Metabolic affinity-based biological system and application thereof in preparation of antitumor drugs and detection kits

Abstract

The invention discloses a biological system based on metabolic affinity and application of the biological system in preparation of antitumor drugs and detection kits. According to the system, selective metabolic affinity screening is carried out based on living microorganisms through an in-vitro adaptability protocol, and selective recognition of a tumor microenvironment, local generation of a bioactive agent and biological restraint are achieved. The system integrates chemotropism detection of tumor metabolic markers, biologic and adaptive decision based on nutritional gradients, controlled production of biologically active metabolites, and a safety architecture. And the self-amplification treatment effect in proportion to the tumor load is realized through a nutrition chemical trend mechanism. The invention aims to eliminate systemic toxicity of traditional chemotherapy through concentration gradient mediated super localized release, reduce production cost through controlled self-replication in continuous flow biological reaction, and guarantee safety through immune clearance. Affinity can be obtained through a metabolic adaptation experiment, the method is suitable for various tumors through a modular reselection protocol, and a new direction is provided for metastatic cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of advanced tumor biotherapy and metabolic affinity-based biological drug delivery systems, and in particular to a metabolic affinity-based biological system and its application in the preparation of anti-tumor drugs and detection kits. BACKGROUND

[0002] The field of tumor therapy has long been faced with the challenge of balancing efficacy and safety, and existing treatment methods have significant limitations in terms of precise targeting, control of side effects, and cost-effectiveness, among others, necessitating innovative technological breakthroughs. Current tumor treatment strategies are mainly based on systemic chemotherapy, radiotherapy, immunotherapy, and surgical methods. Among them, traditional chemotherapy lacks cell selectivity and presents significant systemic limitations, leading to dose-limiting side effects, severe systemic toxicity, and extensive damage to healthy tissues. For example, traditional cytotoxic drugs, although showing efficacy in eliminating tumor cells, lack the mechanism to distinguish between malignant and normal cell populations, causing bone marrow suppression, cardiotoxicity, nephrotoxicity, and neurotoxicity.

[0003] To improve the therapeutic index, second and third generation drug carrier systems for tumor therapy have been developed, including stealth liposomes, polymeric nanoparticles, micelles, dendrimers, antibody-drug conjugates (ADCs), and active targeting systems. However, these methods have key limitations: (1) high development and production costs, 50,000-200,000 euros per kilogram of active ingredient in the formulation; (2) manufacturing complexity and industrial scalability issues; (3) physicochemical instability in biological fluids; (4) accelerated clearance mediated by the reticuloendothelial system; (5) limited tissue penetration due to anatomical and physiological barriers; (6) non-specific accumulation in healthy organs, and the development of drug resistance.

[0004] In recent years, innovative biological approaches have emerged, such as engineered oncolytic viruses, modified bacteria for tumor targeting, adoptive cell therapy (CAR-T, TIL), and combined immunotherapy, but each also faces unique challenges. For example, oncolytic viruses, although showing tumor specificity through transcriptional or receptor targeting, have issues with viral replication control, potential systemic adverse reactions caused by cytokine release, immunogenicity limiting repeated administration, and variability in sensitivity among patients. Engineered bacteria (such as modified Salmonella, Clostridium, Bifidobacterium) show selective colonization ability to hypoxic tumor microenvironments, but are plagued by difficulties in precise control of bacterial growth, risks of systemic infection, development of antibiotic resistance, and rapid immune clearance. At the same time, the application of fungal-derived metabolites in tumor therapy has gradually attracted research attention, and many secondary metabolites derived from fungi have been confirmed to have anti-tumor activity. For example, paclitaxel, originally isolated from the endophyte Taxomyces andreanae, has become a successful case of treatment, with global annual sales exceeding $1 billion. In addition, other fungal compounds such as bioactive metabolites (protein synthesis inhibitors), derivatives that interfere with sphingolipid biosynthesis, cytoskeletal modulators, and bioactive cyclic peptides, etc., have shown significant cytotoxic properties in preclinical studies. However, their clinical application is limited by: (1) expensive extraction and purification requirements; (2) low production yield; (3) complex drug formulation; (4) limited stability, and difficulty in controlling temporal and spatial release. Fungi have a complex chemotaxis system based on transmembrane receptors, involving the G protein-coupled receptor (GPCR) and specific metabolic transporter families. Nutritional chemotaxis in filamentous fungi is mediated by transduction pathways involving: (1) nutrient gradient sensors through high-affinity transporters (amino acids with Km values of 10-100 μM, sugars with 1-10 mM); (2) signal cascades that activate adenylyl cyclase and phosphodiesterase through heterotrimeric G proteins (Gα, Gβγ); (3) intracellular Ca 2+ mobilization through voltage-dependent channels and organelle release; (4) directed growth through cytoskeletal reorganization mediated by motor proteins (dynein, kinesin). These natural mechanisms can be exploited and enhanced through metabolic adaptation protocols to develop specific affinity for tumor-related metabolites such as lactate, glutamine, and other compounds abundant in the tumor microenvironment.

[0005] Notably, a study published in 2022 (PMID 35120601) confirmed that the fungal microbiome is involved in the activation of IL-33 and the induction of type 2 immune responses in pancreatic ductal adenocarcinoma, which are associated with tumor progression. The results of this study support the presence of endogenous fungal communities in the tumor microenvironment and provide scientific evidence for the present invention that fungal strains isolated from tumors or appropriately domesticated exogenous fungal strains can be used to specifically regulate the tumor ecosystem.

[0006] Living organisms exhibit natural molecular recognition capabilities through established chemotactic mechanisms: endophytic fungi naturally develop specificity for host metabolites through evolution of specific transporters and adaptive metabolic pathways; commensal bacteria gain affinity for specific environments through selection of enhanced chemotactic systems; pathogenic microorganisms evolve precise cell targeting capabilities through adhesion and invasion receptors. However, direct therapeutic applications of these natural mechanisms are limited by: (1) extended time (years-decades) for natural evolution of specific affinities; (2) lack of directional control over selection of therapeutically relevant targets; (3) potential development of undesirable virulence or pathogenic properties; (4) instability of chemotactic properties acquired in the absence of sustained selective pressure; (5) inability to selectively target specific tumor markers in the absence of directed adaptation protocols in bioreactors with controlled nutritional composition.

[0007] Bioreactor technology can accelerate natural adaptation processes through precise control of environmental and nutritional conditions. Fed-batch systems with controlled gradients are able to create selective microenvironments that recapitulate specific tumor conditions, including: (1) pH gradients controlled through organic buffers (MES pH 6.2-6.8, HEPES pH 7.0-7.4) to mimic tumor acidosis; (2) tumor-specific metabolite graded concentrations (lactate 8-15 mM, glutamine 3-8 mM, glutamate 1-3 mM) through programmable peristaltic pumps; (3) controlled hypoxic conditions (0.5-5% O2) through calibrated gas mixing; (4) regulated osmolarity (300-350 mOsm / kg) to mimic tumor interstitial environments. These systems theoretically allow for accelerated adaptation cycles, isolating variant strains with specific chemotactic properties for targeted microenvironments through standardized selective enrichment protocols.

