Composition and method for treating tumors using attenuated salmonella and immune checkpoint inhibitor

Abstract

The present invention belongs to the field of biomedicine. Provided are a composition for treating tumors using attenuated Salmonella and an immune checkpoint inhibitor, and a method for treating tumors. In the present invention, an anti-tumor immune response is activated by means of the combined use of attenuated Salmonella and an immune checkpoint inhibitor, which synergistically improves the inhibitory effect on tumor cells. The present invention exhibits excellent therapeutic effects against malignant tumors for which effective control means are currently lacking, and has broad application prospects.

Description

Compositions and methods for treating tumors using attenuated Salmonella and immune checkpoint inhibitors

[0001] This application claims priority to an earlier application filed on March 21, 2025, with the China National Intellectual Property Administration, patent application number 202510343395.3, entitled "Composition and Method for Treating Tumors Using Attenuated Salmonella and Immune Checkpoint Inhibitors". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention relates to compositions and methods for treating tumors using attenuated Salmonella and immune checkpoint inhibitors, and belongs to the field of biomedicine. Background Technology

[0003] Malignant tumors are among the most serious diseases threatening human health worldwide, with widespread and profound harm. They not only severely threaten the physical health of patients but also impose a heavy burden on families and society. Currently, surgical resection, radiotherapy, chemotherapy, and targeted therapy are widely used in clinical practice. However, due to the complexity of tumor biology and the tumor microenvironment, these clinical treatment methods all have certain limitations.

[0004] Over the past two decades, research on anti-tumor drugs using bacteria as gene therapy vectors has made some progress. The tumor microenvironment, with its significantly different physiological state compared to normal tissues, makes it a priority target for certain bacteria, which can be used for bacterial-mediated tumor immunotherapy. In particular, the hypoxic environment of tumors allows anaerobic or facultative anaerobic bacteria (such as Salmonella, Escherichia coli, and Listeria) to selectively target tumors through various mechanisms. By leveraging their inherent immunogenicity and locally releasing effective payloads, they can modulate the tumor's immune microenvironment, thereby achieving the goal of tumor treatment.

[0005] Meanwhile, the emergence of immune checkpoint inhibitors (ICIs) marks a major breakthrough in cancer treatment, including drugs against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1). Anti-PD-1 or anti-PD-L1 antibodies specifically bind to PD-1 or PD-L1, preventing their binding and thus relieving T cell suppression, allowing T cells to be reactivated and exert their anti-tumor activity. Anti-CTLA-4 antibodies block the binding of CTLA-4 to co-stimulatory molecules, promoting T cell activation and proliferation, and enhancing the immune system's ability to attack tumor cells. However, not all patients benefit from immune checkpoint inhibitor therapy; the overall response rate varies across different tumor types, generally ranging from 20% to 40%. Furthermore, with the progression of treatment, some patients develop resistance to immune checkpoint inhibitors, further limiting the efficacy of ICIs.

[0006] Therefore, by combining bacteria with immune checkpoint inhibitors, synergistic effects can be achieved against different biological characteristics of tumor cells or the tumor microenvironment, increasing the sensitivity of tumor cells to immunotherapy, thereby improving the overall tumor treatment response rate and benefiting more patients. Summary of the Invention

[0007] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell and tissue culture, microbiology, immunology, and pharmaceutical terminology and laboratory procedures used in this invention are all widely used terms and routine procedures in their respective fields.

[0008] To address the aforementioned deficiencies in the prior art, this invention provides a pharmaceutical composition for treating tumors, comprising attenuated Salmonella and an immune checkpoint inhibitor.

[0009] Another aspect of the present invention provides the use of attenuated Salmonella and immune checkpoint inhibitors in the preparation of pharmaceutical compositions for treating tumors.

[0010] In order for attenuated Salmonella to survive only under low oxygen concentration conditions, three requirements must be met:

[0011] 1. Control the strength and background leakage expression of the upstream promoter.

[0012] If oxygen receptors leak expression without binding to the upstream promoter, oxygen regulation will be impossible, resulting in bacteria surviving at any oxygen concentration. Alternatively, if the upstream promoter is too weak, downstream gene expression cannot be initiated, causing bacteria to fail to survive at any oxygen concentration.

[0013] 2. Select suitable genes essential for survival.

[0014] By selecting genes essential for survival, it is possible to ensure bacterial death when these genes are not expressed, while rapidly ensuring bacterial survival when expressed under hypoxia.

[0015] 3. Under switching conditions between hypoxia and hyperxia, the upstream promoter can quickly initiate transcription, rapidly achieving the synthesis and expression of genes essential for survival, ensuring the survival of the modified bacteria. However, under hyperxia conditions, the expression of essential survival genes will not be initiated, leading to bacterial death.

[0016] Therefore, in this invention, the attenuated Salmonella can initiate the expression of essential genes under hypoxic conditions. It contains essential genes controlled by hypoxia-inducible promoters and may further contain other regulatory elements required for the expression of these essential genes if necessary.

[0017] In this invention, an "essential gene" refers to a gene that plays a decisive role in the growth and / or survival of bacteria. Bacteria cannot survive, divide, and / or grow normally if they lack this gene or its functional expression product. A typical example of a lack of an essential gene or its functional expression product is an auxotrophic strain, which cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments without the presence of a specific exogenous supplement. Essential genes typically exist as a single copy on the bacterial chromosome.

[0018] Therefore, the requirements for genes essential for survival are as follows:

[0019] 1. It is an essential gene for bacterial reproduction; its absence can lead to the rapid death of bacteria.

[0020] 2. The product of this gene does not exist in the normal environment or in the human body, which ensures that it will not go out of control in the human environment, and the corresponding expression product of this gene can be added to the normal culture environment to facilitate the culture and preparation of bacteria.

[0021] 3. This gene needs to be rapidly activated under the regulation of a hypoxic promoter and can rapidly synthesize products to achieve the regulatory function of the host bacteria.

[0022] For Gram-negative bacteria like Salmonella, the cell wall is an indispensable component. The core component of the cell wall is peptidoglycan. Bacteria require D-alanine as a crucial building block for peptidoglycan synthesis. Without D-alanine, bacteria cannot synthesize a cell wall, leading to cell lysis.

[0023] In nature, all amino acids are L-type. Therefore, Gram-negative bacteria contain two genes: the alar gene and dadX, which are responsible for the biosynthesis of alanine racemic enzyme. They are responsible for converting L-alanine into D-alanine to meet the needs of cell wall synthesis.

[0024] Research has found that simultaneously mutating the alar and dadX genes can result in lethal mutations in Salmonella, and these mutations can be compensated for by supplementing the culture medium with additional D-alanine.

[0025] This invention selects the alar gene and the dadX gene as essential genes for survival. The alar gene and the dadX gene are functionally homologous genes. Therefore, in this invention, the alanine racemic enzyme gene can be the alar gene or the Salmonella dadX gene from Salmonella, or the alar or dadX gene from other Gram-negative bacteria, or genes with equivalent functions.

[0026] In this invention, bacteria with the arr gene knocked out were first constructed, and then another gene, dadX, was modified so that the bacteria became bacteria with defects in both the arr and dadX genes after editing. At the same time, a positive hypoxia promoter and a negative hyperxia promoter were used to regulate additional arr or dadX genes.

[0027] Therefore, in a preferred embodiment of the present invention, the attenuated Salmonella includes a positive hypoxia promoter, a survival-essential gene, and a negative hyperxia promoter. The positive hypoxia promoter can be induced to express under hypoxia, the negative hyperxia promoter functions under normal organ oxygen content conditions, and the survival-essential gene is a gene encoding alanine racemase.

[0028] In a more preferred embodiment of the present invention, the forward hypoxia promoter is selected from the promoter region sequence of yhbU or ynfK, the survival essential gene is selected from alar or dadX, and the reverse hyperxia promoter is selected from the promoter region sequence of cyoA or ydcI.

[0029] In a further preferred embodiment of the present invention, the attenuated Salmonella contains a positive hypoxia promoter yhbU, a survival-essential gene alar, and a negative hyperxia promoter cyoA.

[0030] In a further preferred embodiment of the present invention, the attenuated Salmonella includes a positive hypoxia promoter yhbU, a survival-essential gene alar, and a negative hyperxia promoter ydcI.

[0031] In a further preferred embodiment of the present invention, the attenuated Salmonella contains a positive hypoxia promoter ynfK, a survival-essential gene dadX, and a negative hyperxia promoter cyoA.

[0032] In a further preferred embodiment of the present invention, the attenuated Salmonella includes a positive hypoxia promoter ynfK, a survival-essential gene dadX, and a negative hyperxia promoter ydcI.

[0033] In a particularly preferred embodiment of the present invention, the attenuated Salmonella is selected from SWT5001, SWT5005, SWT6009 and SWT6013.

[0034] In the most preferred embodiment of the present invention, the attenuated Salmonella is SWT5005.

[0035] In a preferred embodiment of the present invention, the attenuated Salmonella is regulated by oxygen concentration.

[0036] For attenuated Salmonella and other Gram-negative bacteria to survive in hypoxic environments, an oxygen concentration of 1% is a crucial threshold, representing pathological hypoxia. This is because an oxygen concentration below 1% is a clear marker of hypoxia in tumors; areas with oxygen concentrations below 1% do not exist in normal organs and tissues. For example, hypoxic areas in various tumors, such as pancreatic cancer, cervical cancer, and prostate cancer, have oxygen concentrations below 0.7%.

[0037] The attenuated Salmonella strain designed in this invention is produced by precisely controlling oxygen levels to enable it to recognize and proliferate in hypoxic tumor regions. The goal of this invention is to allow the modified attenuated Salmonella to survive and proliferate even at oxygen concentrations below 0.8%, and to undergo suicidal lysis in environments with normal oxygen concentrations.

[0038] Therefore, in a more preferred embodiment of the invention, the positive hypoxia promoter functions when the oxygen content is below 1%, but does not function when the oxygen content is above 1%; and / or

[0039] The reverse hyperoxia promoter functions when the oxygen content is above 1%, but cannot function when the oxygen content is below 1%.

[0040] In a further preferred embodiment of the invention, the positive hypoxia promoter functions when the oxygen content is below 0.8%, but does not function when the oxygen content is above 0.8%; and / or

[0041] The reverse hyperoxygen promoter functions when the oxygen content is above 0.8%, but cannot function when the oxygen content is below 0.8%.

[0042] Immune checkpoint inhibitors (ICIs) are a class of drugs used to treat cancer. They activate the immune system by blocking immune checkpoint molecules, causing it to attack tumor cells. Immune checkpoint inhibitors are monoclonal antibodies that counteract immunosuppressive pathways known as checkpoints.

[0043] Currently, the most widely used immune checkpoint inhibitors are PD-1 antibodies, PD-L1 antibodies, and CTLA-4 antibodies. These drugs are not considered to directly kill tumors, but rather to indirectly mediate anti-tumor effects by inducing T cell activation.

[0044] In the process of tumor antigen presentation, two different checkpoints have been studied and developed to obtain therapeutic benefits.

[0045] When antigen-presenting cells (APCs) present tumor antigens to T cells, the checkpoint protein CTLA-4 can inhibit T cell activation by competitively binding to APC co-stimulatory ligands. If T cells are indeed activated, they may encounter a second checkpoint expressing the PD-1 ligand PD-L1 when they encounter tumor cells and / or other tissues in the target cells. Tumors utilize this second checkpoint by overexpressing PD-L1 to avoid T cell-mediated tumor killing, effectively preventing T cell infiltration at the tumor periphery. However, if immunotherapy inhibits PD-1 and / or PD-L1 signaling, theoretically, T cell activation could proceed unimpeded, leading to a re-attack on the tumor by new, undepleted T cells. CTLA-4 and PD-1 are receptors expressed on the surface of cytotoxic T cells, interacting with their ligands CD80 / CD86 / CD28 in the case of CTLA-4 and with PD-L1 in the case of PD-1. These pathways can be used to help cancer cells evade cytotoxic T cell-mediated death. Immune checkpoint inhibitors work by preventing receptors and ligands from binding together, thereby disrupting signaling.

[0046] PD-1 (programmed death receptor 1) is an important immunosuppressive molecule, belonging to the immunoglobulin superfamily, and is a membrane protein with 288 amino acid residues. It was initially cloned from apoptotic mouse T-cell hybridoma 2B4.11. Immunomodulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune disease, and organ transplant survival.

