-inducible promoter. The initiation of gene expression or transcription of a single nucleic acid transcript from a nucleic acid by an inductive promoter may appropriately not occur under oxygen and / or alkaline conditions; in other words, the inductive promoter (e.g., a tumor-inducible promoter) may preferably be a hypoxia-inducible promoter.

[0044] As can be understood, a promoter comprises multiple promoter elements, each containing a distinct nucleotide sequence. Promoter elements may include, or be selected from, an RNA polymerase binding site, a ribosome binding site (RBS), a -35 sequence, a -10 sequence, a Shine-Qalgarno sequence, a Pribno box, an operator sequence, a TATA box, a transcription factor binding site, or any combination thereof. Promoter elements may be arranged in various different ways, and the sequence of each promoter element may be altered to change the promoter's activation specificity (i.e., the conditions under which the promoter initiates or drives the transcription of a single nucleic acid transcript from a nucleic acid downstream of the promoter) or its strength (i.e., the number of copies of a single nucleic acid transcript transcribed under the promoter's control over a given period). Promoter elements may also be arranged, and the sequence of each promoter element may be altered to change the translation initiation specificity or rate. Exemplary promoter elements that may be arranged or altered to alter translation initiation or rate include, for example, a ribosome binding site (RBS).

[0045] For example, to design inductive promoters that are hypoxia-inducible promoters and / or pH-inducible promoters, these can be created by modifying promoter sequences, for example, single nucleotides in specific promoter elements, as disclosed, for example, in Davis et al., 2011, "Design, construction and characterization of a set of insulated bacterial promoters," Nucleic Acids Res., 39(3):1131-1141, and Chen et al., 2018, "Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors," Nat.Commun., 9:64 (each incorporated herein by reference in its entirety). Any promoter element may be modified, and any modification may be made. For example, a single nucleotide modification may include modifications to introduce single nucleotide polymorphisms, transition mutations, transversion mutations, single nucleotide insertions, and / or base deletions.

[0046] In some embodiments, the inducible promoter (such as a tumor-inducible promoter) is a hypoxia-inducible promoter. Any hypoxia-inducible promoter can be incorporated into the nucleic acid, but in some embodiments, the hypoxia-inducible promoter is optionally selected from the group including or consisting of LOR9, LOR7, LOR1, pflE, pepT, YbiY, and pvhb.

[0047] In some embodiments, the hypoxia-inducible promoter is a) Approximately 0% to 2%, approximately 0.2% to 1.8%, approximately 0.4% to 1.6%, approximately 0.6% to 1.4%, approximately 0.8% to 1.2%, approximately 0% to 1%, approximately 0.2% to 0.8%, approximately 0.4% to 0.6%, 1% to 2%, approximately 1.2% to 1.8%, and approximately 1.4% to 1.6%. b)0%~2%, 0.2%~1.8%, 0.4%~1.6%, 0.6%~1.4%, 0.8%~1.2%, 0%~1%, 0.2%~0.8%, 0.4~0.6%, 1%~2%, 1.2%~1.8%, 1.4%~1.6%, c) Percentages less than approximately 2%, less than approximately 1.5%, less than approximately 1.6%, less than approximately 1.4%, less than approximately 1.2%, less than approximately 1%, less than approximately 0.8%, less than approximately 0.6%, less than approximately 0.4%, less than approximately 0.2%, or lower percentages. d) Less than 2%, less than 1.5%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2%, or lower percentages. e) Approximately 2%, approximately 1.8%, approximately 1.6%, approximately 1.4%, approximately 1.2%, approximately 1%, approximately 0.8%, approximately 0.6%, approximately 0.4%, approximately 0.2%, and / or f)2%, 1.8%, 1.6%, 1.4%, 1.2%, 1%, 0.8%, 0.6%, 0.4%, 0.2% It is activated at a certain oxygen concentration and drives the expression of heterologous ECM-degrading polypeptides.

[0048] In some embodiments, the hypoxia-inducible promoter is not activated under normal oxygen conditions and does not drive the expression of heterologous ECM-degrading polypeptides, and optionally, the hypoxia-inducible promoter is... a) Approximately 6% or more, approximately 5% or more, approximately 4% or more, b) Over 6%, over 5%, over 4%, or over 3%, c) Approximately 6%, approximately 5%, approximately 4%, approximately 3%, and / or d) 6%, 5%, 4%, 3% It is not activated at this oxygen concentration and does not drive the expression of heterologous ECM-degrading polypeptides.

[0049] Hypoxic conditions can be simulated in vitro by culturing cells in a liquid culture medium in which the surface of the air-liquid interface is completely or substantially covered with a layer of mineral oil. In some embodiments, a hypoxia-inducible promoter is activated when recombinant cells are incubated in culture medium under mineral oil, driving the expression of heterologous ECM-degrading polypeptides. A suitable method for culturing cells under such conditions can be determined by those skilled in the art, but in some embodiments, the cells are optionally incubated at 37°C for 18 hours.

[0050] In some embodiments, the hypoxia-inducible promoter is the nucleotide sequence of SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, SEQ ID NOs. It includes a ribosome-binding site (RBS) encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105.

[0051] In some embodiments, the hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0052] In some embodiments, the inducible promoter (such as a tumor-inducible promoter) is a pH-inducible promoter. Any pH-inducible promoter can be incorporated into the nucleic acid, but in some embodiments, the pH-inducible promoter is optionally selected from the group including or consisting of LPR7, LPR1, LPR9, Stm1787, hyaA, P170-IL, P170-MG, P1, P2, and P3.

[0053] In some embodiments, the pH-inducible promoter is activated at an acidic pH, driving the expression of heterologous ECM-degrading polypeptides, and optionally, Acidity pH is, a) About pH 5.5 to about pH 7, about pH 5.6 to about pH 6.9, about pH 5.7 to about pH 6.8, about pH 5.8 to about pH 6.7, about pH 5.9 to about pH 6.6, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.2 to about pH 6.3, b) pH 5.5~pH 7, pH 5.6~pH 6.9, pH 5.7~pH 6.8, pH 5.8~pH 6.7, pH 5.9~pH 6.6, pH 6.0~pH 6.5, pH 6.1~pH 6.4, pH 6.2~pH 6.3, c) pH less than approximately 7, less than approximately 6.9, less than approximately 6.8, less than approximately 6.7, less than approximately 6.6, less than approximately 6.5, less than approximately 6.4, less than approximately 6.3, less than approximately 6.2, less than approximately 6.1, less than approximately 6.0, less than approximately 5.9, less than approximately 5.8, less than approximately 5.7, less than approximately 5.6, or a pH lower than these. d) pH less than 7, pH less than 6.9, pH less than 6.8, pH less than 6.7, pH less than 6.6, pH less than 6.5, pH less than 6.4, pH less than 6.3, pH less than 6.2, pH less than 6.1, pH less than 6.0, pH less than 5.9, pH less than 5.8, pH less than 5.7, pH less than 5.6, e) Approximately pH 5.5, approximately pH 5.6, approximately pH 5.7, approximately pH 5.8, approximately pH 5.9, approximately pH 6.0, approximately pH 6.1, approximately pH 6.2, approximately pH 6.3, approximately pH 6.4, approximately pH 6.5, approximately pH 6.6, approximately pH 6.7, approximately pH 6.8, approximately pH 6.9, or approximately pH 7, and / or f) pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, or pH 7 That is the case.

[0054] In some embodiments, the pH-inducible promoter is activated at an acidic pH, driving the expression of heterologous ECM-degrading polypeptides, and optionally, Acidity pH is, a) Approximately pH 5.9 to 6.5, approximately pH 5.95 to 6.45, approximately pH 6.0 to 6.4, approximately pH 6.05 to 6.35, approximately pH 6.1 to 6.3, or approximately pH 6.15 to 6.25. b) pH 5.9~pH 6.5, pH 5.95~pH 6.45, pH 6.0~pH 6.4, pH 6.05~pH 6.35, pH 6.1~6.3, or pH 6.15~pH 6.25 c) Approximately pH 5.9, approximately pH 5.95, approximately pH 6.0, approximately pH 6.05, approximately pH 6.1, approximately pH 6.15, approximately pH 6.2, approximately pH 6.25, approximately pH 6.3, approximately pH 6.35, approximately pH 6.4, approximately pH 6.45, or approximately pH 6.5, and / or d) pH 5.9, pH 5.95, pH 6.0, pH 6.05, pH 6.1, pH 6.15, pH 6.2, pH 6.25, pH 6.3, pH 6.35, pH 6.4, pH 6.45, or pH 6.5 That is the case.

[0055] In some embodiments, the pH-inducible promoter is the nucleotide sequence of SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, SEQ ID NOs. It includes a ribosome-binding site (RBS) encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105.

[0056] In some embodiments, the pH-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, or SEQ ID NO: 61.

[0057] In some embodiments, pH-inducible promoters are not activated at alkaline pH and do not drive the expression of heterologous ECM-degrading polypeptides. The term "alkaline pH" is known to those skilled in the art. A solution has an "alkaline pH" if it has a pH between 7 and 14.

[0058] The inductive promoters disclosed herein (such as tumor-inducible promoters) may be pH-inducible and / or hypoxia-inducible promoters. Therefore, in one embodiment, the inductive promoter (such as tumor-inducible promoter) is a pH-inducible and / or hypoxia-inducible promoter. In some embodiments, the pH-inducible and hypoxia-inducible promoter is L0R9. In some embodiments, a) The L0R9 promoter includes a ribosome-binding site (RBS) encoded by the nucleotide sequence of SEQ ID NO: 73, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 73, and / or b) The LOR9 promoter is encoded by the nucleotide sequence of SEQ ID NO: 55, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55.

[0059] pH-inducible promoters and hypoxia-inducible promoters can be activated at the same pH and O2 concentrations as those provided herein.

[0060] As disclosed herein, promoter elements may be arranged in a variety of different ways, and the arrangement of each promoter element may vary.

[0061] Therefore, in some embodiments, the hypoxia-inducible promoter includes a modified RNA polymerase binding site, a modified ribosome binding site (RBS), a modified -35 sequence, a modified -10 sequence, a modified Shine-Dalgarno sequence, a modified Pribno box, a modified operator sequence, a modified TATA box, a modified transcription factor binding site, or any combination thereof, or a modified regulatory element selected from the group consisting of these.

[0062] In some embodiments, the pH-inducible promoter includes a modified RNA polymerase binding site, a modified ribosome binding site (RBS), a modified -35 sequence, a modified -10 sequence, a modified Shine-Dalgarno sequence, a modified Pribno box, a modified operator sequence, a modified TATA box, a modified transcription factor binding site, or any combination thereof, or a modified regulatory element selected from the group consisting of these.

[0063] The promoter may be identified as a pH-inducible promoter and / or a hypoxia-inducible promoter using methods known in the art. An exemplary method for identifying a promoter as pH-inducible is disclosed in Chen et al., 2018, "Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors," Nat.Commun., 9:64 (which is incorporated herein by reference in its entirety). For example, cells may be provided that contain nucleic acids comprising a nucleotide sequence encoding a promoter sequence operably linked to a nucleotide sequence encoding a detectable marker, such as a luminescent detectable marker. Suitable detectable markers may include, for example, fluorescent proteins (including, but not limited to, GFP, YFP, RFP, mCherry, BFP, iLOV, and any variants or derivatives thereof), luciferases, horseradish peroxidases, alkaline phosphatases, etc. Cells may be cultured under appropriate test conditions, for example, in a medium having a test pH, and / or in a medium that is hypoxic and / or has the hypoxic O2 concentration discussed herein. Appropriate controls may be used, for example, cells may be cultured under appropriate control conditions. When identifying pH-inducible and / or hypoxia-inducible promoters, control conditions may involve culturing cells in a culture medium having a pH different from the test pH, and / or in a medium having oxygen normal or non-hypoxiatric and / or oxygen normal O2 concentrations as discussed herein. The presence or absence of a signal from the cells (e.g., light of a given wavelength) may then be detected under the test conditions and / or control conditions. If a signal is detected under the test conditions, the promoter is determined to be induced under those test conditions, and for example, the promoter may be determined to be a pH-inducible promoter and / or a hypoxia-inducible promoter.Similarly, if, when an appropriate control is used, a signal is detected under the test conditions but not under the control conditions, or is detected at a statistically significantly lower level under the control conditions than under the test conditions, the promoter is determined to be induced under the test conditions, and for example, the promoter may be determined to be a pH-inducible promoter and / or a hypoxia-inducible promoter. The signal may be present at levels 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 700, 800, 900, 10000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or higher under test conditions compared to control conditions. Appropriate methods for detecting detectable markers are known in the art and include, for example, visual inspection, optical microscopy, fluorescence microscopy, flow cytometry (e.g., fluorescence-activated cell sorting (FACS)), and microplate readers.

[0064] It is understood that the promoter may be a dual inducible promoter, which is hypoxia-inducible and pH-inducible. For example, a dual inducible promoter may be induced under hypoxic conditions and may increase in activity at a given pH, for example, the acidic pH provided herein.

[0065] Therefore, in some embodiments, the dual inductive promoter is a hypoxia-inductive promoter whose activity increases under acidic conditions. In some embodiments, the dual inductive promoter is a) Approximately 0% to 2%, approximately 0.2% to 1.8%, approximately 0.4% to 1.6%, approximately 0.6% to 1.4%, approximately 0.8% to 1.2%, approximately 0% to 1%, approximately 0.2% to 0.8%, approximately 0.4% to 0.6%, 1% to 2%, approximately 1.2% to 1.8%, and approximately 1.4% to 1.6%. b)0%~2%, 0.2%~1.8%, 0.4%~1.6%, 0.6%~1.4%, 0.8%~1.2%, 0%~1%, 0.2%~0.8%, 0.4~0.6%, 1%~2%, 1.2%~1.8%, 1.4%~1.6%, c) Percentages less than approximately 2%, less than approximately 1.5%, less than approximately 1.6%, less than approximately 1.4%, less than approximately 1.2%, less than approximately 1%, less than approximately 0.8%, less than approximately 0.6%, less than approximately 0.4%, less than approximately 0.2%, or lower percentages. d) Less than 2%, less than 1.5%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2%, or lower percentages. e) Approximately 2%, approximately 1.8%, approximately 1.6%, approximately 1.4%, approximately 1.2%, approximately 1%, approximately 0.8%, approximately 0.6%, approximately 0.4%, approximately 0.2%, and / or f)2%, 1.8%, 1.6%, 1.4%, 1.2%, 1%, 0.8%, 0.6%, 0.4%, 0.2% It is activated at a certain oxygen concentration and drives the expression of heterologous ECM-degrading polypeptides.

[0066] In some embodiments, the dual inductive promoter is not activated under normal oxygen conditions and does not drive the expression of heterologous ECM-degrading polypeptides, and optionally, the dual inductive promoter is... a) Approximately 6% or more, approximately 5% or more, approximately 4% or more, b) Over 6%, over 5%, over 4%, or over 3%, c) Approximately 6%, approximately 5%, approximately 4%, approximately 3%, and / or d) Activated at oxygen concentrations of 6%, 5%, 4%, and 3%, it drives the expression of heterologous ECM-degrading polypeptides.

[0067] In some embodiments, the dual inducible promoter is activated at an acidic pH to drive the expression and / or increased expression of heterologous ECM-degrading polypeptides, and optionally, the acidic pH is a) About pH 5.5 to about pH 7, about pH 5.6 to about pH 6.9, about pH 5.7 to about pH 6.8, about pH 5.8 to about pH 6.7, about pH 5.9 to about pH 6.6, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.2 to about pH 6.3, b)pH 5.5~pH 7, pH 5.6~pH 6.9, pH 5.7~pH 6.8, pH 5.8~pH 6.7, pH 5.9~pH 6.6, pH 6.0~pH 6.5, pH 6.1~pH 6.4, pH 6.2~pH 6.3 c) pH less than approximately 7, less than approximately 6.9, less than approximately 6.8, less than approximately 6.7, less than approximately 6.6, less than approximately 6.5, less than approximately 6.4, less than approximately 6.3, less than approximately 6.2, less than approximately 6.1, less than approximately 6.0, less than approximately 5.9, less than approximately 5.8, less than approximately 5.7, less than approximately 5.6, or a pH lower than these. d) pH less than 7, pH less than 6.9, pH less than 6.8, pH less than 6.7, pH less than 6.6, pH less than 6.5, pH less than 6.4, pH less than 6.3, pH less than 6.2, pH less than 6.1, pH less than 6.0, pH less than 5.9, pH less than 5.8, pH less than 5.7, pH less than 5.6, e) Approximately pH 5.5, approximately pH 5.6, approximately pH 5.7, approximately pH 5.8, approximately pH 5.9, approximately pH 6.0, approximately pH 6.1, approximately pH 6.2, approximately pH 6.3, approximately pH 6.4, approximately pH 6.5, approximately pH 6.6, approximately pH 6.7, approximately pH 6.8, approximately pH 6.9, or approximately pH 7, and / or f) pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, or pH 7 That is the case.

[0068] In some embodiments, the dual inducible promoter is activated at an acidic pH to drive the expression and / or increased expression of heterologous ECM-degrading polypeptides, and optionally, the acidic pH is a) Approximately pH 5.9 to 6.5, approximately pH 5.95 to 6.45, approximately pH 6.0 to 6.4, approximately pH 6.05 to 6.35, approximately pH 6.1 to 6.3, or approximately pH 6.15 to 6.25. b) pH 5.9~pH 6.5, pH 5.95~pH 6.45, pH 6.0~pH 6.4, pH 6.05~pH 6.35, pH 6.1~6.3, or pH 6.15~pH 6.25 c) Approximately pH 5.9, approximately pH 5.95, approximately pH 6.0, approximately pH 6.05, approximately pH 6.1, approximately pH 6.15, approximately pH 6.2, approximately pH 6.25, approximately pH 6.3, approximately pH 6.35, approximately pH 6.4, approximately pH 6.45, or approximately pH 6.5, and / or d) pH 5.9, pH 5.95, pH 6.0, pH 6.05, pH 6.1, pH 6.15, pH 6.2, pH 6.25, pH 6.3, pH 6.35, pH 6.4, pH 6.45, or pH 6.5 That is the case.

[0069] In some embodiments, the dual inductive promoter is LOR9 or LOR1. In some embodiments, the dual inductive promoter is encoded by the nucleotide sequence of SEQ ID NO: 55 or SEQ ID NO: 56, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55 or SEQ ID NO: 56.

[0070] A dual-inducible promoter can be identified using the same methods described for identifying pH-inducible and hypoxia-inducible promoters. If a promoter is identified as both a pH-inducible promoter and a hypoxia-inducible promoter, it can be identified as a dual-inducible promoter. If a promoter is identified as a hypoxia-inducible promoter, and the activity of the hypoxia-inducible promoter increases under the same conditions as those for identifying the promoter as a pH-inducible promoter, the promoter can be determined to be a dual-inducible promoter.

[0071] In some embodiments, the promoter may be a hybrid promoter. Transcription from a hybrid promoter may be activated by two or more transcription factors, either separately or simultaneously. A hybrid promoter may be constructed, for example, by encoding operator sites for multiple transcription factors within a single promoter, by operably linking a gene to two consecutive promoters, each containing specific regulatory features and transcription initiation sites, or by manipulating different transcription factors that bind to the same operator.

[0072] The optimal activity of heterogeneous extracellular matrix (ECM) degrading polypeptides may depend on their localization to recombinant cells. For example, the expression or localization of ECM degrading polypeptides in cells, or their presentation on the cell surface, restricts ECM degradation to the vicinity of the cell and controls cellular degradation of the ECM. Secretion of ECM degrading polypeptides from cells may allow them to degrade the ECM in areas that are not close to or in contact with the cell. While we do not wish to be constrained by theory, it is conceivable that ECM degrading polypeptides secreted from cells can diffuse through the ECM or tumor and degrade the ECM in areas of the tumor that are not in direct contact with the cell. Polypeptides may not diffuse outside the tumor (e.g., due to polypeptide size and / or short half-life), may not retain extracellular matrix (ECM) degradation activity outside the tumor, and / or may not diffuse outside the tumor in amounts, quantities, or concentrations that effectively degrade the extracellular matrix (ECM) present in the tissues outside the tumor.

[0073] In some embodiments, heterogeneous extracellular matrix (ECM) degrading polypeptides are a) secreted from cells, b) Presented on the cell surface, or c) It is expressed in cells.

[0074] In a preferred embodiment, heterogeneous extracellular matrix (ECM) degrading polypeptides are secreted from the cells.

[0075] "Secreted by cells" means that the polypeptide is actively transported across the cell's plasma membrane (or polypeptide membrane) from the cytoplasm to the extracellular environment via a polypeptide secretory system expressed by the cell. The polypeptide secretory system may be endogenous to the cell or introduced into the cell. Appropriate polypeptide secretion systems may be selected from the group including or comprising type 1 secretion system (T1SS), type 2 secretion system (T2SS), type 3 secretion system (T3SS), flagellar type 3 secretion system (F-T3SS), non-flagellar type 3 secretion system (NF-T3SS), type 4 secretion system (T4SS), type 5 secretion system, type 5 secretion system, type 6 secretion system (T6SS), type 7 secretion system (T7SS), type 8 secretion system (T8SS), type 9 secretion system (T9SS), type 10 secretion system (T10SS), type 11 secretion system (T11SS), autotransporters, Usp45-mediated secretion mechanisms, Sec-dependent secretion systems, ompin-dependent secretion systems, Tat-dependent secretion systems, Lactococcus secretion systems, or any combination thereof.Appropriate secretion systems and homologous signal sequences that can instruct polypeptides for secretion by these systems are described in International Publication No. 2021 / 255480, Burdette et al., 2018, "Developing Gram-negative bacteria for the secretion of heterologous proteins," Microb, Cell Factories, 17:196, Costa et al., 2015, "Secretion systems in Gram-negative bacteria: structural and mechanistic insights," Nat. Rev. Microbiol., 13:343-359, Zhang et al., 2006, "Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli," Nat. Biotechno!, 24:100-104, Borrero et al., 2011, "Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal These are disclosed in “bacteriocins in Lactococcus lactis”, Appl. Microbiol. Biotechnol., 89(1):131-143, and Kim et al., 2014, “Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli”, Biotechnol. Bioeng., 112(2):346-355 (each of which is incorporated herein by reference in its entirety).

[0076] As is known in the art, the secretion of polypeptides from bacterial cells via a specific secretory system requires that the polypeptide contain a secretory signal or secretory domain that targets the polypeptide to the specific secretory system. However, such secretory signals or secretory domains have degenerate sequences, and therefore, secretory signals or secretory domains that are not 100% identical to the consensus sequence or known sequence of the secretory signal or secretory domain can still be secreted by the target secretory system. For example, a secretory signal or secretory domain that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the consensus sequence or known sequence of the secretory signal or domain can be secreted by the target secretory system.

