A modular tumor amino acid metabolism combined treatment platform based on homologous engineering bacteria step-by-step oral and phage cascade amplification

Abstract

The application discloses a modular and flexible configurable tumor amino acid metabolism combined treatment platform. The platform adopts a unified "in vivo drug cascade amplification delivery system" architecture, and by replacing different metabolic enzymes and toxic analog modules, precise "starvation + poisoning" combined attack can be carried out on multiple tumor amino acid metabolism weaknesses. The system core is the same chassis bacteria, and is divided into two stages of oral administration: the first stage of oral administration of the colonizing bacteria, which colonizes in the tumor hypoxic microenvironment and expresses specific amino acid metabolic enzymes; and the second stage of oral administration of the effector bacteria, which carries lysogenic bacteriophages controlled by the tumor microenvironment multiple signal logic "and" gate. Because the two have the same chassis, the bacteriophages can efficiently cross-infect and lyse the colonizing bacteria, cascade amplify the local concentration of specific amino acid metabolic enzymes, and efficiently deplete the target amino acids. Neutralizing antibodies are simultaneously used to remove the enzyme proteins leaked into the blood. After confirming the amino acid depletion, a corresponding low-dose toxic amino acid analog is orally administered, and the "mistaken eating" of the starved tumor cells is utilized to realize precise killing, and the sensitivity of radiotherapy and chemotherapy or drug resistance is reversed. The platform can be applied to multiple amino acid metabolism targets such as arginine, methionine, glutamine and asparagine, and can be used alone or in combination to provide customized combined treatment schemes for tumor patients with different metabolic types.

Description

[Technical Field]

[0001] This invention belongs to the field of synthetic biology and precision tumor therapy, specifically involving a "starvation + poisoning" combined therapy platform that adopts a unified in vivo delivery architecture and can modularly combine various tumor amino acid metabolism weaknesses. [Background Technology]

[0002] Tumor cell metabolism exhibits significant heterogeneity. Tumors from different origins, and even those from the same origin, may rely on different amino acid metabolic pathways to maintain their malignant proliferation. Examples include arginine auxotrophy, methionine dependence, asparagine dependence, and glutamine addiction. Corresponding single-target therapies have been developed to address these metabolic weaknesses, such as ADI-PEG20 and L-asparaginase; however, single-therapies are highly susceptible to drug resistance due to tumor compensatory mechanisms.

[0003] The combined strategy of "starvation + poisoning"—that is, first using enzymes to deplete the target amino acid, and then feeding it its toxic analogue—has been shown to have great potential for synergistic effects, sensitization to radiotherapy and chemotherapy, and reversal of drug resistance at multiple amino acid targets. However, this strategy for different amino acid targets all face the same core obstacle: toxic analogues indiscriminately poison all living systems and cannot be directly produced in vivo using living factories.

[0004] Therefore, there is an urgent need for a universal treatment platform that can perfectly separate the production of "starvation tools" from the delivery of "poison," and can flexibly select different amino acid target modules according to the specific metabolic subtype of tumors to achieve truly personalized precision treatment. [Summary of the Invention]

[0005] The core concept of this invention is to use a proven "homogeneous engineered bacteria-phage cascade amplification and delivery system" as a general-purpose hardware platform, and "metabolic enzymes + toxicity analogs" targeting specific amino acids as plug-and-play software modules. The hardware is responsible for solving the common problems of "safety, efficiency, and local amplification," while the software is responsible for solving the problem of precise targeting of different tumors.

[0006] The technical solution of this invention: General-purpose hardware platform (cascaded amplification and delivery system): Chassis bacteria: Select a safe chassis bacteria.

[0007] Colonizing bacteria: The expression cassette of the target amino acid metabolic enzyme is integrated into the chassis bacteria. After oral administration, it colonizes the tumor and continuously expresses the enzyme at low levels.

[0008] Effector bacteria: Using the same chassis bacteria, an additional lysogenic phage is integrated, controlled by an AND gate logic of the tumor microenvironment (hypoxia, high lactate, low pH). After oral administration, it targets the tumor and activates the phage upon specific signals.

[0009] Cascade amplification: The bacteriophages released by the effector bacteria cross-infect and lyse the colonizing bacteria, causing the concentration of metabolic enzymes to soar exponentially and efficiently deplete the target amino acids.

[0010] Safe neutralization: Systematically use neutralizing antibodies against this metabolic enzyme to remove circulating leaked enzyme.

[0011] Plug-and-play software module (amino acid metabolism intervention module): Each module contains a pair of specific tools: Target 1: Arginine. Metabolic enzymes: Arginine deiminase (ADI) or arginase; Toxicity analog: Canavanilic acid.

[0012] Target 2: Methionine. Metabolic enzyme: Methionase; Toxicity analog: Ethionine.

[0013] Target 3: Asparagine. Metabolic enzyme: Asparaginase; Toxicity analog: N,N-dibenzylasparagine.

[0014] Target 4: Glutamine. Metabolic enzyme: Glutaminase; Toxicity analogue: DON or its prodrug (such as DRP-104).

[0015] Precision poisoning and combined treatment: After completing the "starvation" phase using the corresponding module, administer a low dose of the corresponding toxic analogue orally. Once amino acid depletion is confirmed, administer the corresponding poison.

[0016] Modular combination and personalized treatment: Personalized treatment based on the metabolic dependence of the patient's tumor. A single module can be used alone; alternatively, multiple dependencies can be detected, and the colonizing and effector bacteria of different modules can be combined to achieve multi-target combined attack.