[0008] According to the analysis of the existing patents and documents, the current technology has not described a system that simultaneously integrates the following features: (1) Accelerated metabolic affinity adaptation: obtaining specific chemotaxis to tumor-related metabolites through a standardized protocol of metabolic adaptation and experimental selection protocol in bioreactors with controlled nutritional gradients. (2) Conditional multi-parameter activation through chemotaxis: having the ability to simultaneously recognize multiple tumor metabolic features (lactate + glutamine + acidic pH + hypoxia), which can be obtained through selection pressure under combined conditions. (3) Nutritional dependence adaptation time control: developing a nutritional dependence mechanism through experimental induction of tumor-specific metabolite auxotrophies to control treatment duration. (4) Matrix depletion containment strategy: based on the availability of metabolic-dependent biological clearance systems, activating cell death cascades when tumor substrates are depleted. (5) Modularity of metabolic affinity and re-adaptation: providing an adaptable architecture for different tumor targets through standardized protocols of specific re-selection cycles in bioreactors for different metabolic profiles. (6) Specific affinity-based biological containment: based on metabolic-dependent containment mechanisms, theoretically making survival outside the target tumor microenvironment impossible.

[0009] In summary, there is an unmet clinical and commercial need to develop a therapeutic system that simultaneously overcomes the limitations of the current technology, which should have: super-localized release through selective metabolic affinity to reduce systemic toxicity; time control of the therapeutic window through nutritional dependence on tumor-specific substrates; safety strategies through immune clearance and metabolic deficiency containment; sustainable production costs through controlled self-replication in standardized bioreactors; and modular applicability to different tumor types through guided metabolic affinity re-adaptation protocols. SUMMARY

[0010] The objective of the present invention is to provide a metabolic affinity-based biological system and its application in the preparation of antitumor drugs and detection kits to solve the deficiencies of the prior art. The present invention solves the technical gap of the prior art through a modular metabolic affinity-based biological system based on microorganisms (preferably filamentous fungi, alternatively bacterial variants) modulated through metabolic adaptation protocols in bioreactors with controlled nutritional gradients for the controlled, localized and timed release of cytotoxic agents in specific tumor environments. This system has a multi-layered natural safety architecture based on conditional metabolic dependence, a containment mechanism based on the lack of tumor-specific substrates, biological containment control based on the induction of auxotrophies by metabolites theoretically abundant in the tumor microenvironment, and the key reversibility of the system, allowing the restoration of the original physiological conditions through natural immune clearance without permanent changes to the host organism.

[0011] The present application provides a metabolic affinity-based biological system, characterized in that it comprises:

[0012] (a) Metabolic response module: a living organism composed of endogenous filamentous fungi and / or exogenous filamentous fungi isolated from tumor tissue, and non-pathogenic commensal bacteria, said living organism comprising the following features: (i) selective response capacity to tumor metabolites obtained by adaptive culture in a gradient-controlled bioreactor; (ii) multi-threshold metabolic sensors integrated into an adaptive transduction circuit, which must simultaneously satisfy at least two of the following tumor metabolic characteristics to activate the system through convergent pathways: lactic acid concentration greater than 10 mM, glutamine concentration greater than 5 mM, pH less than 6.8, and hypoxia less than 3% O2; (iii) biosynthetic pathways induced by tumor-specific substrates for the production of secondary metabolites, bioactive peptides, or extracellular lytic enzymes.

[0013] (b) Biological containment module: comprising synergistic redundant mechanisms: (i) nutritional dependence on conditions of lactic acid concentration greater than 8 mM and glutamine concentration greater than 3 mM; (ii) protease-mediated intracellular apoptosis pathway triggered by ATP depletion and reactive oxygen species accumulation; (iii) inducible pharmacological sensitivity to fluconazole, amphotericin B, or 5-fluorocytosine;

[0014] (c) Controlled drug delivery system: comprising image-guided stereotactic injection, surgical implantation of biocompatible hydrogel formulations, or combined immunosuppression.

[0015] Further, the present application provides the use of a metabolic affinity-based biological system in the preparation of antitumor drugs, according to the type of formulation by administration form: (i) isotonic injection suspension for stereotactic administration, with optimized injection viscosity, maintaining cell viability; (ii) biocompatible hydrogel pre-implant for surgical positioning, with release kinetics controlled by programmed degradation; (iii) mucosal adhesive formulation for transdermal / mucosal application by modulating epithelial permeability organisms; (iv) liposome formulation for systemic transport protection, with passive targeting and metabolic capacity maintenance functions. Different types of formulations have controlled microbiological stability and maintain affinity biological activity during storage.

[0016] Further, the anti-tumor drugs are suitable for: (i) HER2-positive breast cancer, systemically adapted to high-lactate environment through metabolic adaptation protocol, potentially adapting dual-affinity to anti-HER+ metabolic; (ii) prostate adenocarcinoma, systemically adapted to hypoxic microenvironment and presence of PSA marker through anaerobic bioreactor modulation protocol; (iii) colorectal cancer, with dual-targeting system to VEGF / CEA+ metabolic affinity to hypoglycemic environment through multiple adaptation protocol; (iv) brain tumor, with potential ability to cross blood-brain barrier through carrier cells loaded with adaptive neurotrophic organisms.

[0017] Further, the present application provides the use of the metabolic affinity-based biological system in the preparation of detection kits, including (i) specific primers for quantitative PCR detection of modulated organisms; (ii) biosensor components for metabolic gradient verification; (iii) metabolic detection reagents for detection of lactic acid, glutamine, pH; (iv) migration detection system for affinity function verification.

[0018] (I) reversible advantage through specific affinity

[0019] The uniqueness of the system lies in the biological reversibility obtained through selective metabolic affinity rather than permanent genetic modification. Unlike genetic engineering systems based on permanent changes to the genome of the organism, the present method uses metabolic adaptation protocols to obtain organisms with acquired affinity but genetic integrity. These can maintain their natural genomic stability by developing metabolic dependence on the tumor microenvironment (high-lactate environment, glutamine-rich, acidic pH, hypoxic conditions). Suspension of temporary immunosuppression can activate natural immune clearance through phagocytosis by macrophages and neutrophils, theoretically eliminating the organism through normal physiological mechanisms. At the same time, elimination of tumor cells can deprive the organism of essential metabolic substrates for survival, activating programmed containment strategies. This redundancy ensures the restoration of the patient's original physiological state without permanent changes to the microbiota or systemic metabolic balance.

[0020] (II) modular architecture of the metabolic affinity-based system

[0021] The system includes metabolic affinity-modulated organisms with integrated functional capabilities:

[0022] a) condition-specific chemotactic module: theoretically, the sensory ability developed through metabolic adaptation protocols in bioreactors with controlled nutrient gradients can recognize a variety of tumor metabolites through potentially adaptable receptors and metabolic transporters. Through selection pressure in alternating growth cycles between normal and tumor media, a reduction in cross-reactivity to normal physiological substrates can be obtained, maintaining the genetic integrity of the organism without producing permanent chromosomal modifications.

[0023] b) Differential response to multiple metabolic thresholds: Theoretically, the developed signal integration system can be regulated by a coordinated response to a combinatorial nutritional gradient, with the condition for the activation of adaptive decisions requiring the simultaneous presence of different tumor metabolic signatures. This system eliminates false activation from single non-specific signals through a convergent transduction circuit requiring multiple simultaneous thresholds. Tumor specificity is obtained through evolution-optimized metabolic pattern recognition algorithms without generating permanent genetic alterations.

[0024] c) Production module of metabolic pathway control: A biosynthetic platform for cytotoxic metabolites with expression control obtained through upregulation of the biosynthetic pathway in response to tumor-specific substrates. Regulation occurs through multiple levels of transcriptional, post-transcriptional, and post-translational control. The biosynthetic pathway is optimized while maintaining the stability of the natural genome.

