An engineered bacterium that targets and colonizes tumors and can be conditionally cleared from the gut and its applications.

Abstract

This invention discloses an engineered bacterium capable of tumor-targeted colonization and conditional clearance from the intestine, and its applications. This engineered bacterium integrates for the first time a "tumor-specific colonization circuit" and a "colitin-conditionally sensitive circuit": through dual locking of a auxotrophic mutation (ΔaroA) and a hypoxia-dependent proliferation circuit (P hypox-ftsZ), it achieves strict tumor-specific colonization within the microenvironment; through the synergy of an exogenous colitin receptor gene and a time-delayed immune protein circuit, it achieves conditional clearance capabilities, exhibiting tolerance to colitin during the intestinal crossing window (approximately 0-4 hours) and regaining sensitivity after crossing. This engineered bacterium solves the core contradiction of oral live bacterial preparations—"both efficiently crossing the intestinal barrier and thoroughly clearing intestinal residues"—providing a safe and controllable living substrate for tumor bacterial therapy. It can be widely used as a colonizing bacterium or effector bacterium in various cascade amplification tumor immunotherapy systems.

Description

[Technical Field]

[0001] This invention belongs to the fields of synthetic biology and in vivo biopharmaceutical technology, specifically relating to an engineered bacterial strain, particularly a dual-function engineered bacterium with both tumor microenvironment-specific colonization ability and intestinal conditional clearance sensitivity, and its application in tumor treatment. [Background Technology]

[0002] Utilizing attenuated bacteria (such as Salmonella and Escherichia coli) as tumor-targeted delivery vectors has become a research hotspot in tumor biotherapy in recent years. These bacteria can specifically colonize and proliferate in tumor tissues, achieving a tumor / normal tissue ratio of over 1000:1, providing an ideal in vivo vector platform for targeted tumor therapy.

[0003] However, oral live bacteria preparations face a core dilemma: ensuring that sufficient live bacteria can safely cross the intestinal barrier and reach the tumor, while preventing the strains from remaining in the intestine for a long time and causing biosafety risks.

[0004] Current technologies primarily rely on single auxotrophic mutations (such as ΔaroA) to limit bacterial proliferation in normal tissues. However, auxotrophic mechanisms are passive clearance mechanisms, with clearance rates dependent on nutrient competition, and cannot achieve active, rapid, and targeted intestinal clearance. Even if a small number of bacterial strains obtain nutritional supplementation in the gut, there remains a risk of prolonged retention.

[0005] Furthermore, the tumor targeting of existing engineered bacteria mainly relies on the bacteria's natural tropism and nutritional deficiencies, and there is still room for improvement in their selectivity between the gut and tumors.

[0006] Therefore, there is an urgent need for a new type of engineered strain that can simultaneously meet the following requirements: (1) efficient colonization in the tumor microenvironment; and (2) active, precise, and time-controlled clearance in the gut. [Summary of the Invention] Purpose of the invention

[0007] The purpose of this invention is to provide a novel engineered bacterial strain that integrates a tumor-specific colonization circuit with an e. coli conditionally sensitive circuit to achieve the following objectives: Through multiple locking mechanisms, the strain is endowed with a strict tumor microenvironment-specific colonization ability; By using a time-delayed immune protein circuit, the strain is endowed with the conditional clearance ability to be resistant to coliform toxins during the intestinal crossing window and to regain sensitivity after crossing. This provides the first controllable "cross-clearance" separation protocol for oral live bacteria formulations; As the "colonizing bacteria" or "effect bacteria" in the tumor immunotherapy system. Technical solution

[0008] This invention provides an engineered bacterium, characterized in that it simultaneously comprises the following genetic modifications: [First Functional Module] Tumor-Specific Engrafting Circuit The tumor-specific colonization circuit comprises a combination of at least two of the following modifications: (a) Auxotrophic mutation: In the genome of the engineered bacteria, the key gene (preferably aroA) of the aromatic amino acid synthesis pathway is knocked out or inactivated, so that it loses the ability to synthesize phenylalanine, tyrosine and tryptophan autonomously, and can only proliferate in an environment rich in exogenous aromatic amino acids.