[0077] Appropriate secretory signals or secretory domains can be identified by methods known in the art. Such methods may include, for example, culturing bacterial cells expressing a fusion polypeptide containing a secretory signal or secretory domain and the second polypeptide of interest in a liquid culture medium under test conditions, separating the bacterial cells from the liquid culture medium, and determining the presence or absence of the fusion polypeptide or the second polypeptide of interest in the separated culture medium. Appropriate controls may be used, for example, by separately culturing bacterial cells expressing a control polypeptide containing the second polypeptide but without a secretory signal or secretory domain, separating the bacterial cells from the liquid culture medium, and determining the presence or absence of the control polypeptide in the separated culture medium. If the fusion polypeptide is under the control of an inducible promoter, further appropriate controls may be used. Such controls may include separately culturing bacterial cells expressing the control polypeptide containing a secretory signal or secretory domain and the second polypeptide, separating the bacterial cells from the liquid culture medium, and determining the presence or absence of the control polypeptide in the separated culture medium. If the fusion polypeptide or the second polypeptide of interest is present in the separated culture medium under test conditions, it is determined that the fusion polypeptide or the second polypeptide of interest is secreted by the bacterial cells. Similarly, if an appropriate control is used and the fusion polypeptide or the second polypeptide of interest is present in the isolated culture medium under the test conditions, but the control polypeptide is not present under the control conditions or is present in statistically significantly lower levels under the control conditions than under the test conditions, then the fusion polypeptide or the second polypeptide of interest is determined to be secreted by bacterial cells. The fused polypeptide or the second polypeptide of interest may be present under test conditions at levels 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 900, 10000, or higher than the level at which the control polypeptide is present under control conditions.Methods for detecting proteins in separated liquid culture media are known to those skilled in the art, and include, for example, Western blotting, HPLC, and ELISA.

[0078] Methods for separating bacterial cells from liquid culture media or liquid culture medium are known to those skilled in the art and include, for example, centrifugation and filtration. For example, in some embodiments, bacterial cells can be separated from liquid culture media or liquid culture medium by centrifugation at a specific time of 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm or longer. In some embodiments, bacterial cells can be separated from liquid culture media or liquid culture medium by centrifugation for 1 minute, 2 minutes, 3 minutes, 4 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or longer. In some embodiments, bacterial cells can be separated from liquid culture media or liquid culture medium by filtration. As those skilled in the art will recognize, bacterial cells from different bacterial species have different sizes. For example, Escherichia coli cells have a diameter of approximately 0.25–1 μm and a length of 1–2 μm, while Lactococcus lactis cells have a length of approximately 0.5–1.5 μm and a diameter of approximately 0.7–1 μm. The appropriate filter size for liquid culture medium or for separating bacterial cells from liquid medium can be determined by those skilled in the art.

[0079] Table 1 shows exemplary secretory domains and exemplary secretory systems in which polypeptides containing said secretory domains can be transported from cells.

[0080] [Table 1]

[0081] Such secretory domains can be used to target fusion polypeptides for secretion by cells. Therefore, in some embodiments, heterogeneous extracellular matrix (ECM) degrading polypeptides are a) ECM degradation domain, and b) Secretory domain It is a fusion polypeptide containing [a specific compound].

[0082] In some embodiments, the heterogeneous extracellular matrix (ECM) degrading polypeptide includes two secretory domains.

[0083] In some embodiments, the secretory domain is selected from the group comprising or consisting of the PelB secretory signal sequence, YebF carrier protein, CtxB signal sequence, AIDA-I autotransporter, FliC signal sequence, and Usp45 signal sequence, or any combination thereof. As understood, if polypeptide secretion requires the presence of a specific homologous secretory system (e.g., FliC signal sequence and T3SS), cells containing such secretory system should be selected. However, as described herein, the polypeptide secretory system may be endogenous to the cell or may be introduced into the cell using methods known in the art.

[0084] In some embodiments, the secretory domain is a) The amino acid sequences of SEQ ID NOs. 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35, or containing or consisting of amino acid sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs. 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35, and / or b) Encoded by the nucleotide sequences of SEQ ID NOs. 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs.

[0085] In some embodiments, the fusion polypeptide is a) Contains an ECM degradation domain including hyaluronidase, and i) The amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or containing or consisting of amino acid sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, and / or ii) Encoded by the nucleotide sequences of SEQ ID NOs. 2, 38, 40, 42, 44, and 46, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs. 2, 38, 40, 42, 44, and 46, or b) comprising an ECM degradation domain including neuraminidase, and i) The amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, or ii) Encoded by the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO: 115.

[0086] In some embodiments, the heterogeneous extracellular matrix (ECM) degrading polypeptide includes a cleavage domain. The term “cleavage domain” means a portion of a polypeptide that can be cleaved or cleaved by a polypeptide-degrading polypeptide, such as a protease or peptidase (e.g., endopeptidase or exopeptidase). The cleavage of the cleavage domain may be site-specific or sequence-specific. For example, the cleavage domain may include an amino acid sequence that is recognized and cleaved or cleaved by a specific protease or peptidase capable of cleaving or cleaving the amino acid sequence, or is recognized and cleaved or cleavable. Such an amino acid sequence may be referred to as a “protease site” or “peptidase site.”

[0087] In some embodiments, the cleavage domain is located between the extracellular matrix (ECM) degradation domain and the secretory domain, thereby enabling cleavage of the ECM degradation domain from the secretory domain. Cleavage of the ECM degradation domain from the secretory domain at the cleavage domain is thought to improve the secretion of extracellular matrix (ECM) degradation polypeptides from the cell. While we do not wish to be bound by theory, some secretory domains provided herein are known to associate with the cell's outer membrane or outer membrane proteins during the secretion of cargo proteins fused to the secretory domain, and cleavage of cargo proteins from the secretory domain (e.g., the extracellular matrix (ECM) degradation domains provided herein) is thought to increase the release of extracellular matrix (ECM) degradation polypeptides from the cell surface into the extracellular environment.

[0088] In some embodiments, the cleavage domain comprises an amino acid sequence that is a protease or peptidase present in the target tissue or tumor described herein, for example, a protease or peptidase naturally produced by a human subject, or that is recognized and cleavable or divisible by it. Human proteases and peptidases are known in the art and may be selected from the group including metalloproteinases (including matrix metalloproteinases (MMPs)), serine proteases, cysteine ​​proteases, threonine proteases, aspartate proteases, glutamate proteases, asparagine proteases, and peptidolyases. In some embodiments, the cleavage domain comprises an amino acid sequence that is a matrix metalloproteinase (MMP), or that is recognized and cleavable or divisible by it, i.e., the cleavage domain comprises a matrix metalloproteinase (MMP) site.

[0089] It may be advantageous to provide a nucleotide sequence encoding a transcription-regulating polypeptide that can regulate transcription from a promoter in response to the presence of a given molecule or specific conditions. Therefore, in one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a transcription-regulating polypeptide.

[0090] Transcriptional regulatory polypeptides may appropriately be transcriptional repressors, transcriptional activators, operator proteins, or any combination thereof. Transcriptional regulatory polypeptides may be functionally related to secretory signals or secretory domains. Therefore, in some embodiments, the secretory domain is a FliC signaling sequence, and the transcriptional regulatory peptide is selected from the group including or consisting of FliC repressors, and optionally, the FliC repressor is a GadE repressor. GadE is thought to repress fliC transcription by directly binding to the fliC promoter and repressing transcription. GadE expression should prevent flagellin protein secretion by T3SS and compete with the secretion of heterologous extracellular matrix (ECM) degrading polypeptides.

[0091] In some embodiments, the transcriptional regulatory peptide is a) The amino acid sequence of SEQ ID NO: 49, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49, and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 50.

[0092] Recombinant bacterial cells can be any suitable bacterial cell. However, cells are advantageously those that are genetically manageable, possess the inherent ability to actively migrate to tumors, and induce immune responses within tumors. Due to the infection risks and side effects associated with administering pathogenic cells to a target (e.g., systemic inflammation, toxic shock syndrome, etc.), it is considered advantageous if the cells are not pathogenic or non-pathogenic cells. "Pathogenic cells" means cells capable of causing disease in a target by presenting toxic factors, such as persistence in immune cells. Therefore, in some embodiments, the cells are non-pathogenic bacterial cells, symbiotic cells, or probiotic cells. As is known in the art, symbiotic cells are, for example, components of the target's healthy microbiome on the surface of the target's body or mucous membranes, and are cells that do not normally cause disease or infection in a healthy target. Therefore, symbiotic cells may be components of the gastrointestinal microbiome, skin microbiome, conjunctival microbiome, urethral and bladder microbiome, vaginal microbiome, uterine microbiome, oral microbiome, nasal microbiome, lung microbiome, and / or bile duct microbiome. Probiotic cells are cells that promote the formation or maintenance of a healthy microbiome in a subject and do not typically cause disease or infection in a healthy subject. Probiotic cells may be cells that are components of the healthy microbiome of a subject.

[0093] As disclosed herein, the tumor environment or microenvironment can be hypoxic, anaerobic, anoxic, or microaerophilic. Therefore, it is advantageous that the recombinant cells according to the present invention are viable and / or replicable in hypoxic, anaerobic, anoxic, or microaerophilic environments. Accordingly, in some embodiments, the cells are facultative anaerobic cells.

[0094] Recombinant bacterial cells can be any suitable bacterial cells. In some embodiments, the cells are a) Gram-negative bacterial cells, or b) Gram-positive bacterial cells.

[0095] In some embodiments, Gram-negative bacterial cells are selected from a group including or consisting of Escherichia coli cells. Optionally, Gram-negative bacterial cells include Escherichia coli K12 cells or Escherichia coli Nissile cells. Optionally, Gram-negative bacterial cells include Escherichia coli K12 cells. Randomly selected Escherichia coli K12 cells, Escherichia coli K12 MG1655 cells, Escherichia coli K12 58 cells, Escherichia coli K12 679 cells, Escherichia coli K12 WG1 cells, Escherichia coli K12 5K cells, Escherichia coli K12 58-161 cells, Escherichia coli K12 AB284 cells, Escherichia coli K12 AB311 cells, Escherichia coli K12 AG1 cells, Escherichia coli K12 C600 cells, and Escherichia coli K12 cells. Escherichia coli K12 Cavalli Hfr cells, Escherichia coli K12 DH1 cells, Escherichia coli K12 DH5-α cells, Escherichia coli K12 DP50 cells, Escherichia coli K12 EMG2 cells, Escherichia coli K12 EPI100-T1R cells, Escherichia coli K12 H1443 cells, Escherichia coli K12 HB101 cells, Escherichia coli K12 Hfr3000 cells, Escherichia coli K12 Hfr 3000 X74 cells, Escherichia coli K12 HMS 174 cells, Escherichia coli K12 JM109 cells, Escherichia coli K12 TGI cells, Escherichia coli K12 TOP10 cells, Escherichia coli K12 W1485 cells, Escherichia coli K12 W208 cells, Escherichia coli K12 W3110 cells, Escherichia coli K12 W945 cells, Escherichia coli K12The cells are selected from the group comprising or consisting of WA704 cells and Escherichia coli K12 WG1 cells. In some embodiments, Escherichia coli K12 cells are Escherichia coli K12 MG1655 cells.

[0096] Escherichia coli (E. coli) K12 (such as E. coli K12 MG1655) is a genetically manageable, non-pathogenic, facultative anaerobic, rod-shaped Gram-negative bacterium with a good safety profile, capable of localizing to tumors. Considered safe by regulatory authorities, E. coli K12 (such as E. coli K12 MG1655) has the ability to home to tumors after intravenous administration, with a maximum tolerated dose of 5 × 10⁻⁶. 7 CFU is high (Kang et al., 2020, "Imaging of tumor colonization by Escherichia coli using 18F-FDS PET", Theranostics, 2020;10(11):4958-4966; the whole is incorporated herein by reference).

[0097] In some embodiments, Gram-positive bacterial cells are lactic acid bacteria cells, and optionally, the lactic acid bacteria cells are selected from the group including or consisting of Lactococcus lactis cells, Lactobacillus cells, and Bifidobacterium cells. Optionally, The Gram-positive bacterial cells are Lactococcus lactis MG1363 cells.

[0098] Lactococcus lactis MG1363 is a plasmid-free lactic acid bacterium considered safe by regulatory authorities. As a single-membrane Gram-positive cocci, molecular biological tools for expressing heterologous proteins are well defined, enabling the use of the strain as a raw vector for expressing therapeutic agents (Jacouton et al., 2019, "Anti-tumoral Effects of Recombinant Lactococcus lactis Strain Secreting IL-17A Cytokine," Front Microbiol., 9:3355; the whole article is incorporated herein by reference).

[0099] As discussed herein, the cells are, advantageously, not pathogenic or non-pathogenic. Pathogenic cells can be attenuated by methods known in the art, but the safety profile of these cells may not be equivalent to that of non-pathogenic cells. Therefore, in some embodiments, the cells are not attenuated bacterial cells.

[0100] "Attenuated bacterial cells" refer to bacterial cells that have reduced toxicity compared to pathogenic bacterial cells of the same strain or species that have not been attenuated. "Attenuation" refers to a procedure that weakens the disease-causing factors (pathogens). Attenuated pathogens are weaker and less active compared to unattenuated ones. Attenuation may result from genetic mutations. Genetic mutations may be manipulated or may arise from passage of pathogens in cell culture.

[0101] In some embodiments, the cells are a) Salmonella cells, optionally including Salmonella bongori cells, Salmonella cholreaesuis cells, Salmonella enterica cells, Salmonella enteritidis cells, Salmonella paratyphi cells, Salmonella typhi cells, or Salmonella typhimurium cells. b) Cells of the genus Vibrio, optionally Vibrio cholerae cells or Vibrio fischeri cells. c) Shigella cells, optionally Shigella boydii cells, Shigella dysenteriae cells, Shigella flexneri cells, or Shigella sonnei cells, d) Lactobacillus cells, optionally Lactobacillus buigaricus cells or Lactobacillus plantarum cells. e) Listeria cells, optionally Listeria monocytogenes cells, f) Enterococcus cells, optionally Enterococcus faecium cells, g) Streptococcus cells, optionally, Streptococcus pyogenes cells, Streptococcus zooepidemic cells, or Streptococcus pneumoniae cells, h) Pathogenic Escherichia coli cells, optionally enteroinvasive Escherichia coli (EIEC) cells, adherent invasive Escherichia coli (AIEC) cells, enteroaggregative Escherichia coli (EAEC) cells, Shiga toxin-producing Escherichia coli (Escherichia coli) (STEC) cells, enterotoxigenic Escherichia coli (ETEC) cells, or enterohemorrhagic Escherichia coli (EHEC) cells, i) Bacillus cells, optionally Bacillus CGMCC cell number 5744 or Bacillus subtilis, j) Arthrobacter cells, optionally Arthrobacter sphaeroides cells, k) Cells of the genus Streptomyces, optionally Streptomyces koganeiensis cells, Streptomyces pristinaespiralis cells, or Streptomyces zooepidemic cells, l) Cells of the genus Bacteroides, optionally Bacteroides uniformis cells, isn't it.

[0102] The cells provided herein are bacterial cells. Therefore, as should be understood, the cells are not eukaryotic cells such as fungal cells, mammalian cells, or plant cells. For example, the cells are not cells of the genus Pichia, such as Pichia pastoris cells (also known as Komagataella pastoris cells).

[0103] In some embodiments, heterogeneous extracellular matrix (ECM) degrading polypeptides do not localize to the intracellular compartment of the cell.

[0104] In some embodiments, heterogeneous extracellular matrix (ECM) degrading polypeptides do not localize to the cell membrane or cell wall.

[0105] It may be desirable for nucleic acids to contain transcriptional regulatory elements. Therefore, in some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a transcriptional regulatory element. In some embodiments, a nucleotide sequence encoding a heterogeneous extracellular matrix degradation (ECM) polypeptide is operably ligated to the transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is selected from the group comprising or consisting of a terminator sequence, a repressor-binding sequence, an insulator, an operator sequence, or any combination thereof.

[0106] In some embodiments, a) The terminator is selected from the group including or consisting of rrnB T1 / T2, and is arbitrarily selected. The terminator sequence has the sequence of sequence number 51. b) The repressor binding sequence is selected from the group including or consisting of FNRs, and is selected arbitrarily. The repressor binding sequence has the sequence of SEQ ID NO: 52, and / or c) The insulator sequence is selected from the group including or consisting of RiboJ, and is selected arbitrarily. The insulator sequence has the sequence corresponding to sequence number 53.

[0107] Suitable nucleic acids are known in the art. In some embodiments, the nucleic acid is selected from the group including or consisting of plasmids, phagemids, and bacterial artificial chromosomes (BACs). In some embodiments, optionally, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is incorporated into the cell genome.

[0108] In some embodiments, the nucleic acid may include a detectable marker, such as an antibiotic resistance marker or a colorimetrically detectable marker such as LacZ, or a luminescent detectable marker. Suitable detectable markers include, for example, fluorescent proteins (including, but not limited to, GFP, YFP, RFP, mCherry, BFP, and any variants or derivatives thereof), luciferase, horseradish peroxidase, iLOV, etc. In preferred embodiments, the nucleic acid does not include an antibiotic resistance marker. Therefore, in preferred embodiments, the recombinant bacterial cells provided herein are not resistant to antibiotics.

[0109] Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, electroporation, heat shock transformation, conjugation, or any combination thereof. Methods for incorporating nucleic acids into the genome of cells are known in the art and include, but are not limited to, homologous recombination, non-homologous end joining (NHEJ), transposition, retrotransposition, and chromosome editing methods, such as CRISPR-Cas editing, zinc finger nuclease (ZNF) editing, TALEN editing, and any combination thereof. Such methods are described, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, 4. th This is described in Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2012.

[0110] As discussed above, bacteria (including those provided herein) are known to accumulate in tumors. Therefore, in some embodiments, cells can accumulate in target tissue or tumors. "Accumulating in target tissue or tumor" means that cells are present in the target tissue or tumor at a higher level or concentration compared to the surrounding area of ​​the target tissue or tumor. For example, cells may be determined to have "accumulated in target tissue or tumor" if a greater number of colony-forming units (CFUs) of cells are recoverable from a given volume or mass of target tissue or tumor than are recoverable from an equivalent volume or mass of the surrounding area of ​​the target tissue or tumor. CFUs are determined by methods known in the art, which may include plate counting and microscopy. Such methods may be carried out in vitro or in vivo.

[0111] When this method is performed in vivo, cells can be administered to a subject. For example, if a greater number of colony-forming units (CFUs) of cells are recoverable from a given volume or mass of target tissue or tumor in the subject than are recoverable from an equivalent volume or mass of non-target tissue or tumor in the same subject, then the cells may be determined to have "accumulated in the target tissue or tumor." Methods for recovering tissue or tumor samples for CFU determination are known in the art.

[0112] Similarly, the cells provided herein may include detectable markers, such as luminescent detectable markers. Suitable detectable markers include, for example, fluorescent proteins (including, but not limited to, GFP, YFP, RFP, mCherry, BFP, and any variants or derivatives thereof), luciferase, horseradish peroxidase, etc. Such cells may be administered to a subject. For example, if a higher level of signal (e.g., light of a given wavelength) produced by a detectable marker is detectable or detected in the target tissue or tumor in the subject than is detectable or detected in the target tissue or tumor in the same subject, then the cells may be determined to have "accumulated in the target tissue or tumor." Methods for detecting the accumulation of luminescent and / or fluorescent cells in a subject are known in the art, including, for example, the IVIS Spectrum In Vivo Imaging System, which is described, for example, in Warawa and Lawrenz, 2013, "Bioluminescent Imaging of Bacteria During Mouse Infection," In: Badr, C. (eds) Bioluminescent Imaging. Methods in Molecular Biology, vol 1098. Humana Press, Totowa, NJ (the entire article is incorporated herein by reference).

[0113] To be understood, the term “target tissue,” as used herein, refers to tissue including tumors or cancers.

[0114] The promoter, nucleic acid, and cell of the present invention In a second aspect, the present invention provides a hypoxia-inducible promoter, which is a promoter provided herein. In some embodiments, the hypoxia-inducible promoter is optionally a manipulated hypoxia-inducible promoter.

[0115] In some embodiments, a) The hypoxia-inducible promoter is the LOR9, LOR7, or LOR1 promoter, and / or b) The hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0116] In a preferred embodiment, the hypoxia-inducible promoter is a LOR9 promoter, for example, a promoter encoded by the nucleotide sequence of SEQ ID NO: 55, or a sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55.

[0117] In a preferred embodiment, the hypoxia-inducible promoter is a LOR1 promoter, for example, a promoter encoded by the nucleotide sequence of SEQ ID NO: 55, or a sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55.

[0118] In further embodiments, the present invention provides a pH-inducible promoter, which is a promoter described herein. In some embodiments, the pH-inducible promoter is optionally an engineered pH-inducible promoter.

[0119] In some embodiments, a) The pH-inducible promoter is the LPR9, LPR7, or LPR1 promoter, and / or b) The hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59.

[0120] "Manipulated hypoxia-inducible promoter" and "manipulated pH-inducible promoter" (each also referred to herein as "manipulated promoter") mean that the nucleotide sequence of the promoter is not the natural promoter sequence. However, a manipulated promoter may be derived from a natural promoter and may be created by modifying any promoter element or transcriptional regulatory element described herein, for example, a promoter element selected from the group including or consisting of a ribosome binding site (RBS), operator binding site, and RNA polymerase binding site. For example, a natural promoter may be modified by replacing a promoter element or transcriptional regulatory element that is endogenous to the natural promoter with a heterologous promoter element or transcriptional regulatory element. A manipulated promoter may be created by mutagenesis of a natural promoter or another manipulated promoter, for example, by error-prone PCR, site-directed mutagenesis, chemical mutagenesis, or any combination thereof. Methods for determining whether a promoter is a hypoxia-inducible promoter and / or a pH-inducible promoter are provided herein and may be applied to determine whether a promoter is a manipulated hypoxia-inducible promoter and / or a manipulated pH-inducible promoter.

[0121] In further embodiments, tumor-inducible promoters are provided herein. In some embodiments, the tumor-inducible promoter is a hypoxia-inducible promoter provided herein. In some embodiments, the tumor-inducible promoter is a pH-inducible promoter provided herein. In some embodiments, the tumor-inducible promoter is a hypoxia-inducible and pH-inducible promoter provided herein.

[0122] In a further embodiment, the present invention provides nucleic acids comprising nucleotide sequences of hypoxia-inducible promoters and / or pH-inducible promoters provided herein.

[0123] In a further embodiment, the present invention provides nucleic acids comprising nucleotide sequences of tumor-inducible promoters provided herein.