[0025] d) Suppression system based on metabolic dependency: Theoretically, the developed safety architecture can be regulated by nutritional dependency, including: conditional nutritional dependency on tumor metabolites obtained through controlled adaptation protocols, substrate depletion-based suppression strategies through a programmed cascade activated by nutritional deficiency, controlled sensitivity to antifungal agents, programmable immune clearance through the suspension of immunosuppression, and suppression mechanisms based on the nutritional deficiency of the tumor microenvironment, which can prevent survival under normal physiological conditions.

[0026] (III) Integrated operational mode through specific affinity reversibility

[0027] System operation can foresee controlled and monitored sequential phases with the possibility of interruption and resumption through natural mechanisms:

[0028] Phase 1 - Controlled inoculation: Metabolic affinity-regulated organisms are administered locally in the tumor area through direct intervention methods. A temporary immunosuppression protocol is optimized through the use of corticosteroids to prevent acute rejection while maintaining immune surveillance, with the possibility of interruption and clearance through the suspension of immunosuppressive agents.

[0029] Phase 2 - Metabolic detection and affinity verification: Theoretically obtained chemotactic ability is activated through the identification of tumor metabolic gradients with activation thresholds calibrated through bioreactor regulation. Metabolic sensors verify the presence of the tumor microenvironment through receptors that only activate a convergent transduction cascade when the target parameters are simultaneously present.

[0030] Phase 3 - Therapeutic activation controlled by metabolic gradient: The production of cytotoxic metabolites is controlled induced through biosynthetic pathways upregulated in response to tumor-specific substrate concentrations. Localized release occurs through modified transporters secreted in proportion to the metabolic gradient, with the possibility of interruption through depletion of the target nutritional substrate.

[0031] Phase 4 - Selective Tumor Elimination via Metabolic Affinity: Targeted cytotoxic action against tumor cells through diverse molecular mechanisms activated by specific tumor microenvironment recognition. Locally generated cytotoxic metabolites act via multiple pathways. Protection of surrounding healthy tissues is ensured through concentration gradients and action specificity based on tumor metabolic microenvironment affinity.

[0032] Phase 5 - Programmed containment and recovery of metabolic deficiency: Activation of biological clearance mechanisms through sequential cascade: The gradual reduction of tumor-specific nutrients due to tumor cell death, activation of internal metabolic sensors, and the triggering of a programmed cascade when endogenous reserves are depleted, ensuring the clearance of biological debris and the restoration of original tissue homeostasis through active immune clearance by pausing immunosuppression.

[0033] (iv) Unique technological innovations through the reversibility of natural affinity

[0034] Metabolic affinity modulation: By implementing metabolic adaptation protocols in a bioreactor, specific chemotaxis to tumor-associated metabolites is acquired over generations while maintaining natural genomic integrity. The bioreactor can reproduce tumor conditions, allowing the isolation of variants with enhanced affinity while preserving natural genetic stability.

[0035] Multi-parameter activation logic based on metabolic sensors: Theoretically, biological decision-making systems developed through the integration and regulation of multiple metabolic signals require the simultaneous presence of different tumor characteristics for activation. Integration occurs through convergent transduction circuits, eliminating spurious activation from a single normal physiological signal.

[0036] Dynamic dosing control based on metabolic feedback: a theoretically achievable self-regulating mechanism for modulating cytotoxicity production based on available tumor substrate concentrations. The system can implement feedback loops to prevent overdosing and optimize efficacy through natural regulation proportional to tumor burden.

[0037] Natural metabolic-dependent biocures: The implementation of biological barriers theoretically developed through multiple auxotrophic modulations makes survival outside the tumor microenvironment difficult. Dependence includes auxotrophic dependence on tumor-specific substrates and adaptation to specific microenvironmental conditions.

[0038] Modularity of Metabolic Reregulation: An adaptable architecture for different tumor metabolic profiles through modular reregulation protocols in configurable bioreactors. For tumor targets with different metabolic characteristics, the system allows for reregulation by modifying bioreactor conditions without redesigning the entire approach.

[0039] Through the unique competitive advantage of reversible affinity safety

[0040] The system provides therapeutic, economic, and operational benefits with the possibility of reversibility through natural mechanisms:

[0041] Therapeutic effect based on reversible affinity: super-localized release through selective concentration in the tumor microenvironment by specific chemical tropism, potentially reducing systemic toxicity.

[0042] In situ therapeutic production with the possibility of therapeutic window time control by pausing the metabolic gradient through the selective activation of metabolic biosensors, potentially optimized for tumor-specific metabolic profiles.

[0043] Intrinsic safety and reversibility through natural dependence: natural biological containment system of multiple nutritional deficiencies based on metabolic deficiency containment mechanisms, real-time monitoring protocols, and system reversibility without permanent changes to the organism, allowing retreatment without accumulation of toxicity.

[0044] Economically sustainable through natural regulation: potential reduction in production costs through the use of genetically intact but affinity-regulated organisms, scalability through regulatory processes in bioreactors, reduction in medical costs associated with systemic side effects, reduction in monitoring costs due to reversibility, and the possibility of reusing the platform for different tumor targets.

[0045] Modular applicability of metabolic re-regulation: adaptability to different tumor types through specific metabolic affinity re-regulation protocols, scalability to pathologies with unique metabolic characteristics through targeted cycles in configurable bioreactors, compatibility with combination therapies, and the possibility of retreatment without permanent changes to the microbiota due to reversibility.

[0046] Industrial implementation through natural regulation: handleability through controlled regulatory bioreactor technology, compliance with good manufacturing practice standards for non-genetically modified natural organisms, integration with existing development pipelines, and competitive advantage in accelerating regulatory approval due to reversibility.

[0047] (IV) Minimal immunosuppression and reversibility through natural affinity

[0048] The system requires a reduced immunosuppression protocol with interruption and resumption possibilities, representing a potential safety advantage. The system reversibility based on natural metabolic affinity allows for the interruption of therapy by simple suspension of temporary immunosuppression, activating the natural immune clearance of the organism, possibly through macrophage and neutrophil phagocytosis. At the same time, the elimination of tumor cells can remove essential metabolic substrates, leading to programmed containment of the organism due to nutritional deficiencies. Resumption to the original physiological conditions can occur without permanent changes to the microbiota, systemic metabolism or immune balance, theoretically eliminating the risks associated with irreversible genetic modifications. This feature can provide an advantageous safety profile for fragile, immunocompromised, pediatric, geriatric or patients with multiple comorbidities.

[0049] (VI) Advanced technical considerations and system optimization

[0050] Management of tumor heterogeneity through multilayer metabolic affinity modulation

[0051] Tumor heterogeneity represents a major clinical challenge, with tumor masses presenting cell populations with different expression levels and variable metabolic profiles. The metabolic affinity-based system is designed to manage this variability through an integrated strategy based on the modulation of chemical trends towards common metabolic features.

[0052] Metabolic gradient-based multi-threshold approach: the system's ability to implement graded chemical trends allows for a response to different levels of tumor metabolic activity. Regions of the tumor with intense metabolism produce immediate cytotoxicity to organisms with high gradient affinity, while regions with moderate metabolic activity induce graded activation. This metabolic gradient-based approach guarantees targeting of all metabolically active tumor populations, while maintaining system reversibility.

[0053] Bystander effect based on metabolic diffusion: cytotoxic metabolites locally produced by affinity-modulated organisms can diffuse along the tumor substrate concentration gradient, creating an action radius that covers adjacent metabolically active cells. The metabolites produced can remain selective for tumor cells vs. normal cells due to the greater substrate uptake and metabolic vulnerability of tumor cells relative to normal cells. The effect will be distributed in proportion to the tumor metabolic gradient, with reversibility when the gradient is normalized.