[0047] PD-1's ligand PD-L1 can also serve as a target, and corresponding antibodies can achieve the same effect. The binding of PD-1 and PD-L1 initiates programmed cell death in T cells, allowing tumor cells to escape the immune system. PD-1 has at least two ligands: PD-L1 and PD-L2. PD-L1 has at least two ligands: PD-1 and CD80. PD-L2 has at least two ligands: PD-1 and RGMB.

[0048] PD-1 prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells).

[0049] PD-1 antibodies are a new class of drugs that block PD-1, which can activate the immune system to attack tumors and are used to treat certain types of cancer.

[0050] PD-L1 (programmed cell death ligand 1) is a cell-mediated immune antigen on the surface of tumors. When PD-L1 binds to PD-1, it can participate in tumor-associated immune responses, inhibit the body's immune signals, affect T cell proliferation, and lead to impaired immune cell function. This allows tumor cells to escape the surveillance and killing of the immune system, and tumor cells can continue to proliferate.

[0051] PD-L1 antibodies can competitively bind to PD-L1 in the patient's body, block PD-L1 pathway signals, restore the immune killing function of T cells, and enable the body to exert good immune efficacy, thereby playing a role in killing tumor cells.

[0052] CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a leukocyte differentiation antigen and a transmembrane receptor on T cells. It shares a B7 molecule ligand with CD28. CTLA-4 binding to the B7 molecule induces T cell unresponsiveness and participates in the negative regulation of the immune response.

[0053] CTLA-4 antibodies can effectively and specifically inhibit cellular and humoral immune responses in vitro and in vivo, and have significant therapeutic effects on transplant rejection and various autoimmune diseases. They have extremely low toxicity and side effects, and are currently considered to be promising new immunosuppressive drugs.

[0054] Therefore, in a preferred embodiment of the present invention, the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

[0055] In a more preferred embodiment of the present invention, the PD-1 antibody is selected from pembrolizumab, nivolumab, simiprelimab, camrelizumab, tislelizumab, sintilimab, toripalimab, penamprolium, and edoximumab. The PD-L1 antibody is selected from atezolizumab, durvalumab, avelumab, adebelimumab, sugemalimab, and envorimab. The CTLA-4 antibody is selected from ipilimumab and cantalonilmab.

[0056] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0057] As used in this invention, a "pharmaceutically acceptable carrier" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tension agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). For example, depending on the route of administration, the bacteria of this invention may be encapsulated in a material to protect the bacteria from acids and other natural conditions that could inactivate them. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the provisional preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any ranges incompatible with the active compound, may be used in the pharmaceutical compositions of this invention.

[0058] In a more preferred embodiment of the invention, the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tension agents, stabilizers, antioxidants, surfactants, and lubricants.

[0059] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors.

[0060] In a more preferred embodiment of the present invention, the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, liver cancer, bile duct cancer, melanoma, breast cancer, bladder cancer, thyroid cancer, testicular cancer, gastric cancer, prostate cancer, ovarian cancer, esophageal cancer, kidney cancer, uterine cancer, neuroblastoma, glioma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, and head and neck cancer.

[0061] In the most preferred embodiment of the present invention, the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, and liver cancer.

[0062] In this invention, for therapeutic purposes, the term "object" is preferably an object for which a target pathological condition, such as a tumor, needs to be treated. For preventative purposes, the object is preferably an object at risk of developing or prone to developing the target pathological condition. The term "object" includes living organisms, such as prokaryotes and eukaryotes. Examples of objects include mammals, such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, rats, and transgenic non-human animals. In a particular embodiment of the invention, the object is a human.

[0063] As used in this invention, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing the proliferation (or destroying) of neoplastic or cancerous cells, inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating malignant tumors, reducing symptoms caused by malignant tumors, improving the quality of life of patients with malignant tumors, reducing the dosage of other drugs required to treat malignant tumors, delaying the progression of malignant tumors, curing malignant tumors, and / or prolonging the survival of patients with malignant tumors.

[0064] As used in this invention, an "effective amount" or "effective dose" of attenuated Salmonella, a drug, or a pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of disease, reducing disease severity, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in a patient. For example, relative to an untreated subject, an "effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in animal model systems that predict the efficacy of treatment against human tumors. Alternatively, the efficacy can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro using assays known to those skilled in the art. A therapeutically effective amount of the therapeutic compound can reduce tumor size or otherwise alleviate the subject's symptoms. Those skilled in the art can determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0065] In a preferred embodiment of the present invention, the attenuated Salmonella, drug, or drug composition of the present invention is administered via the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0066] In a preferred embodiment of the present invention, the attenuated Salmonella, drug, or pharmaceutical composition of the present invention can be formulated for administration via the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0067] The dosage forms of the attenuated Salmonella, pharmaceuticals, or pharmaceutical compositions of the present invention may be solutions, emulsions, freeze-dried preparations, or suspensions; for oral administration, the dosage forms may be tablets or capsules; for intranasal dosage forms, the dosage forms may be powders, nasal drops, or aerosols; for topical administration, the dosage forms may be aqueous solutions, suspensions, ointments, creams, or gels; for rectal or vaginal administration, the dosage forms may be suppositories, enemas, or delivered as part of an endoscopic or colonoscopy procedure.

[0068] The attenuated Salmonella, pharmaceuticals, or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as microbial growth in a fermenter followed by centrifugation and concentration, washing, filtration or dialysis, conventional granulation, mixing, dissolving, encapsulation, lyophilization or emulsification processes, and other methods. The bacteria, pharmaceuticals, or pharmaceutical compositions of the present invention can be produced in various forms, including granules, precipitates or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions, or solutions. The formulation may optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0069] The attenuated Salmonella, drug, or pharmaceutical composition of the present invention can be administered alone or in combination with other compounds or compositions in the presence of a carrier. In a preferred embodiment of the invention, the attenuated Salmonella, drug, or pharmaceutical composition can be administered in combination with other malignant tumor therapies (including, but not limited to, radiotherapy, chemotherapy, and surgery).

[0070] In another aspect, the present invention provides a method for treating tumors, comprising administering the pharmaceutical composition of the present invention to a subject.

[0071] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors.

[0072] In a more preferred embodiment of the present invention, the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, liver cancer, bile duct cancer, melanoma, breast cancer, bladder cancer, thyroid cancer, testicular cancer, gastric cancer, prostate cancer, ovarian cancer, esophageal cancer, kidney cancer, uterine cancer, neuroblastoma, glioma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, and head and neck cancer.

[0073] In the most preferred embodiment of the present invention, the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, and liver cancer.

[0074] In a preferred embodiment of the present invention, after a pre-treatment cycle of administration of an immune checkpoint inhibitor, a combined treatment of attenuated Salmonella and an immune checkpoint inhibitor is administered.

[0075] In a more preferred embodiment of the present invention, the administration route of the attenuated Salmonella is selected from intravenous, intratumoral, and interventional perfusion.

[0076] In a more preferred embodiment of the present invention, the administration route of the immune checkpoint inhibitor is selected from intraperitoneal, intravenous, intratumoral, oral, and subcutaneous.

[0077] By adopting the above technical solution, the present invention achieves the following beneficial effects:

[0078] This invention utilizes a combination of attenuated Salmonella and immune checkpoint inhibitors to observe CD8 within tumor tissue. + Significant aggregation of cytotoxic T cells and macrophages was observed. This combined therapy significantly enhanced the activation of the anti-tumor immune response by synergistically regulating the infiltration of immune cells in the tumor microenvironment, thereby achieving a synergistic enhancement of the inhibitory effect on tumor cell growth. Experimental results show that this treatment strategy demonstrates significant efficacy against malignant tumors for which there are currently no effective clinical control methods, providing important theoretical basis and application foundation for the development of novel tumor treatment methods, and has broad application prospects.

[0079] This invention provides a novel combination therapy approach, which involves administering an immune checkpoint inhibitor one dosing cycle in advance, followed by combined treatment with attenuated Salmonella and the immune checkpoint inhibitor. This approach demonstrates superior antitumor efficacy compared to using immune checkpoint inhibitors alone or in combination therapy without prior administration of the immune checkpoint inhibitor. Attached Figure Description

[0080] Figure 1. Agarose gel electrophoresis diagram for the cloning and verification of the positive hypoxia promoters yhbU-S and ynfK-S.

[0081] Figure 2. Agarose gel electrophoresis diagram for the verification and amplification of the essential gene library.

[0082] Figure 3. Agarose gel electrophoresis identification and verification of the reverse hyperoxia promoters cyoA-S and ydcI-S.

[0083] Figure 4. Schematic diagram of hypoxia-specific gene expression cassette.

[0084] Figure 5. Schematic diagram of lambda RED recombinase and CRE recombinase system.

[0085] Figure 6. Identification of the Salmonella aroA gene knockout experiment.

[0086] (A) Verification diagram of PCR amplification products of aroA gene knockout fragment identified by agarose gel electrophoresis;

[0087] (B) Verification diagram of PCR amplification products of target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0088] Figure 7. Identification of the Salmonella alar gene knockout experiment.

[0089] (A) Verification diagram of PCR amplification products of alar gene knockout fragment identified by agarose gel electrophoresis;

[0090] (B) Verification diagram of PCR amplification products of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0091] Figure 8. Agarose gel electrophoresis verification of strains SWT1001 and SWT1005 after insertion into a hypoxia-specific gene expression cassette (alr is an essential gene).

[0092] Figure 9. Verification diagram of PCR amplification products of SWT007 by agarose gel electrophoresis.

[0093] Figure 10. Agarose gel electrophoresis verification of strains SWT2009 and SWT2013 after insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene).

[0094] Figure 11 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1001.

[0095] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0096] (B) Cultured under aerobic conditions on LB plates without D-alanine.

[0097] Figure 12 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1005.

[0098] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0099] (B) Cultured under aerobic conditions on LB plates without D-alanine.

[0100] Figure 13 shows the results of the oxygen adaptability verification test for Salmonella strains SWT2009 and SWT2013.

[0101] (A) SWT2009 was cultured on LB plates without D-alanine, with the top layer under anaerobic conditions and the bottom layer under aerobic conditions;

[0102] (B) SWT2013 was cultured on LB plates without D-alanine, with the top layer under anaerobic conditions and the bottom layer under aerobic conditions.

[0103] Figure 14 shows the results of the oxygen adaptability verification test for various Salmonella strains at an oxygen concentration below 0.8%.

[0104] (A) Salmonella strain SWT1001 cultured on LB plates;

[0105] (B) Salmonella strain SWT1005 cultured on LB plates.

[0106] Figure 15 is a schematic diagram containing the chloramphenicol resistance gene, lacUV5, and T7 RNA polymerase gene cassette pPRO005.

[0107] Figure 16. Enzyme digestion identification of plasmid pPRO005.

[0108] The arrow points to the cut T7 RNA polymerase gene cassette fragment.

[0109] Figure 17 shows the identification of lacUV5-controlled T7 RNA polymerase constitutive expression cassette recombination into the Salmonella chromosome.

[0110] (A) PCR amplification fragments used for recombination (primers SWTO102 and SWTO103);

[0111] (B) PCR identification diagrams of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P after construction.

[0112] Figure 18. Schematic diagram of the construction of plasmid expression vector pPRO010.

[0113] Figure 19 shows the PCR identification of plasmid pPRO010 in strains SWT5001, SWT5005, SWT6009 and SWT6013.

[0114] Figure 20A shows the low-dose (3.75 × 10⁻⁶) of attenuated Salmonella SWT5005. 6Figure 1. Statistical results of tumor volume in CT26 tumor-bearing mice administered CFU / mouse intravenously and in combination with PD-1 antibody;

[0115] Figure 20B shows the high dose (3.75 × 10⁻⁶) of attenuated Salmonella SWT5005. 7 Figure 1. Statistical results of tumor volume in CT26 tumor-bearing mice administered CFU / mouse intravenously and in combination with PD-1 antibody;

[0116] Figure 20C shows the statistical results of body weight of CT26 tumor-bearing mice from groups 1 to 8;

[0117] Figure 20D shows actual photographs of tumors in CT26 tumor-bearing mice that received low-dose intravenous administration of attenuated Salmonella SWT5005 and combined with PD-1 antibody.

[0118] Figure 20E shows actual photographs of tumors in CT26 tumor-bearing mice that received high-dose intravenous administration of attenuated Salmonella SWT5005 and combined with PD-1 antibody.

[0119] Figure 20F shows the individual tumor volume results of CT26 tumor-bearing mice in Group 1.