[0124] In further embodiments, the present invention provides nucleic acids comprising a nucleotide sequence encoding an extracellular matrix (ECM) degrading polypeptide. In some embodiments, the extracellular matrix (ECM) degrading polypeptide encoded by the nucleotide sequence is a heterogeneous extracellular matrix (ECM) degrading polypeptide provided herein. In some embodiments, the nucleic acid further comprises a promoter, optionally the promoter being a promoter provided herein. In some embodiments, the nucleotide sequence encoding the extracellular matrix (ECM) degrading polypeptide is operably ligated to the promoter. In some embodiments, the nucleic acid further comprises a transcriptional regulatory element, optionally the transcriptional regulatory element being a transcriptional regulatory element provided herein. In some embodiments, the nucleotide sequence encoding the extracellular matrix (ECM) degrading polypeptide is operably ligated to the transcriptional regulatory element.

[0125] In some embodiments, the nucleic acid is selected from the group comprising or consisting of plasmids, phagemids, and bacterial artificial chromosomes (BACs), and optionally, the nucleic acid is a plasmid.

[0126] In a further embodiment, the present invention provides cells comprising an engineered hypoxia-inducible promoter, an engineered pH-inducible promoter, and / or nucleic acids provided herein.

[0127] In some embodiments, nucleic acids are a) Located outside the chromosome, or b) It is incorporated into the cell's genome.

[0128] In some embodiments, the cells are recombinant bacterial cells, and optionally, the cells are recombinant bacterial cells provided herein.

[0129] Medical methods and medical uses of recombinant bacterial cells provided herein As discussed herein, the recombinant bacterial cells of the present invention may be useful in medicine, particularly in the treatment of cancer.

[0130] Accordingly, in one embodiment, the present invention provides recombinant bacterial cells provided herein for use in medicine.

[0131] In related embodiments, the present invention provides recombinant bacterial cells provided herein for use as pharmaceuticals.

[0132] In a further embodiment, the present invention provides recombinant bacterial cells provided herein for use in treating cancer.

[0133] In a further related aspect, the present invention provides the use of recombinant bacterial cells provided herein in the manufacture of pharmaceuticals for the treatment of cancer.

[0134] In one embodiment, the present invention provides a method for treating cancer using recombinant bacterial cells provided herein.

[0135] In one embodiment, the present invention provides cells provided herein for use in medicine.

[0136] In related embodiments, the present invention provides cells provided herein for use as pharmaceuticals.

[0137] In a further embodiment, the present invention provides cells provided herein for use in treating cancer.

[0138] In a further relevant aspect, the present invention provides the use of cells provided herein in the manufacture of pharmaceuticals for the treatment of cancer.

[0139] In one embodiment, the present invention provides a method for treating cancer using cells provided herein.

[0140] In some embodiments, the medical and / or therapeutic uses and methods provided herein involve administering an effective dose of recombinant bacterial cells or cells to a subject. The subject may be any suitable subject, but in some preferred embodiments, the subject is human. Preferably, the subject may have cancer, be diagnosed with cancer, be suspected of having cancer, or have recovered from cancer.

[0141] In some embodiments, the cancer is a tumor solid tumor. In preferred embodiments, the cancer is an immune-excluded solid tumor cancer and / or an immune-excluded tumor. As used herein, the terms “immune-excluded solid tumor cancer” and “immune-excluded tumor” are interchangeable. As discussed herein, an immune-excluded tumor is characterized by a tumor microenvironment (TME) that physically and biochemically excludes immune cells from the tumor core, thereby limiting the effectiveness of cancer immunotherapy. Other characteristics of an immune-excluded tumor include, but are not limited to, high levels of stromal deposition that prevent effective infiltration of different subpopulations of immune cells into the tumor microenvironment; vascular breakdown, low levels of oxygen, and acidic pH.

[0142] Cancers and tumors that can be appropriately treated with recombinant bacterial cells or cells provided herein are based on Wilkinson et al. (2017) Oxford Handbook of Clinical Medicine (10th Ed), Oxford University Press, and Cassidy et al. (2015) Oxford Handbook of Oncology (4 th Disclosed in (ed.), Oxford University Press (each incorporated herein by reference in its entirety). In some embodiments, cancer includes an extracellular matrix. In some embodiments, cancer is a cancer selected from or including triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer. Cancer can be primary or secondary cancer, such as metastatic cancer. In some embodiments, cancer is not a cancer that does not include an extracellular matrix. In some embodiments, cancer is not a hematological cancer. In some embodiments, cancer is not a cancer selected from or including leukemia, myeloma, and lymphoma.

[0143] In one embodiment, the present invention provides compositions comprising recombinant bacterial cells or cells provided herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, salts, etc. Suitable pharmaceutical compositions are known in the art. Exemplary pharmaceutical compositions are, for example, Tovey, 2018, Pharmaceutical Formulation: The Science and Technology of Dosage Forms: Volume 64 (Drug Discovery), Royal Society of Chemistry, and Niazi, 2019, Handbook of Pharmaceutical Manufacturing Formulations (3 rd (Ed.), CRC Press (each of which is incorporated herein by reference in its entirety).

[0144] In related embodiments, the present invention provides compositions provided herein for use in medicine.

[0145] In a further relevant aspect, the present invention provides compositions provided herein for use as pharmaceuticals.

[0146] In a further relevant aspect, the present invention provides compositions provided herein for use in methods of treating cancer.

[0147] In a further relevant aspect, the present invention provides the use of the compositions provided herein in the manufacture of pharmaceuticals for the treatment of cancer.

[0148] In one embodiment, the present invention provides a method for treating cancer using compositions provided herein.

[0149] In some embodiments, the medical and / or therapeutic uses and methods provided herein involve administering an effective dose of the composition to a subject.

[0150] In some embodiments, the subject has cancer.

[0151] In some embodiments, the cancer is a tumor solid tumor. In preferred embodiments, the cancer is an immunoexcluded solid tumor cancer and / or an immunoexcluded tumor. As used herein, the terms “immune excluded solid tumor cancer” and “immune excluded tumor” are interchangeable. As discussed herein, an immunoexcluded (or “immunologically cold”) tumor is characterized by a tumor microenvironment (TME) that physically excludes immune cells from the tumor core, thereby limiting the effectiveness of cancer immunotherapy. Other characteristics of an immunoexcluded (or “immunologically cold”) tumor include, but are not limited to: high levels of stromal deposition, vascular breakdown, low levels of oxygen, and acidic pH, which prevent effective infiltration of different subpopulations of immune cells into the tumor microenvironment.

[0152] Cancers and tumors that can be appropriately treated with recombinant bacterial cells or cells provided herein are based on Wilkinson et al. (2017) Oxford Handbook of Clinical Medicine (10th Ed), Oxford University Press, and Cassidy et al. (2015) Oxford Handbook of Oncology (4 th Disclosed in (ed.), Oxford University Press (each of which is incorporated herein by reference in its entirety). In some embodiments, the cancer is a cancer selected from or including triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer. The cancer may be a primary cancer or a secondary cancer, such as a metastatic cancer.

[0153] In one embodiment, the present invention provides a method for degrading the extracellular matrix (ECM) in a tissue, comprising contacting the tissue with recombinant bacterial cells, cells, or compositions provided herein.

[0154] The method may be an in vitro method or an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method, e.g., an in vivo medical method. In some embodiments, the method comprises administering recombinant bacterial cells, cells, or compositions to a subject. The subject may be any subject discussed herein. In some embodiments, the subject has a disease. In preferred embodiments, the disease is cancer. The cancer may be any cancer discussed herein, but in some embodiments, the cancer includes an extracellular matrix. In some embodiments, the cancer is a solid tumor cancer, and optionally, the cancer is an immunoexcluded solid tumor. In some embodiments, the cancer is a cancer selected from or including triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer. The cancer may be a primary cancer or a secondary cancer, e.g., metastatic cancer. In some embodiments, the cancer is not a cancer that does not include an extracellular matrix. In some embodiments, the cancer is not a hematological cancer. In some embodiments, the cancer is not a cancer selected from or including leukemia, myeloma, and lymphoma.

[0155] As disclosed herein, the subject may be any suitable subject, but in some embodiments, the subject is a human subject.

[0156] In one embodiment, the present invention provides the use of recombinant bacterial cells, cells, or compositions provided herein for the degradation of the extracellular matrix (ECM). In some embodiments, the use is in vitro or in vivo. In some embodiments, the use involves administering recombinant bacterial cells, cells, or compositions to a subject. The subject may be any subject provided herein.

[0157] In embodiments relating to the administration of recombinant bacterial cells, cells, or compositions provided herein, in some embodiments, the recombinant bacterial cells, cells, or compositions are administered to a target by a route selected from or including the group consisting of oral administration, intraocular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intratumoral administration, buccal administration, nasal administration, and pulmonary administration. In some embodiments, the recombinant bacterial cells, cells, or compositions are administered by injection, for example, by intravenous injection, intratumoral injection, intraperitoneal injection, intraocular injection, intra-arterial injection, or intramuscular injection. In some embodiments, the recombinant bacterial cells, cells, or compositions are administered as tablets, capsules, powders, boluses, suspensions, or solutions, for example, by oral administration. In some embodiments, the recombinant bacterial cells, cells, or compositions are administered as a spray, for example, by nasal administration and / or pulmonary administration.

[0158] In some embodiments, after administration, recombinant bacterial cells or cells accumulate in the target tissue or tumor. The terms “accumulation in target tissue or tumor” or “accumulation in target tissue or tumor” are discussed herein.

[0159] In some embodiments, recombinant bacterial cells or cells degrade the extracellular matrix (ECM) of a target tissue or tumor. For example, recombinant bacterial cells or cells may degrade any extracellular matrix (ECM) component disclosed herein by expressing, for example, an extracellular matrix (ECM) degrading polypeptide provided herein. The extracellular matrix (ECM) degrading polypeptide may be any extracellular matrix (ECM) degrading polypeptide provided herein, but in preferred embodiments, the extracellular matrix (ECM) degrading polypeptide is secreted by a cell. While we do not wish to be bound by theory, such degradation of the extracellular matrix (ECM) in a target tissue or tumor allows for the entry of cancer therapeutics, such as immune cells and / or immunotherapeutic agents, into the tissue or tumor. While we do not wish to be bound by theory, the entry of immune cells into an immune-exclusionary tumor is thought to enable the effectiveness of immunotherapy strategies that activate such immune cells to eliminate cancer cells. The entry of cancer therapeutics into a tissue or tumor allows the cancer therapeutic to have a relevant therapeutic effect on the tissue or tumor, or improves the therapeutic effect of the cancer therapeutic on the tissue or tumor. In other words, when administered concurrently, the recombinant bacterial cells provided herein or the cell and cancer therapeutics provided herein have a synergistic therapeutic effect on target tissues, tumors, and / or cancer.

[0160] As can be understood, it may be advantageous to administer recombinant bacterial cells, cells, or compositions provided herein to a subject simultaneously with or nearly simultaneously with the administration of a therapeutic agent to the subject. The therapeutic agent may be any suitable therapeutic agent, but is preferably a cancer therapeutic agent. Therefore, in one embodiment, recombinant bacterial cells, cells, or compositions provided herein are, a) administered prior to subsequent administration of cancer treatment drugs, and / or b) It is administered concurrently with cancer treatment drugs.

[0161] The cancer treatment drug can be any suitable cancer treatment drug, but in a preferred embodiment, the cancer treatment drug is cancer immunotherapy. In some embodiments, the cancer treatment drug is selected from the group comprising or comprising antibodies (optionally selected from the group comprising or comprising immune checkpoint inhibitors (ICIs), T cell engagers, and bispecific antibodies or antibody-drug conjugates), hormone therapy, targeted therapy, immunotherapy, cell therapy (optionally selected from the group comprising or comprising T cells, CAR-T cells, and T4 cells), chemotherapeutic agents (optionally selected from the group comprising or comprising FOLFOX, gemcitabine, paclitaxel, cisplatin, epirubicin, and irinotecan), immunomodulatory agents (optionally selected from the group comprising or comprising cytokines and chemokines), vaccines (optionally selected from the group comprising mRNA vaccines), viruses (optionally selected from the group comprising oncolytic viruses), and second live biotherapeutic agents (optionally selected from the group comprising or comprising engineered live biotherapeutic agents and recombinant bacterial cells). In some embodiments, the immune checkpoint inhibitor is selected from the group comprising or consisting of anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti-PDL1 antibodies, and anti-TIGIT antibodies, anti-TIM3 antibodies, anti-NKG2A antibodies, anti-BTLA antibodies, and anti-GITR antibodies, anti-ICOS antibodies, and anti-VISTA antibodies. In some embodiments, the cell therapy is selected from the group comprising or consisting of tumor-infiltrating lymphocytes (TILs), T cells with engineered T cell receptors (TCRs), CAR T cells, and natural killer cells. In some embodiments, chemotherapy includes or is selected from the group comprising alkylating agents (optionally temozolomide and carboplatin), antimetabolites (optionally 5-FU or gemcitabine), antitumor antibiotics (optionally doxorubicin), topoisomerase inhibitors (optionally irinotecan), mitotic inhibitors (optionally taxanes, optionally paclitaxel or docetaxel), and corticosteroids (optionally dexamethasone).

[0162] In one embodiment, the present invention provides a pharmaceutical composition comprising recombinant bacterial cells or cells provided herein and a cancer drug for use in treating cancer. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, salts, etc. Suitable pharmaceutical compositions are known in the art. Exemplary pharmaceutical compositions are, for example, Tovey, 2018, Pharmaceutical Formulation: The Science and Technology of Dosage Forms: Volume 64 (Drug Discovery), Royal Society of Chemistry, and Niazi, 2019, Handbook of Pharmaceutical Manufacturing Formulations (3 rd (Ed.), CRC Press (each of which is incorporated herein by reference in its entirety).

[0163] In one embodiment, the present invention provides a combination of recombinant bacterial cells or cells provided herein and a cancer drug for use in treating cancer.

[0164] In one embodiment, the present invention provides a combination of a cancer therapeutic agent and recombinant bacterial cells or cells provided herein for use in treating cancer.

[0165] In one embodiment, the combination may be provided as a composition, and optionally the composition is a pharmaceutical composition described herein.

[0166] In one embodiment, a) The cancer is a cancer containing extracellular matrix, and optionally, the cancer is selected from the group including or consisting of triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer, and / or b) Cancer is not a cancer that does not contain extracellular matrix, and selectively, cancer is not a hematological cancer, and selectively, cancer is not a cancer that includes or is selected from the group including leukemia, myeloma, and lymphoma.

[0167] In one embodiment, the cancer treatment agent is a cancer immunotherapy agent, which optionally includes or is selected from the group comprising antibodies (optionally selected from the group comprising or comprising immune checkpoint inhibitors (ICIs), T cell engagers, and bispecific antibodies or antibody-drug conjugates), cell therapies (optionally selected from the group comprising or comprising T cells, CAR-T cells, and T4 cells), chemotherapeutic agents (optionally selected from the group comprising or comprising FOLFOX, gemcitabine, paclitaxel, cisplatin, epirubicin, and irinotecan), immunomodulatory agents (optionally selected from the group comprising or comprising cytokines and chemokines), vaccines (optionally selected from the group comprising mRNA vaccines), viruses (optionally selected from the group comprising oncolytic viruses), and a second live biotherapy agent (optionally selected from the group comprising or comprising engineered live biotherapy agents and recombinant bacterial cells). In some embodiments, the immune checkpoint inhibitor is selected from the group comprising or consisting of anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti-PDL1 antibodies, and anti-TIGIT antibodies, anti-TIM3 antibodies, anti-NKG2A antibodies, anti-BTLA antibodies, and anti-GITR antibodies, anti-ICOS antibodies, and anti-VISTA antibodies. In some embodiments, the cell therapy is selected from the group comprising or consisting of tumor-infiltrating lymphocytes (TILs), T cells with engineered T cell receptors (TCRs), CAR T cells, and natural killer cells. In some embodiments, chemotherapy includes or is selected from the group comprising alkylating agents (optionally temozolomide and carboplatin), antimetabolites (optionally 5-FU or gemcitabine), antitumor antibiotics (optionally doxorubicin), topoisomerase inhibitors (optionally irinotecan), mitotic inhibitors (optionally taxanes, optionally paclitaxel or docetaxel), and corticosteroids (optionally dexamethasone).

[0168] In one embodiment, the composition or combination for use is administered to the subject. In one embodiment, a) Recombinant bacterial cells or cells are administered before or after the administration of cancer drugs, and / or b) Recombinant bacterial cells or cells are administered concurrently with cancer treatment drugs.

[0169] In one embodiment, the composition or combination for use is administered to the target by a route selected from or including the group consisting of oral administration, intraocular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intratumoral administration, buccal administration, nasal administration, and pulmonary administration. Appropriate methods of administration via these routes are provided herein.

[0170] In one embodiment, after administration, recombinant bacterial cells or cells accumulate in the target tissue or tumor. The terms “accumulation in target tissue or tumor” or “accumulation in target tissue or tumor” are discussed herein.

[0171] In one embodiment, recombinant bacterial cells or cells degrade the extracellular matrix (ECM) of a target tissue or tumor. For example, recombinant bacterial cells or cells may degrade any extracellular matrix (ECM) component disclosed herein by expressing, for example, an extracellular matrix (ECM) degrading polypeptide provided herein. The extracellular matrix (ECM) degrading polypeptide may be any extracellular matrix (ECM) degrading polypeptide provided herein, but in a preferred embodiment, the extracellular matrix (ECM) degrading polypeptide is secreted by a cell. While we do not wish to be bound by theory, such degradation of the extracellular matrix (ECM) in a target tissue or tumor allows for the entry of immune cells and / or cancer therapeutics such as immunotherapeutic agents into the tissue or tumor.

[0172] Therefore, in one embodiment, degradation of the extracellular matrix (ECM) of the target tissue or tumor allows host immune cells or administered cancer therapeutics to come into contact with and / or enter the tumor. In one embodiment, such contact with and / or entry into the tumor is necessary for or enhances the therapeutic effect of the cancer therapeutic. In other words, when administered concurrently, the recombinant bacterial cells or cells and cancer therapeutics provided herein have a synergistic therapeutic effect on the target tissue, tumor, and / or cancer.

[0173] As should be understood, the recombinant bacterial cells and cells provided herein, upon administration to a subject, will not persist in and / or colonize the subject before, during, or after localization to the target tissue or tumor and / or degradation of the extracellular matrix (ECM) in the target tissue or tumor. For example, recombinant bacterial cells may dissolve and be removed by the immune system during and / or after localization to the target tissue or tumor and / or degradation of the extracellular matrix (ECM) in the target tissue or tumor, or they may be consumed during and / or after localization to the target tissue or tumor and / or degradation of the extracellular matrix (ECM) in the target tissue or tumor. Thus, recombinant bacterial cells or cells, when administered alone or in combination with cancer drugs, including when administered as part of a composition, are consumed or "used up" in the course of their therapeutic and / or medical use. As described herein, in preferred embodiments, the nucleic acids provided herein do not contain antibiotic resistance markers. Therefore, in preferred embodiments, the recombinant bacterial cells provided herein are not resistant to antibiotics. The recombinant bacterial cells or cells provided herein may also be removed from a subject by antibiotic treatment. Treatment with appropriate antibiotics is well known to those skilled in the art.

[0174] In one embodiment, the present invention is a kit of components, a) Recombinant bacterial cells provided herein, b) The manipulated hypoxia-inducible promoter provided herein, c) A manipulated pH-inducible promoter provided herein, d) Nucleic acids provided herein, e) Cells provided herein, f) Compositions provided herein, g) Therapeutic agents, or optional anticancer drugs We provide a kit of components including [the specified component].

[0175] In one embodiment, the present invention provides recombinant bacterial cells, cells, pH-inducible promoters, hypoxia-inducible promoters, nucleic acids, uses, compositions, methods, and kits substantially described herein.