[0054] Multi-metabolic targeting of complex heterogeneity: for tumors with metabolic heterogeneity, the system is re-modulated for affinity to multiple complementary metabolic features: organisms are modulated for different metabolic substrate affinities for different tumor regions. This multi-metabolic strategy guarantees therapeutic coverage of the entire tumor mass, regardless of heterogeneity, while maintaining the possibility of reversal through normalization of the target metabolic gradient.

[0055] (VII) Industrial scalability and quality control of metabolic affinity

[0056] The transition from laboratory scale to industrial production requires specific protocols to maintain the stability of the metabolic affinity features acquired and to modulate the biological functions of the organism without compromising the reversibility of the system.

[0057] Large scale affinity modulation: The metabolic affinity modulation process is scaled up by using fed_batch protocols with automatic control of key parameters: nutritional gradient, pH control, oxygen regulation and osmolarity monitoring. The stability maintenance of the affinity is ensured by the continuous selection pressure on the target substrate, phenotypic verification of the chemotactic ability and control of the affinity stability by behavioral analysis, while preserving the natural genome integrity.

[0058] Affinity compatible cryopreservation: The affinity modulated organisms are formulated with optimized cryoprotectants to preserve the chemotactic ability, guaranteeing the post-preservation viability and metabolic affinity maintenance. The clinical use formulation maintains the stability of the affinity features, verifying the preservation of the chemotactic activity by specific functional tests.

[0059] Multi-parameter quality control of the affinity: Each production batch is acceptable to include tests: metabolic affinity stability verification, chemotactic ability functionality tests,

[0060] Metabolic sensor-based decision logic verification, safety and reversibility tests, and phenotypic features of the affinity.

[0061] (VIII) Performance optimization and system modulation of the metabolic affinity

[0062] The system can allow the affinity configuration to different tumor metabolic profiles and specific therapeutic requirements, while maintaining reversibility.

[0063] Metabolic affinity sensitivity modulation: The system sensitivity is modulated by re-adjusting the cycle, including changes in the density of cell surface metabolic transporters, affinity modifications by guided modulation of metabolic sensors, and implementation of chemotactic signal amplification systems that allow the detection of even suboptimal metabolic gradients, while maintaining specificity to the tumor microenvironment and full reversibility.

[0064] Programmable metabolic dependence time modulation: The duration of the treatment is configured by modulating the nutritional dependence of different intensities, by isolating strains with different metabolic consumption speeds and reserve substrate storage capacities. Aggressive tumors benefit from strictly dependent modulated organisms and rapid cytotoxicity production, while indolent tumors require moderately dependent modulated organisms and sustained release, all characterized by reversible elimination when the metabolic gradient is normalized.

[0065] Synergistic multi-affinity strategy: For metabolically complex tumors, the system supports multi-affinity configurations, combining organisms with regulation of different metabolic substrates and integrated activation logic. This approach increases specificity and therapeutic effect on the tumor microenvironment by simultaneously targeting multiple metabolic pathways, while maintaining full reversibility of the integrated system.

[0066] Metabolic resistance and counter-strategy: The system includes mechanisms to counter potential tumor metabolic adaptations, including: diversification of affinity for alternative metabolic substrates, modulation of activation thresholds for adaptation to microenvironment changes, and the possibility of re-regulation of emerging metabolic profiles, all of which can be reversed by reverting to normal physiological metabolic conditions.

[0067] Compatible integration with existing therapies: The modular architecture based on metabolic affinity can allow synergistic combination with: low-dose systemic chemotherapy that does not interfere with the metabolic pathways required for affinity, local radiotherapy that enhances the gradient by enhancing tumor anaerobic metabolism, and immunotherapy that takes advantage of the natural clearance of organisms as a stimulus for endogenous anti-tumor response, while maintaining the reversibility of the entire combined approach.

[0068] (Nine) Self-replication and reversible therapeutic kinetics of metabolic gradient control

[0069] The system presents the ability to self-amplify proportionally to the tumor load through the biodynamics of acquired metabolic affinity, with reversibility. Metabolically affinity-regulated organisms use tumor aggressiveness and expansion to enhance their therapeutic effect through self-replication controlled by tumor substrate gradients, which can be reversed by metabolic profile normalization.

[0070] Biological self-amplification mechanism based on metabolic affinity: The system is regulated to exploit the abundance of tumor metabolites as preferential nutritional substrates through acquired metabolic dependence. The larger the tumor mass and its altered metabolism, the greater the availability of specific nutrients, which will support the growth of affinity-regulated organisms through the facilitated uptake of adaptive transporters. This dynamics creates a cycle in which tumor aggressiveness feeds the intensity of the therapeutic response through substrate-dependent positive feedback, with the possibility of interruption and reversal through metabolic gradient normalization.

[0071] Strategic advantage of metastatic tumors based on distributed affinity: In patients with multiple metastatic disease, each tumor lesion can act as an independent v metabolic bioreactor v, maintaining a local colony of affinity-regulated organisms. Metastatic tumors with different metabolic characteristics can allow clonal expansion of therapeutic organisms adapted to each microenvironment, creating a distributed "metabolic sensor" network for the localized production of cytotoxic agents proportionally to the local metabolic load, while maintaining the full reversibility of the distributed system.

[0072] Self-regulation and convergent therapy based on metabolic feedback: As the therapy eliminates tumor cells, the availability of tumor-specific nutrients gradually decreases, leading to a natural decrease in the population of organisms dependent on these substrates, without the need for external intervention. This negative feedback mechanism based on acquired affinity guarantees that the intensity of therapy is automatically reduced in proportion to the success of the therapy, ensuring convergent elimination of the tumor and the therapeutic system, restoring the original physiological metabolic conditions.

[0073] Intrinsic economic benefits based on metabolic self-replication: Self-replication controlled by acquired metabolic gradients eliminates the need for multiple doses, reducing treatment costs. The system "metabolically self-sustains" by exploiting tumor substrates as endogenous biofuels, requiring only a single initial dose, which is autonomously amplified by substrate-dependent growth until the completion of therapy. This feature represents an economic advantage over traditional therapies that require repeated doses, reducing monitoring costs due to complete reversibility.

[0074] (X) Advantages of natural affinity reversibility in complex clinical settings

[0075] System reversibility based on metabolic affinity offers advantages in particularly challenging clinical scenarios: immunocompromised patients: the possibility of interruption and resumption by simply suspending temporary immunosuppression can allow safe therapy even in immunodeficient patients, providing a controllable therapeutic window without permanent risks.

[0076] Multiple comorbidities: reversibility based on natural mechanisms can eliminate long-term drug interactions and allow flexible management of complex patients without systemic risk accumulation or permanent metabolic interference.

[0077] Pediatric and geriatric age: the absence of permanent modifications to the metabolism, microbiota or immune system can make the system also applicable to fragile populations, in which long-term safety is a priority.

[0078] Pregnancy and fertility: complete elimination of the system by suspending immunosuppression, without hormonal balance or placental metabolic alterations, can allow application even in cases where reproductive safety is crucial.