[0017] The beneficial effects of this invention are as follows: Platformization and scalability: Standardizing complex in vivo therapeutic systems. Any new amino acid metabolism targets discovered in the future can be quickly integrated into the platform by developing new "software modules."

[0018] True personalized precision treatment: Based on diagnostic results, a "starvation + poisoning" cocktail therapy targeting a single or combined target is customized for the patient, overcoming tumor heterogeneity and drug resistance in one fell swoop.

[0019] Unified safety and efficiency architecture: All modules share the same optimized cascade amplification system and safety mechanism (neutralizing antibody), greatly simplifying the R&D and clinical translation process.

[0020] Strong patent barriers: By combining a general platform architecture with multiple target modules for comprehensive protection, a highly defensive patent barrier is formed, building a solid competitive moat.

Detailed Implementation Methods

[0021] (1) Diagnosis: Biopsy confirmed that U87MG glioma is methionine dependent.

[0022] (2) Assembly: Select a hardware platform and insert the "methionine metabolism intervention module" (metabolic enzyme: methionine enzyme; toxic analog: ethionine).

[0023] (3) Treatment: Oral administration of colonizing bacteria (expressing methionine enzyme), effector bacteria (carrying bacteriophages), and subsequent oral administration of ethionine, in accordance with the aforementioned standard sequence, combined with radiotherapy for synergistic treatment. Example 2: Multi-module combined application – Treatment of arginine-deficient and methionine-dependent mixed-type colorectal cancer

[0024] (1) Diagnosis: Biopsy confirmed that the tumor has both arginine deficiency (ASS1 negative) and methionine dependence characteristics.

[0025] (2) Assembly: Select the hardware platform and insert the “arginine metabolism intervention module” and the “methionine metabolism intervention module”.

[0026] (3) Combination therapy: The colonizing bacteria corresponding to the two modules were mixed 1:1 and administered orally; 5-7 days later, the corresponding effector bacteria were mixed 1:1 and administered orally. Simultaneously, a mixed antibody against ADI and methionine was used. After the local arginine and methionine in the tumor were simultaneously depleted, a mixed solution containing low doses of canavonine and ethionine was administered orally. Combined with chemotherapy, the synergistic effect of multi-target combined attack was evaluated.

Claims

1. A modular tumor amino acid metabolism combined therapy platform, characterized in that, The invention comprises a universal in vivo drug cascade amplification and delivery system and at least one replaceable amino acid metabolism intervention module; the universal in vivo drug cascade amplification and delivery system comprises: (a) a first engineered bacterium (colonizing bacterium), which is a genetically modified bacterium capable of colonizing and expressing and secreting a target amino acid metabolic enzyme in the hypoxic microenvironment of a tumor; (b) a second engineered bacterium (effective bacterium), which uses the same chassis bacterium as the first engineered bacterium, but carries and maintains a genetically modified lysogenic bacteriophage, the lysis cycle of which is initiated by a logical AND gate controlled by one or more tumor-specific microenvironment signals; the amino acid metabolism intervention module comprises: (i) a target amino acid metabolic enzyme expressed by the colonizing bacterium; and (ii) a toxic amino acid analog corresponding to the target amino acid.

2. The platform according to claim 1, characterized in that, The target amino acid is selected from one or more of arginine, methionine, glutamine, and asparagine.

3. The platform according to claim 2, characterized in that: When the target amino acid is arginine, the metabolic enzyme is arginine deiminase (ADI) or arginase, and the toxic analogue is canavanine; when the target amino acid is methionine, the metabolic enzyme is methionase, and the toxic analogue is ethionine; when the target amino acid is asparagine, the metabolic enzyme is asparaginase, and the toxic analogue is N,N-dibenzylasparagine; when the target amino acid is glutamine, the metabolic enzyme is glutaminase, and the toxic analogue is DON or its prodrug (such as DRP-104).

4. The platform according to claim 1, characterized in that, The one or more tumor-specific microenvironment signals are selected from low oxygen, high lactate concentration, low pH, or any combination thereof.

5. The platform according to claim 1, characterized in that, The colonizing bacteria and effector bacteria are both selected from the same species among attenuated Salmonella Typhimurium, probiotic Escherichia coli Nissle 1917, or Lactococcus lactis.

6. The platform according to claim 1, characterized in that, It also contains one or more neutralizing antibodies, each antibody used to neutralize the activity of corresponding amino acid metabolic enzymes in circulating blood, said antibodies being administered before, during or after the administration of effector bacteria.

7. The platform according to claim 1, characterized in that, When two or more amino acid metabolism intervention modules are included, the colonizing bacteria of the modules can be mixed and applied, and the corresponding effector bacteria can also be mixed and applied to achieve a multi-target combined "starvation + poisoning" effect on tumors.

8. A kit for combination therapy of tumors, comprising components of the platform according to any one of claims 1-7, and instructions for use.

9. The use of the platform according to any one of claims 1-7 in the preparation of a medicament for treating malignant tumors, characterized in that, The formulation of the drug is based on molecular diagnostic results regarding the amino acid metabolism dependence of the patient's tumor.

10. The use according to claim 9, characterized in that, The drug is used in combination with radiotherapy and / or chemotherapy to enhance the sensitivity of tumors to radiotherapy and chemotherapy or to reverse their resistance.