[0176] The present invention also provides embodiments of the following numbered paragraphs: 1. Recombinant bacterial cells capable of expressing heterologous extracellular matrix (ECM)-degrading polypeptides that degrade extracellular matrix components. 2. Recombinant bacterial cells as described in paragraph 1, comprising nucleic acids containing a nucleotide sequence encoding a heterogeneous extracellular matrix degradation (ECM) polypeptide. 3. Extracellular matrix-degrading polypeptides a) Hyaluronidase, optionally microbial hyaluronidase or mammalian hyaluronidase, Selectively, microbial hyaluronidase is bacterial hyaluronidase. b) Chondroitin ABC lyase, optionally microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase, Selectively, microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase. c) Neuraminidase, optionally, microbial neuraminidase or mammalian neuraminidase, Selectively, microbial neuraminidase is bacterial neuraminidase, and / or d) PNGase, optionally, microbial PNGase or mammalian PNGase, optionally, microbial PNGase is bacterial PNGase, Selectively, the extracellular matrix component is proteoglycan. Recombinant bacterial cells as described in paragraph 1 or 2. 4. a) The ECM component is hyaluronic acid (HA), and the ECM-degrading polypeptide is hyaluronidase, optionally microbial hyaluronidase, or mammalian hyaluronidase. Selectively, microbial hyaluronidase is bacterial hyaluronidase. b) The ECM component is versican, and the ECM-degrading polypeptide is chondroitin ABC lyase, which can be optionally microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase. Selectively, microbial chondroitin ABC lyase is bacterial chondroitin ABC lyase. c) The ECM component is versican, and the ECM-degrading polypeptide is neuraminidase, optionally microbial neuraminidase, or mammalian neuraminidase. Selectively, microbial neuraminidase is bacterial neuraminidase, and / or d) The ECM component is versican, and the ECM-degrading polypeptide is PNGase, optionally microbial PNGase or mammalian PNGase. Selectively, microbial PNGase is bacterial PNGase. Recombinant bacterial cells as described in any of paragraphs 1-3. 5. Heterogeneous extracellular matrix (ECM) degrading polypeptides a) Hyaluronidase containing or consisting of the amino acid sequence of SEQ ID NO: 1, chondroitin ABC lyase containing or consisting of the amino acid sequence of SEQ ID NO: 3 or 47, neuraminidase containing or consisting of the amino acid sequence of SEQ ID NO: 5, or PNGase containing or consisting of the amino acid sequence of SEQ ID NO: 7, or containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 47, and / or b) Recombinant bacterial cells as described in any of paragraphs 1 to 3, encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 48, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 / SEQ ID NO: 8, or SEQ ID NO: 48. 6. The heterogeneous extracellular matrix (ECM) degrading polypeptide is bacterial hyaluronidase, and selectively, Heterogeneous extracellular matrix (ECM) degrading polypeptides a) The amino acid sequence of SEQ ID NO: 1, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, and / or b) Recombinant bacterial cells as described in any of paragraphs 1 to 4, encoded by the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. 7. Recombinant bacterial cells as described in paragraphs 2-6, wherein the nucleic acid further contains a promoter. 8. Recombinant bacterial cells as described in paragraph 7, wherein a nucleotide sequence encoding a heterogeneous extracellular matrix degradation (ECM) polypeptide is operably linked to a promoter. 9. Recombinant bacterial cells as described in paragraph 7 or 8, wherein the promoter is an inducible promoter. 10. Recombinant bacterial cells as described in paragraph 9, wherein the inducible promoter is a hypoxia-inducible promoter, and the hypoxia-inducible promoter is optionally selected from the group comprising or consisting of LOR9, LOR7, LOR1, pflE, pepT, YbiY, and pvhb. 11. Hypoxia-inducible promoters a) Approximately 0% to 2%, approximately 0.2% to 1.8%, approximately 0.4% to 1.6%, approximately 0.6% to 1.4%, approximately 0.8% to 1.2%, approximately 0% to 1%, approximately 0.2% to 0.8%, approximately 0.4% to 0.6%, 1% to 2%, approximately 1.2% to 1.8%, and approximately 1.4% to 1.6%. b)0%~2%, 0.2%~1.8%, 0.4%~1.6%, 0.6%~1.4%, 0.8%~1.2%, 0%~1%, 0.2%~0.8%, 0.4~0.6%, 1%~2%, 1.2%~1.8%, 1.4%~1.6%, c) Percentages less than approximately 2%, less than approximately 1.5%, less than approximately 1.6%, less than approximately 1.4%, less than approximately 1.2%, less than approximately 1%, less than approximately 0.8%, less than approximately 0.6%, less than approximately 0.4%, less than approximately 0.2%, or lower percentages. d) Less than 2%, less than 1.5%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2%, or lower percentages. e) Approximately 2%, approximately 1.8%, approximately 1.6%, approximately 1.4%, approximately 1.2%, approximately 1%, approximately 0.8%, approximately 0.6%, approximately 0.4%, approximately 0.2%, and / or f)2%, 1.8%, 1.6%, 1.4%, 1.2%, 1%, 0.8%, 0.6%, 0.4%, 0.2% Recombinant bacterial cells as described in paragraph 10, which are activated at a certain oxygen concentration and drive the expression of heterologous ECM-degrading polypeptides. 12. The hypoxia-inducible promoter is not activated under normal oxygen conditions, does not drive the expression of heterologous ECM-degrading polypeptides, and selectively... The hypoxia-inducible promoter a) Approximately 6% or more, approximately 5% or more, approximately 4% or more, b) Over 6%, over 5%, over 4%, or over 3%, c) Approximately 6%, approximately 5%, approximately 4%, approximately 3%, and / or d) 6%, 5%, 4%, 3% Recombinant bacterial cells as described in paragraph 10 or 11, which are not activated at oxygen concentrations and do not drive the expression of heterologous ECM-degrading polypeptides. 13. Recombinant bacterial cells as described in any of paragraphs 10-12, wherein a hypoxia-inducible promoter is optionally activated when the recombinant cells are incubated in culture medium under mineral oil, at 37°C for 18 hours, and drives the expression of heterologous ECM-degrading polypeptides. 14. a) The hypoxia-inducible promoter is a ribosome-binding site (RBS) encoded by the nucleotide sequence of SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, The sequence includes a ribosome-binding site (RBS) encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs. 69, SEQ ID NOs. 70, SEQ ID NOs. 71, SEQ ID NOs. 72, SEQ ID NOs. 73, SEQ ID NOs. 84, SEQ ID NOs. 85, SEQ ID NOs. 86, SEQ ID NOs. 87, SEQ ID NOs. 88, SEQ ID NOs. 89, SEQ ID NOs. 90, SEQ ID NOs. 91, SEQ ID NOs. 92, SEQ ID NOs. 93, SEQ ID NOs. 94, SEQ ID NOs. 95, SEQ ID NOs. 96, SEQ ID NOs. 97, SEQ ID NOs. 98, SEQ ID NOs. 99, SEQ ID NOs. 100, SEQ ID NOs. 101, SEQ ID NOs. 102, SEQ ID NOs. 103, SEQ ID NOs. 104, or SEQ ID NOs. 105, and / or b) Recombinant bacterial cells as described in any of paragraphs 10 to 13, wherein the hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. 15. Recombinant bacterial cells as described in paragraph 9, wherein the inducible promoter is a pH-inducible promoter, and optionally, the pH-inducible promoter is selected from the group comprising or consisting of LPR7, LPR1, LPR9, Stml787, hyaA, P1, P2, and P3. 16. The pH-inducible promoter is activated at acidic pH, driving the expression of heterologous ECM-degrading polypeptides, and selectively, Acidity pH, a) About pH 5.5 to about pH 7, about pH 5.6 to about pH 6.9, about pH 5.7 to about pH 6.8, about pH 5.8 to about pH 6.7, about pH 5.9 to about pH 6.6, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.2 to about pH 6.3, b)pH 5.5~pH 7, pH 5.6~pH 6.9, pH 5.7~pH 6.8, pH 5.8~pH 6.7, pH 5.9~pH 6.6, pH 6.0~pH 6.5, pH 6.1~pH 6.4, pH 6.2~pH 6.3 c) pH less than approximately 7, less than approximately 6.9, less than approximately 6.8, less than approximately 6.7, less than approximately 6.6, less than approximately 6.5, less than approximately 6.4, less than approximately 6.3, less than approximately 6.2, less than approximately 6.1, less than approximately 6.0, less than approximately 5.9, less than approximately 5.8, less than approximately 5.7, less than approximately 5.6, or a pH lower than these. d) pH less than 7, pH less than 6.9, pH less than 6.8, pH less than 6.7, pH less than 6.6, pH less than 6.5, pH less than 6.4, pH less than 6.3, pH less than 6.2, pH less than 6.1, pH less than 6.0, pH less than 5.9, pH less than 5.8, pH less than 5.7, pH less than 5.6, e) Approximately pH 5.5, approximately pH 5.6, approximately pH 5.7, approximately pH 5.8, approximately pH 5.9, approximately pH 6.0, approximately pH 6.1, approximately pH 6.2, approximately pH 6.3, approximately pH 6.4, approximately pH 6.5, approximately pH 6.6, approximately pH 6.7, approximately pH 6.8, approximately pH 6.9, or approximately pH 7; and / or f) pH 5.5 / pH 5.6 / pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5 / pH 6.6, pH 6.7, pH 6.8, pH 6.9, or pH 7 The recombinant bacterial promoter described in paragraph 15. 17. The pH-inducible promoter is activated at acidic pH, driving the expression of heterologous ECM-degrading polypeptides, and selectively, Acidity pH, a) Approximately pH 5.9 to 6.5, approximately pH 5.95 to 6.45, approximately pH 6.0 to 6.4, approximately pH 6.05 to 6.35, approximately pH 6.1 to 6.3, or approximately pH 6.15 to 6.25. b) pH 5.9~pH 6.5, pH 5.95~pH 6.45, pH 6.0~pH 6.4, pH 6.05~pH 6.35, pH 6.1~6.3, or pH 6.15~pH 6.25 c) Approximately pH 5.9, approximately pH 5.95, approximately pH 6.0, approximately pH 6.05, approximately pH 6.1, approximately pH 6.15, approximately pH 6.2, approximately pH 6.25, approximately pH 6.3, approximately pH 6.35, approximately pH 6.4, approximately pH 6.45, or approximately pH 6.5, and / or d) pH 5.9 / pH 5.95 / pH 6.0, pH 6.05, pH 6.1, pH 6.15, pH 6.2, pH 6.25, pH 6.3, pH 6.35, pH 6.4, pH 6.45, or pH 6.5 The recombinant bacterial promoter described in paragraph 15 or 16. 18. a) The pH-inducible promoter is the nucleotide sequence of SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, and / or 0, which include a ribosome-binding site (RBS) encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 0, 71, 72, 73, 74, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, and / or b) Recombinant bacterial cells as described in any of paragraphs 15-17, wherein the pH-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61. 19. Recombinant bacterial cells as described in any of paragraphs 15-18, wherein the pH-inducible promoter is not activated at alkaline pH and does not drive the expression of heterologous ECM-degrading polypeptides. 20. The inductive promoter is a pH-inducible and / or hypoxia-inducible promoter, and optionally, The pH-inducible and hypoxia-inducible promoter is LOR9, and optionally, a) The LOR9 promoter includes a ribosome-binding site (RBS) encoded by the nucleotide sequence of SEQ ID NO: 73, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 73, and / or b) Recombinant bacterial cells as described in paragraphs 9-19, wherein the LOR9 promoter is encoded by the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55. 21. Recombinant bacterial cells according to any one of paragraphs 10-14 and 20, wherein the hypoxia-inducible promoter comprises a modified RNA polymerase binding sequence, a modified operator sequence, or any combination thereof, or a modified regulatory element selected from the group consisting of these. 22. Recombinant bacterial cells as described in any of paragraphs 15-19 and 20, wherein the pH-inducible promoter comprises a modified RNA polymerase binding sequence, a modified operator sequence, or any combination thereof, or a modified regulatory element selected from the group consisting of these. 23. Heterogeneous extracellular matrix (ECM) degrading polypeptides, a) secreted from cells, b) Presented on the cell surface, or c) Recombinant bacterial cells expressed in cells as described in any of paragraphs 1-22. 24. Recombinant bacterial cells described in paragraphs 1-23, from which heterogeneous extracellular matrix (ECM) degrading polypeptides are secreted. 25. Heterogeneous extracellular matrix (ECM) degrading polypeptides, a) ECM degradation domain, and b) Secretory domain, optionally, The heterogeneous extracellular matrix (ECM) degrading polypeptide contains two secretory domains. Optionally, c) A cleavage domain, optionally, where the cleavage domain is located between the ECM degradation domain and the secretory domain. Recombinant bacterial cells as described in paragraphs 1-24, which are fusion polypeptides containing [the specified compound]. 26. a) The secretory domain is selected from the group consisting of or comprising the PelB secretory signal sequence, YebF carrier protein, CtxB signal sequence, AIDA-I autotransporter, FliC signal sequence, and Usp45 signal sequence, or any combination thereof, and / or b) Recombinant bacterial cells as described in paragraph 24 or 25, wherein the cleavage domain includes a matrix metalloproteinase (MMP) cleavage site. 27. i) The secretory domain, a) Amino acid sequences of SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35. Or containing or consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% the same amino acid sequence as SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and / or b) Encoded by the nucleotide sequences of SEQ ID NOs. 18, SEQ ID NOs. 20, SEQ ID NOs. 22, SEQ ID NOs. 24, SEQ ID NOs. 26, SEQ ID NOs. 28, SEQ ID NOs. 30, SEQ ID NOs. 32, SEQ ID NOs. 34, SEQ ID NOs. 36, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs. And / or ii) The cleavage domain is a) The amino acid sequence of SEQ ID NO: 116, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 116, and / or b) Recombinant bacterial cells as described in any of paragraphs 24-26, encoded by the nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 117. 28. The fusion polypeptide, a) Contains an ECM degradation domain including hyaluronidase, and i) The amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or the ii) Encoded by the nucleotide sequences of SEQ ID NOs. 38, 40, 42, 44, and 46, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs. 38, 40, 42, 44, and 46, or b) comprising an ECM degradation domain including neuraminidase, and i) The amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114, and / or ii) Recombinant bacterial cells as described in any of paragraphs 24-27, encoded by the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO: 115. 29. Recombinant bacterial cells as described in any of paragraphs 2-28, wherein the nucleic acid further comprises a nucleotide sequence encoding a transcription-regulating polypeptide. 30. Recombinant bacterial cells as described in paragraph 29, wherein, if the secretory domain is a FliC signaling sequence, the transcriptional regulatory peptide is selected from the group comprising or consisting of a FliC repressor, and optionally the FliC repressor is a GadE repressor. 31. Transcriptional regulatory peptides, a) The amino acid sequence of SEQ ID NO: 49, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49, and / or b) Recombinant bacterial cells as described in paragraph 30, encoded by the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 50. 32. Recombinant bacterial cells as described in any of paragraphs 1 to 31, which are non-pathogenic bacterial cells, symbiotic cells, or probiotic cells. 33. Recombinant bacterial cells, which are facultative anaerobic cells, as described in any of paragraphs 1 to 32. 34. a) Gram-negative bacterial cells, or b) Recombinant bacterial cells as described in any of paragraphs 1-33, which are Gram-positive bacterial cells. 35. Gram-negative bacterial cells are selected from a group that includes or consists of Escherichia coli cells. Optionally, Gram-negative bacterial cells include Escherichia coli K12 cells. Recombinant bacterial cells as described in paragraph 34, in which, for arbitrary selection, Escherichia coli K12 cells are Escherichia coli K12 MG1655 cells. 36. Gram-positive bacterial cells are lactic acid bacteria cells, and optionally, lactic acid bacteria cells are selected from a group that includes or consists of Lactococcus lactis cells, Lactobacillus cells, and Bifidobacterium cells. Optionally, Recombinant bacterial cells as described in paragraph 34, wherein the Gram-positive bacterial cells are Lactococcus lactis MG1363 cells. 37. Recombinant bacterial cells described in any of paragraphs 1-36, which are not attenuated bacterial cells. 38. a) Salmonella cells, optionally including Salmonella bongori cells, Salmonella cholreaesuis cells, Salmonella enterica cells, Salmonella enteritidis cells, Salmonella paratyphi cells, Salmonella typhi cells, or Salmonella typhimurium cells. b) Cells of the genus Vibrio, optionally Vibrio cholerae cells or Vibrio fischeri cells. c) Shigella cells, optionally Shigella boydii cells, Shigella dysenteriae cells, Shigella flexneri cells, or Shigella sonnei cells, d) Lactobacillus cells, optionally Lactobacillus buigaricus cells, e) Listeria cells, optionally Listeria monocytogenes cells, f) Enterococcus cells, optionally Enterococcus faecium cells, g) Streptococcus cells, optionally, Streptococcus pyogenes cells, and / or h) Pathogenic Escherichia coli cells, enteroinvasive Escherichia coli (EIEC) cells, adherent invasive Escherichia coli (AIEC) cells, Enterotactic Escherichia coli (EAEC) cells, Shiga toxin-producing Escherichia coli (STEC) cells, enterotoxin-producing Escherichia coli (ETEC) cells, or enterohemorrhagic Escherichia coli (EHEC) cells Not the recombinant bacterial cells described in any of paragraphs 1-37. 39. Recombinant bacterial cells as described in any of paragraphs 1 to 38, wherein heterologous extracellular matrix (ECM) degrading polypeptides do not localize into the intracellular compartment of the cell. 40. Recombinant bacterial cells of any of paragraphs 1-39, in which heterogeneous extracellular matrix (ECM) degrading polypeptides do not localize to the cell membrane or cell wall. 41. The recombinant bacterial cell according to any one of paragraphs 2-40, wherein the nucleic acid further comprises a nucleotide sequence encoding a transcriptional regulatory element. 42. The recombinant bacterial cell according to paragraph 41, wherein the nucleotide sequence encoding the heterologous extracellular matrix degrading (ECM) polypeptide is operably linked to a transcriptional regulatory element. 43. The recombinant bacterial cell according to paragraphs 41-42, wherein the transcriptional regulatory element is selected from the group consisting of or comprising a terminator sequence, a repressor binding sequence, an insulator, an operator sequence, or any combination thereof. 44. a) The terminator is selected from the group consisting of or comprising rrnB T1 / T2, and optionally, the terminator sequence has the sequence of SEQ ID NO: 51. b) The repressor binding sequence is selected from the group consisting of or comprising FNR, and optionally, the repressor binding sequence has the sequence of SEQ ID NO: 52, and / or c) The insulator sequence is selected from the group consisting of or comprising RiboJ, and optionally, the insulator sequence has the sequence of SEQ ID NO: 53, the recombinant bacterial cell according to paragraph 43. 45. The nucleic acid is selected from the group consisting of or comprising a plasmid, a phagemid, and a bacterial artificial chromosome (BAC), and optionally, the nucleic acid is a plasmid, the recombinant bacterial cell according to any one of paragraphs 2-44. 46. The recombinant bacterial cell according to any one of paragraphs 2-44, wherein the nucleic acid is integrated into the genome of the cell. 47. The recombinant bacterial cell according to any one of paragraphs 1-46, which is capable of accumulating in a target tissue or tumor. 48. A hypoxia-inducible promoter which is the promoter according to any one of paragraphs 10-14 and 20, and optionally, an engineered hypoxia-inducible promoter. 49. a) The hypoxia-inducible promoter is the LOR9, LOR7 promoter, or LOR1 promoter, and / or b) The hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, the hypoxia-inducible promoter according to paragraph 48. 50. A pH-inducible promoter that is the promoter according to any one of paragraphs 15 to 19 and 21, and optionally, an engineered pH-inducible promoter. 51. a) The pH-inducible promoter is the LPR9, LPR7 promoter, or LPR1 promoter, and / or b) The pH-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59, the pH-inducible promoter according to paragraph 50. 52. A nucleic acid comprising the nucleotide sequence of the hypoxia-inducible promoter according to paragraphs 48 to 49 and / or the pH-inducible promoter according to paragraphs 50 to 51. 53. A nucleic acid comprising a nucleotide sequence encoding an extracellular matrix (ECM)-degrading polypeptide. 54. The nucleic acid according to paragraph 53, wherein the extracellular matrix (ECM)-degrading polypeptide is the heterologous extracellular matrix (ECM)-degrading polypeptide according to any one of paragraphs 1 to 6 and 19 to 28. 55. The nucleic acid according to paragraph 53 or 54, further comprising a promoter, wherein optionally the promoter is a promoter described in any of paragraphs 7-18 and 48-51. 56. The nucleic acid described in paragraph 55, wherein a nucleotide sequence encoding an extracellular matrix (ECM) degrading polypeptide is operably linked to a promoter. 57. A nucleic acid according to any one of paragraphs 52 to 56, further comprising a transcriptional regulatory element, wherein optionally the transcriptional regulatory element is a transcriptional regulatory element described in any one of paragraphs 41 to 44. 58. The nucleic acid described in paragraph 57, wherein a nucleotide sequence encoding an extracellular matrix (ECM) degrading polypeptide is operably linked to a transcriptional regulatory element. 59. Selected from the group comprising or consisting of plasmids, phagemids, and bacterial artificial chromosomes (BACs), The nucleic acid is a plasmid, as described in any of paragraphs 52-58. 60. Cells comprising a modified hypoxia-inducible promoter as described in any of paragraphs 48-49, a modified pH-inducible promoter as described in any of paragraphs 50-51, and / or a nucleic acid as described in any of paragraphs 52-59. 61. Nucleic acids, a) Located outside the chromosome, or b) Cells described in paragraph 60, which are incorporated into the cell genome. 62. Recombinant bacterial cells, wherein the cells are optionally, the recombinant bacterial cells described in any of paragraphs 1 to 47, as described in paragraphs 60 to 61. 63. Recombinant bacterial cells, as described in any of paragraphs 1-47, for use in medicine. 64. Recombinant bacterial cells as described in any of paragraphs 1-47, for use as a pharmaceutical. 65. Recombinant bacterial cells, as described in any of paragraphs 1-47, for use in treating cancer. 66. Cells described in any of paragraphs 60-62 for use in medicine. 67. Cells described in any of paragraphs 60-62, for use as a medicine. 68. Cells described in any of paragraphs 60-62 for use in treating cancer. 69. Recombinant bacterial cells for use as described in any of paragraphs 63-65 or cells for use as described in any of paragraphs 66-68, wherein the use comprises administering an effective dose of cells to a subject having cancer. 70. Recombinant bacterial cells for use as described in paragraph 65 or 69, or cells for use as described in either paragraph 68 or 69, wherein the cancer is a tumor solid carcinoma, and optionally, the cancer is an immune-excluded solid tumor carcinoma and / or an immune-excluded tumor. 71. a) The cancer is a cancer containing extracellular matrix, and optionally, the cancer is selected from the group including or consisting of triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer, and / or b) Recombinant bacterial cells for use as described in paragraphs 65, 69, or 70, or cells for use as described in any of paragraphs 68-70, wherein the cancer is not a cancer that does not contain an extracellular matrix, and optionally, the cancer is not a hematological cancer, and optionally, the cancer is not a cancer selected from the group including or consisting of leukemia, myeloma, and lymphoma. 72. A composition comprising recombinant bacterial cells from paragraphs 1 to 47 or cells from paragraphs 60 to 62, wherein the composition is optionally a pharmaceutical composition. 73. The composition described in paragraph 72 for use in medicine. 74. The composition described in paragraph 72 for use as a pharmaceutical. 75. The composition described in paragraph 72 for use in a method of treating cancer. 76. Use A composition for use as described in any of paragraphs 73 to 75, comprising administering an effective dose of the composition to a target. 77. A composition for use according to any of paragraphs 73 to 76, wherein the cancer is a solid tumor carcinoma, and optionally, the cancer is an immunoexcludable solid tumor. 78. A composition for use according to any of paragraphs 73 to 77, wherein the cancer is a cancer selected from or including triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer. 79. A method for degrading the extracellular matrix (ECM) in a tissue, comprising contacting the tissue with recombinant bacterial cells described in any of paragraphs 1 to 47, cells described in paragraphs 60 to 61, or a composition described in paragraph 72. 80. The in vivo method described in paragraph 79. 81. The method according to paragraph 79 or 80, comprising administering recombinant bacterial cells, cells, or compositions. 82. The method described in paragraph 81, wherein the subject has a disease. 83. The method according to paragraph 82, wherein the disease is cancer, and optionally, the cancer is a solid tumor carcinoma, and optionally, the cancer is an immunoexclusionary solid tumor. 84. a) The cancer is a cancer containing extracellular matrix, and optionally, the cancer is selected from the group including or consisting of triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer, and / or b) The method according to paragraph 83, wherein the cancer is not a cancer that does not contain extracellular matrix, and optionally, the cancer is not a hematological cancer, and optionally, the cancer is not a cancer selected from the group including or consisting of leukemia, myeloma, and lymphoma. 85. The in vitro method described in paragraph 79. 86. Recombinant bacterial cells for use as described in any of paragraphs 63-65 or 69-71, cells for use as described in any of paragraphs 66-71, compositions for use as described in any of paragraphs 73-78, or methods as described in any of paragraphs 79-85, for which the subject is a human subject. 87. Use of recombinant bacterial cells described in any of paragraphs 1 to 47, cells described in paragraphs 60 to 62, or compositions described in paragraph 72 for degrading the extracellular matrix (ECM). 88. Use as described in paragraph 87, either in vitro or in vivo. 89. Uses as described in paragraphs 87-88, including administration to recombinant bacterial cells, cells, or compositions. 90. Recombinant bacterial cells, cells, or compositions are administered to a subject by a route selected from or including oral administration, intraocular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intratumoral administration, buccal administration, nasal administration, or pulmonary administration, wherein the recombinant bacterial cells, cells, or compositions are administered to the subject by a route including or selected from the group consisting of oral administration, intraocular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intratumoral administration, buccal administration, nasal administration, or pulmonary administration. Recombinant bacterial cells for use as described in any of paragraphs 63-65, 69-71, and 86, cells for use as described in any of paragraphs 66-71 or 86, compositions for use as described in any of paragraphs 73-78 and 86, methods as described in any of paragraphs 79-86, or uses as described in any of paragraphs 87-89. 91. Recombinant bacterial cells for use, composition for use, method, or use described in paragraph 90, wherein, after administration, recombinant bacterial cells or cells accumulate in the target tissue or tumor of the subject. 92. Recombinant bacterial cells or cells that degrade the extracellular matrix (ECM) of a target tissue or tumor, as described in paragraph 91, a composition for use, a method for use, or a use. 93. Recombinant bacterial cells, cells, or compositions a) administered before or after administration of cancer treatment drugs, and / or b) Recombinant bacterial cells for use as described in any of paragraphs 63-65, 69-71, 86 and 90-92, cells for use as described in any of paragraphs 66-71, 86 and 90-92, compositions for use as described in any of paragraphs 73-78, 86 and 90-92, the method described in any of paragraphs 79-86 and 90-92, or the use described in any of paragraphs 87-92 and 76-77, administered concurrently with a cancer drug. 94. Cancer treatment drugs, a) Antibodies (optionally selected from the group including or comprising immune checkpoint inhibitors (ICIs), T cell engagers, and bispecific antibodies or antibody-drug conjugates), cell therapies (optionally selected from the group including or comprising T cells, CAR-T cells, and T4 cells), chemotherapeutic agents (optionally selected from the group including or comprising FOLFOX, gemcitabine, paclitaxel, cisplatin, epirubicin, and irinotecan), immunomodulatory agents (optionally selected from the group including or comprising cytokines and chemokines), vaccines (optionally, mRNA vaccines), viruses (optionally selected from the group including oncolytic viruses), and second live biotherapeutic agents (optionally selected from the group including or comprising engineered live biotherapeutic agents and recombinant bacterial cells), and / or b) Antibodies (optionally, immune checkpoint inhibitor antibodies, bispecific antibodies, or antibody-drug conjugates), hormone therapy, targeted therapy, immunotherapy, cell therapy (optionally, tumor-infiltrating lymphocytes (TILs), lymphocytes with engineered T cell receptors (TCRs), CARs) T cells or natural killer cells), immunomodulatory agents (optionally, chemokines or cytokines), vaccines (optionally, mRNA vaccines), viruses (optionally, oncolytic viruses), other live biotherapeutic agents (optionally, engineered live biotherapeutic agents), chemotherapy (optionally, alkylating agents (optionally, temozolomide and carboplatin), antimetabolites (optionally, 5-FU or gemcitabine), antitumor antibiotics (optionally, doxorubicin), topoisomerase inhibitors (optionally, irinotecan), mitotic inhibitors (optionally, taxanes such as paclitaxel or docetaxel), corticosteroids (optionally, dexamethasone) Recombinant bacterial cells for use, compositions for use, methods, or uses described in paragraph 93, comprising or selected from the group consisting of the above. 95. A pharmaceutical composition for use in treating cancer, comprising a recombinant bacterial cell as described in any one of paragraphs 1 to 47 or a cell as described in any one of paragraphs 60 to 62, and a cancer therapeutic agent. 96. A combination for use in treating cancer, comprising a recombinant bacterial cell as described in any one of paragraphs 1 to 47 or a cell as described in paragraphs 60 to 62, and a cancer therapeutic agent. 97. A combination for use in treating cancer, comprising a cancer therapeutic agent and a recombinant bacterial cell as described in any one of paragraphs 1 to 47 or a cell as described in paragraphs 60 to 62. 98. a) The cancer is a cancer containing an extracellular matrix, optionally the cancer is selected from the group consisting of or comprising triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer, and / or b) The cancer is not a cancer that does not contain an extracellular matrix, optionally the cancer is not a blood cancer, optionally the cancer is not a cancer selected from the group consisting of or comprising leukemia, myeloma, and lymphoma. The pharmaceutical composition for use according to paragraph 95, or the combination for use according to paragraph 96 or 97. 99. The cancer therapeutic agent is a cancer immunotherapy agent, optionally the cancer immunotherapy agent is a) An antibody (optionally selected from the group consisting of or comprising an immune checkpoint inhibitor (ICI), a T cell engager, and a bispecific antibody or an antibody-drug conjugate), a cell therapy (optionally selected from the group consisting of or comprising T cells, CAR-T cells, and T4 cells), a chemotherapeutic agent (optionally selected from the group consisting of or comprising FOLFOX, gemcitabine, paclitaxel, cisplatin, epirubicin, and irinotecan), an immunomodulatory agent (optionally selected from the group consisting of or comprising cytokines and chemokines), a vaccine (optionally an mRNA vaccine), a virus (optionally an oncolytic virus), and a second live biotherapeutic agent (optionally selected from the group consisting of or comprising an engineered live biotherapeutic agent and a recombinant bacterial cell), and / or b) Antibodies (optionally, immune checkpoint inhibitor antibodies, bispecific antibodies, or antibody-drug conjugates), hormone therapy, targeted therapy, immunotherapy, cell therapy (optionally, tumor-infiltrating lymphocytes (TILs), lymphocytes with engineered T cell receptors (TCRs), CARs) T cells or natural killer cells), immunomodulatory agents (optionally, chemokines or cytokines), vaccines (optionally, mRNA vaccines), viruses (optionally, oncolytic viruses), other live biotherapeutic agents (optionally, engineered live biotherapeutic agents), chemotherapy (optionally, alkylating agents (optionally, temozolomide and carboplatin), antimetabolites (optionally, 5-FU or gemcitabine), antitumor antibiotics (optionally, doxorubicin), topoisomerase inhibitors (optionally, irinotecan), mitotic inhibitors (optionally, taxanes such as paclitaxel or docetaxel), corticosteroids (optionally, dexamethasone) A pharmaceutical composition for use as described in paragraph 95 or 98, or a combination for use as described in any of paragraphs 96 to 98, comprising or selected from the group consisting of the above. 100. Recombinant bacterial cells or cells are administered to the target, and optionally, a) Recombinant bacterial cells or cells are administered before or after the administration of cancer drugs, and / or b) Recombinant bacterial cells or cells are administered concurrently with cancer drugs. A composition for use as described in any of paragraphs 95, 98, or 99, or a combination for use as described in any of paragraphs 96 to 99. 101. A composition for use as described in paragraph 95 or 98-100, or a combination for use as described in paragraph 96-100, which is administered to a subject by a route selected from or including oral administration, intraocular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intratumoral administration, buccal administration, nasal administration, or pulmonary administration. 102. A composition for use described in any of paragraphs 95 or 98-101, or a combination for use described in any of paragraphs 96-101, wherein, after administration, recombinant bacterial cells or cells accumulate in the target tissue or tumor. 103. A composition or combination for use described in paragraph 102, wherein recombinant bacterial cells or cells degrade the extracellular matrix (ECM) of a target tissue or tumor. 104. Degradation of the extracellular matrix (ECM) of the target tissue or tumor allows the cancer drug to come into contact with and / or enter the tumor. Recombinant bacterial cells for use, compositions for use, methods or uses described in paragraph 93, or compositions or combinations for use described in paragraph 103, wherein such contact with and / or entry into a tumor is required for the therapeutic effect of the cancer drug. 105. A kit of components, a) Recombinant bacterial cells as described in any of paragraphs 1 to 39, b) The manipulated hypoxia-inducible promoter described in paragraph 40, c) The manipulated pH-inducible promoter described in paragraph 41, d) Nucleic acids as described in any of paragraphs 42-49, e) Cells described in any of paragraphs 50-59, f) A composition described in any of paragraphs 61 to 64, g) A kit of components, including a therapeutic agent, optionally containing an anticancer drug. [Brief explanation of the drawing]