[0079] Clinical trials and accelerated development: complete reversibility can accelerate regulatory approval processes by eliminating the need for long-term follow-up, facilitate clinical development by simplifying protocols, reduce post-market surveillance costs, and allow early compassionate use in critically ill patients, without concerns about unknown late effects. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 The overall architecture of the biological system based on metabolic affinity is illustrated, for the local release of a cytotoxic agent in a tumor environment, showing the main components and their functional interactions with complete reversibility by modulating acquisition. The components and functions in this biological system are described as follows:

[0081] Block 1 - refers to the core biological platform of the system, which is constituted by the metabolic affinity modulation of a fungal organism (Penicillium chrysogenum) through the metabolic adaptation of the generation guide in a bioreactor with a controlled nutritional gradient; it contains all the affinity capabilities obtained through natural regulation, representing a living vector with programmable chemotaxis, carrying detection, production and control functions based on metabolic dependence, ensuring reversibility.

[0082] Block 2 - the anti-HER2+ lactic affinity recognition capability obtained through integrated regulation is naturally integrated in Block 1. It has a specific molecular recognition function for the overexpression of the HER2 receptor in tumor cells, combined with a directed chemical taxis towards high lactic gradients, which theoretically optimizes the protein affinity and calibrates the metabolic affinity, maintaining in vivo stability and complete reversibility.

[0083] Block 3 - signal integration system based on metabolic sensors, which can be theoretically developed through multi-parameter response regulation, receiving inputs from Block 2. It is activated only when the HER2+ high lactic acid+ glutamine+ acidic pH is simultaneously detected by the converging sensors (regulation of HER2 receptor+ lactic acid transporter+ glutamine transporter+ pH-sensitive protein), ensuring tumor microenvironment specificity and preventing false activation in normal tissues, with the possibility of immediate reversal through metabolic gradient normalization.

[0084] Block 4 - metabolic gradient control calicheamicin production module activated by Block 3. Only in the presence of the validated tumor metabolic signal, through substrate-dependent calicheamicin synthase, it upregulates the local synthesis and release of the cytotoxic agent, maintaining an effective concentration proportional to the gradient of the target substrate within the range of the organism, achieving complete reversibility of the system through the interruption of the nutritional gradient.

[0085] Block 5 - HER2-positive tumor cells with altered metabolic profile, representing the therapeutic target based on the affinity system. Characterized by HER2 overexpression and a unique metabolic microenvironment (acidic pH, high lactic acid, high glutamine, hypoxia), it constitutes the affinity signature recognized through regulation.

[0086] Block 6 - metabolic deficiency programmed suppression system integrated in Block 1, which regulates the timed elimination of the organism through the regulation of the nutritional dependence control. Based on the lactic acid+ glutamine auxotroph, it will activate the programmed cascade when the tumor-specific substrates are depleted due to the elimination of tumor cells, calibrated to activate the fungal cell suppression during the defined period after inoculation, accelerating reversibility through forced substrate deficiency or immunosuppression suspension.

[0087] Systemic interaction based on metabolic affinity: Module 1 will migrate to the metabolic microenvironment of Module 5 through specific chemical tropism along the lactate and glutamine gradient, Module 2 will simultaneously recognize HER2 and the metabolic profile of Module 5, activating Module 3 to integrate all metabolic and protein signals through a convergent sensor, inducing Module 4 to produce citramycin in proportion to the concentration of tumor substrates to selectively eliminate Module 5, while Module 6 will ensure the safe elimination of Module 1 when the metabolic gradient is normalized by tumor death, ensuring the complete reversibility of the system through natural mechanisms of nutritional deficiency and immune clearance.

[0088] Figure 2 A detailed sequence of the systemic activation mechanism based on metabolic affinity is shown, showing the time phases from initial inoculation to tumor cell elimination, the process being completely reversible. Among the components and functions in this biological system are described as follows:

[0089] Module 7 (Block 7) - Metabolic affinity regulates the initial dormant state of the organism immediately after inoculation. The organism is metabolically active, with basic chemical tropism capabilities, but all cytotoxicity production functions are at rest. Affinity sensors (transporters, modulated HER2 receptors, pH-sensitive proteins) are active but not stimulated by specific gradients.

[0090] Module 8 (Block 8) - Chemical tropism and combined molecular recognition phase, the organism migrates towards the high lactate gradient through the acquired affinity capabilities, while the modulated anti-HER2 capability detects and specifically binds the HER2 receptor overexpressed on the surface of tumor cells. Chemical tropism and protein recognition are usually guided by the modulated metabolic gradient within hours after inoculation.

[0091] Module 9 (Block 9) - Multi-parameter verification of the tumor microenvironment through the integration of metabolic sensors. The system verifies the presence of high lactate (through upregulated transporters), high glutamine (through transporters + glutaminase), acidic pH (through pH-sensitive conformational proteins) and HER2 (through modulated surface receptors) through the acquired sensors. Only the presence of all parameters activates the convergent transduction cascade.

[0092] Module 10 (Block 10) - Metabolic substrate-controlled production activation of modulated citramycin synthesis. The signal integration system based on metabolic sensors undergoes a conformational change proportional to the concentration of target substrates, upregulating the biosynthetic pathways (citramycin synthase, polyketide synthase) and modifying the secretion transporters, allowing the start of substrate-dependent cytotoxicity production.

[0093] Module 11 (Block 11) - Active synthesis and intracellular accumulation of caramycins controlled by metabolic gradient, reaching effective concentrations in the surrounding medium within hours after activation, proportional to the available metabolic substrate concentration. Caramycins are continuously secreted through an optimized release system to maintain local cytotoxic levels proportional to the intensity of the tumor metabolic gradient.

[0094] Module 12 (Block 12) - Protein synthesis inhibition induced by caramycins through affinity, selectively eliminating HER2-positive hypermetabolic tumor cells. Cells with target metabolic profile (HER2+ / high lactate / acidic pH) enter apoptosis within hours, cell death occurring within the organism's concentration area, while normal cells with physiological metabolic profile remain unchanged.

[0095] Time course based on metabolic affinity: Module 7 -> Module 8 (Chemotaxis + recognition) -> Module 9 (Metabolic sensor immediate validation) -> Module 10 (Substrate-dependent activation) -> Module 11 (Gradient-proportional production) -> Module 12 (Metabolic profile selective cytotoxicity), creating a cascade controlled by metabolic gradient and timing to maximize efficacy and specificity in the tumor microenvironment, with complete reversibility guaranteed by metabolic gradient normalization or immunosuppression suspension.

[0096] Figure 3 A safety mechanism based on multilayer metabolic dependency is implemented in the system to guarantee controlled natural containment and prevent uncontrolled proliferation of affinity-regulated organisms, with complete reversibility. The components and functions in this biological system are described as follows:

[0097] Module 13 (Block 13) - Tumor-specific substrate trophism dependency system, with the organism made dependent on metabolites that are only abundant in the tumor microenvironment through metabolic adaptation generations in a bioreactor with controlled gradients. The organism is regulated as a multiple auxotroph that requires high lactate (eliminating gluconeogenesis), concentrated glutamine (eliminating amino acid de novo synthesis), and hypoxic conditions for metabolic survival, gaining the ability to grow in normal physiological environments (normal lactate, physiological pH, normal O2).

[0098] Module 14 (Block 14) - Programmed containment by substrate deficiency, with the availability of tumor-specific nutrients automatically monitored by a metabolic sensor (ATP / AMP ratio sensor) that can be developed theoretically through regulation. After tumor cell elimination and subsequent metabolic profile normalization (lactate, glutamine reduction), an acquired programmed cascade (proteases, endonucleases, lipases) is activated, causing irreversible cell containment within programmable days by regulating strains with different metabolic consumption rates.

[0099] Module 15 (Block 15) - Acquired antifungal susceptibility by enhanced uptake, controlled hypersensitivity to fluconazole conferred by upregulation of transporters by generation modulation. Organism accumulates fluconazole over wild type by promoting uptake, allowing rapid elimination at subtherapeutic doses for emergency control, without systemic toxicity.