[0009] (b) Hypoxia-dependent proliferation circuit: In the genome of the engineered bacteria, the endogenous gene essential for cell division (preferably ftsZ) is removed from the control of its natural promoter and placed under the control of a strict hypoxia-inducible promoter.

[0010] The strictly hypoxia-inducible promoter is preferably the pPepT promoter, FF+20* promoter or its functional variants, which initiates transcription when the oxygen concentration is below 1% (preferably below 0.5%) and has virtually no transcriptional activity at intestinal physiological oxygen concentrations.

[0011] [The synergistic effect of modifications (a) and (b)]: In the gut: the concentration of free aromatic amino acids is extremely low and the oxygen concentration does not reach the hypoxia-induced threshold. Modification (a) causes the strain to starve, and modification (b) causes cell division to stop. The dual lockout ensures that the strain cannot proliferate in the gut.

[0012] In tumors: The necrotic area of ​​the tumor is rich in free amino acids and the core area is severely hypoxic. Modification (a) provides nutritional support and modification (b) removes proliferation inhibition, and the strain gains a huge growth advantage relative to the intestine.

[0013] [Second Functional Module] Coliformin Conditional Sensitive Circuit The coliform conditional sensitivity circuit includes: (c) Escherichia coli receptor gene: The genome of the engineered bacteria is integrated with an exogenous escherichia coli receptor gene (preferably btuB or cirA), which enables the strain to be recognized and attacked by specific escherichia coli.

[0014] (d) Time-delayed immune protein loop: The engineered bacteria carry a time-delayed gene encoding an immune protein that can specifically neutralize the toxicity of the specific coliform toxin.

[0015] The gene encoding the immune protein is controlled by a delayed decay expression system, which highly expresses the immune protein when the bacteria are in their initial state, making the strain resistant to coliform toxins; as time goes by and / or the number of cell divisions increases, the expression of the immune protein gradually decays, and the strain regains sensitivity to coliform toxins.

[0016] [Optional Feature] The delay decay expression system is implemented through one or more of the following mechanisms: The C-terminus of the immunoprotein is fused with an ssrA degradation tag, which gives the immunoprotein a short half-life. Immunoprotein genes are placed under promoters controlled by negative feedback from cumulative transcriptional repressors; Immunoprotein genes are placed under promoters controlled by cell division counters.

[0017] Furthermore, the engineered bacteria may also include one or more of the following optional modifications: (e) Genome toxin island knockout: Knockout of endogenous toxin synthesis gene clusters such as pks gene islands to eliminate the risk of genotoxicity.

[0018] (f) Bile salt-induced self-destruction circuit: Introduce a toxin gene driven by a bile salt response promoter as a second redundancy guarantee for intestinal clearance.

[0019] Key innovative features Dual-targeting tumor-specific colonization: For the first time, "auxotrophic" and "hypoxia-driven proliferation" are integrated into the essential proliferation pathway of the same strain, forming an "AND" logic gate, which significantly improves tumor / gut selectivity.

[0020] Conditional colicin sensitivity: The first time-sequence control logic of "initial tolerance → delayed sensitivity" was proposed and implemented, which protects the strain during the intestinal crossing window and allows it to be actively cleared by colicin after crossing.

[0021] Crossing-Clearing Spatiotemporal Separation: By designing delayed decay immune proteins, the inherent contradiction of oral live bacteria "must both efficiently cross and thoroughly clear" has been solved for the first time.

[0022] Multiple safety redundancies: nutritional deficiency type + hypoxia dependence + conditional colicin sensitivity + optional bile salt self-destruction constitute at least three layers of intestinal clearance protection, providing sufficient safety redundancy for clinical translation.

[0023] [Chassis Strains] The chassis strain of the engineered bacteria is selected from attenuated Salmonella typhimurium, Escherichia coli, or Bifidobacterium.

[0024] Preferably, the attenuated Salmonella is the VNP20009 strain or its derivative with the msbB and / or purI gene knocked out.

[0025] Preferably, the Escherichia coli is the probiotic strain Nissle 1917 (EcN).

[0026] Application in tumor immunotherapy systems The aforementioned engineered bacteria can be used as "colonizing bacteria" and / or "effect bacteria" in a multi-component synergistic cascade amplification tumor immunotherapy system.