[0177] [Figure 1] A) Luminescence measurement of bioluminescent Escherichia coli (E. coli) K12 MG1655 using the Photoimager Optima In Vivo Imaging System, demonstrating the successful generation of a bioluminescent strain. B) Body weight of mice intravenously administered bioluminescent Escherichia coli (E. coli) K12 MG1655, heat-sterilized bacteria, or vehicle alone at doses of 106, 107, and 108 CFU. [Figure 2] Bioluminescence imaging using the Photoimager Optima In Vivo Imaging System, demonstrating the localization of bioluminescent Escherichia coli (E. coli) K12 MG1655 to tumors in mice. [Figure 3] A) Bioluminescence imaging using the Photoimager Optima In Vivo Imaging System, showing the localization of bioluminescent Escherichia coli (E. coli) K12 MG1655 to tumors in mice. B) Quantification of bacterial load by CFU in four tissue samples (liver, spleen, heart, and tumor) from eight mice, showing accumulation of Escherichia coli (E. coli) K12 MG1655 in tumors. [Figure 4] Functional analysis of Escherichia coli K12 MG1655 PelB-bH. Error bars = standard error of the mean. All data are representative of more than three experiments. [Figure 5] Bacterial infestation in patient-derived tissue samples. GFP-expressing bacteria are shown in green (white) and identified by white arrows, while the extracorporeal membrane (ECM) is shown in blue (gray). [Figure 6] Degradation of hyaluronic acid (HA) in patient exgraft tissue by Escherichia coli K12 MG1655 PelB-bH. IHC staining of HA. Untreated tissue represents tissue incubated with culture medium only. [Figure 7] HA degradation by bH-expressing bacteria has a synergistic effect with CAR-T cell-mediated cancer cell killing in patient-derived tissue explants. The bar graph shows the relative cell killing by each treatment group in the assay. [Figure 8-1]ECM degradation by secreted bacterial enzymes. A) Functional analysis of secreted bacterial hyaluronidase. Bar graphs show the level of hyaluronic acid in the culture medium after each treatment group in the assay. B) Western blot for amino-terminal Flag tags fused to bH. Predicted bH size approx. 90 kDa (arrow). Ctrl - No induction. Rh - Induction with rhamnose. GFP - Bacteria expressing GFP. 1B - Bacteria expressing bacterial hyaluronidase. Error bars = standard error of the mean. All data are representative of more than three experiments. C) Level of neuraminidase activity secreted by Escherichia coli (E. coli) K12 MG1655 PelB-Nh. Negative control Escherichia coli (E. coli) K12 MG1655 GFP. Error bars = standard error of the mean. All data are representative of more than three experiments. D) Evaluation of the secretory system by measuring neuraminidase as a payload using a neuraminidase assay kit (Abeam, UK). The bar graph shows the level of neuraminidase (NH) in the culture medium after each treatment group in the assay. Evec = No payload. MMP = Cleavage site for releasing payloads for the YebF, FliC, and AidaC secretory systems. Error bars = Standard error of the mean. All data are representative of three or more experiments. [Figure 8-2]ECM degradation by secreted bacterial enzymes. A) Functional analysis of secreted bacterial hyaluronidase. Bar graphs show the level of hyaluronic acid in the culture medium after each treatment group in the assay. B) Western blot for amino-terminal Flag tags fused to bH. Predicted bH size approx. 90 kDa (arrow). Ctrl - No induction. Rh - Induction with rhamnose. GFP - Bacteria expressing GFP. 1B - Bacteria expressing bacterial hyaluronidase. Error bars = standard error of the mean. All data are representative of more than three experiments. C) Level of neuraminidase activity secreted by Escherichia coli (E. coli) K12 MG1655 PelB-Nh. Negative control Escherichia coli (E. coli) K12 MG1655 GFP. Error bars = standard error of the mean. All data are representative of more than three experiments. D) Evaluation of the secretory system by measuring neuraminidase as a payload using a neuraminidase assay kit (Abeam, UK). The bar graph shows the level of neuraminidase (NH) in the culture medium after each treatment group in the assay. Evec = No payload. MMP = Cleavage site for releasing payloads for the YebF, FliC, and AidaC secretory systems. Error bars = Standard error of the mean. All data are representative of three or more experiments. [Figure 9-1]A) and B) GFP fluorescence levels under normal and hypoxic conditions at pH 7 and pH 6. GFP = Positive control E. coli K12 MG1655 constitutively expressing GFP. LO / LP R7, R9, R1 = E. coli K12 MG1655 expressing GFP under a manipulated hypoxia-inducible promoter. Fluorescence levels were corrected using E. coli K12 MG1655 expressing non-fluorescent proteins under the control of a rhamnose-inducible promoter. Error bars = Standard error of the mean. C) HA degradation by secreted bacterial hyaluronidase under normal and hypoxic conditions at pH 7 and pH 6.1. B+ = E. coli K12 MG1655 expressing bacterial hyaluronidase under the control of a rhamnose-inducible promoter, induced by 0.2% rhamnose. LOR7, R9 = Escherichia coli (E. coli) K12 MG1655 expressing GFP under the control of an engineered hypoxia-inducible promoter. Degradation levels were corrected using E. coli (E. coli) K12 MG1655 constitutively expressing the fluorescent protein. Error bars = standard error of the mean. [Figure 9-2]A) and B) GFP fluorescence levels under normal and hypoxic conditions at pH 7 and pH 6. GFP = Positive control E. coli K12 MG1655 constitutively expressing GFP. LO / LP R7, R9, R1 = E. coli K12 MG1655 expressing GFP under a manipulated hypoxia-inducible promoter. Fluorescence levels were corrected using E. coli K12 MG1655 expressing non-fluorescent proteins under the control of a rhamnose-inducible promoter. Error bars = Standard error of the mean. C) HA degradation by secreted bacterial hyaluronidase under normal and hypoxic conditions at pH 7 and pH 6.1. B+ = E. coli K12 MG1655 expressing bacterial hyaluronidase under the control of a rhamnose-inducible promoter, induced by 0.2% rhamnose. LOR7, R9 = Escherichia coli (E. coli) K12 MG1655 expressing GFP under the control of an engineered hypoxia-inducible promoter. Degradation levels were corrected using E. coli (E. coli) K12 MG1655 constitutively expressing the fluorescent protein. Error bars = standard error of the mean. [Figure 10-1] Treatment of mice with 4T1 mammary tumors with Escherichia coli (E. coli) K12 MG1655 expressing hyaluronidase under the control of LOR7 and LOR9 promoters reduces tumor stiffness just 3 days after administration, as indicated by a significant decrease in hyaluronan levels in ultrasonic shear elastography (SWE) measurements (A) and TME (C). Vascular perfusion, as measured by contrast-enhanced ultrasound (CEUS), is also improved upon administration of E. coli (Hase) (B). Error bars = standard error of the mean. N = 6 mice. [Figure 10-2]Treatment of mice with 4T1 mammary tumors with Escherichia coli (E. coli) K12 MG1655 expressing hyaluronidase under the control of LOR7 and LOR9 promoters reduces tumor stiffness just 3 days after administration, as indicated by a significant decrease in hyaluronan levels in ultrasonic shear elastography (SWE) measurements (A) and TME (C). Vascular perfusion, as measured by contrast-enhanced ultrasound (CEUS), is also improved upon administration of E. coli (Hase) (B). Error bars = standard error of the mean. N = 6 mice. [Figure 11] Escherichia coli (E. coli) K12 MG1655, which expresses hyaluronidase under the control of the LOR7 promoter, enhances the efficacy of immunotherapy in a mouse model of triple-negative mammary cancer. Tumor growth curves of treated orthotopic (A)4T1 and (B)E0771 tumors are shown (n=7-10 mice per treatment group). When the tumors reached an average size of 150 mm3, Escherichia coli (E. coli) expressing hyaluronidase under the control of the LO R7 promoter (LO R7 cells) was administered intravenously at a dose of 106 CFU, followed by 3 cycles of immunotherapy cocktail consisting of 10 mg / kg anti-PD-1 and 5 mg / kg anti-CTLA-4 (ICI, immune checkpoint inhibitor). Data are shown as mean ± SE. Statistical analysis was performed by comparing the means between independent groups using a two-way ANOVA test (for the 4T1 trial: PBS vs. LO R7, p=0.0004; LO R7 vs. LO R7+ICI, p=0.0045; ICI vs. LO R7+ICI, p=0.0007; for the E0771 trial: PBS vs. ICI, p=0.036; PBS vs. LO R7+ICI, p=0.0008; LO R7 vs. ICI, p=0.0199; LOR 7 vs. LOR 7+ICI, p=0.0016; ICI vs. LO R7+ICI, p=0.0401). [Figure 12] Survival curves for 4T1 and E0771 tumor models. Survival curves for orthotopic (A)4T1 and (B)E0771 tumors treated as shown (n=7-10 mice per treatment group). [Figure 13-1](A-G) Flow cytometry analysis of 4T1 tumors. (A) Percentage of CD45+ lymphocytes among the viable cells of treated 4T1 tumors as shown. (B) Percentage of lymphocytes from the myeloid-derived suppressor cell (MDSC) population defined as CD45+GR1+CD11b+, and (C) Tumor-associated macrophages (TAMs) defined as CD45+GR1_CD11b+F4 / 80+. (D) Ratio of immunosupportive M1-like TAMs (CD45+GR1-CD11b+F4 / 80+CD38+CD206-) to immunosuppressive M2-like TAMs (CD45+GR1-CD11b+F4 / 80+CD38-CD206+). (E) Percentage of CD4+ T cells (CD3+CD4SP+) and (F) CD8+ T cells (CD3+CD8SP+) gated to lymphocytes. (G) Ratio of cytotoxic CD8+ T cells to regulatory T cells, defined as CD3+CD4SP+CD25hiCD127loFoxp3+ (n=7-10 mice per treatment group). (H-N) Flow cytometry analysis of E0771 tumors. (H) Percentage of CD45+ lymphocytes in viable cells. (I) Percentage of MDSCs and (J) TAMs gated to lymphocytes. (K) Ratio of M1-like TAMs to M2-like TAMs. (L) Percentage of CD4+ T cells (TCRB+CD4SP+) and (M) CD8+ T cells gated to lymphocytes (TCRB+CD8SP+). (N)Ratio of cytotoxic CD8+ T cells to Tregs (n=7-10 mice for PBS, LO R7, and ICI treatment groups; n=4 for the LO R7 + ICI combination group due to complete tumor regression in 5 out of 10 mice in this group). Data are shown as mean ± SE. Statistical analysis was performed by comparing the means between independent groups using a one-way ANOVA test. [Figure 13-2](A-G) Flow cytometry analysis of 4T1 tumors. (A) Percentage of CD45+ lymphocytes among the viable cells of treated 4T1 tumors as shown. (B) Percentage of lymphocytes from the myeloid-derived suppressor cell (MDSC) population defined as CD45+GR1+CD11b+, and (C) Tumor-associated macrophages (TAMs) defined as CD45+GR1_CD11b+F4 / 80+. (D) Ratio of immunosupportive M1-like TAMs (CD45+GR1-CD11b+F4 / 80+CD38+CD206-) to immunosuppressive M2-like TAMs (CD45+GR1-CD11b+F4 / 80+CD38-CD206+). (E) Percentage of CD4+ T cells (CD3+CD4SP+) and (F) CD8+ T cells (CD3+CD8SP+) gated to lymphocytes. (G) Ratio of cytotoxic CD8+ T cells to regulatory T cells, defined as CD3+CD4SP+CD25hiCD127loFoxp3+ (n=7-10 mice per treatment group). (H-N) Flow cytometry analysis of E0771 tumors. (H) Percentage of CD45+ lymphocytes in viable cells. (I) Percentage of MDSCs and (J) TAMs gated to lymphocytes. (K) Ratio of M1-like TAMs to M2-like TAMs. (L) Percentage of CD4+ T cells (TCRB+CD4SP+) and (M) CD8+ T cells gated to lymphocytes (TCRB+CD8SP+). (N)Ratio of cytotoxic CD8+ T cells to Tregs (n=7-10 mice for PBS, LO R7, and ICI treatment groups; n=4 for the LO R7 + ICI combination group due to complete tumor regression in 5 out of 10 mice in this group). Data are shown as mean ± SE. Statistical analysis was performed by comparing the means between independent groups using a one-way ANOVA test.