[0120] Figure 20G shows the individual tumor volume results of CT26 tumor-bearing mice in Group 2.

[0121] Figure 20H shows the individual tumor volume results of CT26 tumor-bearing mice in group 3.

[0122] Figure 20I shows the individual tumor volume results of CT26 tumor-bearing mice in group 4.

[0123] Figure 20J shows the individual tumor volume results of CT26 tumor-bearing mice in group 5.

[0124] Figure 20K shows the individual tumor volume results of CT26 tumor-bearing mice in group 6.

[0125] Figure 20L shows the individual tumor volume results of CT26 tumor-bearing mice in group 7.

[0126] Figure 20M shows the individual tumor volume results of CT26 tumor-bearing mice in group 8.

[0127] Figure 20N shows the immunohistochemical staining results of Salmonella, neutrophils (ly6G), T cells (CD3), cytotoxic T cells (CD8), and macrophages (F4 / 80) in tumor tissue of mice in the PBS control group.

[0128] Figure 20O shows the immunohistochemical staining results of Salmonella, neutrophils, T cells, cytotoxic T cells, and macrophages in tumor tissues of mice in the single PD-1 antibody administration group.

[0129] Figure 20P shows a high dose (3.75 × 10⁻⁶) of single attenuated Salmonella SWT5005. 7 Immunohistochemical staining results of Salmonella, neutrophils, T cells, cytotoxic T cells, and macrophages in tumor tissues of mice in the CFU / mouse intravenous administration group;

[0130] Figure 20Q shows the immunohistochemical staining results of Salmonella, neutrophils, T cells, cytotoxic T cells, and macrophages in tumor tissues of mice in the high-dose intravenous administration of attenuated Salmonella SWT5005 combined with PD-1 antibody.

[0131] Figure 20R shows the immunohistochemical staining results of Salmonella, neutrophils, T cells, cytotoxic T cells, and macrophages in tumor tissues of mice in the high-dose intravenous administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody.

[0132] Where * represents a significance level P<0.05, ** represents a significance level P<0.01, and *** represents a significance level P<0.001.

[0133] Figure 21A shows the low-dose (3.5 × 10⁻⁶) of attenuated Salmonella SWT5005. 6 Figure 1. Statistical results of tumor volume in CT26 tumor-bearing mice with intratumoral administration of CFU / mouse and combined with PD-1 antibody;

[0134] Figure 21B shows the high dose (3.5 × 10⁻⁶) of attenuated Salmonella SWT5005. 7 Figure 1. Statistical results of tumor volume in CT26 tumor-bearing mice with intratumoral administration of CFU / mouse and combined with PD-1 antibody;

[0135] Figure 21C shows the statistical results of body weight of CT26 tumor-bearing mice from group 1 to group 8;

[0136] Figure 21D shows actual photographs of tumors in CT26 tumor-bearing mice after low-dose intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1 antibody.

[0137] Figure 21E shows actual photographs of tumors in CT26 tumor-bearing mice that received high-dose intratumoral administration of attenuated Salmonella SWT5005 and combined with PD-1 antibody.

[0138] Figure 21F shows the individual tumor volume results of CT26 tumor-bearing mice in Group 1.

[0139] Figure 21G shows the individual tumor volume results of CT26 tumor-bearing mice in Group 2.

[0140] Figure 21H shows the individual tumor volume results of CT26 tumor-bearing mice in group 3.

[0141] Figure 21I shows the individual tumor volume results of CT26 tumor-bearing mice in group 4.

[0142] Figure 21J shows the individual tumor volume results of CT26 tumor-bearing mice in group 5.

[0143] Figure 21K shows the individual tumor volume results of CT26 tumor-bearing mice in group 6.

[0144] Figure 21L shows the individual tumor volume results of CT26 tumor-bearing mice in group 7.

[0145] Figure 21M shows the individual tumor volume results of CT26 tumor-bearing mice in group 8.

[0146] Where * represents a significance level P<0.05, ** represents a significance level P<0.01, *** represents a significance level P<0.001, and **** represents a significance level P<0.0001.

[0147] Figure 22A shows the tumor volume statistics of LLC tumor-bearing mice in the groups of intravenous administration of attenuated Salmonella SWT5005 and combined PD-1 antibody;

[0148] Figure 22B shows the tumor volume statistics of LLC tumor-bearing mice in the groups of intravenous administration of attenuated Salmonella SWT5005 and combined PD-L1 antibody.

[0149] Figure 22C shows the weight statistics of LLC tumor-bearing mice in the group that received intravenous administration of attenuated Salmonella SWT5005 and combined with PD-1 / PD-L1 antibody;

[0150] Figure 22D shows the tumor volume statistics of LLC tumor-bearing mice in the groups of intratumoral administration of attenuated Salmonella SWT5005 and combined PD-1 antibody.

[0151] Figure 22E shows the tumor volume statistics of LLC tumor-bearing mice in the groups of intratumoral administration of attenuated Salmonella SWT5005 and combined PD-L1 antibody.

[0152] Figure 22F shows the tumor volume statistics of LLC tumor-bearing mice in the groups of intratumoral administration of attenuated Salmonella SWT5005 and combined administration of CTLA-4 antibody.

[0153] Figure 22G shows the weight statistics of LLC tumor-bearing mice in the groups of intratumoral administration of attenuated Salmonella SWT5005 and combined PD-1 / PD-L1 / CTLA-4 antibody group.

[0154] Figure 22H shows the individual tumor volume results of LLC tumor-bearing mice in Group 1.

[0155] Figure 22I shows the individual tumor volume results of LLC tumor-bearing mice in Group 2.

[0156] Figure 22J shows the individual tumor volume results of LLC tumor-bearing mice in group 3.

[0157] Figure 22K shows the individual tumor volume results of LLC tumor-bearing mice in group 4.

[0158] Figure 22L shows the individual tumor volume results of LLC tumor-bearing mice in group 5.

[0159] Figure 22M shows the individual tumor volume results of LLC tumor-bearing mice in group 6.

[0160] Figure 22N shows the individual tumor volume results of LLC tumor-bearing mice in group 7.

[0161] Figure 22O shows the individual tumor volume results of LLC tumor-bearing mice in group 8.

[0162] Figure 22P shows the individual tumor volume results of LLC tumor-bearing mice in group 9;

[0163] Figure 22Q shows the individual tumor volume results of LLC tumor-bearing mice in group 10.

[0164] Figure 22R shows the individual tumor volume results of LLC tumor-bearing mice in group 11.

[0165] Figure 22S shows actual photographs of tumors in LLC tumor-bearing mice that were given attenuated Salmonella SWT5005 intravenously and combined with PD-1 antibody.

[0166] Figure 22T shows actual photographs of tumors in LLC tumor-bearing mice that were intravenously administered with attenuated Salmonella SWT5005 and combined with PD-L1 antibody.

[0167] Figure 22U shows actual photographs of tumors in LLC tumor-bearing mice that received intratumoral administration of attenuated Salmonella SWT5005 and combined with PD-1 antibody.

[0168] Figure 22V shows actual photographs of tumors in LLC tumor-bearing mice that received intratumoral administration of attenuated Salmonella SWT5005 and combined with PD-L1 antibody.

[0169] Figure 22W shows actual photographs of tumors in LLC tumor-bearing mice that received intratumoral administration of attenuated Salmonella SWT5005 and combined with CTLA-4 antibody.

[0170] Figure 22X shows the immunohistochemical staining results of Salmonella, neutrophils (ly6G), cytotoxic T cells (CD8), and macrophages (F4 / 80) in tumor tissues of mice in the PBS control group.

[0171] Figure 22Y shows the immunohistochemical staining results of Salmonella, neutrophils, cytotoxic T cells, and macrophages in tumor tissues of mice in the single PD-1 antibody administration group.

[0172] Figure 22Z shows the immunohistochemical staining results of Salmonella, neutrophils, cytotoxic T cells, and macrophages in tumor tissues of mice in the single attenuated Salmonella SWT5005 intravenous administration group.

[0173] Figure 22AA shows the immunohistochemical staining results of Salmonella, neutrophils, cytotoxic T cells, and macrophages in tumor tissues of mice in the group that received intravenous administration of attenuated Salmonella SWT5005 combined with PD-1 antibody.

[0174] Where * represents a significance level P<0.05, ** represents a significance level P<0.01, *** represents a significance level P<0.001, and **** represents a significance level P<0.0001.

[0175] Figure 23A shows the statistical results of tumor volume in all WEHI-164 tumor-bearing mice from group 1 to group 4;

[0176] Figure 23B shows the statistical results of the body weight of all WEHI-164 tumor-bearing mice in groups 1 to 4;

[0177] Figure 23C shows the individual tumor volume results of WEHI-164 tumor-bearing mice in Group 1.

[0178] Figure 23D shows the individual tumor volume results of WEHI-164 tumor-bearing mice in Group 2.

[0179] Figure 23E shows the individual tumor volume results of WEHI-164 tumor-bearing mice in Group 3.

[0180] Figure 23F shows the individual tumor volume results of WEHI-164 tumor-bearing mice in group 4.

[0181] Figure 23G shows actual photographs of tumors in all WEHI-164 tumor-bearing mice from groups 1 to 4.

[0182] * indicates a significance level (P < 0.05).

[0183] Figure 24A shows the statistical results of tumor volume in Pan02 tumor-bearing mice.

[0184] Figure 24B shows the statistical results of body weight in Pan02 tumor-bearing mice.

[0185] Figure 24C shows the individual tumor volume results of Pan02 tumor-bearing mice in the single CTLA-4 antibody administration group.

[0186] Figure 24D shows the individual tumor volume of Pan02 tumor-bearing mice in the group that received intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody.

[0187] Figure 24E shows actual photographs of tumors in Pan02 tumor-bearing mice in the single CTLA-4 antibody administration group and the group receiving attenuated Salmonella SWT5005 intravenous combined with CTLA-4 antibody administration group.

[0188] Where ** represents a significance level P<0.01.

[0189] Figure 25A shows the statistical results of tumor volume in Hepa1-6 tumor-bearing mice;

[0190] Figure 25B shows the statistical results of body weight in Hepa1-6 tumor-bearing mice.

[0191] Figure 25C shows the individual tumor volume results of Hepa1-6 tumor-bearing mice in the single CTLA-4 antibody administration group;

[0192] Figure 25D shows the tumor volume of individual tumor-bearing mice in the group treated with intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody. Detailed Implementation

[0193] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0194] Example 1: Construction of attenuated Salmonella strains SWT5001, SWT5005, SWT6009, and SWT6013

[0195] Attenuated Salmonella strains SWT5001, SWT5005, SWT6009, and SWT6013 were constructed according to the description in patent application 202311480978.8. All details described in that patent application are incorporated herein by reference. The specific process for constructing the attenuated Salmonella strains is as follows.

[0196] I. Construction of a hypoxia-specific gene expression cassette with alar and dadX genes as essential survival genes

[0197] The positive hypoxia promoter used Salmonella yhbU (yhbU-S) (SEQ ID No. 1) and the essential gene used Salmonella alar gene (SEQ ID No. 2). The negative hyperxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 3) and Salmonella ydcI (ydcI-S) (SEQ ID No. 4). The positive hypoxia promoter used Escherichia coli ynfK (ynfK-E) (SEQ ID No. 5) and the essential gene used Salmonella dadX gene (SEQ ID No. 6). The negative hyperxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 3) and Salmonella ydcI (ydcI-S) (SEQ ID No. 4). Hypoxia-specific gene expression cassettes were constructed.

[0198] The combinations of hypoxia-specific gene expression cassettes are shown in Table 1.

[0199] Table 1. Combinations of positive hypoxia promoters and negative hyperxia promoters.

[0200] (Among them, the essential genes for survival are the alar gene or dadX gene of Salmonella.)

[0201] (I) Construction of positive hypoxia promoter cloning library

[0202] 1. Single colonies of wild-type Salmonella Typhimurium (strain SWT001, purchased from CICC China Industrial Microbial Culture Collection Center) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) were picked and inoculated into 5 ml LB liquid medium and incubated at 37°C and 220 rpm for 16 hours. OD 600 The reading is between 2 and 3;

[0203] 2. Using a pipette tip, take 2 μl of the corresponding strain culture medium and mix it into the corresponding primer and high-fidelity PCR amplification enzyme (purchased from TAKARA), and then place the mixture into the PCR amplification device;

[0204] The amplification program was: 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0205] 3. The amplified PCR products are purified and recovered using a DNA gel recovery system;

[0206] 4. Mix the recovered product with the restriction enzyme digestion system (purchased from NEB) and incubate at 37°C for 1 hour;

[0207] 5. The enzyme digestion products are purified and recovered using a DNA gel recovery system.