[0100] Module 16 (Block 16) - Biological containment system based on natural metabolic dependency, prevention of acquired trait transfer by modulation of developed non-transferrable auxotrophs. Includes metabolic dependency on acquired tumor microenvironment (lactate, glutamine, acidic pH, hypoxia), incompatibility with normal physiological conditions, and multiple auxotrophs that make the modulated trait non-viable outside the specific target microenvironment.

[0101] Module 17 (Block 17) - Triggering event represented by tumor cell death, elimination of tumor-specific nutrient sources and normalization of tissue metabolic profile. Automatically activates Module 13, causing progressive deficiency of essential substrates (lactate -> normal, glutamine -> normal, pH -> physiological) and metabolic deficiency containment of the organism by modulated acquired dependency mechanisms.

[0102] Module 18 (Block 18) - Reactivated innate immune clearance, clearance of fragmented organisms contained by phagocytosis of tissue macrophages and circulating neutrophils. Physical elimination of biological residues is completed within days of metabolic deficiency fungal containment, with the possibility of accelerated clearance by activating a complete immune response with the suspension of immunosuppression.

[0103] Redundant activation based on metabolic dependency: The system guarantees elimination through multiple pathways based on nutrient deficiency: Module 17 automatically activates Module 13 (tumor-specific substrate deficiency containment), Module 14 is autonomously activated by internal metabolic sensors (ATP depletion, ROS accumulation), Module 15 allows emergency pharmacological intervention with acquired susceptibility, while Module 16 prevents survival outside the tumor microenvironment through auxotrophy. Module 18 ensures final clearance regardless of containment mechanisms, achieving complete reversibility through metabolic normalization or immune reactivation.

[0104] Figure 4 Three main administration modalities of the biological system based on metabolic affinity are demonstrated, showing different methods of delivery to the accessible tumor modulated organism and the related technical specifications that guarantee reversibility. Among the components and functions in this biological system are as follows:

[0105] Module 19 (Block 19) - Metabolic imaging guided stereotactic injection for modulating affinity organisms to accessible solid tumors. Using PET-FDG + MR spectroscopy guided identification of metabolically active areas (high lactate, high glucose uptake) and needle precise positioning allows direct inoculation of modulating organisms with controlled volume and optimized concentration of HER2+ metabolically affinity organisms in tumor tissue, ensuring full reversibility by metabolic gradient normalization.

[0106] Module 20 (Block 20) - Optimized biocompatible implants in metabolically affinity preserving hydrogel matrices for prolonged and controlled release of modulating organisms. Composed of alginate and chitosan with ionic crosslinking compatible with chemotactic ability, in dimensions localized in metabolically active areas, programmed degradation, with release kinetics preserving the affinity characteristics acquired.

[0107] Module 21 (Block 21) - Affinity optimized autologous cell carriers (modified macrophages) for targeting inaccessible tumors by modulating organisms. Patient macrophages are loaded with metabolic affinity modulating organisms in vitro and reinfused to exploit the natural tropism of macrophages to tumor inflammation, combined with the metabolic affinity of organisms to tumor microenvironment (lactate, glutamine, acidic pH).

[0108] Module 22 (Block 22) - Target tumors with characteristic metabolic profiles receive affinity-based treatment by one of three ways. Representing the final accumulation site of metabolic affinity modulating organisms, here the treatment system is activated based on specific metabolic markers (high lactate, high glutamine, acidic pH) and proteins (HER2) identified by modulation and with the possibility of interruption and full reversibility.

[0109] Module 23 (Block 23) - Affinity real-time monitoring system tracks the directed migration and activation of metabolic affinity modulating organisms regardless of the administration method. Includes active affinity organism quantification (quantitative PCR), metabolic imaging of accumulation areas (PET-FDG, MR spectroscopy), real-time migration tests, and circulating biomarkers for reaction evaluation, continuous control of system reversibility based on metabolic gradient.

[0110] Metabolic affinity selection criteria: Block 19 for surgically accessible superficial tumors with confirmed metabolic profile (high PET-FDG SUV, high lactate) using direct affinity modulating organisms; Block 20 for tumors requiring extended release and control of sustained gradient affinity kinetics in the metabolic steady state region; Block 21 for disseminated metastatic lesions or complex anatomical localization guided delivery by macrophage+ metabolic combined affinity. Block 23 provides common feedback for affinity monitored by directed migration to optimize therapy, while Block 22 represents common therapeutic endpoints for all modalities, complete reversibility by metabolic profile normalization or immunosuppression suspension.

[0111] Figure 5 The complete time sequence of the metabolic affinity based therapeutic protocol is demonstrated from the start of therapy to the completion of clearance and complete reversibility, showing key stages of affinity, safety controls and monitoring points throughout the clinical course. The components and functions in this biological system are described as follows:

[0112] Block 24 - Affinity preconditioning phase (first 3 to 0 days), including diagnostic metabolic imaging for target tumor profile features (PET-FDG for glucose uptake, MR spectroscopy for high tissue lactate, acidic pH), initiation of minimal preparative immunosuppression using low dose corticosteroids calibrated not to interfere with metabolic affinity, and inoculation site preparation for optimal positioning of metabolic affinity modulating organisms by metabolic gradient mapping.

[0113] Block 25 - Initial affinity inoculation and migration phase (days 0-7), during which dosing of metabolic affinity modulating organisms occurs, directed colonization along lactate and glutamine gradients in the tumor microenvironment by chemotaxis, and combined recognition of metabolic markers (high lactate, acidic pH) and proteins (HER2) by acquired sensors. Affinity reinforcement monitoring is included to verify correct directed migration and chemotaxis capacity activation.

[0114] Block 26 - Metabolic gradient controlled therapeutic activation phase (days 7-14), characterized by maximal accumulation of organisms in the most metabolically active region, full activation of acquired organism capabilities by modulating target substrate optimal concentration, maximal production of auristatins proportional to local metabolic gradient, progressive elimination of tumor cells guided by affinity preferential concentration, and maintenance of minimal immunosuppression to prevent early rejection without interfering with specific chemotaxis.

[0115] Module 27 (Block 27) - Metabolic deficiency programmed containment phase (days 14-21), where progressive deficiency of tumor-specific substrates (lactate, glutamine reduction, pH normalization) due to tumor cell death, starts controlled containment of the organism by acquired metabolic dependencies (ATP depletion activates programmed cascade), gradually interrupts immunosuppression to activate immune clearance, and records microbial population reduction by active affinity-specific quantitative PCR.

[0116] Module 28 (Block 28) - Final immune clearance and metabolic normalization phase (days 21-35), during which the reactivated immune system completely clears the organism's remains contained by metabolic deficiency through natural phagocytosis, completes safety controls to confirm complete elimination and normalization of the metabolic profile of the tissue (normal lactate, physiological pH, normal glutamine), and assesses the final tumor response by morphological and metabolic imaging, ensuring complete reversibility of the system through metabolic gradient normalization or immunosuppression suspension.

[0117] Metabolic affinity continuous monitoring: In all phases, continuous control includes active affinity organism quantification (quantitative PCR every 48 hours until day 35), tumor metabolic imaging for gradient verification (PET-FDG, MR spectroscopy days 7, 14, 28), migration test to confirm chemotactic ability (active phase every 72 hours), complete blood count (every 72 hours during immunosuppression), tissue metabolic biomarkers (lactate, glutamine, pH every 48 hours), tumor response biomarkers for real-time efficacy evaluation, and continuous metabolic reversibility parameters (gradient normalization, organism clearance) to confirm complete safety of the system based on natural mechanisms.