[0178] The sequences referred to herein Sequence ID: l_bacteria_HA_AA MTYRIKKWQKLSTITLMAGVITLNGGEFRSVDKHQIAVADTNVQTPDYEKLRNTWLDVNYGYDKYDENNPDMKKKFDATEKEATNLLKEMKTESGRKYLWSGAETLETNSSHMTRTYRNIEKIAEAMRNPPKTTLNTDENKKKVKDALEWLHKNAYGKEPDKKVKELSENFTKTTGKNTNLNWWDYEIGTPKSLTNTLILLNDQFSNEEKKKFTAPIKTFAPDSDKILSSVGKAELAKGGNLVDISKVKLLECIIEEDKDMMKKSIDSFNKVFTYVQDSATGKERNGFYKDGSYIDHQDVPYTGAYGWLLEGISQMMPMIKETPFNDKTQNDTTLKSWIDDGFMPLIYKGEMMDLSRGRAISRENETSHSASATVMKSLLRLSDAMDDSTKAKYKKIVKSSV ESDSSYKQNDYLNSYSDIDKMKSLMTDNSISKNGLTQQLKIYNDMDRVTYHNKDLDFAFGLSMTSKNVARYESINGENLKWHTGAGMSYLYNSDVKHYHDNFWVTADMKRLSGTTTLDNEILKDTDDKKSSKTFVGGTKVDDQHASIGMDFENQDKTLTAKKSYFILNDKIVFLGTGIKSTDSSKNPVTTIENRKANGYTLYTDDKQTTNSDNQENNSVFLESTDTKKNIGYHFLNKPKITVKKESHTGKWKEINKSQKDTQKTDEYYEVTQKHSNSDNKYGYVLYPGLSKDVFKTKKDEVTWKQEDDFHWKDNESVWAGVNYSNSTQTFDINNTKEVKAKGMFILKKKDDNTYECSFYNPESTNSASDIESKISMTGYSITNKNTSTSNESGVHFELTK > sequence number_2_ bacteria_HA_DNA >Sequence ID 3 Chondroitin ABC Lyase AA >Sequence ID 4 Chondroitin ABC Lyase DNA >Sequence ID 5: Arcanobacterium neuraminidase AA >Sequence ID 6: Arcanobacterium Neuraminidase DNA >Sequence_7_PNGase_AA MRKLLIFSISAYLMAGIVSCKGVDSATPVTEDGLALNAVNAPADNTVNIKTFDKVKNAFGDGLSQSAEGTFTFPADVTTVKTIKMFIKNECPNKTCDEWDRYANVYVKNKTTGEWYEIGRFITPYWVGTEKLPRGLEIDVTDFKSLLSGNTELKIYTETWLAKGREYSVDFDIVYG TPDYKYSAWPVIQYNKSSIDGVPYGKAHTLGLKKNIQLPTNTEKAYLRTTISGWGHAKPYDAGSRGCAEWCFRTHTIAINNANTFQHQLGALGCSANPINNQSPGIWAPDRAGWCPGMAVPTRIDVLNNSLTGSTFSYEYKFQSWTNNGTNGDAFYAISSFVIAKSNTPISAPWTN >Sequence_8_PNGase_DNA >Sequence number_9_E_coli_pf1E CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCaaccatcctcaaaatgtactttttgtctgaatgtagactatcacgggaaaactctctgaacaGTTGACGCACATCAAtcTAACTTtcattcgaaagTATTTTaatgcttgaacgaaaAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >Sequence number_10_Salmonella_pf1E CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCccactttacggataaacataccaaagcacggtttatggccgtgctgtatcaacagataattactgtcatcagacgcaaagccagcttacccaagctccgactgacgatcggctcaagcagcgcggcatcatcttaaacgccataccacaacctcaaaccgtgatgttgtacctcatttaacgcctctttgtcagaacctctccattcgttgacgcacatcaagatagctttcattcgaaagtaatttaatctttatatgaaaAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >Sequence number_11_E_coli_pepT CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCgtaaacgcaacggatggcttaccgatgcggggtttgtggttaaccaccttggtgactcttaatgagggcggtaattctacggcaaaccgcttgaatcgccaatctttgttgtgaattactggcttagctttatattcattaaggtaatgctgataaatattcccgcttgcaggggtaaaagtgacctgacgcaatatttgtcttttcttgcttcttaataatgttgtcacaaaaagAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >SEQ ID No._12_Vitreoscilla_pvhb CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCtgtggattaagttttaagaggcaataaagattataataagtgctgctacaccatactgatgtatggcaaaaccataataatgaacttAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >SEQ ID No._13_stm1787 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCgccctctttcaaacagtctcttttttgcattcctttaaaaccagcatcactattttatataaaaatcatcacgaagtatgcttcttttaacgatgacctcaaatcctccccccttttgcatcaacttacgcatccctgaaatggcgagaacaggctaaatctacccgaggtcactcgctaaaaacctcatcctggaacaagctcaaccgcccttccccgctacggccctttcgccgaaaaaccgataatgggtataaatataccgctataagctatacccacacacttcacattgtttcttatcgttaatccgatacgcttttttcacattatttctggcggtatacgccggtgcaaagtgaatcgggctcgtttccgcgctcgcgcgtaacacaatgatttaccttgctggcggaacgtcaacggacccgcaaaagcgccttatgcccctggcggagcgtggtAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >SEQ ID NO: 14_hyaA_MG1655 Tgagtcactctctatgacagatgtaattaattaagcagcataatgataatgcgtaagggcacccagaagttttacccatctttacgcatttgatctggaacaggtttaacagcggattatcaggtcattaagcaaatataacgccctgagaatttcgacaggcaaaagaaaaaggggtta gcatttagctaaccccttatcttatttggcggaagcgcagagattcgaactctggaaccctttcgggtcgccggtttcaagaccggtgccttcaaccgctcggccacacttccggaatgacgcgcactataaaacatcccgatgcggcgtgtaaacccctaatttgtttgtttgcctgaa aaacagccaaaagtgcattgatagcgtgaaataacagcagattgatcatttcatcaccatgaattccttctcttttactcgtttagcaaccggctaaacatccccaccgccccggccaaaagaaaaataggtccatttttatcgctaaagataaatccacacagtttgtattgttttgtt caaaagtttcactacgctttattaacaatactttctggcgacgtgcgccagtgcagaaggatgagctttcgttttcagcatctcacgtgaagcgatggtttgccttgctacagggacgtcgcttgccgaccataagcgccccggtgtcctgccggtgtcgcaaggaggagagacgtgcgat > அக்க்கியுக்க்கு_15_P2 tacagatctggaagtaattctagagctttgcatgtctataaaaaaaatgggattccaaattaccgatatcaatatgcgaaaagaactatgaatatccactccatttttggtgccatttgttaacgctgcctcctctccctagtgctataataaaaaatggcccattttggaacagactttactattttgttgtctagtaggagctc > குற்றுக்க்கு_16_P1 tacagatctggaagtaattctagagctttgcatgtctataaaaaaatgggattccaaattaccgatatcaatatgcgaaaagaactatgaatatccactccatttttggttgccatttgttaacgctgcctcctctccctagtgctataataaaaatggcccattttggaacgagctc >SEQ ID NO:_17_PelB_AA MKYLLPTAAAGLLLLAAQPAMA >SEQ ID NO:_18_PelB_DNA Atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggccatggcc >SEQ ID NO:_19_YebF_AA MKKRGAFLGLLLVSACASVFAANNETSKSVTFPKCEDLDAAGIAASVKRDYQQNRVARWADDQKIVGQADPVAWVSLQDIQGKDDKWSVPLTVRGKSADIHYQVSVDCKAGMAEYQRR >SEQ ID NO:_20_YebF_DNA atgaaaaaaagaggggcgtttttagggctgttgttggtttctgcctgcgcatcagttttcgctgccaataatgaaaccagcaagtcggtcactttcccaaagtgtgaagatctggatgctgccggaattgccgcgagcgtaaaacgtgattatcaacaaaatcgcgtggcgcgttgggcagatgatcaaaaaattgtcggtcaggccgatcccgtggcttgggtcagtttgcaggacattcagggtaaagatgataaatggtcagtaccgctaaccgtgcgtggtaaaagtgccgatattcattaccaggtcagcgtggactgcaaagcgggaatggcggaatatcagcggcgt >SEQ ID NO:_2l_YebF_MMP_AA MKKRGAFLGLLLVSACASVFAANNETSKSVTFPKCELDDAAGIASVKRDYQQNRVARWADDQKIVGQADPVAWVSLQDIQGKDDKWSVPLTVRGKSADIHYQVSVDCKAGMAEYQRRPGLWA >Sequence_22_YebF_MMP_DNA Atgaaaaaaagaggggcgtttttagggctgttgttggtttctgcctgcgcatcagttttcgctgccaataatgaaaccagcaagtcggtcact ttcccaaagtgtgaagatctggatgctgccggaattgccgcgagcgtaaaacgtgattatcaacaaaatcgcgtggcgcgttgggcagatgat caaaaaattgtcggtcaggccgatcccgtggcttgggtcagtttgcaggacattcagggtaaagatgataaatggtcagtaccgctaaccgtg cgtggtaaaagtgccgatattcattaccaggtcagcgtggactgcaaagcgggaatggcggaatatcagcggcgtCCATTAGGATTATGGGCA >Sequence_23_CtxB_AA MIKLKFGVFFTVLLSSAYANGTPQNITDL >Sequence_24_CtxB_DNA Atgattaaattaaaatttggtgtttttttacagttttactatcttcagcatatgcaaatggaacacctcaaaatattactgatttg >Array_25_AIDA_AA LNPTKESAGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGINIISVEGNSDAEFSLKNRWAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSGKLNDGQNKTTT NQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFWLQ PHLQAVWMGVTPDTHQEDNGTWQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSNAISGALGIKYSF >Array_26_AIDA_DNA >SEQ ID NO: 27 MMP-AIDA AA PLGLWALNPTKESAGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGINIISVEGNSDAEFSLKNRVVAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDTHQEDNGTWQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSNAISGALGIKYSF >SEQ ID NO: 28 MMP-AIDA DNA >SEQ ID No._29_FLAG-MMP-AIDA_AA DYKDDDDKPLGLWALNPTKESAGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGINIISVEGNSDAEFSLKNRWAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDTHQEDNGTWQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSNAISGALGIKYSF >SEQ ID No._30_FLAG-MMP-AIDA_DNA >Sequence_3l_FliC_AA MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAG >Sequence_32_FliC_DNA atggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaacaagaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaacagcgcgaaggatgacgcagcgggt >Sequence_33_FliC-MMP_AA MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGPLGLWA >Sequence_34_FliC-MMP_DNA atggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaacaagaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaacagcgcgaaggatgacgcagcgggtCCATTAGGATTATGGGCA >Sequence_35_Usp45_AA MKKKIISAILMSTVILSAAAPLSGVYA >Sequence_36_Usp45_DNA atgaagaaaaagattatttcagcaattttgatgtctacagttatcttatcagcagctgctccactttcaggagtttatgca >Sequence_37_PelB-bH_AA MKYLLPTAAAGLLLLAAQPAMADTNVQTPDYEKLRNTWLDVNYGYDKYDENNPDMKKKFDATEKEATNLKEMKTESGRKYLWSGAETLETNSSHMTRTYRNIEKIAEAMRNPKTTLNTDENKKKVKDALEWLHKNAYGKEPDKKVKELSENFTKTTGKNTNLNWWDYEIGTPKSLTNTLILLNDQFSNEEKKKFTAP IKTFAPDSKILSVGKAELAKGGNLVDISKVKLLECIIEEDKDMMKKSIDSFNKVFTYVQDSATGKERNGFYKDGSYIDHQVPYTGAYGWLLEGISQMMPMIKETPFNDKTQNDTTLKSWIDDGFMPLIYKGEMMDLSRGRAISRENETSHSASATVMKSLLRLSDAMDDSTKAKYKKIVKSSVESDSSYKQNDYLN SYSDIDKMKSLMTDNSISKNGLTQQLKIYNDMDRVTYHNKDLDFAFGLSMTSKNVARYESINGENLKGWHTGAGMSYLYNSDVKHYHDNFWVTADMKRLSGTTTLDNEILKDTDDKKSSKTFVGGTKVDDQHASIGMDFENQDKTLTAKKSYFILNDKIVFLGTGIKSTDSSKNPVTTIENRKANGYTDDKQTTN SDNQENNSVFLESTDTKKNIGYHFLNKPKITVKKESHTGKWKEINKSQKDTQKTDEYYEVTQKHSNSDNKYGYVLYPGLSKDVFKTKKDEVTWKQEDDFHWKDNESVWAGVNYSNSTQTFDINNTKVEVKAKGMFILKKKDDNTYECSFYNPESTNSASDIESKISMTGYSITNKNTSTSNESGVHFELTKDYKDDDDK > sequence number_38_PelB-bH_DNA >Array number_39_YebF-bH_AA MKKRGAFLGLLLVSACASVFAANNETSKSVTFPKCEDLDAAGIAASVKRDYQQNRVARWADDQKIVGQADPVAWVSLQDIQGKDDKWSVPLTVRGKSADIHYQVSVDCKAGMAEYQRRPLGLWATYRIKKWQKLSTITLLMAGVITLNGGEFRSVDKHQIAVADTNVQTPDYEKLRNTWLDVNYGYDKYDENNPDMKKKFDATEKEATNLLKEMKTESGRKYLWSGAETLETNSSHMTRTYRNIEKIAEAMRNPKTTLNTDENKKKVKDALEWLHKNAYGKEPDKKVKELSENFTKTTGKNTNLNWWDYEIGTPKSLTNTLILLNDQFSNEEKKKFTAPIKTFAPDSDKILSSVGKAELAKGGNLVDISKVKLLECIIEEDKDMMKKSIDSFNKVFTYVQDSATGKERNGFYKDGSYIDHQDVPYTGAYGWLLEGISQMMPMIKETPFNDKTQNDTTLKSWIDDGFMPLIYKGEMMDLSRGRAISRENETSHSASATVMKSLLRLSDAMDDSTKAKYKKIVKSSVESDSSYKQNDYLNSYSDIDKMKSLMTDNSISKNGLTQQLKIYNDMDRVTYHNKDLDFAFGLSMTSKNVARYESINGENLKGWHTGAGMSYLYNSDVKHYHDNFWVTADMKRLSGTTTLDNEILKDTDDKKSSKTFVGGTKVDDQHASIGMDFENQDKTLTAKKSYFILNDKIVFLGTGIKSTDSSKNPVTTIENRKANGYTLYTDDKQTTNSDNQENNSVFLESTDTKKNIGYHFLNKPKITVKKESHTGKWKEINKSQKDTQKTDEYYEVTQKHSNSDNKYGYVLYPGLSKDVFKTKKDEVTWKQEDDFHWKDNESVWAGVNYSNSTQTFDINNTKVEVKAKGMFILKKKDDNTYECSFYNPESTNSASDIESKISMTGYSITNKNTSTSNESGVHFELTKDYKDDDDK >Sequence ID_40_YebF-bH_DNA >Sequence_41_AIDA-bH_AA >Sequence_42_AIDA-bH_DNA >SEQ ID NO:_43_FliC-bH_AA MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGPLGLWADTNVQTPDYEKLRNTWLDVNYGYDKYDENNPDMKKKFDATEKEATNLLKEMKTESGRKYLWSGAETLETNSSHMTRTYRNIEKIAEAMRNPKTTLNTDENKKKVKDALEWLHKNAYGKEPDKKVKELSENFTKTTGKNTNLNWWDYEIGTPKSLTNTLILLNDQFSNEEKKKFTAPIKTFAPDSDKILSSVGKAELAKGGNLVDISKVKLLECIIEEDKDMMKKSIDSFNKVFTYVQDSATGKERNGFYKDGSYIDHQDVPYTGAYGWLLEGISQMMPMIKETPFNDKTQNDTTLKSWIDDGFMPLIYKGEMMDLSRGRAISRENETSHSASATVMKSLLRLSDAMDDSTKAKYKKIVKSSVESDSSYKQNDYLNSYSDIDKMKSLMTDNSISKNGLTQQLKIYNDMDRVTYHNKDLDFAFGLSMTSKNVARYESINGENLKGWHTGAGMSYLYNSDVKHYHDNFWVTADMKRLSGTTTLDNEILKDTDDKKSSKTFVGGTKVDDQHASIGMDFENQDKTLTAKKSYFILNDKIVFLGTGIKSTDSSKNPVTTIENRKANGYTLYTDDKQTTNSDNQENNSVFLESTDTKKNIGYHFLNKPKITVKKESHTGKWKEINKSQKDTQKTDEYYEVTQKHSNSDNKYGYVLYPGLSKDVFKTKKDEVTWKQEDDFHWKDNESVWAGVNYSNSTQTFDINNTKVEVKAKGMFILKKKDDNTYECSFYNPESTNSASDIESKISMTGYSITNKNTSTSNESGVHFELTKDYKDDDDK >SEQ ID NO:_44_FliC-bH_DNA >Sequence_45_Usp45-bH_AA >Sequence_46_Usp45-bH_DNA >Sequence ID 47 Chondroitin ABC Lyase AA >Sequence ID 48 Chondroitin ABC Lyase DNA >Sequence_49_GadE_AA MIFLMTKDSFLLQGFWQLKDNHEMIKINSLSEIKKVGNKPFKVIIDTYHNHILDEEAIKFLEKLDAERIIVLAPYHISKLKAKAPIYFVSRKESIKNLLEITYGKHLPHKNSQLCFSHNQFKIMQLILKNKNESNITSTLNISQQTLKIQKFNIMYKLKLRRMSDIVTLGITSYF >Sequence_50_GadE_DNA Atgatttttctcatgacgaaagattcttttcttttacagggcttttggcagttgaaagataatcacgaaatgataaaaatcaattccctgtcagagatcaaaaaagtaggcaataaacccttcaaggttatc attgatacctatcacaatcatatccttgatgaagaagcgattaaatttctggagaaattagatgccgagagaattattgttttggcaccttatcacatcagtaaactaaaagctaaagcgcctatttatttt gttagccgcaaagaaagtatcaaaaatcttcttgagattacttatggtaaacacttgccccataagaattcacaattatgtttttcacataatcagttcaaaattatgcaactgattctgaaaaataaaaat gaaagcaatatcacgtcgacgctcaatatttcgcaacaaacattaaagattcagaaattcaacattatgtacaagctgaaactaagacgtatgagcgacatcgtcaccctgggtatcacatcttatttttag >Sequence_51_rrnB_T1 / T2 Gcttgattaagtccaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccggga gcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttt >Sequence_52_FNR GTTGACGCACATCAA >Sequence_53_RiboJ AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >Sequence_54_PromoterLOR7 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCccactttacggataaacataccaaagcacggtttatggccgtgctgtatcaa cagataattactgtcatcagacgcaaagccagcttacccaagctccgactgacgatcggctcaagcagcgcggcatcatcttaaacgccataccacaacctcaaaccgtgatgt tgtacctcatttaacgcctctttgtcagaacctctccattcgttgacgcacatcaagatagctttcattcgaaagtaatttaatctttatatgaaaAGCTGTCACCGGATGTG CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatctaaggaggtagtcc >Sequence_55_PromoterLOR9 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCccactttacggataaacataccaaagcacggtttatggccgtgctgtatcaacagataattactgtcatcagacgcaaagccagcttacccaagctccgactgacgatcggctcaagcagcgcggcatcatcttaaacgccataccacaacctcaaaccgtgatgttgtacctcatttaacgcctctttgtcagaacctctccattcgttgacgcacatcaagatagctttcattcgaaagtaatttaatctttatatgaaaAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatctcaggaggtagtcc >SEQ ID NO: 56 Promoter LOR1 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCccactttacggataaacataccaaagcacggtttatggccgtgctgtatcaacagataattactgtcatcagacgcaaagccagcttacccaagctccgactgacgatcggctcaagcagcgcggcatcatcttaaacgccataccacaacctcaaaccgtgatgttgtacctcatttaacgcctctttgtcagaacctctccattcgttgacgcacatcaagatagctttcattcgaaagtaatttaatctttatatgaaaAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAActcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatccaaggaggtagtcc >SEQ ID NO: 57 Promoter LPR7 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCgccctctttcaaacagtctcttttttgcattcctttaaaaccagcatcactattttatataaaaatcatcacgaagtatgcttcttttaacgatgacctcaaatcctccccccttttgcatcaacttacgcatccctgaaatggcgagaacaggctaaatctacccgaggtcactcgctaaaaacctcatcctggaacaagctcaaccgcccttccccgctacggccctttcgccgaaaaaccgataatgggtataaatataccgctataagctatacccacacacttcacattgtttcttatcgttaatccgatacgcttttttcacattatttctggcggtatacgccggtgcaaagtgaatcgggctcgtttccgcgctcgcgcgtaacacaatgatttaccttgctggcggaacgtcaacggacccgcaaaagcgccttatgcccctggcggagcgtggtAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatctaaggaggtagtcc >SEQ ID NO: 58 Promoter LPR1 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCgccctctttcaaacagtctcttttttgcattcctttaaaaccagcatcactattttatataaaaatcatcacgaagtatgcttcttttaacgatgacctcaaatcctccccccttttgcatcaacttacgcatccctgaaatggcgagaacaggctaaatctacccgaggtcactcgctaaaaacctcatcctggaacaagctcaaccgcccttccccgctacggccctttcgccgaaaaaccgataatgggtataaatataccgctataagctatacccacacacttcacattgtttcttatcgttaatccgatacgcttttttcacattatttctggcggtatacgccggtgcaaagtgaatcgggctcgtttccgcgctcgcgcgtaacacaatgatttaccttgctggcggaacgtcaacggacccgcaaaagcgccttatgcccctggcggagcgtggtAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAActcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatccaaggaggtagtcc >SEQ ID NO: 59 Promoter LPR9 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCgccctctttcaaacagtctcttttttgcattcctttaaaaccagcatcactattttatataaaaatcatcacgaagtatgcttcttttaacgatgacctcaaatcctccccccttttgcatcaacttacgcatccctgaaatggcgagaacaggctaaatctacccgaggtcactcgctaaaaacctcatcctggaacaagctcaaccgcccttccccgctacggccctttcgccgaaaaaccgataatgggtataaatataccgctataagctatacccacacacttcacattgtttcttatcgttaatccgatacgcttttttcacattatttctggcggtatacgccggtgcaaagtgaatcgggctcgtttccgcgctcgcgcgtaacacaatgatttaccttgctggcggaacgtcaacggacccgcaaaagcgccttatgcccctggcggagcgtggtAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatctcaggaggtagtcc >SEQ ID NO: 60_hyaA_stm1787 CTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCtgagtcactctctatgacagatgtaattaattaagcagcataatgataatgcgtaagggcacccagaagttttacccatctttacgcatttgatctggaacaggtttaacagcggattatcaggtcattaagcaaatataacgccctgagaatttcgacaggcaaaagaaaaaggggttagcatttagctaaccccttatcttatttggcggaagcgcagagattcgaactctggaaccctttcgggtcgccggttttcaagaccggtgccttcaaccgctcggccacacttccggaatgacgcgcactataaacatcccgatgcggcgtgtaaacccctaatttgtttgtttgcctgaaaaacagccaaaagtgcattgatagcgtgaaataacagcagattgatcatttcatcaccatgaattccttctcttttactcgtttagcaaccggctaaacatccccaccgcccggccaaaagaaaaataggtccatttttatcgctaaaagataaatccacacagtttgtattgttttgtgcaaaagtttcactacgctttattaacaatactttctggcgacgtgcgccagtgcagaaggatgagctttcgttttcagcatctcacgtgaagcgatggtttgccttgctacagggacgtcgcttgccgaccataagcgcccggtgtcctgccggtgtcgcAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA >SEQ ID NO: 61_P3 tacagatctggaagtaattctagagctttgcatgtctataaaaaaatgggattccaaattaccgatatcaatatgcgaaaagaactatgaatatccactccatttttggttgccatttgttaacgctgcctcctctccctagtgctataataaaaatggccaaaaaaaaacgagctc >Sequence ID_62_U1-RBS1 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatcacacaggacTAGTCC >Sequence ID_63_U1-RBS2 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAaaagaggggaaaTAGTCC >Sequence ID_64_U1-RBS3 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAaaagaggagaaaTAGTCC >Sequence ID_65_U1-RBS-AO1 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAatccaaggaggtagtcc >Sequence ID_66_U1-RBS-AO2 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCCAGGGAGGTAGTCC >Sequence ID_67_U1-RBS-AO3 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCCCAGGAGGTAGTCC >Sequence ID_68_U1-RBS-A04 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCCCGGGAGGTAGTCC >Sequence ID_69_U1-RBS-AO5 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCCGAGGAGGTAGTCC >Sequence ID_70_U1-RBS-A06 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCCGGGGAGGTAGTCC >Sequence ID_71_U1-RBS-A07 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTAAGGAGGTAGTCC >Sequence ID_72_U1-RBS-A08 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTAGGGAGGTAGTCC >Sequence ID_73_U1-RBS-A09 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTCAGGAGGTAGTCC >Sequence ID_74_U1-RBS-A10 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTCGGGAGGTAGTCC >Sequence ID_75_U1-RBS-A11 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTGAGGAGGTAGTCC >Sequence ID_76_U1-RBS-A12 ctcgttgaacaccgtcTCAGGTAAGTATCAGTTGTAAATCTGGGGAGGTAGTCC >Sequence ID_77_U2-RBS1 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAatcacacaggacTAGTCC >Sequence_78_U2-RBS2 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAaaagaggggaaaTAGTCC >Sequence ID_79_U2-RBS3 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAaaagaggagaaaTAGTCC >Sequence ID_80_U2-RBS-A01 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAatccaaggaggtagtcc >Sequence ID_81_U2-RBS-A02 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCCAGGGAGGTAGTCC >Sequence ID_82_U2-RBS-A03 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCCCAGGAGGTAGTCC >Sequence ID_83_U2-RBS-A04 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCCCGGGAGGTAGTCC >Sequence ID_84_U2-RBS-A05 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCCGAGGAGGTAGTCC >Sequence ID_85_U2-RBS-A06 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCCGGGGAGGTAGTCC >Sequence ID_86_U2-RBS-A07 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTAAGGAGGTAGTCC >Sequence ID_87_U2-RBS-A08 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTAGGGAGGTAGTCC >Sequence ID_88_U2-RBS-A09 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTCAGGAGGTAGTCC >Sequence ID_89_U2-RBS-A10 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTCGGGAGGTAGTCC >Sequence ID_90_U2-RBS-A11 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTGAGGAGGTAGTCC >Sequence ID_91_U2-RBS-A12 CTCGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAATCTGGGGAGGTAGTCC >Sequence ID_92_U3-RBS1 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAatcacacaggacTAGTCC >Sequence ID_93_U3-RBS2 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAaaagaggggaaaTAGTCC >Sequence ID_94_U3-RBS3 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAaaagaggagaaaTAGTCC >Sequence ID_95_U3-RBS-A01 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAatccaaggaggtagtcc >Sequence ID_96_U3-RBS-A02 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCCAGGGAGGTAGTCC >Sequence ID_97_U3-RBS-A03 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCCCAGGAGGTAGTCC >Sequence ID_98_U3-RBS-A04 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCCCGGGAGGTAGTCC >Sequence ID_99_U3-RBS-A05 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCCGAGGAGGTAGTCC >Sequence ID_100_U3-RBS-A06 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCCGGGGAGGTAGTCC >Sequence ID_101_U3-RBS-A08 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCTAGGGAGGTAGTCC >Sequence ID_102_U3-RBS-A09 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCTCAGGAGGTAGTCC >Sequence ID_103_U3-RBS-A10 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCTCGGGAGGTAGTCC >Sequence ID_104_U3-RBS-A11 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCTGAGGAGGTAGTCC >Sequence ID_105_U3-RBS-A12 CTCGGTATCTCGTGGTCTGACGGTAAAATCTATTGTAATCTGGGGAGGTAGTCC >Sequence ID_106_Neuraminidase_PelB_AA MKYLLPTAAAGLLLLAAQPAMAAPSTAEPPASGTATTAAPTTNSPVTASSFKILNPKAEGESFEEEIRFQVTLKNDTDVQRAFAFQSSNLNDYQKCKWWKVEPGETKEDCKFPHHKVTADDVATGSFTPRVTWIMRESTGHASPVEKTGEAVGKPVPVTKLAKLSPITVTSGEGKASYSAGDQFGYDYTVTSLSKDKISVEGECPAKELDPEKSMKCSAKYAVTEEDLERGEAELTAKVKVSD GKRNVTLTETKSVKTPRVWPQAKAFKTPNADPNLGARLTDLNILDEKTSEYNIRIPAIAVASNGDILASYDLRPNLGAWHGGDSPNESIVQRRSTDGGKTWGPRTTVAEGKVAGQGKRFGWSDPSYWDHTTGEIFNFHVGSLDAGLPNNPSYRLVNGKVDDTHRQTMNFALSSSSTDNGRTWNSRLITNDVLSTSGTHIDEIKGFATSGAGIQKMHEPFKGRLLQQAACKFNTGRFRAITIFSDDHG KTWQGGHFTSDTEGAPAGKHWNFDENKIAELSDGRMLNSRIPGNSYGTGYRLVAYSADGGETWGGYHIEKQLPDSTNNAQLIRAFPSANNGTLRAKVLLFSNTEKPNNRVDGKVKMSYDDGKSWPIAKQIRNGHTGYTTMAVQPDGSIGLLMEPTSQSVGYVNFTLKTLAENLPFEVALDKIGDVKATDGTPIAPIKVTSTGNDPALADTYSAEGLPAGLKINAETGQIEGTPAVGNTDVKSFDVKVTLTEAEDGTGIPRTSSQTFKITLAPNPTPAPAPEPKPEPKPEPTPKPEPKPEPTPKPEPKPPEPKVEVVVPNAPVFPDASDPVTCTVKPFVTLQPTKGVSYSVTVDGKELDWVEGNPSRFEYDYGKTVVVKAKAVEGFELAKGAKTQWSWTAPTLDELGCTTPAPDPKPTPDPKPTPDPKPTPEPKPTPDPKPPTIPKPTPKPTIIAQAKAPKSSLVHTGATWGLSVAAAVLLLAGGAIAIIRRRQGDYKDDDDK >Sequence ID_107_Neuraminidase_PelB_DNA >Sequence ID_108_Neuraminidase_Aida_AA MIKLKFGVFFTVLLSSAYANGTPQNITDLAAPSTAEPPASGTATTAAPTTNSPVTASSFKILNPKAEGESFEEGEEIRFQVTLKNDTDVQRAFAFQSSNLNDYQKCKWWKVEPGETKEDCKFPHHKVTADDVATGSFTPRVTWIMRESTGHASPVEKTGEAVGKPPVTTKLAKLSPITVTSGEGKASYSAGDQFGYDYTVTSLSKDKISVEGCPAKELDPEKSMKCSAKYAVTEEDLERGEAELTA KVKVSDGKRNVTLTETKSVKTPRVWPQAKAFKTPNADPNLGARLTDLNILDEKTSEYNIRIPAIAVASNGDILASYDLRPNLGAWHGGDSPNESIVQRRSTDGGKTWGPRTTVAEGKVAGQGKRFGWSDPSYWDHTTGEIFNFHVGSLDAGLPNNPSYRLVNGKVDDTHRQTMNFALSSSSTDNGTRNSRLITNDVLSTSGTHIDEIKGCFATSGAGIQKMHEPFKGRLLQQAACKFNTGRITIFS DDHGKTWQGGHFTSDTEGAPAGKHWNFDENKIAELSDGRMLNSRIPGNSYGTGYRLVAYSADGGETWGGYHIEKQLPDSTNNAQLIRAFPSANNGTLRAKVLLFSNTEKPNNRVDGKVKMSYDDGKSWPIAKQIRNGHTGYTTMAVQPDGSIGLLMEPTSQSVGYVNFTLKTLAENLPFEVALDKIGDVKATDGTPIAPIKVTSTGNDPALADTYSAEGLPAGLKINAETGQIEGTPAVGNTDVKSFDVKVTLTEAEDGTGIPRTSSQTFKITLAPNPTPAPAPEPKPEPKPEPTPKPEPKPEPTPKPEPKPPEPKPVEVVVPNAPVFPDASDPVTCTVKPFVTLQPTKGVSYSVTVDGKELDWVEGNPSRFEYDYGKTVVVKAKAVEGFELAKGAKTQWSWTAPTLDELGCTTPAPDPKPTPDPKPTPDPKPTPEPKPTPDPKPPTIPKPTPKPPTIIAQAKAPKSSLVHTGATWGLSVAAAVLLLAGGAIAIIRRRQGDYKDDDDK >Sequence ID 109: Neuraminidase Aida DNA >Sequence ID_110_Neuraminidase_yebF_AA >Sequence ID_111_Neuraminidase_YebF_DNA >Sequence ID_112_Neuraminidase_FliC_AA >Sequence ID 113: Neuraminidase FliC DNA >Sequence ID_114_Neuraminidase_Usp45_AA * >Sequence ID_115_Neuraminidase_Usp45_DNA >SEQ ID NO: 116_MMP_AA GSGSGSPLGLWAGSGSGS >SEQ ID NO: 117_MMP_DNA ggCTCGGGCTCGGGCTCCCCATTAGGATTATGGGCAGGATCGGGCTCCGGGTCC