[0208] The primers and endonucleases used for different positive hypoxia promoters are as follows:

[0209] 1. yhbU-S (SEQ ID No. 1): Strain SWT001 was amplified using primers SWTO1 and 2, and the product was double-digested with NotI and HindIII and then recovered.

[0210] 2. ynfK-E (SEQ ID No. 5): Escherichia coli strain DH10B was amplified using primers SWTO3 and 4, and the product was double-digested with NotI and HindIII and then recovered.

[0211] The results of validating the positive hypoxia promoter clone library by agarose gel electrophoresis are shown in Figure 1.

[0212] (II) Construction of a survival-essential gene library

[0213] 1. Select a single clone of wild-type Salmonella Typhimurium (strain SWT001, purchased from CICC China Industrial Microbial Culture Collection Center) and inoculate it into 5 ml LB liquid medium, and incubate it in a constant temperature shaker at 37℃ and 220 rpm for 16 hours.

[0214] 2. Using a pipette tip, aspirate 2 μl of the corresponding strain culture medium and mix it into the corresponding primer and high-fidelity PCR amplification enzyme system, then place the mixture into the PCR amplification device;

[0215] The amplification program was: 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0216] 3. The amplified PCR products were purified and recovered using a DNA gel recovery system;

[0217] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees Celsius for 1 hour;

[0218] 5. The enzyme digestion products are purified and recovered using a DNA gel recovery system.

[0219] The essential genes for survival from different sources are as follows:

[0220] 1. Salmonella alar gene (SEQ ID No. 2): The strain SWT001 was amplified using primers SWOT5 and 6, and the product was double-digested with HindIII and XhoI and then recovered.

[0221] 2. Salmonella dadX gene (SEQ ID No. 6): The strain SWT001 was amplified using primers SWOT7 and 8, and the product was double-digested with HindIII and XhoI and then recovered.

[0222] The results of validating the survival-essential gene library by agarose gel electrophoresis are shown in Figure 2.

[0223] (III) Construction of the reverse hyperoxia promoter library

[0224] 1. Select a single clone of wild-type Salmonella Typhimurium (strain SWT001, purchased from CICC China Industrial Microbial Culture Collection Center) and inoculate it into 5 ml LB liquid medium, and place it in a constant temperature shaker at 37℃ and 220 rpm for 16 hours.

[0225] 2. Using a pipette tip, aspirate 2 μl of the corresponding strain culture medium and mix it into the corresponding primer and high-fidelity PCR amplification enzyme system, then place the mixture into the PCR amplification device;

[0226] The amplification program was: 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0227] 3. The amplified PCR products were purified and recovered using a DNA gel recovery system;

[0228] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0229] 5. The enzyme digestion products are purified and recovered using a DNA gel recovery system.

[0230] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0231] 1. cyoA-S (SEQ ID No. 3): Strain SWT001 was amplified using primers SWOT9 and 10, and the product was double-digested with XhoI and PstI and then recovered.

[0232] 2. ydcI-S (SEQ ID No. 4): Strain SWT001 was amplified using primers SWTO11 and 12, and the product was double-digested with XhoI and PstI and then recovered.

[0233] The results of the verification of the reverse hyperoxia promoter clone library by agarose gel electrophoresis are shown in Figure 3.

[0234] (iv) Construction of a combined library of positive hypoxia promoters, essential genes for survival, and negative hyperxia promoters

[0235] The hypoxia-specific gene expression cassette is composed of a positive hypoxia promoter, essential genes for survival, and a negative hyperxia promoter in sequence, as shown in Figure 4.

[0236] As shown in Table 1, a cloned library was formed by combining sequences of a forward hypoxia promoter library digested with NotI and HindIII, a Salmonella alar gene (SEQ ID No. 2) or a Salmonella dadX gene (SEQ ID No. 6) digested with HindIII and XhoI, and a reverse hyperxia promoter library digested with XhoI and PstI. This library was then ligated into the pSWT003 vector (vector backbone, pBlueScript SK(+), purchased from BioWind) digested with SpeI and PstI, and the pSWT007 vector (containing bilateral unidirectional loxp sequences and a DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 7)) digested with SpeI and NotI.

[0237] The products obtained by amplifying Salmonella SWT001 or Escherichia coli DH10B with primers and recovering them by enzyme digestion were combined and ligated by enzyme ligation reaction, transformed into DH10B bacteria, and plated on LB plates containing 25 μg / ml chloramphenicol to obtain the corresponding plasmids as shown in Table 1.

[0238] (V) Construction of the corresponding strain of the present invention

[0239] 1. Wild-type Salmonella Typhimurium (SWT001)

[0240] Wild-type Salmonella Typhimurium (SWT001) was purchased from CICC China Industrial Microbial Culture Collection Center.

[0241] 2. Construction of Salmonella (SWT002) containing temperature-induced lambda-RED recombinase and loxp-CRE enzyme system.

[0242] The Lambda-RED recombination system is widely used for homologous recombination in Gram-negative bacteria. In this invention, this system consists of plasmid pSWT001.

[0243] As shown in Figure 5, plasmid pSWT001 contains a lambda-RED recombinase module (SEQ ID No. 8) (functionally similar to the plasmid vector psim6 from BioWind) and a loxp-Cre recombinase module (SEQ ID No. 8) (functionally similar to the plasmid vector 705-Cre from Gene Bridges). The lambda-RED recombinase module consists of three recombinases: EXO, BET, and GAM, and the recombinases are controlled by the CI857 temperature regulator. Therefore, expression does not occur at 32°C and only occurs at temperatures above 37°C. Under temperature-induced conditions, the recombinase EXO cleaves the 5' end of double-stranded linear DNA, creating a 3' protruding single-stranded DNA. This single-stranded DNA is bound to the BET protein and protected from degradation by other nucleases. GAM inhibits the activity of endogenous bacterial nucleases. The homologous arms of homologous recombination, approximately 35-50 bp, are added to both sides of the DNA fragment requiring recombination via PCR. The advantage of this technique is that it precisely targets the desired region of the bacterial chromosome without causing additional mutations. The outermost flanking layers of the recombinant double-stranded DNA fragment include homologous arm sequences at the desired recombination site, followed by two unidirectional loxp sequences totaling 34 bp. The unidirectional double loxp sites contain the chloramphenicol resistance gene, used for screening recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature regulator) specifically recognizes the loxp sequence and cleaves the middle sequence of the unidirectional loxp, leaving one loxp sequence, thus eliminating the chloramphenicol resistance gene. Repeating this process allows for continuous gene knockout and knock-in.

[0244] The specific operating steps are as follows:

[0245] (1) Streak strain SWT001 on LB plates and incubate statically overnight in a 37°C incubator;

[0246] (2) Pick a single clone and inoculate it into 5 ml of LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0247] (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 =0.3 Place on ice and let stand for 1 hour;

[0248] (4) Wash the bacteria three times with sterilized purified water;

[0249] (5) The recovered bacterial cells were mixed with 10 ng of plasmid pSWT001 and electroporated using a voltage of 1.8 kV.

[0250] (6) The electroporated bacterial cells were spread on LB plates containing 100 μg / ml ampicillin sodium and incubated overnight in a constant temperature incubator at 32°C until a single clone colony grew, which was named SWT002.

[0251] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0252] (1) Pick single clones of SWT002 and inoculate them into 5 ml of LB liquid medium containing 100 μg / ml ampicillin sodium, and place them in a constant temperature shaker at 32℃ and 220 rpm for 16 hours.

[0253] (2) Inoculate the culture at a ratio of 1:100 into fresh LB liquid medium containing 100 μg / ml ampicillin sodium, and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 When the α=0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0254] (3) Wash the bacteria three times with sterilized purified water.

[0255] (4) Prepare PCR products of SWTO13 and SWTO14.

[0256] Primers SWTO13 and SWTO14 were mixed with plasmid pSWT002 (containing bilaterally aligned loxp sequences with the chloramphenicol resistance gene SEQ ID No. 9 in the middle) and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0257] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0258] The amplified PCR products were verified by agarose gel electrophoresis to identify the PCR amplification products of the aroA gene knockout fragment SWTO13 and SWTO14. The verification results are shown in Figure 6A.

[0259] The DNA was purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR product were determined by nanodrop.

[0260] (5) The recovered bacterial cells were mixed with 100 ng of PCR products of SWTO13 and SWTO14 and electroporated at 1.8 kV.

[0261] (6) Spread the electroporated bacterial cells on a plate containing 25 μg / ml chloramphenicol and incubate overnight in a constant temperature incubator at 32°C until a single clone colony grows.

[0262] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified by SWTO15 and SWTO16, SWTO17 and SWTO18.

[0263] The PCR amplification product of the target fragment of strain SWT003 was verified by agarose gel electrophoresis. The verification results are shown in Figure 6B.

[0264] (8) Inoculate positive monoclonal antibodies into 5 ml of LB medium and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0265] 4. Construction of attenuated bacteria with alar gene knockout (SWT004)

[0266] (1) Pick a single clone of SWT003 and inoculate it into 5ml LB liquid medium and place it in a constant temperature shaker at 32℃ and 220rpm for 16 hours.

[0267] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 When the α=0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0268] (3) Wash the bacteria three times with sterilized purified water.

[0269] (4) Prepare PCR products of SWTO19 and SWTO20.

[0270] Primers SWTO19 and SWTO20 were mixed with plasmid pSWT002 and high-fidelity PCR amplification enzyme system and placed into the PCR amplification device.

[0271] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0272] The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were then determined using nanodrop.

[0273] (5) The recovered bacterial cells were mixed with 100 ng of PCR products of SWTO19 and SWTO20 and electroporated at 1.8 kV.

[0274] The PCR amplification products of the alar gene knockout fragments SWTO19 and SWTO20 were verified by agarose gel electrophoresis. The verification results are shown in Figure 7A.

[0275] (6) Spread the electroporated bacterial cells on a plate containing 25 μg / ml chloramphenicol and incubate overnight in a constant temperature incubator at 32°C until a single clone colony grows.

[0276] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified by using SWTO21 and SWTO18, SWTO16 and SWTO22.

[0277] The PCR amplification product of the target fragment of strain SWT004 was verified by agarose gel electrophoresis. The verification results are shown in Figure 7B (the primers used were SWTO21, SWTO18, SWTO16, and SWTO22).

[0278] (8) Inoculate positive monoclonal antibodies into 5 ml of LB medium and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0279] 2. Integrating a hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of strain SWT004 (disrupting its function).

[0280] Construction of ALR-deficient attenuated bacteria containing hypoxia-specific gene expression cassettes

[0281] 1. Select a single clone of the attenuated bacteria SWT004 with the ar gene knocked out and inoculate it into 5 ml of fresh LB liquid medium. Incubate in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0282] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100, and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 When the α=0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0283] 3. Wash the bacteria three times with sterilized purified water.

[0284] 4. Prepare PCR products for constructing strains containing hypoxia-specific expression cassettes. Mix primers SWTO23 and SWTO24 with hypoxia-specific gene expression cassette plasmids pOL1001 and pOL1005, and a high-fidelity PCR amplification enzyme system and place them into a PCR amplification device.

[0285] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 120 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0286] The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were then determined using nanodrop.

[0287] 5. Mix the recovered bacterial cells with 100 ng of PCR product and electroporate using a voltage of 1.8 kV.

[0288] 6. Spread the electroporated bacterial cells onto plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubate overnight at 32°C until single clonal colonies grow. The resulting strains are SWT1001 and SWT1005, respectively.

[0289] 7. Inoculate the single clone into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and culture in a constant temperature shaker at 37℃ and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0290] 8. Using primers SWOT25,2 and SWOT26,2, the clones were identified by colony PCR. The identification results of strain SWT1001 (yhbU-S+alr+cyoA-S combination) and strain SWT1005 (yhbU-S+alr+ydcI-S combination) are shown in Figure 8.

[0291] III. Construction of a hypoxia-specific gene expression cassette with dadX gene as an essential survival gene

[0292] As alanine racemic enzyme, bacteria such as Salmonella and Escherichia coli have two isoenzyme genes in their genomes: the alar gene and the dadX gene. To verify the effect, the essential genes for survival of some hypoxia-specific gene expression cassettes mentioned above were adapted using the corresponding homologous genes dadX, pOL2009 and pOL2013.