[0027] When used as "colonizing bacteria", the engineered bacteria do not carry the target bacteriophage and are used to establish a high-density bacterial reservoir around tumor blood vessels.

[0028] When used as an "effect bacterium," the engineered bacteriophage carries an engineered phage genome that integrates genes encoding immune checkpoint inhibitors (such as anti-PD-L1 nanobodies), and the activation and lysis cycle of the phage is controlled by tumor microenvironment-specific logic gates.

Detailed Implementation Methods

[0029] 1.1 Selection of strains for chassis The attenuated Salmonella VNP20009 (ΔmsbB, ΔpurI) was used as the starting strain.

[0030] 1.2 Introduction of auxotrophic mutations The VNP20009 ΔaroA strain was obtained by knocking out the aroA gene (encoding 5-enolpyruvylshikimate-3-phosphate synthase) using the λRed recombination system. Its growth defect was verified on M9 basal medium lacking exogenous aromatic amino acids.

[0031] 1.3 Construction of Hypoxia-Dependent Proliferative Circuit The endogenous ftsZ gene (encoding the key cell division protein FtsZ) was knocked out from its natural chromosomal locus, and a ftsZ gene expression cassette driven by the strictly hypoxia-inducible promoter pPepT was inserted into another neutral chromosomal locus (such as the phoB locus). After construction, the growth curves of the strain were measured under normoxic (21% O2) and hypoxic (<0.5% O2) conditions to verify the specific unlocking of proliferation under hypoxic conditions.

[0032] 1.4 Integration of colicin receptor gene At chromosomal neutral sites (such as the araBAD site), the expression cassette of the codon-optimized E. coli receptor gene btuB is integrated, driven by a constitutive promoter, to ensure stable expression of the receptor on the bacterial cell surface.

[0033] 1.5 Construction of Time-Delayed Immune Protein Circuits Construct an expression plasmid containing the following elements: The E. coli E3 immune protein gene (immE3) has an ssrA degradation tag (AANDENYALAA) fused to its C-terminus. The immune protein gene is driven by a constitutive promoter, but the promoter region contains the lacO operon sequence; At the same time, the lacI gene, driven by a weak constitutive promoter, is integrated into the chromosome, and the LacI repressor protein gradually accumulates with cell division.

[0034] In the initial stage of transformation, the LacI concentration is low and immunoglobulin expression is high; as cells divide, the LacI concentration increases, gradually inhibiting immunoglobulin transcription. Existing immunoglobulins are rapidly degraded due to the ssrA tag.

[0035] 1.6 Optional Modifications The pks gene island is knocked out using the λRed recombination system (e.g., when using EcN as the chassis).

[0036] A bile salt-induced self-destruction circuit was constructed as an enhancement module for intestinal clearance. Example 2: In vitro functional verification

[0037] 2.1 Validation of Nutritional Deficiency The engineered bacteria and wild-type control were inoculated into M9 basal medium containing or without aromatic amino acids (Phe, Tyr, Trp 40 μg / mL each), and cultured at 37°C for 24 hours. OD600 was measured. The engineered bacteria grew normally only when aromatic amino acids were supplemented.

[0038] 2.2 Validation of Hypoxia-Dependent Proliferation The engineered bacteria were cultured in LB medium containing aromatic amino acids under normoxic (21% O2) and hypoxic (<0.5% O2) conditions, respectively, and CFU were measured every 2 hours. The engineered bacteria only exhibited exponential proliferation under hypoxic conditions.

[0039] 2.3 Verification of temporal changes in coliform susceptibility The engineered bacteria were cultured in LB medium, and samples were taken at different time points (0h, 2h, 4h, 6h, 8h, 12h, 24h). The samples were co-incubated with supernatant containing coliformin E3 for 1 hour, and the viable bacteria were counted by plating. The time curve of the "tolerance → sensitivity" transition was plotted.

[0040] Expected results: No significant decrease in viable bacterial count within 0-4 hours (tolerance); >99% decrease in viable bacterial count after 8 hours (recovery of sensitivity). Example 3: In vivo verification in animals

[0041] 3.1 Validation of tumor targeting and normal tissue clearance A subcutaneous CT26 colorectal cancer model was established in BALB / c mice. The engineered bacteria (1×10⁻⁶) were administered orally. 7 Mice were sacrificed on days 1, 3, 5, and 7 after administration, and tumors, liver, spleen, mesenteric lymph nodes, and intestinal contents were collected, homogenized, and plated for counting.