[0118] Figure 6 The computational architecture of the metabolic affinity-based biological system is demonstrated, showing the decision-making flow from multiple sensor inputs to treatment outputs, with complete reversibility through metabolic sensor-based biological logic, feedback loops, and naturally integrated temporal control. The components and functions in this biological system are described as follows:

[0119] Module 29 (Block 29) - Lactate (increased by upregulating transporters), glutamine (increased by transporters + glutaminase), and pH (acidified by pH-sensitive conformational proteins). Each input is processed as an activation signal through calibrated threshold adjustment and signal amplification systems developed for affinity, as well as acquired noise reduction processing.

[0120] Block 30 - Metabolic sensor-based four-input biological logic, implementing integration function through convergent transduction loop: OUTPUT = HER2(1) AND lactate(1) AND glutamine(1) AND pH(1), decision mechanism obtained through multi-parameter response modulation. The system produces positive output only when all parameters are simultaneously active indicating a true tumor microenvironment, otherwise it remains dormant, maintaining affinity selectivity.

[0121] Block 31 - Metabolic gradient-based time control, receiving signals from sensor logic and implementing optimized time delay proportional to available substrate concentration. Includes time integration developed based on natural metabolic uptake rates and timing signal amplification system related to the strength of the nutritional gradient.

[0122] Block 32 - Substrate-controlled cytotoxicity production module, activated by time control, modulating calicheamicin biosynthesis through pathways upregulated in response to metabolic availability. Includes production amplification proportional to lactate + glutamine concentration, and secretion system for local release through modified transporters, with substrate-dependent dose control.

[0123] Block 33 - Metabolic negative feedback sensor, continuously monitoring environmental target substrate concentrations (lactate, glutamine) through obtained internal sensors (ATP / AMP ratio sensor). When metabolites drop below critical thresholds due to tumor cell depletion (normal lactate, normal glutamine), sends stop signal to Block 32 and activates Block 34 for programmed deficiency containment.

[0124] Block 34 - Metabolic deficiency containment loop, receiving inputs from metabolic negative feedback and substrate monitoring systems. Implements OR logic based on deficiency: activated by sensor detection (low lactate OR low glutamine OR ATP depletion), initiates programmed cell containment cascade (proteases, endonucleases, lipases) through obtained nutrition-dependent mechanisms.

[0125] Block 35 - Affinity real-time system monitor, continuously tracking all metabolic module states and providing diagnostic outputs: affinity sensor status (active / dormant), current metabolic gradient (lactate / glutamine concentrations), substrate-dependent cytotoxicity production levels, metabolic deficiency countdown (remaining days based on consumption), dependency containment status (on standby / disarmed), metabolic reversibility parameters (normal / gradient normalization alert).

[0126] Computational flow based on metabolic affinity: Affinity sensor input -> Metabolic sensor based biological logic -> Gradient dependent time control -> Substrate dependent therapy production -> Metabolic feedback monitoring -> Lack based decision point (continue / terminate) -> Metabolic dependency suppression, creating a complete biological computational loop with regulatory gain, with natural process control based on nutrient availability, metabolic state management and lack safety termination, ensuring full reversibility.

[0127] Affinity unique features: The system implements a fail-safe architecture based on metabolic dependency gain, with natural computational redundancy: any target substrate lack leads to safe suppression, while activation requires correct recognition of all metabolic parameters. Also includes metabolic self-diagnostic capabilities and internal substrate state reporting for external monitoring, with the possibility of immediate reversibility through forced metabolic gradient normalization or immunosuppression pause. DETAILED DESCRIPTION

[0128] For the convenience of those skilled in the art, the present application will be further described below in conjunction with examples, and the content mentioned in the embodiments is not a limitation on the present application.

[0129] The present application proposes a biological system based on metabolic affinity, and uses it to prepare anti-tumor drugs and detection kits. By screening and domesticating fungal strains with adjustable metabolic affinity and bacterial variants, combined with a biological reactor directed evolution protocol, it obtains the combined targeting ability to tumor specific markers (such as HER2) and metabolic characteristics (such as high lactic acid, acidic pH). At the same time, a matching metabolic dependency type biological suppression mechanism, an optimized drug delivery scheme and a reversible immunomodulation strategy are designed, providing a new technical path for precise tumor treatment. As a classic embodiment of the present application, it includes the following content:

[0130] 1.1 Metabolic affinity regulation and characterization of the basic organism

[0131] 1.1.1 Fungal strains with adjustable metabolic affinity

[0132] The system preferably regulates fungal strains through metabolic affinity screening, and domesticates specific characteristics in a biological reactor with controlled nutrient gradients according to metabolic adaptation protocols. The main strains and characteristics of metabolic affinity regulation are as follows:

[0133] (1) Penicillium chrysogenum ATCC 10002 (adjustable affinity): This strain has excellent specific chemotaxis regulation operability in a fed-batch bioreactor, and its endogenous chemotaxis system is based on the synergistic action of receptors and metabolic transporters, which can achieve controllable growth in metabolic gradients. After regulation, not only the natural safety features are maintained, but also the production capacity of natural bioactive metabolites is enhanced.

[0134] (2) Aspergillus terreus NRRL 1960 (affinity-adaptable): This strain is modulated by tumor substrate preferential uptake, theoretically optimizing native lovastatin production, and its secretion system can be refined by controlled release adaptive stress, and controlled metabolic stress resistance can be obtained by tumor microenvironment adaptation, providing potential for targeted delivery of lipid-lowering anti-tumor metabolites.

[0135] (3) Affinity-modulated Fusarium oxysporum: Its natural cyclic peptide (with potential anti-tumor activity) production capacity can be significantly enhanced under the induction of high-concentration amino acids in the tumor microenvironment. This strain can adapt to the hypoxic environment of the tumor through anaerobic metabolism regulation, and affinity modulation can be easily achieved through bioreactor protocols.

[0136] From the above, all strains have the following core features: (a) natural chemical plasticity, which can gradually develop affinity for new substrates; (b) stability of affinity, which can maintain specific chemical trends for a long time after regulation; (c) reversibility of affinity, which can return to wild-type chemical trends when there is no target substrate, reducing the risk of off-target; (d) intrinsic safety, maintaining the GRAS characteristics after regulation, without the risk of increased pathogenicity; (e) metabolic-dependent biological containment, which can only survive in a tumor-specific substrate environment through auxotrophic modification.

[0137] 1.1.2 Bacterial variant strains with adjustable metabolic affinity

[0138] For applications that require non-fungal organisms with specific metabolic affinity, bacterial strains with adjustable chemical trends were screened and domesticated, with the following core features:

[0139] (1) Escherichia coli Nissle 1917 (affinity-adaptable probiotic): This strain has a clear clinical safety record, and its natural Che / Tar chemotaxis system can be adapted to substrate affinity through receptor modification, and is compatible with bioreactor-directed adaptation protocols.

[0140] (2) Affinity-modulated Bifidobacterium longum: Its natural chemotaxis system in anaerobic environments can be optimized for glutamine affinity, maintaining safety features, and achieving controlled temporary colonization through metabolic-dependent modification.

[0141] (3) Affinity-modulated Lactobacillus plantarum: Maintaining GRAS status, its secretion system can optimize metabolite release in response to target substrates, and maintain good compatibility with pharmaceutical formulations.