[0293] Hypoxia-specific gene expression cassette was integrated into the alar gene locus on the chromosome of strain SWT007.

[0294] 1. Construction of attenuated bacteria with dadX gene knockout (SWT007)

[0295] (1) Pick a single clone of SWT003 and inoculate it into 5ml LB liquid medium and place it in a constant temperature shaker at 32℃ and 220rpm for 16 hours.

[0296] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 When the α=0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0297] (3) Wash the bacteria three times with sterilized purified water.

[0298] (4) Prepare PCR products of SWTO23 and SWTO24.

[0299] Primers SWTO23 and SWTO24 were mixed with plasmid pSWT002 (as described above) and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0300] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0301] The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were then determined using nanodrop.

[0302] (5) The recovered bacterial cells were mixed with 100 ng of PCR products of SWTO23 and SWTO24 and electroporated at 1.8 kV.

[0303] (6) Spread the electroporated bacterial cells on a plate containing 25 μg / ml chloramphenicol and incubate overnight in a constant temperature incubator at 32°C until a single clone colony grows.

[0304] (7) Positive clones were identified by colony PCR. SWOT27, SWOT18, SWOT16, and SWOT28 were used to identify the insertion of the chloramphenicol resistance gene, as shown in Figure 9.

[0305] (8) Inoculate positive monoclonal antibodies into 5 ml of LB medium and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0306] 2. Construction of a defective attenuated bacterium containing a hypoxia-specific gene expression cassette and whose essential survival gene is the Salmonella dadX gene.

[0307] (1) Select a single clone of the attenuated bacteria SWT007 with the dadX gene knocked out and inoculate it into 5 ml of LB liquid medium and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0308] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 When the α=0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0309] (3) Wash the bacteria three times with sterilized purified water.

[0310] (4) Prepare PCR products for constructing strains containing hypoxia-specific expression cassettes. Mix primers SWTO29 and SWTO30 with hypoxia-specific gene expression cassette plasmids pOL2009 and pOL2013 and a high-fidelity PCR amplification enzyme system and place them into a PCR amplification device.

[0311] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 120 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0312] The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were then determined using nanodrop.

[0313] (5) The recovered bacterial cells were mixed with 100 ng of PCR product and electroporated at 1.8 kV.

[0314] (6) Spread the electroporated bacterial cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubate overnight in a constant temperature incubator at 32°C until a single clone colony grows.

[0315] (7) Inoculate positive monoclonal antibodies into 5 ml of LB medium and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0316] (8) Using the corresponding primers SWTO31, 4; SWTO32, 4, the clones were identified by colony PCR. The identification results of strain SWT2013 (ynfK-E+dadX+ydcI-S) and strain SWT2009 (ynfK-E+dadX+cyoA-S) are shown in Figure 10.

[0317] IV. Validation of Oxygen Adaptability of Salmonella Strains SWT1001, SWT1005, SWT2009, and SWT2013 Containing Hypoxia-Specific Gene Expression Cascades

[0318] 1. Select single clones of strains SWT1001, SWT1005, SWT2009, and SWT2013 containing hypoxia-specific expression cassettes and inoculate them into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine. Incubate at 37°C and 200 rpm for 16 hours in a constant temperature shaker.

[0319] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600 nm.

[0320] 3. Calculate the bacterial culture volume of 1OD bacteria according to the following formula, and add deionized water to 1ml.

[0321] 4. Take 10 μl of the above bacterial culture and spot it onto two LB plates that do not contain D-alanine. Label them as “1” and repeat three times, labeling them as “a”, “b” and “c”.

[0322] 5. After serial dilution by 10 times, take another 10 μl of the diluted solution and spot it onto the above culture medium, labeling it "2".

[0323] 6. Continue this 10-fold dilution gradient until you reach the mark "8".

[0324] 7. One plate was incubated in an anaerobic environment at 37°C, and the other plate was incubated in an atmospheric environment at 37°C (21% oxygen concentration).

[0325] The results of the oxygen adaptability verification test of strains containing hypoxia-specific expression cassettes are shown in Figures 11-13. The results demonstrate that Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013, which contain the aforementioned hypoxia-specific gene expression cassettes, exhibit hypoxia regulation capabilities.

[0326] V. Simulation of oxygen concentration in strain libraries containing hypoxia-specific gene expression cassettes

[0327] By using anaerobic gas-generating bags to consume oxygen in a sealed culture vessel, and using an oxygen analyzer to measure the oxygen concentration in the sealed culture vessel, the oxygen concentration in the sealed culture vessel can be stabilized within a specific range after using the anaerobic gas-generating bags for a certain period of time. The specific method is as follows:

[0328] The anaerobic gas-generating bag (brand: Mitsubishi Japan, item number: D-119) was placed into a 7.0L sealed culture tank (brand: Mitsubishi Japan, item number: D-112), and an oxygen analyzer (brand: Meicheng Electrochemical, item number: OX-100A) was also placed inside.

[0329] The above-mentioned anaerobic gas-generating bag combination was used to verify the growth of the genetically modified strain at oxygen concentrations below 0.8%.

[0330] Adaptability verification of strain libraries containing hypoxia-specific gene expression cassettes at oxygen concentrations below 0.8%.

[0331] Since the pathological hypoxic zone of a tumor is characterized by an oxygen concentration of less than 1%, this invention aims to demonstrate that Salmonella modified with a hypoxia-specific gene expression cassette can still grow normally when the oxygen concentration is less than 1% (or close to 1%).

[0332] Oxygen concentration was regulated by an anaerobic gas-generating bag. After one hour of consumption by the anaerobic gas-generating bag, the oxygen meter reading showed an oxygen concentration of 0.8-1%. Therefore, this experiment simulated the hypoxic environment inside a tumor to verify the growth of Salmonella on a culture dish after one hour of consumption by the anaerobic gas-generating bag in a sealed culture vessel, and to simulate the growth of Salmonella strains containing hypoxia-specific gene expression cassettes in the hypoxic environment inside a tumor.

[0333] The procedure for the spot plate experiment is as follows:

[0334] (1) Select a single clone of the strain containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0335] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0336] (3) Calculate the bacterial culture volume of 1OD bacteria according to the following formula, and add deionized water to 1ml.

[0337] (4) Take 10 μl of the above bacterial culture and spot it onto an LB plate containing D-alanine for culture. Mark it as “1” and repeat three times, marking it as “a”, “b” and “c”.

[0338] (5) At the same time, take 10 μl of the above bacterial solution and spot it onto another LB plate that does not contain D-alanine for culture. Mark it as “1” and spot three replicates, labeled as “a”, “b” and “c”.

[0339] (6) After dilution by 10 times, take 10 μl of the diluted solution and spot it onto the above culture medium, and mark it as "2".

[0340] (7) Continue this 10-fold dilution gradient until you reach the mark "8".

[0341] (8) Place the two plates in a 37°C environment with 0.8% oxygen for incubation.

[0342] As shown in Figure 14, this invention compares the growth of different strains (Figure 14(A) SWT1001, Figure 14(B) STW1005) on LB agar plates without added D-alanine to determine the growth status of each strain at oxygen concentrations below 0.8%. Because the medium without added D-alanine requires the corresponding Salmonella strains to rely on their own oxygen regulation system for normal growth, the results confirm that strains containing hypoxia-specific expression cassettes that can grow normally in anaerobic environments can also grow normally at oxygen concentrations below 0.8%.

[0343] VI. Construction of Salmonella strains with homologous substitution of the asd gene locus for T7 RNA polymerase (SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P)

[0344] (I) Construction of constitutive expression plasmid pPRO005 for lacUV5-controlled T7 RNA polymerase

[0345] To construct a constitutively expressed T7 RNA polymerase, the vector pPRO005 was constructed. The process is shown in Figure 15. The constitutive expression promoter lacUV5 was placed upstream of the RBS sequence (SEQ ID No. 10) and the T7 RNA polymerase (SEQ ID No. 11). Furthermore, the chloramphenicol resistance gene cm (SEQ ID No. 7), flanked by loxp sequences, was added upstream of lacUV5. This was to facilitate recombination into the Salmonella chromosome for subsequent screening.

[0346] 1. The chloramphenicol resistance gene cm, consisting of approximately 1100 bp fragment containing flanking loxp sequences, was recovered by digesting plasmid pSWT007 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) with SpeI and NotI enzymes.

[0347] 2. NotI and HindIII nicks were generated by direct annealing with primers SWTO33 and SWTO34, and the recovered fragments were approximately 70 bp, including the promoter lacUV5.

[0348] 3. The plasmid pPRO004 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with HindIII and XhoI enzymes, and a fragment of approximately 2690 bp, including the T7 RNA polymerase gene, was recovered.

[0349] 4. The plasmid pSWT003 (plasmid backbone) was digested with SpeI and XhoI enzymes, and a fragment of about 3000 bp was recovered.

[0350] 5. The above 5 fragments were ligated using T4 DNA ligase, transformed into DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), plated on LB plates with double resistance to ampicillin and chloramphenicol, and single clones were screened and named pPRO005.

[0351] 6. Extract plasmid pPRO005 and mix 100 ng with NotI and XhoI restriction enzyme system, incubate at 37℃ for 1 h, and identify by agarose gel electrophoresis, as shown in Figure 16.

[0352] (ii) Integration of the recombinant lacUV5-controlled T7 RNA polymerase constitutive expression cassette into the Salmonella chromosome

[0353] 1. Pick single clones of SWT1001, SWT1005, SWT2009 and SWT2013 respectively and inoculate them into 5ml LB liquid medium containing 100μg / ml D-alanine, and place them in a constant temperature shaker at 32℃ and 220rpm for 16 hours.

[0354] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD. 600 =0.3, place the culture flask in a 42℃ water bath, shake and incubate for 15 minutes, then place it on ice and let it stand for 1 hour.

[0355] 3. Wash the bacteria three times with sterilized purified water.

[0356] 4. Prepare PCR products of SWOT35 and SWOT36.

[0357] Primers SWTO35 and SWTO36 were mixed with plasmid pPRO005 and high-fidelity PCR amplification enzyme system and placed into the PCR amplification device.

[0358] The amplification program was 95℃ for 2 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 60 seconds, repeated 30 times; 72℃ for 10 minutes; 4℃ for 5 minutes.

[0359] The amplified PCR products were verified by agarose gel electrophoresis. The PCR amplification products of SWOT35 and SWOT36 were verified. The verification results are shown in Figure 17A.

[0360] The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were then determined using nanodrop.

[0361] 5. The recovered bacterial cells were mixed with 100 ng of PCR products of SWOT35 and SWOT36 and electroporated at 1.8 kV.

[0362] 6. Spread the electroporated bacterial cells onto plates containing 25 μg / ml chloramphenicol, 100 μg / ml D-alanine, and 100 μg / ml DAP (diaminopimelic acid), and incubate overnight at 32°C until a single colony grows.

[0363] 7. Inoculate the single clone into 5 ml of LB medium containing 100 μg / ml DAP and 100 μg / ml D-alanine, and culture in a constant temperature shaker at 37℃ and 220 rpm for 16 hours. The chloramphenicol resistance gene is eliminated by the action of CRE enzyme.

[0364] 8. Positive clones were identified by colony PCR using SWTO37, SWTO38, SWTO39, and SWTO40.

[0365] The PCR amplification products of the target fragment of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P were verified by agarose gel electrophoresis. The verification results are shown in Figure 17B.

[0366] VII. Construct strains (SWT5001, SWT5005, SWT6009, SWT6013) carrying the three-in-one expression vector pPRO010.

[0367] In this embodiment, a plasmid vector without antibiotic resistance selection was constructed, as shown in Figure 18. The pUC replicon (SEQ ID No. 12) and the Salmonella asd gene (SEQ ID No. 13) were used as balanced lethal control mechanisms. Furthermore, a T7 promoter containing biphasic phages was used to control the expression of the drug protein exotoxin DTA fragment (SEQ ID No. 14) and the membrane perforation protein gene LLO (SEQ ID No. 15), respectively. The constructed plasmid pPRO010 was transformed into SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, respectively, generating the corresponding strains SWT5001, SWT5005, SWT6009, and SWT6013.

[0368] The specific operating steps are as follows:

[0369] 1. Plasmid pPRO006 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with XhoI and NotI enzymes, and a fragment of approximately 820 bp containing the pUC replicon was recovered;

[0370] 2. The plasmid pPRO007 (synthesized by Beijing Liuhe Huada Genomics Co., Ltd.) was digested with PstI and XhoI enzymes, and a fragment of about 1270 bp containing the Salmonella asd gene was recovered.