Examples

[0179] Study on the in vivo tolerance and biodistribution of candidate Escherichia coli K12 strains in T1 tumor-bearing mice in Examples 1-4 The candidate Escherichia coli K12 MG1655 strain was engineered to express a bioluminescence bioreporter using the bacterial luxCDABE luciferase operon as described in Fedorec et al., 2019, "Two New Plasmid Post-segregational Killing Mechanisms for the Implementation of Synthetic Gene Networks in Escherichia coli", iScience, 14:323-334 (which is hereby incorporated by reference in its entirety).

[0180] Tumors were induced in healthy female BALB / c mice, 6-7 weeks old at the time of receipt, obtained from Charles River. The BALB / c mice were anesthetized with isoflurane and 1×10 5 cells of 4T1 breast cells suspended in RPMI 1640 medium at a volume of 50 μL were injected into the MFP tissue (upper right breast).

[0181] The candidate Escherichia coli K12 MG1655 strain was administered to 4T1 tumor-bearing mice by intravenous injection into the tail vein in doses of 10 6 CFU, 10 7 CFU and 10 8 CFU per mouse in PBS. All three doses were tolerated during the 6-day observation period after administration. One mouse had 10 8Due to tumor necrosis in heat-sterilized bacteria in group 5 of the CFU, the cells were sacrificed one day after administration.

[0182] As can be seen in Figure 1, candidate Escherichia coli K12 MG1655 strain was selected in 10 6 CFU and 10 7 Mice administered with CFU doses maintained an average body weight comparable to that of mice administered with vehicle alone. 8 Higher doses of CFU resulted in greater weight loss.

[0183] Mice were imaged at 24, 48, and 72 hours (pa) after administration using the Photoimager Optima in vivo imaging system. Throughout all acquisitions, mice were sequentially anesthetized using isoflurane (Minerve, France) in an oxygen mixture via a nosepiece. As shown in Figure 2, 10 6 and 10 7 In mice administered CFU doses, luminescence localized to the tumor 48 and 72 hours after administration and could be observed 6 days after administration (data not shown). 10 8 In mice administered with CFU doses, luminescence localized to the tumors at 24-hour pa.

[0184] 10 8 Considering the weight loss in the group administered with the candidate CFU strain, the level of colony formation in tumors and secondary organs was assessed at 10 72 hours after administration. 7 The CFU was quantified in mice administered a dose of CFU. 10 candidate strains were selected. 7 The drug was administered by intravenous injection into the tail vein at a CFU dose. Mice were sacrificed and imaged 72 hours after administration. (Kang et al, 2020, "Imaging of tumor colonization by Escherichia coli using 18Organs were collected for CFU counting according to "F-FDS PET", Theranostics, 10(11):4958-4966 (the entire text is incorporated herein by reference). As shown in Figure 3A, the luminescence was localized to the tumor. CFU counting was performed using high concentrations in the tumor (10 per gram of tissue). 8 This shows the bacteria in CFU (concentrated force purchase) and the residual bacterial levels in secondary organs (approximately 10 per gram of tissue). 4 CFU also showed this.

[0185] This study demonstrates that the candidate Escherichia coli K12 MG1655 strain is well-tolerated, localizes to tumors in a mouse tumor model, and accumulates within them.

[0186] Example 2 - Development of an extracellular matrix (ECM) dearadina bacterial strain Several components of the extracellular matrix (ECM) have been identified as crucial for preventing immune cell infiltration into solid tumors. Hyaluronic acid (HA) is particularly important in this context. It is a highly hygroscopic glycosaminoglycan that swells upon hydration, leading to high interstitial pressure and vascular collapse. It is broken down by various forms of naturally occurring enzymes called hyaluronidases. Therefore, this study focused on generating hyaluronidase-producing bacterial strains that can be deployed in vivo.

[0187] Hyaluronidase-producing bacteria in candidate Escherichia coli strain K12 MG1655 were manipulated using BASIC, a modular DNA assembly method developed by Professor Geoff Baldwin of Imperial College London. This method uses oligolinkers to join multiple DNA bioparts together at once (Storch et al., 2015, "BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology," ACS Synth. Biol., 4:781-787; the entire text is incorporated herein by reference).

[0188] The hyaluronic acid degradation ability of candidate Escherichia coli strain K12 MG1655 was evaluated using the turbidimetric cetyltrimethylammonium method disclosed by Oueslati et al. 2014, "CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form," Carbohydrate Polymers, 112:102-108 (the entire work is incorporated herein by reference). In this method, cetyltrimethylammonium bromide interacts with hyaluronic acid present in solution, causing turbidity of the solution and resulting in high OD when analyzed spectrophotometrically. 600 It brings value. When hyaluronic acid is broken down, the solution becomes clearer, OD 600 The measured value will decrease.

[0189] Candidate Escherichia coli K12 MG1655, manipulated as discussed in Example 3 below to express secretory bacterial hyaluronidase (e.g., SEQ ID NO: 1 or SEQ ID NO: 37) (Escherichia coli K12 MG1655 PelB-bH) under the control of a rhamnose-inducible promoter, was cultured in LB medium supplemented with 0.4 mg / mL purified hyaluronan ("no induction" condition) or LB medium supplemented with 0.4 mg / mL purified hyaluronan and 0.2% (w / v) rhamnose ("induction" condition). The third condition ("medium") contained only LB medium supplemented with 0.4 mg / mL purified hyaluronan. OD of the medium under each condition 600 The values ​​were measured over a period of 6 hours. As shown in Figure 4, purified hyaluronic acid was degraded by Escherichia coli K12 MG1655 bH when bacterial hyaluronidase expression was induced.

[0190] Next, Escherichia coli K12 MG1655 bH was tested in patient-derived tissue explants (maintaining the natural extracellular matrix (ECM) structure and composition) as disclosed in Puttock et al., 2023, "Extracellular matrix educates an immunoregulatory tumor macrophage phenotype found in ovarian cancer metastasis," Nat. Common., 14:2514 (the entire explant is incorporated herein by reference).

[0191] Escherichia coli K12 MG1655, which constitutively expresses GFP, was inoculated into decellularized tissue explants and incubated for up to 5 hours. The tissue explants were imaged using a fluorescence microscope at 1-hour intervals up to 5 hours to measure the depth of invasion into the tissue. As shown in Figure 5, Escherichia coli K12 MG1655 was able to invade the extracellular matrix for 5 hours. 3D z-stacks show bacterial invasion into the matrix (indicated by white arrows; white; stained with fibronectin (FN1), a major component of fibrous tissue, to visualize general tissue structure).

[0192] To test the ability of hyaluronidase-producing bacteria to degrade the extracellular matrix (ECM) in a model, 10 7 CFU Escherichia coli K12 MG1655 bH was incubated with decellularized tissue explant material in or without the presence of rhamnose, a bH expression-inducing molecule. After 4 hours, the explant material was stained with hyaluronic acid-binding protein (IHC; Sigma Aldrich, catalog number 385911) and imaged using a light microscope. As shown in Figure 5, tissue explants incubated with Escherichia coli K12 MG1655 bH completely lost all hyaluronic acid staining.

[0193] Finally, the ability of hyaluronidase-producing bacteria to enhance immunotherapy was tested using a tumor-killing assay developed in a university laboratory. Briefly, patient-derived tissue containing cancer cells was treated with either Escherichia coli K12 MG1655 bH in culture medium (DMEM supplemented with 10% FBS, 1% L-glucose, 1× kanamycin, and 0.2% rhamnose) or the culture medium alone. Cancer-reactive T cells (T4), non-reactive T cells (UT), or the culture medium alone were then added. As shown in Figure 7, treatment of tissue with Escherichia coli K12 MG1655 bH resulted in a significant increase in cancer cell death by cancer-reactive T cells (labeled "T4+Bac") compared to cases where no bacterial treatment step was applied.

[0194] Therefore, this study demonstrates that the inventors have successfully generated a bacterial strain capable of inducibly expressing hyaluronidase, an extracellular matrix (ECM) degrading polypeptide. This bacterium can invade and degrade the extracellular matrix in decellularized tissue explants and exhibit synergistic effects with cancer-responsive T cells.

[0195] Example 3 - Development of secreted extracellular matrix (ECM) degradation polypeptides The bacterial secretory system consists of protein complexes located on the cell membrane. These can be classified into two distinct mechanisms. First, there is a one-step mechanism in which proteins from the bacterial cytoplasm are directly transported to the outside of the bacterial cell and secreted; this is found in both Gram-negative and Gram-positive bacteria. Second, there is a two-step mechanism found only in Gram-negative bacteria, in which proteins are first transported from the intracellular membrane, then deposited in the periplasm, and finally secreted outside the bacterial cell.

[0196] A fusion protein containing bacterial hyaluronidase fused to the PelB signal sequence at its N-terminus was cloned into Escherichia coli K12 MG1655 (Escherichia coli K12 MG1655 PelB-bH) under the control of a rhamnose-inducible promoter. Escherichia coli K12 MG1655 PelB-bH was incubated for 4 hours in a medium containing or without 0.2% (w / v) rhamnose. Escherichia coli K12 MG1655 expressing GFP was incubated under the same conditions as a negative control. After incubation, the supernatant and pellet were separated, and 0.8 mg / mL of hyaluronic acid was added to each sample. The mixtures were incubated at 37°C for 18 hours, and the level of hyaluronan degradation was evaluated using the cetyltrimethylammonium bromide turbidimetric method (CTM) as described above. As shown in Figure 8A, the supernatant recovered from Escherichia coli K12 MG1655 PelB-bH incubated with 0.2% rhamnose ("inducing" condition) was able to degrade hyaluronic acid, but hyaluronic acid added to the supernatant recovered from Escherichia coli K12 MG1655 PelB-bH incubated under "non-inducing" conditions was not degraded. Figure 8B shows that bacterial hyaluronidase was detectable by Western blotting in the supernatant of Escherichia coli K12 MG1655 PelB-bH incubated under "inducing" conditions, but not under "non-inducing" conditions.

[0197] A fusion protein containing bacterial neuraminidase fused to the PelB signal sequence at its N-terminus was cloned into Escherichia coli K12 MG1655 (Escherichia coli K12 MG1655 PelB-Nh) under the control of a rhamnose-inducible promoter. Escherichia coli K12 MG1655 PelB-Nh was incubated for 4 hours in a medium containing 0.2% (w / v) rhamnose. Escherichia coli K12 MG1655 expressing GFP was incubated under the same conditions as a negative control. After incubation, the supernatant and pellet were separated, and the supernatant was tested for neuraminidase detection using the Neuraminidase Assay Kit (Abeam, ab138888). As shown in Figure 8C, the supernatant recovered from Escherichia coli K12 MG1655 PelB-Nh was capable of degrading terminal sialic acid residues and neuraminic acid, but Escherichia coli K12 MG1655 GFP did not show neuraminidase activity.

[0198] A fusion protein containing a bacterial neuraminidase (NH) fused at the N-terminus to a secretory signal sequence selected from the PelB signal sequence, the carrier protein YebF, and T3SS(FliC), and an autotransporter (AIDAc), was cloned into Escherichia coli K12 MG1655 under the control of a rhamnose-inducible promoter. Escherichia coli K12 MG1655 PelB-NH, Escherichia coli MG1655 yebF-NH, Escherichia coli MG1655 FliC-NH, and Escherichia coli MG1655 AidaC-NH were incubated for 4 hours in a medium supplemented with 0.2% (w / v) rhamnose and 2.5 μg / mL human matrix metalloproteinase 1 (MMP1), respectively. As a negative control, Escherichia coli K12 MG1655 transformed with an empty vector (Evec) was incubated under the same conditions. After incubation, the supernatant and pellet were separated, and the supernatant was tested for neuraminidase detection using the Neuraminidase Assay Kit (Abeam, ab138888). As shown in Figure 8D, YebF, FliC, and AidaC resulted in the secretion of active neuraminidase into the supernatant. By treating with MMP to release NH secreted from the relevant signals, the neuraminidase activity of NH secreted under the control of YebF, FliC, and AidaC was increased.

[0199] Example 4 - Development of pH-inducible and hypoxia-inducible promoters Manipulating bacteria as vectors for therapeutic payload delivery necessitates the development of tumor microenvironment biosensors. Some bacteria can naturally sense the hypoxic and acidic pH levels present in the tumor microenvironment. Several promoters have been identified as being activated only when the bacteria are inside the tumor core. Exemplary such promoters are shown in Table 2.

[0200] [Table 2]

[0201] Candidate pH-inducible and hypoxia-inducible promoters were prepared by synthesizing promoter DNA sequences with linkers (including a linker encoding RBS) using the method described in Storch et al., 2015, "BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology," ACS Synth. Biol., 4:781-787 (the entire sequence is incorporated herein by reference). This approach resulted in five test promoters—LOR7, LOR9, LOR1, LPR7, and LPR1—being tested for hypoxia-inducible and pH-inducible promoter activity.

[0202] A test construct encoding GFP was cloned into Escherichia coli K12 MG1655 under the control of a test promoter to create a control strain constitutively expressing GFP. The bacteria were cultured in medium until the metaphase of the logarithmic growth phase. The bacteria were then resuspended in mediums at pH 7 and pH 6 and added to 96-well microplates. Hypoxic conditions were created by adding mineral oil to the top of the wells. The plates were incubated at 37°C for 18 hours, and fluorescence levels were measured using a microplate reader.

[0203] As shown in Figures 9A and 9B, the LOR9, LOR1, and LPR1 promoters are induced under hypoxic conditions. Of these, LOR9 and LOR1 show strong suppression of GFP expression under normal oxygen conditions and increased GFP expression under hypoxic conditions at pH 6. Therefore, LOR9 and LOR1 represent promoters that are specifically induced in the tumor microenvironment.

[0204] PelB-bH was placed under the control of LOR7 and LOR9 in Escherichia coli K12 MG1655 to determine whether these promoters can drive hyaluronic acid degradation under conditions that replicate the tumor microenvironment. E. coli K12 MG1655 LOR7-PelB-bH, E. coli K12 MG1655 LOR9-PelB-bH, and E. coli K12 MG1655, all constitutively expressing GFP, were cultured in medium until metaphase logarithmic growth. They were then resuspended in pH 7 and pH 6 medium supplemented with 0.4 mg / mL of hyaluronic acid and placed in 96-well microplates. Cells were cultured under normal oxygen conditions or under hypoxic conditions induced by adding mineral oil to the top of the wells. The plates were incubated at 37°C for 18 hours, and the level of hyaluronan degradation was assessed using cetyltrimethylammonium bromide turbidimetry (CTM). As shown in Figure 9C, LOR9 activity increased under tumor physiological conditions (hypoxia and low pH).

[0205] Example 5 - In vivo activity of tumor-induced secretory hyaluronidase Candidate Escherichia coli K12 derivatives were engineered to secrete hyaluronidase under the control of tumor-inducible promoters LO_R7 and LO_R9, which respond to low levels of oxygen in the tumor microenvironment provided herein. Further control strains were engineered to secrete hyaluronidase under constitutive promoters.

[0206] The tumors were 6-7 weeks old at the time of receipt and were located 5 × 10 cm each beneath the mammary fat bed. 4 4T1 and E0771 cells were orthotopically injected into healthy female BALB / c and C57BL / 6 mice to induce tumors of 150 mm. 3 After reaching the average size, mice were randomized to the following treatment groups according to tumor size: PBS (control group), LO R7 10 6 CFU (raw BioThx group), LO R9 10 6CFU (raw BioThx group), constructive 10 6 CFU (raw BioThx group), ICI (immunotherapy group), and LO R7 10 6 CFU + ICI (combination therapy group). Mice were given raw BioThx (10 per mouse). 6 Mice received a single intravenous injection of CFU or PBS, followed by the first cycle of immunotherapy via intravenous injection (10 mg / kg anti-PD-1 + 5 mg / kg anti-CTLA-4) 3 days later (for the 4T1 study) or 4 days later (for the E0771 study). In total, mice received three cycles of ICI treatment, with a 3-day interval between each cycle. Tumors were collected and processed as appropriate for flow cytometry experiments, and mouse survival was monitored daily.