[0042] Expected results: Bacterial load in tumor tissue peaks on days 3-5 (>10). 8 CFU / g); The bacterial load in the liver and spleen was significantly lower than that in tumors (ratio > 1000:1). The number of viable bacteria in the intestinal contents decreased to the detection limit within 48 hours after administration.

[0043] 3.2 Validation of conditional clearance of coliform bacteria Based on the above model, the following groups are set: G1 (control group): Oral administration of engineered bacteria only.

[0044] G2 (Cleanup Group): Oral administration of engineered bacteria, followed by oral administration of a scavenger strain producing E. coli E3 4-8 hours later.

[0045] The number of engineered bacteria in intestinal contents and intestinal mucosa was detected separately.

[0046] Expected results: Within 24 hours after administration of Scavenger bacteria, the clearance rate of engineered bacteria in the intestine of group G2 was significantly higher than that of group G1 (>99%).

[0047] 3.3 Security Assessment On days 1, 7, 14, and 28 after drug administration, mouse body weight, complete blood count, liver and kidney function (ALT, AST, Cr, BUN), serum inflammatory factors (IL-6, TNF-α), and pathological sections of major organs were monitored.

Claims

1. An engineered bacterium, characterized in that, Also includes: (a) Tumor-specific colonization circuits, comprising: (i) auxotrophic mutations, namely, knockout or inactivation of the aroA gene; and (ii) hypoxia-dependent proliferation circuits, wherein the endogenous cell division essential gene ftsZ is under strict hypoxia-inducible promoter control. (b) A conditionally sensitive colicin-containing loop comprising: (i) an exogenous colicin receptor gene that sensitizes the strain to a specific colicin; and (ii) a time-delayed immunoprotein loop, wherein the expression level of the immunoprotein gradually decreases over time and / or with cell division while neutralizing the toxicity of the specific colicin, thereby enabling the strain to tolerate the colicin during the initial intestinal crossing window (approximately 0-4 hours) and regain sensitivity after crossing.

2. The engineered bacteria according to claim 1, characterized in that, The strictly hypoxia-inducible promoter is selected from the pPepT promoter, the FF+20* promoter or a functional variant thereof, which initiates transcription when the oxygen concentration is below 1%.

3. The engineered bacteria according to claim 1, characterized in that, The time-delayed immune protein circuit is implemented through one or more of the following mechanisms: C-terminus of the immune protein is fused with an ssrA degradation tag; Immunoprotein genes are placed under promoters controlled by negative feedback from cumulative transcriptional repressors; Immunoprotein genes are placed under promoters controlled by cell division counters.

4. The engineered bacteria according to claim 1, characterized in that, It also includes genomic toxin island knockout mutations (Δpks) and / or bile salt-induced self-destruction circuits.

5. The engineered bacteria according to any one of claims 1 to 4, characterized in that, The strain in its chassis was selected from attenuated Salmonella ( Salmonella typhimurium ), Escherichia coli ( Escherichia coli ) or Bifidobacterium ( Bifidobacterium ).

6. The engineered bacteria according to claim 5, characterized in that, The attenuated Salmonella strain is VNP20009 or a derivative thereof, and the Escherichia coli strain is Nissle 1917.

7. A method for constructing engineered bacteria as described in any one of claims 1 to 6, characterized in that, This includes the steps of performing the gene modification sequentially or simultaneously.

8. The use of an engineered bacterium as described in any one of claims 1 to 6 in the preparation of a medicament for the treatment of tumors.

9. The application according to claim 8, characterized in that, The engineered bacteria, as colonizing and / or effector bacteria, are used in a multi-component synergistic cascade amplification tumor immunotherapy system.

10. A staged oral administration regimen, characterized in that, include: (a) Preparation period: The subject is first orally administered a first probiotic strain that does not produce coliformin; (b) Treatment period: The subject is orally administered the engineered bacteria according to any one of claims 1 to 6; (c) Cleanup period: At a predetermined time window after the treatment period, the subject is orally administered a second probiotic strain that produces high coliformin in order to selectively clear the engineered bacteria remaining in the intestinal lumen.