[0371] 3. The plasmid pPRO008 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with PstI enzyme, and the approximately 660bp fragment containing the Salmonella codon-optimized gene DTA controlled by the T7 promoter was recovered.

[0372] 4. The plasmid pPRO009 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NotI and PstI enzymes, and the approximately 1660bp fragment containing the Salmonella codon-optimized gene LLO controlled by the T7 promoter was recovered.

[0373] 5. The above four fragments were ligated using T4 DNA ligase, transformed into competent cells of SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, plated on 100 μg / ml D-alanine LB plates, and single clones were selected and named SWT5001, SWT5005, SWT6009, and SWT6013.

[0374] 6. Select individual clones and amplify them using primers SWTO41 and SWTO42; SWTO43 and SWTO44; SWTO45 and SWTO46; SWTO47 and SWTO48; SWTO49 and SWTO50; SWTO51 and SWTO52; SWTO53 and SWTO54; SWTO55 and SWTO56; SWTO57 and SWTO58; identify them by agarose gel electrophoresis, as shown in Figure 19.

[0375] Information on the corresponding strains of this invention is shown in Table 2.

[0376] Table 2. Construction table of strains of the present invention

[0377] Information on the corresponding tool plasmids of this invention is shown in Table 3.

[0378] Table 3. Information on the tool plasmids used in this invention.

[0379] The sequences of promoters, gene coding regions, proteins, etc. used in this invention are shown in Table 4.

[0380] Table 4. Specific sequences of promoters, gene coding regions, proteins, etc., used in this invention.

[0381] The specific sequences of all primers used in this invention are shown in Table 5.

[0382] Table 5. Specific sequences of all primers used in this invention.

[0383] Example 2: Application of intravenous administration of attenuated Salmonella SWT5005 combined with PD-1 antibody in colorectal cancer

[0384] (I) Establishment of a mouse subcutaneous xenograft model of colon cancer CT26

[0385] Female BALB / c mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0386] The specific animal experiment protocol is as follows:

[0387] Mice of similar weight and activity were selected for the experiment, with an expected weight range of 18-20g at the time of cell inoculation. CT26 colon cancer cells (purchased from Gaining Biotechnology, CM-M109) from mice in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 5 × 10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse at a dose of 100 cells / ml.

[0388] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into eight groups based on tumor size and body weight.

[0389] 1. PBS control group (represented as PBS in the figure);

[0390] 2. Low dose (3.75 × 10⁻⁶) of single-attenuated Salmonella SWT5005 6 The intravenous administration group (CFU / animal) is represented in the figure as SWT5005 (3.75 × 10⁻⁶) 6 cfu,iv));

[0391] 3. High dose (3.75 × 10⁻⁶) of single-attenuated Salmonella SWT5005 7 The intravenous administration group (CFU / animal) is represented in the figure as SWT5005 (3.75 × 10⁻⁶) 7 cfu,iv));

[0392] 4. Single PD-1 antibody administration group (represented in the figure as PD-1 (200 μg));

[0393] 5. Low-dose intravenous administration of attenuated Salmonella SWT5005 combined with PD-1 antibody (shown in the figure as SWT5005 (3.75×10) 6 cfu, IV) + PD-1 (200 μg);

[0394] 6. High-dose intravenous administration of attenuated Salmonella SWT5005 combined with PD-1 antibody (shown in the figure as SWT5005 (3.75×107cfu, iv) + PD-1 (200μg));

[0395] 7. Low-dose intravenous administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody (represented in the figure as Pre-PD-1, SWT5005 3.75×10⁻⁶) 6 cfu, IV) + PD-1 (200 μg);

[0396] 8. High-dose intravenous administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody (represented in the figure as Pre-PD-1, SWT5005 3.75×10⁻⁶) 7 cfu, iv) + PD-1 (200 μg)).

[0397] Two groups of mice that had been pretreated with PD-1 were given intraperitoneal administration of PD-1 antibody (200 μg / mouse) on the day of grouping (Day-2), and then administered the drug to mice in different experimental groups according to Table 6.

[0398] Table 6. Grouping and Dosing Regimens of CT26 Tumor-Bearing Mice

[0399] The PD-1 antibody (InVivoPlus anti-mouse PD-1(CD279), Bio X Cell, BP0146) was purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0400] (II) Pharmacodynamic Evaluation

[0401] During the experiment, animals were observed every two days, including but not limited to the ulceration of tumor nodules, their mental state, and their diet. All abnormal appearances and behaviors were recorded.

[0402] 1. Evaluation Indicators

[0403] (1) Tumor volume

[0404] For mouse tumor volume, after grouping and until the end of the experiment, the tumor volume was measured 3-4 times per week using vernier calipers. The major and minor diameters of the tumor were measured, and the volume was calculated using the formula: Tumor volume = Major diameter × Minor diameter. 2 ×0.52.

[0405] (2)Weight

[0406] Regarding the weight of the mice, after grouping, the weight of the mice was measured and recorded 3-4 times per week until the end of the experiment.

[0407] During the experiment, drug administration was stopped when the mouse's body weight decreased by ≥15%, and the withdrawal period should be long enough for the mouse to recover its weight. Only one mouse was discontinued, while the rest of the mice were given normal medication. The experiment continued when the mouse's body weight recovered to the standard of ≤10% at the time of drug withdrawal.

[0408] (3) Actual photographs of the tumor

[0409] During the experiment, the tumor volume was measured and the mouse tumor was photographed simultaneously to ensure that the photos corresponded to the volume data.

[0410] (4) Immunohistochemical staining

[0411] When the tumor volume of a single animal exceeds 3000 mm 3 At the designated time, the experiment was terminated, and the animals were euthanized. Mouse tumor tissue was fixed with 4% paraformaldehyde fixative, then embedded in paraffin, sectioned, and immunohistochemically stained to analyze the immune microenvironment of the mouse tumor tissue.

[0412] (5) Tumor growth inhibition rate TGI

[0413] Tumor growth curves were plotted based on different days in mice and their corresponding tumor sizes. The tumor growth inhibition rate (TGI) was calculated based on the initial and final tumor sizes.

[0414] TGI% = (1 - (Tt - T0) / (Ct - C0)) × 100%. Where Tt and Ct are the average tumor volumes of the experimental group and the control group at a specific time point, respectively, and T0 and C0 are the average tumor volumes of the experimental group and the control group on day 0, respectively.

[0415] 2. Evaluation Results

[0416] The results of the calculation of tumor growth inhibition rate (TGI) are shown in Table 7.

[0417] Table 7 Tumor Growth Inhibition Rate (TGI)

[0418] As can be seen from Figures 20A, 20B, 20D, 20E and Table 7:

[0419] (1) The tumors in the PBS control group mice grew rapidly. The tumor growth inhibition rate (TGI) of the mice treated with PD-1 alone was 35.98%, which was not significantly different from that of the control group.

[0420] (2) Single intravenous administration of attenuated Salmonella SWT5005 to mice in the low-dose and high-dose groups resulted in good tumor control, with TGI reaching 66.64% and 86.69%, respectively.

[0421] (3) When attenuated Salmonella SWT5005 was used in combination with PD-1 antibody, the TGI of the low-dose group and the high-dose group increased to 74.85% and 100.08%, respectively.

[0422] (4) After intravenous administration of PD-1 antibody to pre-treated attenuated Salmonella SWT5005, the TGI of the low-dose group and the high-dose group reached 83.23% and 108.40%, respectively, and the average tumor volume of mice in the high-dose group had decreased to about 150 mm. 3 The tumors in mice were strongly suppressed.

[0423] Figure 20C shows that statistical analysis of the body weight of all mice in groups 1 to 8 indicates that mice in the single intravenous administration of SWT5005 and the combined administration group experienced a transient decrease in body weight, which then gradually recovered.

[0424] Figures 20F to 20M show that, through individual analysis of each individual mouse in each group, the results indicate that the combined administration of pre-PD-1 treated attenuated Salmonella SWT5005 intravenously with PD-1 antibody, especially at high doses of SWT5005, significantly inhibited mouse tumors compared to the untreated group. Tumor volume remained relatively stable during treatment without significant increase.

[0425] As shown in Figures 20N to 20R, the immunohistochemical results indicate that no significant CD8 activity was observed in the tumors of mice treated with PD-1 antibody alone. + An increase in the number of T cells was observed, but macrophage aggregation was higher and primarily located at the tumor periphery. High dose (3.75 × 10⁻⁶) of single-attenuated Salmonella SWT5005 was administered. 7 In the combined intravenous administration groups (CFU / mouse), the high-dose intravenous administration group of attenuated Salmonella SWT5005 combined with PD-1 antibody, and the pre-PD-1 treated group of attenuated Salmonella SWT5005 combined with PD-1 antibody, a large number of Salmonella bacteria were detected at the tumor site, accompanied by a significant increase in neutrophils. Furthermore, mice in the combined administration group receiving PD-1 pretreatment showed significantly higher levels of CD8+ within the tumor compared to mice in the untreated group. +More pronounced aggregation of cytotoxic T cells and macrophages enhances the anti-tumor effect.

[0426] The above results indicate that intravenous administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody significantly enhanced the inhibitory effect of PD-1 antibody alone or untreated attenuated Salmonella SWT5005 combined with PD-1 antibody on CT26 subcutaneous colon cancer xenograft tumors in mice. + T cells and macrophages can infiltrate deeper into the tumor microenvironment, thereby better stimulating the body's innate and adaptive immune responses and thus exerting a better anti-tumor effect.

[0427] Example 3: Intratumoral administration of attenuated Salmonella SWT5005 and its combined use with PD-1 antibody in colorectal cancer

[0428] (I) Establishment of a mouse subcutaneous xenograft model of colon cancer CT26

[0429] Female BALB / c mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0430] The specific animal experiment protocol is as follows:

[0431] Mice of similar weight and activity were selected for the experiment, with an expected weight range of 18-20g at the time of cell inoculation. CT26 colon cancer cells (purchased from Gaining Biotechnology, CM-M109) from mice in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 5 × 10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse at a dose of 100 cells / ml.

[0432] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into eight groups based on tumor size and body weight.

[0433] 1. PBS control group (represented as PBS in the figure);

[0434] 2. Low dose (3.5 × 10⁻⁶) of single-attenuated Salmonella SWT5005 6 CFU / animal intratumoral administration group (shown in the figure as SWT5005 (3.5×10) 6 cfu, it));

[0435] 3. High dose (3.5 × 10⁻⁶) of single-attenuated Salmonella SWT5005 7 CFU / animal intratumoral administration group (shown in the figure as SWT5005 (3.5×10) 7cfu, it));

[0436] 4. Single PD-1 antibody administration group (represented in the figure as PD-1 (200 μg));

[0437] 5. Low-dose intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1 antibody (shown in the figure as SWT5005 (3.5×10) 6 cfu,it)+PD-1(200μg));

[0438] 6. High-dose intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1 antibody (shown in the figure as SWT5005 (3.5×10) 7 cfu,it)+PD-1(200μg));

[0439] 7. Low-dose intratumoral administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody (represented in the figure as Pre-PD-1, SWT5005 3.5×10⁻⁶) 6 cfu,it)+PD-1(200μg));

[0440] 8. Pre-PD-1 treated attenuated Salmonella SWT5005 high-dose intratumoral administration combined with PD-1 antibody administration group (represented in the figure as Pre-PD-1, SWT5005 (3.5×10) 7 cfu,it)+PD-1(200μg)).

[0441] Two groups of mice that had been pretreated with PD-1 were given intraperitoneal administration of PD-1 antibody (200 μg / mouse) on the day of grouping (Day-3), and then administered the drug to mice in different experimental groups according to Table 8.

[0442] Table 8 Grouping and Dosing Regimens of CT26 Tumor-Bearing Mice

[0443] The PD-1 antibody (InVivoPlus anti-mouse PD-1(CD279), Bio X Cell, BP0146) was purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0444] (II) Pharmacodynamic Evaluation

[0445] 1. Evaluation Indicators

[0446] The evaluation indicators were measured in the same manner as in Example 2.

[0447] 2. Evaluation Results

[0448] The results of the calculation of tumor growth inhibition rate (TGI) are shown in Table 9.