[0207] Tumor stiffness and perfusion were measured at 0, 3, and 6 days after administration using shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS), respectively, which are ultrasound-based methods. Administration of bacteria constitutively expressing hyaluronidase or expressing hyaluronidase under the control of the LOR7 or LOR9 promoter to tumor-carrying mice has been shown to reduce ECM stiffness and increase vascular perfusion in tumors (Figure 10A-B), and a significant decrease in hyaluronan levels in the TME was also observed (Figure 10C).

[0208] For both 4T1 and E0771 combination studies, tumor dimensions were measured every 2-3 days using calipers until their surgical excision two days after the last immunotherapy cycle. Results showed that Escherichia coli (E. coli) expressing hyaluronidase under the control of the LO R7 promoter (also indicated as C35R7) enhanced the efficacy of immunotherapy in an unresponsive mouse model of triple-negative breast cancer after three cycles of ICI administered every 3 days (Figures 11 and 12). After analyzing the immune profiles of the TME after treatment by flow cytometry, results showed a reduction in the myeloid-derived suppressor (MDSC, CD45+GR1+CD11b+) population in both 4T1 and E0771 tumors treated with ICI alone or in combination, while total lymphocyte levels remained unchanged across different treatment groups, suggesting a shift from an immunosuppressive microenvironment to an immunosupportive microenvironment (Figure 13).

[0209] Equal portions The embodiments, examples, and cases described above are applicable to and should be interpreted as applicable to any aspect of the present disclosure.

[0210] While this disclosure describes various aspects, embodiments, and examples, it will be understood that variations, improvements, and equivalents will be conceivable to those skilled in the art. Such variations, improvements, and equivalents are contemplated in this disclosure and fall within the scope of what is disclosed and claimed herein.

[0211] References All references made in this application to other documents are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein.

Claims

1. Recombinant bacterial cells capable of expressing heterologous extracellular matrix (ECM) degrading polypeptides that degrade extracellular matrix components, The nucleic acid comprises a nucleotide sequence and promoter that encode the heterogeneous extracellular matrix (ECM) degradation polypeptide. The nucleotide sequence encoding the heterogeneous extracellular matrix (ECM) degrading polypeptide is operably linked to the promoter, Recombinant bacterial cells in which the promoter is a tumor-inducing promoter.

2. The recombinant bacterial cell according to claim 1, wherein the heterogeneous extracellular matrix (ECM) degrading polypeptide is secreted from the cell.

3. Recombinant bacterial cells capable of expressing heterologous extracellular matrix (ECM) degrading polypeptides that degrade extracellular matrix components, comprising nucleic acids including a nucleotide sequence encoding the heterologous extracellular matrix (ECM) degrading polypeptide, Recombinant bacterial cells from which the aforementioned heterogeneous extracellular (ECM) degrading polypeptide is secreted.

4. The nucleic acid further comprises a promoter, optionally, The recombinant bacterial cell according to claim 3, wherein the nucleotide sequence encoding the heterogeneous extracellular matrix degradation (ECM) polypeptide is operably linked to the promoter, and the promoter is a tumor-inducible promoter.

5. i) The extracellular matrix-degrading polypeptide is a) Hyaluronidase, b) Microbial hyaluronidase or mammalian hyaluronidase, c) Bacterial hyaluronidase, d) Chondroitin ABC lyase, e) Microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase, e) Bacterial chondroitin ABC lyase, f) Neuraminidase, g) Microbial neuraminidase or mammalian neuraminidase, h) Bacterial neuraminidase, i) PNGase, j) Microbial PNGase or mammalian PNGase, k) It is the bacterium PNGase, Selectively, the extracellular matrix component is a proteoglycan. ii) The ECM component is hyaluronic acid (HA), and the ECM-degrading polypeptide is a) Hyaluronidase, b) Microbial hyaluronidase or mammalian hyaluronidase, and / or c) Bacterial hyaluronidase, iii) The ECM component is versican, and the ECM-degrading polypeptide is a) Chondroitin ABC lyase, b) Microbial chondroitin ABC lyase or mammalian chondroitin ABC lyase, and / or c) Bacterial chondroitin ABC lyase, iv) The ECM component is versican, and the ECM-degrading polypeptide is a) Neuraminidase, b) Microbial neuraminidase or mammalian neuraminidase, and / or c) Bacterial neuraminidase, v) The ECM component is versican, and the ECM-degrading polypeptide is a) PNGase, b) Microbial PNGase or mammalian PNGase, and / or c) It is the bacterium PNGase. vi) The heterogeneous extracellular matrix (ECM) degrading polypeptide a) Hyaluronidase containing or consisting of the amino acid sequence of SEQ ID NO: 1, chondroitin ABC lyase containing or consisting of the amino acid sequence of SEQ ID NO: 3 or 47, neuraminidase containing or consisting of the amino acid sequence of SEQ ID NO: 5, or PNGase containing or consisting of the amino acid sequence of SEQ ID NO: 7, or containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 47, and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 48, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 48, vii) The heterogeneous extracellular matrix (ECM) degrading polypeptide is a bacterial hyaluronidase, and / or viiii) The heterogeneous extracellular matrix (ECM) degrading polypeptide, a) The amino acid sequence of SEQ ID NO: 1, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, and / or b) A recombinant bacterial cell according to any one of claims 1 to 4, which is a bacterial hyaluronidase encoded by the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

2.

6. i) a) The tumor-inducible promoter is a hypoxia-inducible promoter, b) The tumor-inducible promoter is a hypoxia-inducible promoter selected from the group comprising or consisting of LOR9, LOR7, LOR1, pflE, pepT, YbiY, and pvhb, or ii) a) The tumor-inducible promoter is a pH-inducible promoter, and / or b) Recombinant bacterial cell according to any one of claims 1, 2, 4, and 5, wherein the tumor-inducing promoter is a pH-inducing promoter selected from the group comprising or consisting of LPR7, LPR1, LPR9, Stm1787, hyaA, P170-IL, P170-MG, P1, P2, and P3.

7. a) The hypoxia-inducible promoter is a ribosome binding site (RBS) encoded by the nucleotide sequences of SEQ ID NOs: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs: 62, 63, 64, 65, 6 6. A nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105, b) The hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO:

56. c) The pH-inducible promoter is the nucleotide sequence of SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, or SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 69, SEQ ID NOs. 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105, comprising a ribosome binding site (RBS) encoded by a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or at least 99%, and / or d) Recombinant bacterial cell according to any one of claims 1, 2, and 4 to 6, wherein the pH-inducible promoter is encoded by a nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO:

61.

8. a) The tumor-inducing promoter is a pH-inducible and hypoxia-inducible promoter. b) The tumor-inducible promoter is a pH-inducible and hypoxia-inducible promoter that is LOR9. c) The tumor-inducible promoter is LOR9 and is a pH-inducible and hypoxia-inducible promoter comprising a ribosome-binding site (RBS) encoded by the nucleotide sequence of SEQ ID NO: 73, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 73, and / or b) Recombinant bacterial cells according to any one of claims 1, 2, and 4 to 7, wherein the tumor-inducible promoter is LOR9 and is a pH-inducible and hypoxia-inducible promoter encoded by the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

55.

9. The tumor-inducing promoter described above, i) a) Approximately 0% to approximately 2%, approximately 0.2% to approximately 1.8%, approximately 0.4% to approximately 1.6%, approximately 0.6% to approximately 1.4%, approximately 0.8% to approximately 1.2%, approximately 0% to approximately 1%, approximately 0.2% to approximately 0.8%, approximately 0.4% to approximately 0.6%, 1% to 2%, approximately 1.2% to approximately 1.8%, and approximately 1.4% to approximately 1.6%. b) 0% to 2%, 0.2% to 1.8%, 0.4% to 1.6%, 0.6% to 1.4%, 0.8% to 1.2%, 0% to 1%, 0.2% to 0.8%, 0.4 to 0.6%, 1% to 2%, 1.2% to 1.8%, 1.4% to 1.6%, c) Percentages less than approximately 2%, less than approximately 1.5%, less than approximately 1.6%, less than approximately 1.4%, less than approximately 1.2%, less than approximately 1%, less than approximately 0.8%, less than approximately 0.6%, less than approximately 0.4%, less than approximately 0.2%, or lower percentages. d) Less than 2%, less than 1.5%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2%, or lower percentages. e) Approximately 2%, approximately 1.8%, approximately 1.6%, approximately 1.4%, approximately 1.2%, approximately 1%, approximately 0.8%, approximately 0.6%, approximately 0.4%, approximately 0.2%, and / or f) 2%, 1.8%, 1.6%, 1.4%, 1.2%, 1%, 0.8%, 0.6%, 0.4%, 0.2% Activated at the oxygen concentration, it drives the expression of the heterologous ECM-degrading polypeptide. ii) Not activated under normal oxygen conditions and not driving the expression of the heterologous ECM-degrading polypeptide, and / or iii) Not activated under normal oxygen conditions, and does not drive the expression of the heterologous ECM-degrading polypeptide, a) Approximately 6% or more, approximately 5% or more, approximately 4% or more, b) More than 6%, more than 5%, more than 4%, or more than 3%, c) Approximately 6%, approximately 5%, approximately 4%, approximately 3%, and / or d) 6%, 5%, 4%, 3% Recombinant bacterial cells according to any one of claims 1, 2, and 4 to 8, wherein the promoter is hypoxia-inducible and is not activated at oxygen concentrations and does not drive the expression of the heterologous ECM-degrading polypeptide.

10. i) The tumor-inducible promoter is a pH-inducible promoter that is activated at an acidic pH and drives the expression of the heterologous ECM-degrading polypeptide. ii) The tumor-inducing promoter is acidic pH, i.e., a) about pH 5.5 to about pH 7, about pH 5.6 to about pH 6.9, about pH 5.7 to about pH 6.8, about pH 5.8 to about pH 6.7, about pH 5.9 to about pH 6.6, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.2 to about pH 6.3, b) pH 5.5 to pH 7, pH 5.6 to pH 6.9, pH 5.7 to pH 6.8, pH 5.8 to pH 6.7, pH 5.9 to pH 6.6, pH 6.0 to pH 6.5, pH 6.1 to pH 6.4, pH 6.2 to pH 6.3 c) pH less than approximately 7, less than approximately 6.9, less than approximately 6.8, less than approximately 6.7, less than approximately 6.6, less than approximately 6.5, less than approximately 6.4, less than approximately 6.3, less than approximately 6.2, less than approximately 6.1, less than approximately 6.0, less than approximately 5.9, less than approximately 5.8, less than approximately 5.7, less than approximately 5.6, or a pH lower than these. d) pH less than 7, pH less than 6.9, pH less than 6.8, pH less than 6.7, pH less than 6.6, pH less than 6.5, pH less than 6.4, pH less than 6.3, pH less than 6.2, pH less than 6.1, pH less than 6.0, pH less than 5.9, pH less than 5.8, pH less than 5.7, pH less than 5.6 e) Approximately pH 5.5, approximately pH 5.6, approximately pH 5.7, approximately pH 5.8, approximately pH 5.9, approximately pH 6.0, approximately pH 6.1, approximately pH 6.2, approximately pH 6.3, approximately pH 6.4, approximately pH 6.5, approximately pH 6.6, approximately pH 6.7, approximately pH 6.8, approximately pH 6.9, or approximately pH 7, and / or f) pH 5.5 / pH 5.6 / pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3 / pH 6.4, pH 6.5 / pH 6.6, pH 6.7, pH 6.8, pH 6.9, or pH 7 A pH-inducible promoter that is activated and drives the expression of the heterologous ECM-degrading polypeptide, and / or iii) The tumor-inducing promoter is acidic pH, i.e., a) Approximately pH 5.9 to approximately pH 6.5, approximately pH 5.95 to approximately pH 6.45, approximately pH 6.0 to approximately pH 6.4, approximately pH 6.05 to approximately pH 6.35, approximately pH 6.1 to approximately pH 6.3, or approximately pH 6.15 to approximately pH 6.

25. b) pH 5.9 to pH 6.5, pH 5.95 to pH 6.45, pH 6.0 to pH 6.4, pH 6.05 to pH 6.35, pH 6.1 to 6.3, or pH 6.15 to pH 6.25 c) Approximately pH 5.9, approximately pH 5.95, approximately pH 6.0, approximately pH 6.05, approximately pH 6.1, approximately pH 6.15, approximately pH 6.2, approximately pH 6.25, approximately pH 6.3, approximately pH 6.35, approximately pH 6.4, approximately pH 6.45, or approximately pH 6.5, and / or d) pH 5.9 / pH 5.95 / pH 6.0, pH 6.05, pH 6.1, pH 6.15, pH 6.2, pH 6.25, pH 6.3, pH 6.35, pH 6.4 / pH 6.45, or pH 6.5 Recombinant bacterial cells according to any one of claims 1, 2, and 4 to 9, wherein the promoter is activated by and drives the expression of the heterologous ECM-degrading polypeptide, and is a pH-inducible promoter.

11. a) The tumor-inducible promoter is a hypoxia-inducible promoter that includes a modified RNA polymerase binding sequence, a modified operator sequence, or any combination thereof, or a modified regulatory element selected from the group consisting of these, or b) The recombinant bacterial cell according to any one of claims 1, 2, and 4 to 10, wherein the tumor-inducible promoter is a pH-inducible promoter comprising a modified RNA polymerase binding sequence, a modified operator sequence, or any combination thereof, or a modified regulatory element selected from the group consisting of these.

12. The aforementioned heterologous extracellular matrix (ECM) degrading polypeptide has an ECM degradation domain and a) Secretory domain, b) Two secretory domains, c) Cutting domains, and / or d) A recombinant bacterial cell according to any one of claims 1 to 11, which is a fusion polypeptide comprising a cleavage domain located between the ECM degradation domain and the secretory domain.

13. i) The secretory domain is selected from the group comprising or consisting of a PelB secretory signal sequence, a YebF carrier protein, a CtxB signal sequence, an AIDA-I autotransporter, a FliC signal sequence, and a Usp45 signal sequence, or any combination thereof. ii) The secretory domain, a) The amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or containing or consisting of amino acid sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and / or b) Encoded by the nucleotide sequences of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, iii) The cleavage domain includes a matrix metalloproteinase (MMP) cleavage site, and optionally the cleavage domain is a) The amino acid sequence of SEQ ID NO: 116, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 116, and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 117 And / or iv) The fusion polypeptide, a) Containing an ECM degradation domain including hyaluronidase, and i) The amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or containing or consisting of amino acid sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, and / or ii) Encoded by the nucleotide sequences of SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, or at least 99% identical, b) comprising an ECM degradation domain including neuraminidase, and i) The amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or the amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114, or consisting of or comprising the same amino acid sequence as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or SEQ ID NO: 114, and / or ii) Recombinant bacterial cells according to claim 12, which are encoded by the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, or by nucleotide sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO:

115.

14. The recombinant bacterial cell according to any one of claims 1 to 13, wherein the nucleic acid further comprises a nucleotide sequence encoding a transcription-regulating polypeptide.

15. The recombinant bacterial cell according to claim 14, wherein, if the secretory domain is a FliC signal sequence, the transcriptional regulatory peptide is selected from the group comprising or consisting of a FliC repressor, and optionally the FliC repressor is a GadE repressor.

16. The aforementioned transcriptional regulatory peptide a) The amino acid sequence of SEQ ID NO: 49, or a sequence containing or consisting of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49, and / or b) Recombinant bacterial cells according to claim 15, which are encoded by the nucleotide sequence of SEQ ID NO: 50, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

50.

17. i) Non-pathogenic bacterial cells, symbiotic cells, or probiotic cells, ii) It is a facultative anaerobic cell, and / or iii) a) Gram-negative bacterial cells, which are selected randomly. The aforementioned Gram-negative bacterial cells are selected from a group that includes or consists of Escherichia coli cells. Optionally, The aforementioned Gram-negative bacterial cells are Escherichia coli K12 cells. Selectively, the Escherichia coli K12 cells are Escherichia coli K12 MG1655 cells, or b) Gram-positive bacterial cells, which are selected randomly. The Gram-positive bacterial cells are lactic acid bacteria cells, and the lactic acid bacteria cells are optionally selected from a group that includes or consists of Lactococcus lactis cells, Lactobacillus cells, and Bifidobacterium cells. Optionally, Recombinant bacterial cells according to any one of claims 1 to 16, wherein the Gram-positive bacterial cells are Lactococcus lactis MG1363 cells.

18. a) The nucleic acid is selected from the group comprising plasmids, phagemids, and bacterial artificial chromosomes (BACs), b) Whether the nucleic acid is a plasmid, or c) The recombinant bacterial cell according to any one of claims 1 to 17, wherein the nucleic acid is incorporated into the genome of the cell.

19. Recombinant bacterial cells according to any one of claims 1 to 18, which are capable of accumulating in target tissue or tumors.

20. A hypoxia-inducible promoter, which is the promoter according to any one of claims 6 to 19.

21. The hypoxia-inducible promoter according to claim 20, which is an engineered hypoxia-inducible promoter.

22. a) is a LOR9, LOR7 promoter, or LOR1 promoter, and / or b) The hypoxia-inducible promoter according to claim 20 or 21, wherein the hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO:

56.

23. A pH-inducible promoter, which is the promoter according to any one of claims 6 to 19.

24. The pH-inducible promoter according to claim 23, which is an manipulated pH-inducible promoter.

25. a) The pH-inducible promoter is the LPR9, LPR7, or LPR1 promoter, and / or b) The pH-inducible promoter according to claim 23 or 24, wherein the hypoxia-inducible promoter is encoded by the nucleotide sequence of SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59, or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO:

59.

26. A nucleic acid comprising the nucleotide sequence of a hypoxia-inducible promoter according to any one of claims 20 to 22 and / or a pH-inducible promoter according to any one of claims 23 to 25.

27. A nucleic acid containing a nucleotide sequence that encodes an extracellular matrix (ECM) degradation polypeptide.

28. i) The extracellular matrix (ECM) degrading polypeptide is a heterogeneous extracellular matrix (ECM) degrading polypeptide according to any one of claims 1 to 19. ii) The nucleic acid further comprises a promoter, iii) The nucleic acid is, The promoter further comprises the promoter described in any one of claims 1, 2, and 4 to 26. iv) The nucleic acid further comprises a promoter, and the nucleotide sequence encoding the extracellular matrix (ECM) degrading polypeptide is operably linked to the promoter. v) Further comprising a transcriptional regulatory element, wherein the transcriptional regulatory element is selected from the group comprising or consisting of a terminator sequence, a repressor binding sequence, an insulator, an operator sequence, or any combination thereof. vi) further comprising a transcriptional regulatory element, optionally, the transcriptional regulatory element being selected from the group comprising or consisting of a terminator sequence, a repressor binding sequence, an insulator, an operator sequence, or any combination thereof, wherein the nucleotide sequence encoding the extracellular matrix (ECM) degrading polypeptide is operably linked to the transcriptional regulatory element, and / or vii) The nucleic acid is selected from the group comprising or consisting of plasmids, phagemids, and bacterial artificial chromosomes (BACs), viiii) The nucleic acid according to claim 27, wherein the nucleic acid is a plasmid.

29. A cell comprising an engineered hypoxia-inducible promoter according to any one of claims 20 to 22, an engineered pH-inducible promoter according to any one of claims 23 to 25, and / or a nucleic acid according to any one of claims 26 to 28.

30. The cell according to claim 29, which is a recombinant bacterial cell, and optionally is a recombinant bacterial cell according to any one of claims 1 to 19.

31. Recombinant bacterial cells according to any one of claims 1 to 19 or cells according to claim 29 or 30, for use in medicine.

32. Recombinant bacterial cells according to any one of claims 1 to 19 or cells according to claim 29 or 30, for use in treating cancer.

33. Recombinant bacterial cells for use according to any one of claims 31 to 32, wherein the use comprises administering an effective dose of the cells to a subject having cancer.

34. i) The cancer is a tumor solid carcinoma, and optionally, the cancer is an immune elimination solid tumor carcinoma and / or an immune elimination tumor, ii) The cancer is a cancer that includes extracellular matrix, iii) The cancer includes an extracellular matrix and is selected from the group including or comprising triple-negative breast cancer, microsatellite-stable colon cancer, and pancreatic cancer, and / or iv) The cancer is not a cancer that does not contain extracellular matrix. v) The cancer is not a cancer that does not contain extracellular matrix, It is not blood cancer. vi) The cancer is not a cancer that does not contain extracellular matrix, is not a hematological cancer, is not a cancer selected from the group including or consisting of leukemia, myeloma, and lymphoma, and / or vii) The recombinant bacterial cells or cells, a) Administered before or after the administration of cancer treatment drugs, b) Administered concurrently with cancer treatment drugs, c) A cancer drug administered concurrently with a cancer drug, wherein the cancer drug is selected from the group comprising or consisting of antibodies (optionally selected from the group comprising or consisting of immune checkpoint inhibitors (ICIs), T cell engagers, and bispecific antibodies or antibody-drug conjugates), cell therapies (optionally selected from the group comprising or consisting of T cells, CAR-T cells, and T4 cells), chemotherapeutic agents (optionally selected from the group comprising or consisting of FOLFOX, gemcitabine, paclitaxel, cisplatin, epirubicin, and irinotecan), immunomodulators (optionally selected from the group comprising or consisting of cytokines and chemokines), vaccines (optionally selected from the group comprising mRNA vaccines), viruses (optionally selected from the group comprising oncolytic viruses), and a second live biotherapy drug (optionally selected from the group comprising or consisting of engineered live biotherapy drugs and recombinant bacterial cells), or d) Concurrent administration with cancer treatment drugs, wherein the cancer treatment drug is an antibody (optionally selected, an immune checkpoint inhibitor antibody, a bispecific antibody, or an antibody-drug conjugate), hormone therapy, targeted therapy, immunotherapy, cell therapy (optionally selected, tumor-infiltrating lymphocytes (TILs), lymphocytes with engineered T cell receptors (TCRs), CARs) Recombinant bacterial cells or cells according to any one of claims 29 to 33, comprising or selected from the group consisting of T cells or natural killer cells), immunomodulatory agents (optionally, chemokines or cytokines), vaccines (optionally, mRNA vaccines), viruses (optionally, oncolytic viruses), other live biotherapeutic agents (optionally, engineered live biotherapeutic agents), chemotherapy (optionally, alkylating agents (optionally, temozolomide and carboplatin), antimetabolites (optionally, 5-FU or gemcitabine), antitumor antibiotics (optionally, doxorubicin), topoisomerase inhibitors (optionally, irinotecan), mitotic inhibitors (optionally, taxanes, optionally, paclitaxel or docetaxel), and corticosteroids (optionally, dexamethasone).

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