[0449] Table 9 Tumor Growth Inhibition Rate (TGI)

[0450] As can be seen from Figures 21A, 21B, 21D, 21E and Table 9:

[0451] (1) The tumors in the PBS control group mice grew rapidly, while the tumor growth inhibition rate (TGI) of the mice treated with PD-1 alone was 34.48%, which was not statistically different from that of the control group. This is consistent with the results of our Example 2.

[0452] (2) Intratumoral administration of attenuated Salmonella SWT5005 to mice in both low-dose and high-dose groups resulted in good tumor control, with TGI of 59.14% and 74.80%, respectively.

[0453] (3) After further combination with PD-1 antibody, the TGI of the low-dose and high-dose combination groups of SWT5005 were 68.40% and 76.83%, respectively;

[0454] (4) After intratumoral administration of PD-1 antibody-treated attenuated Salmonella SWT5005, the TGI of the low-dose and high-dose SWT5005 combination groups were 64.46% and 100.73%, respectively.

[0455] Figure 21C shows that statistical analysis of the body weight of all mice in groups 1 to 8 showed no significant difference in body weight among the groups after drug treatment.

[0456] Figures 21F to 21M, through individual analysis of each mouse in each group, show that the combination of pre-PD-1 treated attenuated Salmonella SWT5005 intratumoral administration with PD-1 antibody, especially at high doses of SWT5005, significantly inhibited tumor growth in mice compared to the untreated group. Tumor volume remained relatively stable during treatment without significant increase. Similarly, the overall efficacy was good in the pre-PD-1 treated attenuated Salmonella SWT5005 low-dose intratumoral administration with PD-1 antibody, with only one mouse out of five exhibiting a tumor volume of approximately 2000 mmHg. 3 The tumor volume of the remaining four mice was controlled at approximately 500 mm. 3 .

[0457] The above results indicate that intratumoral administration of pre-PD-1 treated attenuated Salmonella SWT5005 combined with PD-1 antibody significantly enhanced the inhibitory effect of PD-1 antibody alone or intratumoral administration of untreated attenuated Salmonella SWT5005 combined with PD-1 antibody on CT26 subcutaneous xenograft tumors in mice. The tumor volume was significantly smaller, and there was no significant effect on the body weight of the mice.

[0458] Example 4: Application of attenuated Salmonella SWT5005 intravenous / intratumoral administration and combined PD-1 / PD-L1 / CTLA-4 antibody in lung cancer

[0459] (I) Establishment of a mouse model of subcutaneous xenograft of LLC lung cancer

[0460] Female C57BL / 6J mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0461] The specific animal experiment protocol is as follows:

[0462] Mice of similar weight and activity were selected for the experiment, with an expected weight range of 18-20g at cell inoculation. Mouse lung cancer LLC cells (purchased from Enzyme Research Institute, CC-Y2052) in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 3×10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse at a dose of 100 cells / ml.

[0463] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into 11 groups based on tumor size and body weight.

[0464] 1. PBS control group (represented as PBS in the figure);

[0465] 2. Single PD-1 antibody administration group (represented in the figure as PD-1 (200 μg));

[0466] 3. Single PD-L1 antibody administration group (represented in the figure as PD-L1 (200μg));

[0467] 4. Intravenous administration of single-attenuated Salmonella SWT5005 (represented in the figure as SWT5005 (4.7×10) 6 cfu,iv));

[0468] 5. Attenuated Salmonella SWT5005 intravenous combined with PD-1 antibody administration group (represented in the figure as Pre-PD-1, SWT5005 (4.7×10) 6 cfu, IV) + PD-1 (200 μg);

[0469] 6. The group receiving intravenous administration of attenuated Salmonella SWT5005 combined with PD-L1 antibody (represented in the figure as Pre-PD-L1, SWT5005 (4.7×10⁻⁶)) 6 cfu, iv) + PD-L1 (200 μg);

[0470] 7. Intratumoral administration group of single attenuated Salmonella SWT5005 (shown in the figure as SWT5005 (4.7×10) 6 cfu, it));

[0471] 8. Intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1 antibody (represented in the figure as Pre-PD-1, SWT5005 (4.7×10) 6 cfu,it)+PD-1(200μg));

[0472] 9. Intratumoral administration of attenuated Salmonella SWT5005 combined with PD-L1 antibody (represented in the figure as Pre-PD-L1, SWT5005 (4.7×10⁻⁶)) 6 cfu,it)+PD-L1(200μg));

[0473] 10. Single CTLA-4 antibody administration group (represented in the figure as CTLA-4 (200 μg));

[0474] 11. Intratumoral administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody (represented in the figure as Pre-CTLA-4, SWT5005 (4.7×10) 6 cfu,it)+CTLA-4(200μg)).

[0475] All groups treated with attenuated Salmonella SWT5005 combined with immune checkpoint inhibitors underwent pretreatment with immune checkpoint inhibitors. Specifically, on Day 2, the immune checkpoint inhibitor was administered intraperitoneally (200 μg / animal) once every 3 days. Attenuated Salmonella SWT5005 was administered intravenously once a week, followed by intratumoral administration. Detailed protocols are shown in Table 10.

[0476] Table 10 Grouping and Dosing Regimens of LLC Tumor-Bearing Mice

[0477] PD-1 antibody (InVivoPlus anti-mouse PD-1(CD279), Bio X Cell, BP0146), PD-L1 antibody (InVivoPlus anti-mouse PD-L1(B7-H1), Bio X Cell, BP0101), and CTLA-4 antibody (InVivoMAb anti-mouse CTLA-4(CD152), Bio X Cell, BE0131) were purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0478] (II) Pharmacodynamic Evaluation

[0479] 1. Evaluation Indicators

[0480] The evaluation indicators were measured in the same manner as in Example 2.

[0481] 2. Evaluation Results

[0482] The results of the calculation of tumor growth inhibition rate (TGI) are shown in Table 11.

[0483] Table 11 Tumor Growth Inhibition Rate (TGI)

[0484] As can be seen from Figures 22A, 22B, 22D, 22E, 22F, 22S, 22T, 22U, 22V, 22W, 22H-22R and Table 11:

[0485] (1) Tumors grew rapidly in the PBS control group mice;

[0486] (2) In the monotherapy group, the PD-1 antibody monotherapy group had a certain inhibitory effect on tumors, with a TGI of 52.32%; no significant tumor inhibitory effect was observed in the PD-L1 antibody monotherapy group and the CTLA-4 antibody monotherapy group, with TGIs of 24.47% and 19.02%, respectively.

[0487] (3) The intratumoral administration group of single attenuated Salmonella SWT5005 showed a significant reduction in tumor volume compared with the PBS control group, with a TG1 of 65.97%; the intravenous administration group of single attenuated Salmonella SWT5005 achieved better antitumor effect, with a TG1 of 89.46%.

[0488] (4) In the treatment groups where attenuated Salmonella SWT5005 was pretreated with an immune checkpoint inhibitor and administered in combination with an immune checkpoint inhibitor, significant antitumor effects were observed in all combination therapy groups. Specifically, the tumor growth inhibition rates (TGIs) in mice treated with intravenous attenuated Salmonella SWT5005 in combination with PD-1 / PD-L1 antibodies were 104.89% and 101.69%, respectively, significantly higher than the 52.32% and 24.47% of the PD-1 antibody-only group and the PD-L1 antibody-only group, respectively. The tumor growth inhibition rates (TGIs) in mice treated with intratumoral attenuated Salmonella SWT5005 in combination with PD-1 / PD-L1 / CTLA-4 antibodies were 78.63%, 79.23%, and 98.79%, respectively, also significantly higher than the 52.32% and 24.47% of the PD-1 antibody-only group and the 19.02% of the CTLA-4 antibody-only group, respectively.

[0489] Figure 22C shows a statistical analysis of the body weight of mice in the single PD-1 antibody administration group, the single PD-L1 antibody administration group, the single attenuated Salmonella SWT5005 intravenous administration group, the attenuated Salmonella SWT5005 intravenous combined PD-1 antibody administration group, and the attenuated Salmonella SWT5005 intravenous combined PD-L1 antibody administration group. The results indicate that the body weight of mice in the single intravenous SWT5005 administration group and the combined administration group experienced a transient decrease, which then gradually recovered.

[0490] Figure 22G shows a statistical analysis of the body weights of mice in the following groups: single CTLA-4 antibody administration group, single intratumoral administration of attenuated Salmonella SWT5005, intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1 antibody, intratumoral administration of attenuated Salmonella SWT5005 combined with PD-L1 antibody, and intratumoral administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody. The results indicate that there was no significant difference in body weight among the groups after drug treatment.

[0491] The immunohistochemical results shown in Figures 22X to 22AA indicate that no significant CD8 activity was observed in the tumor when PD-1 antibody was used alone. + An increase in T cell count was observed, but macrophages were present at the tumor periphery. In the intravenous administration of attenuated Salmonella SWT5005 alone, and in the combination of pre-PD-1 treated attenuated Salmonella SWT5005 and PD-1 antibody, a large number of Salmonella bacteria were detected at the tumor site, accompanied by a significant increase in neutrophils. Furthermore, mice in the combined administration group receiving PD-1 pretreatment exhibited CD8+ expression within the tumor. + Significant aggregation of cytotoxic T cells and macrophages enhances antitumor effects by synergistically regulating immune cell infiltration and function in the tumor microenvironment (TME).

[0492] The above results indicate that pretreatment with immune checkpoint inhibitors (PD-1 / PD-L1 / CTLA-4 antibodies) followed by treatment with attenuated Salmonella SWT5005 in combination with immune checkpoint inhibitors (PD-1 / PD-L1 / CTLA-4 antibodies) significantly enhanced the inhibitory effect of PD-1, PD-L1, or CTLA-4 antibodies alone on subcutaneous xenograft tumors in LLC lung cancer mice. Compared with PD-1, PD-L1, or CTLA-4 antibodies alone, intravenous or intratumoral administration of attenuated Salmonella SWT5005 combined with PD-1, PD-L1, or CTLA-4 antibodies resulted in superior antitumor efficacy and significantly smaller tumor volume.

[0493] Example 5: Intratumoral administration of attenuated Salmonella SWT5005 and its combined use with PD-L1 antibody in fibrosarcoma

[0494] (I) Establishment of a mouse fibrosarcoma WEHI-164 subcutaneous xenograft model

[0495] Female C57BL / 6J mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0496] The specific animal experiment protocol is as follows:

[0497] Mice with similar weight and activity levels were selected for the experiment, with an expected weight range of 18-20g at the time of cell inoculation. Mouse fibrosarcoma WEHI-164 cells (purchased from Enzyme Research Institute, CC-Y2162) in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 7.5 × 10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse at a dose of 100 cells / ml.

[0498] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into four groups based on tumor size and body weight.

[0499] 1. PBS control group (represented as PBS in the figure);

[0500] 2. Single PD-L1 antibody administration group (represented in the figure as PD-L1 (200μg));

[0501] 3. Intratumoral administration group of single attenuated Salmonella SWT5005 (shown in the figure as SWT5005 (4.7×10) 6 cfu, it));

[0502] 4. Intratumoral administration of attenuated Salmonella SWT5005 combined with PD-L1 antibody (represented in the figure as Pre-PD-L1, SWT5005 (4.7×10) 6 cfu,it)+PD-L1(200μg)).

[0503] All groups receiving attenuated Salmonella SWT5005 combined with PD-L1 antibody were pretreated with PD-L1 antibody, meaning they received intraperitoneal administration of PD-L1 antibody (200 μg / animal) on Day 3 of grouping, once every 3 days. The attenuated Salmonella SWT5005 was administered intratumorally once a week. Detailed protocols are shown in Table 12.

[0504] Table 12 Grouping and Dosing Regimens of WEHI-164 Tumor-Bearing Mice

[0505] The PD-L1 antibody (InVivoPlus anti-mouse PD-L1(B7-H1), Bio X Cell, BP0101) was purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0506] (II) Pharmacodynamic Evaluation

[0507] 1. Evaluation Indicators

[0508] The evaluation indicators were measured in the same manner as in Example 2.

[0509] 2. Evaluation Results

[0510] The results of the calculation of tumor growth inhibition rate (TGI) are shown in Table 13.

[0511] Table 13 Tumor Growth Inhibition Rate (TGI)

[0512] As can be seen from Figures 23A and 23G and Table 13:

[0513] (1) Tumors grew rapidly in the PBS control group mice;

[0514] (2) In the monotherapy group, the PD-L1 monotherapy group had a certain inhibitory effect on the tumor, with a TGI of 67.20%; the SWT5005 intratumoral administration group also showed a certain reduction in tumor volume, with a TG1 of 72.19%.

[0515] (3) Significant antitumor effects were observed in the treatment group that received intratumoral administration of pre-PD-L1 treated attenuated Salmonella SWT5005 combined with PD-L1 antibody administration, with a TGI of 87.07%.

[0516] Figure 23B shows that statistical analysis of the body weight of all mice in groups 1 to 4 showed no significant difference in body weight among the groups after drug treatment.

[0517] Figures 23C to 23F show that by analyzing each individual mouse in each group, the results indicate that the five mice in the treatment group that received pre-PD-L1-treated attenuated Salmonella SWT5005 intratumorally in combination with PD-L1 antibody administration showed consistent antitumor performance, and their inhibition of mouse tumors was superior to that of the PD-L1 antibody administration group alone.

[0518] The above results indicate that intratumoral administration of pre-PD-L1 treated attenuated Salmonella SWT5005 combined with PD-L1 antibody significantly enhanced the inhibitory effect of PD-L1 antibody alone on WEHI-164 fibrosarcoma subcutaneous xenograft tumors in mice, without significantly affecting the body weight of the mice.

[0519] Example 6: Application of attenuated Salmonella SWT5005 intravenous administration combined with CTLA-4 antibody in pancreatic cancer

[0520] (I) Establishment of a subcutaneous pancreatic cancer Pan02 xenograft model in mice

[0521] Female C57BL / 6J mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0522] The specific animal experiment protocol is as follows:

[0523] Mice of similar weight and activity were selected for the experiment, with an expected weight range of 18-20g at the time of cell inoculation. Pancreatic cancer Pan02 cells (purchased from Enzyme Research Institute, CC-Y2144) from mice in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 5×10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse, with a concentration of cells / m.

[0524] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into two groups based on tumor size and body weight:

[0525] 1. Single CTLA-4 antibody administration group (represented in the figure as CTLA-4 (200 μg));

[0526] 2. The group receiving intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody (represented in the figure as Pre-CTLA-4, SWT5005 (4.7×10) 6 cfu, IV) + CTLA-4 (200 μg)).

[0527] The group receiving attenuated Salmonella SWT5005 combined with CTLA-4 antibody pretreatment received CTLA-4 antibody via intraperitoneal administration (200 μg / animal) on Day 3, once every 3 days. Attenuated Salmonella SWT5005 was administered intravenously as a single dose. Detailed protocols are shown in Table 14.

[0528] Table 14 Grouping and Dosing Regimens of Pan02 Tumor-Bearing Mice

[0529] CTLA-4 antibody (InVivoMAb anti-mouse CTLA-4(CD152), Bio X Cell, BE0131) was purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0530] (II) Pharmacodynamic Evaluation

[0531] 1. Evaluation Indicators

[0532] The evaluation indicators were measured in the same manner as in Example 2.

[0533] 2. Evaluation Results

[0534] The mean tumor volume at the start and end of treatment for the two groups of mice is shown in Table 15.

[0535] Table 15 Tumor volume at the start and end points of treatment in Pan02 tumor-bearing mice

[0536] Figures 24A to 24E and Table 15 show that, with similar average tumor volumes before treatment, the tumors in all four Pan02 tumor-bearing mice in the group receiving intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody disappeared by the treatment endpoint Day 19. This demonstrated a stronger tumor treatment effect compared to the group receiving CTLA-4 antibody alone. Furthermore, there was no significant difference in body weight between the two groups after treatment.

[0537] The above results indicate that in the Pan02 mouse model of subcutaneous pancreatic cancer xenografts, the combination of pre-treated attenuated Salmonella SWT5005 with CTLA-4 antibody and CTLA-4 antibody can effectively inhibit tumor growth and even achieve complete remission.

[0538] Example 7: Application of attenuated Salmonella SWT5005 intravenous combined with CTLA-4 antibody in hepatocellular carcinoma

[0539] (I) Establishment of a mouse Hepa1-6 subcutaneous xenograft model of liver cancer

[0540] Female C57BL / 6J mice (6-8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., and experiments were conducted in the SPF-grade animal facility of Shanghai Qishang Biomedical Technology Co., Ltd.

[0541] The specific animal experiment protocol is as follows:

[0542] Mice with similar weight and viability were selected for the experiment, with an expected weight range of 18-20g at the time of cell inoculation. Hepa1-6 hepatocellular carcinoma cells (purchased from Enzyme Research Institute, CC-Y2041) in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 2.6 × 10⁻⁶. 6 100 μl per mouse was injected subcutaneously into the right breast pad of the mouse at a dose of 100 cells / ml.

[0543] Four days after tumor implantation, tumor measurements were taken. When the tumors reached a certain size, the mice were randomly divided into two groups based on tumor size and body weight:

[0544] 1. Single CTLA-4 antibody administration group (represented in the figure as CTLA-4 (200 μg));

[0545] 2. The group receiving intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody (represented in the figure as Pre-CTLA-4, SWT5005 (4.7×10) 6 cfu, IV) + CTLA-4 (200 μg)).

[0546] The group receiving attenuated Salmonella SWT5005 combined with CTLA-4 antibody pretreatment received CTLA-4 antibody via intraperitoneal administration (200 μg / animal) on Day 3, once every 3 days. Attenuated Salmonella SWT5005 was administered intravenously as a single dose. Detailed protocols are shown in Table 16.

[0547] Table 16 Grouping and Dosing Regimens of Hepa1-6 Tumor-Bearing Mice

[0548] CTLA-4 antibody (InVivoMAb anti-mouse CTLA-4(CD152), Bio X Cell, BE0131) was purchased from Shanghai Qifa Laboratory Reagent Co., Ltd.

[0549] (II) Pharmacodynamic Evaluation

[0550] 1. Evaluation Indicators

[0551] The evaluation indicators were measured in the same manner as in Example 2.

[0552] 2. Evaluation Results

[0553] The mean tumor volume at the start and end of treatment for the two groups of mice is shown in Table 17.

[0554] Table 17 Tumor volume at the starting and ending points of treatment in Hepa1-6 tumor-bearing mice

[0555] Figures 25A to 25D and the results in Table 17 show that, with the average tumor volume being essentially the same in both groups of mice before treatment, the average tumor volume of the four Hepa1-6 tumor-bearing mice in the group receiving intravenous administration of attenuated Salmonella SWT5005 combined with CTLA-4 antibody was 9.92 mm at the treatment endpoint Day 17. 3 Compared to the CTLA-4 antibody-only group, the CTLA-4 antibody-only group had a CTLA-4 antibody-only dose of 40.03 mmHg at the treatment endpoint Day 17. 3 The average tumor volume was reduced to some extent, demonstrating a stronger therapeutic effect on tumors, and there was no significant difference in body weight between the two groups of mice after drug treatment. These results indicate that in the mouse Hepa1-6 subcutaneous hepatocellular carcinoma xenograft model, the combination of pre-treated attenuated Salmonella SWT5005 and CTLA-4 antibody can effectively inhibit tumor growth.

[0556] The present application has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present application based on these embodiments, all of which fall within the protection scope of the present application.

Claims

1. A pharmaceutical composition for treating tumors, comprising attenuated Salmonella and an immune checkpoint inhibitor.

2. Use of attenuated Salmonella and immune checkpoint inhibitors in the preparation of pharmaceutical compositions for the treatment of tumors.

3. The pharmaceutical composition or use according to claim 1 or 2, wherein the attenuated Salmonella comprises a positive hypoxia promoter, a survival-essential gene, and a negative hyperxia promoter.

4. The pharmaceutical composition or use according to claim 3, wherein the positive hypoxia promoter is selected from the promoter region sequence of yhbU or ynfK, the survival-essential gene is selected from alar or dadX, and the negative hyperxia promoter is selected from the promoter region sequence of cyoA or ydcI.

5. The pharmaceutical composition or use according to claim 4, wherein the attenuated Salmonella comprises a positive hypoxia promoter yhbU, a survival-essential gene alar, and a negative hyperxia promoter cyoA.

6. The pharmaceutical composition or use according to claim 4, wherein the attenuated Salmonella comprises the positive hypoxia promoter yhbU, the survival-essential gene alar, and the negative hyperxia promoter ydcI.

7. The pharmaceutical composition or use according to claim 4, wherein the attenuated Salmonella comprises a positive hypoxia promoter ynfK, a survival-essential gene dadX, and a negative hyperxia promoter cyoA.

8. The pharmaceutical composition or use according to claim 4, wherein the attenuated Salmonella comprises a positive hypoxia promoter ynfK, a survival-essential gene dadX, and a negative hyperxia promoter ydcI.

9. The pharmaceutical composition or use according to claim 4, wherein the attenuated Salmonella is SWT5001, SWT5005, SWT6009 or SWT6013.

10. The pharmaceutical composition or use according to claim 9, wherein, The attenuated Salmonella strain is SWT5005.

11. The pharmaceutical composition or use according to any of the preceding claims, wherein the attenuated Salmonella is regulated by oxygen concentration.

12. The pharmaceutical composition or use according to any of the preceding claims, wherein the positive hypoxia promoter functions when the oxygen content is below 1% and does not function when the oxygen content is above 1%; and / or the reverse hyperxia promoter functions when the oxygen content is above 1% and does not function when the oxygen content is below 1%.

13. The pharmaceutical composition or use according to claim 12, wherein the positive hypoxia promoter functions when the oxygen content is below 0.8% and does not function when the oxygen content is above 0.8%; and / or the reverse hyperxia promoter functions when the oxygen content is above 0.8% and does not function when the oxygen content is below 0.8%.

14. The pharmaceutical composition or use according to any of the preceding claims, wherein the immune checkpoint inhibitor is selected from PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

15. The pharmaceutical composition or use according to claim 14, wherein: PD-1 antibodies were selected from pembrolizumab, nivolumab, cimipril, camrelizumab, tislelizumab, sintilimab, toripalimab, penexilimab, and edoximilab. The PD-L1 antibody was selected from atezolizumab, durvalumab, avelumab, adebelimab, sugemalimab, and envorimab. The CTLA-4 antibody was selected from ipilimumab and cantulimumab.

16. The pharmaceutical composition or use according to any of the preceding claims further comprises a pharmaceutically acceptable carrier.

17. The pharmaceutical composition or use according to claim 16, wherein the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tension agents, stabilizers, antioxidants, surfactants, and lubricants.

18. The pharmaceutical composition or use according to any one of the preceding claims, for the treatment of solid tumors.

19. The pharmaceutical composition or use according to claim 18, wherein the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, liver cancer, bile duct cancer, melanoma, breast cancer, bladder cancer, thyroid cancer, testicular cancer, gastric cancer, prostate cancer, ovarian cancer, esophageal cancer, kidney cancer, uterine cancer, neuroblastoma, glioma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, and head and neck cancer, preferably selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, and liver cancer.

20. The pharmaceutical composition or use according to any one of the preceding claims, wherein, After a pre-treatment cycle of immune checkpoint inhibitors, combined treatment with attenuated Salmonella and immune checkpoint inhibitors is administered.

21. The pharmaceutical composition or use according to any one of the preceding claims, wherein, The administration routes for attenuated Salmonella are selected from intravenous, intratumoral, and interventional perfusion.

22. The pharmaceutical composition or use according to any one of the preceding claims, wherein, Immune checkpoint inhibitors can be administered via intraperitoneal, intravenous, intratumoral, oral, or subcutaneous routes.

23. A method of treating a tumor, comprising administering to a subject the pharmaceutical composition of any one of claims 1-22.

24. The method according to claim 23, wherein the tumor is a solid tumor.

25. The method according to claim 24, wherein the solid tumor is selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, liver cancer, bile duct cancer, melanoma, breast cancer, bladder cancer, thyroid cancer, testicular cancer, gastric cancer, prostate cancer, ovarian cancer, esophageal cancer, kidney cancer, uterine cancer, neuroblastoma, glioma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, and head and neck cancer, preferably selected from colon cancer, lung cancer, fibrosarcoma, pancreatic cancer, and liver cancer.

26. The method according to any one of claims 23-25, wherein, After a pre-treatment cycle of immune checkpoint inhibitors, combined treatment with attenuated Salmonella and immune checkpoint inhibitors is administered.

27. The method according to any one of claims 23-26, wherein, The administration routes for attenuated Salmonella are selected from intravenous, intratumoral, and interventional perfusion.

28. The method according to any one of claims 23-27, wherein, Immune checkpoint inhibitors can be administered via intraperitoneal, intravenous, intratumoral, oral, or subcutaneous routes.

Images