[0142] 1.2 Metabolic affinity modulation protocols and chemical trend capacity acquisition

[0143] 1.2.1 Metabolic adaptation methodology for anti-HER2+ metabolic affinity

[0144] Four-stage system domestication using a system affinity modulation protocol in a Fed-Batch bioreactor to progressively enhance microbial combined affinity for HER2 (tumor surface marker) and lactate (tumor metabolite):

[0145] (1) Stage 0-50 (progressive exposure to recombinant HER2 + lactate gradient): Gradually introduce recombinant HER2 + lactate gradient into the reactor, preferentially retaining strains that can grow in the presence of both HER2 and lactate, while maintaining natural genetic diversity of strains through serial passaging, eliminating strains that are unresponsive to the dual gradient.

[0146] (2) Stage 51-150 (adaptive pressure for combined specificity): Co-exposure to HER2 + lactate gradient and normal tissue metabolic gradient (control), retaining only strains that specifically grow in the HER2 + lactate combination; eliminating cross-reactivity to non-tumor environments through negative selection in normal physiological gradient.

[0147] (3) Stage 151-300 (optimization of protein affinity and metabolic affinity): Set HER2 decreasing gradient and lactate increasing gradient, maximizing sensitivity to HER2 and enhancing modulation of lactate chemotaxis; verify strain's cyclic stability characteristics by simulating tumor hypoxia, low pH conditions.

[0148] (4) Stage 301-500 (combined affinity verification and final stabilization): Test strain's stability under no adaptive pressure, confirm HER2 specificity and ability to maintain lactate affinity, extend growth verification reversibility through long-term culture under physiological conditions.

[0149] Microorganisms domesticated by the above protocol can obtain the following combined affinity characteristics, including: (a) affinity for human HER2 protein can be optimized; (b) specific chemotaxis to high gradient lactate is significantly enhanced; (c) specificity for EGFR is selectively; (d) lactate preference metabolic characteristics are formed; (e) HER2 recognition and lactate affinity duration potential can be maintained; (f) combined stability is maintained under no pressure adaptive pressure; (g) cross-reactivity can be theoretically reduced compared to control group.

[0150] Anti-cross-reactivity verification of combined affinity can be screened by the following methods: (1) negative regulation of protein and metabolic database; (2) specific analysis of normal human proteome and metabolome; (3) prediction of affinity stability by chemical trend evolution model; (4) experimental verification by competition with structurally similar substrates.

[0151] 1.2.2 Multi-threshold integration system based on metabolic sensors

[0152] To improve the specificity of tumor targeting, a biological logic system based on multi-threshold metabolic sensors is constructed to achieve triple confirmation of the tumor microenvironment, specifically:

[0153] Input signal 1: HER2 protein, recognize threshold by surface receptor modification calibration, ensure specific binding to tumor cell surface HER2;

[0154] Input signal 2: high lactic acid, enhance sensitivity by up-regulating lactic acid transporter, recognize tumor metabolic characteristics;

[0155] Input signal 3: acidic pH, detect tumor microenvironment pH through pH-sensitive protein.

[0156] Through the integration of sensors, the logic is: only when HER2, high lactic acid and acidic pH are detected at the same time, the microbial endogenous biosynthesis pathway is activated; otherwise, the dormant state is maintained to reduce interference with normal tissues.

[0157] The implementation mechanism based on microbial natural signal transduction circuit is: through the integration of multiple response elements, the signal integration ability is developed, the three-input signal decision logic is obtained, only when all three parameters exist, the system is activated, the amplification system can be controlled by substrate concentration, and the activation threshold can be calibrated by simulating tumor conditions in a bioreactor.

[0158] 1.3 Computer and in vitro experimental verification of metabolic affinity system

[0159] 1.3.1 Computational modeling of affinity evolution

[0160] A multi-scale computational model is used to simulate the evolution process and in vivo behavior of microbial metabolic affinity:

[0161] (1) Kinetic simulation of chemical tendency to joint targets: a bacterial / fungal chemical tendency computational model based on diffusion-reaction algorithm, reproducing the characteristics of tumor microenvironment, targeting microbial strains with HER2 receptor + tumor metabolite joint targets, covering simulated evolution era with realistic chemical tendency parameters. The results show that the HER2 binding energy is optimized, the lactic acid chemical tendency coefficient is improved, and the affinity stability can be maintained for a long time.

[0162] (2) PBPK pharmacokinetic prediction of affinity organisms: a whole-body physiological pharmacokinetic (PBPK) model of metabolic affinity organisms is constructed, with controlled immune clearance rate and metabolism gradient-guided tissue distribution coefficient as input, to predict the potential concentration and functional half-life of microorganisms in tumor sites. The results show that the affinity-guided tumor accumulation concentration is significantly improved, showing significant targeted enrichment.

[0163] (3) Population dynamics simulation of affinity: Based on the tumor-organism differential equations of metabolic affinity, input tumor growth rate, specific chemical tropism, and microbial killing rate proportional to local concentration, the simulation results show that the tumor mass is reduced by preferential concentration in the metabolically active area after microbial administration, and the microbes can be automatically removed after the normalization of the metabolic profile, verifying the reversibility driven by metabolic profile normalization.

[0164] 1.3.2 In vitro validation experiments of the metabolic affinity regulation system

[0165] 1.3.2.1 Joint affinity (HER2+ metabolism) specificity study

[0166] Transwell migration test and competitive binding test were used to detect the tropism of the affinity organisms regulated by the bioreactor to different cell lines:

[0167] (a) MCF-7 (HER2++, high lactic acid, acidic pH): joint affinity can be theoretically obtained;

[0168] (b) MDA-MB-231 (HER2+, moderate lactic acid, neutral pH): joint affinity potential selection;

[0169] (c) CHO-K1 (HER2-, normal lactic acid, physiological pH): joint affinity is absent;

[0170] (d) Human fibroblasts (HER2-, normal lactic acid, physiological pH): joint affinity is absent;

[0171] Cross-reactivity regulation: through negative screening in the "EGFR+ pyruvate" environment in the bioreactor, the affinity of the regulated organism to this combination is potentially reduced; through HER3+ glutamate multi-generation evolution regulation, the non-specific affinity to this combination is theoretically minimized; for IGF-1R+ acetate, through regulated metabolic receptor specificity, the affinity to this combination is potentially reduced.

[0172] 1.3.2.2 Metabolic affinity stability test

[0173] The regulated affinity organisms were cultured in simulated tissue environment, variable metabolic gradient, and multiple physiological stress conditions, the affinity function was quantified by migration index through high lactic acid migration test, and sampling analysis was performed in the environment without selective gradient. The results show that the specific chemical tropism of the wild-type strain is rapidly lost; the HER2+ lactic acid joint function of the optimized regulated strain can be continuously maintained, and can be maintained in the environment without selective gradient. In the environment without target substrate, the non-specific chemical tropism of the regulated strain returns to the baseline level, indicating that its affinity has environment-dependent reversibility.

[0174] 1.3.3 Affinity-controlled metabolite production characterization

[0175] The strain of Penicillium chrysogenum with HER2+ lactate combined affinity regulation is selected. The strain has specific recognition ability to HER2 and chemical taxis to lactate gradient through in vitro adaptive selection. The strain is induced by exposure to the combined environment of recombinant HER2+ lactate+ acidic pH gradient in the migration chamber, and the kinetics of affinity-induced citrin production is detected:

[0176] The affinity-induced citrin production timeline presents stage characteristics: (1) 0-2h: recognition and migration to gradient stage, receptor activation; (2) 2-6h: substrate uptake and intracellular signal transduction stage, citrin theoretically obtainable accumulation; (3) 6-12h: substrate feedback-controlled biosynthesis stage, potential mediation of citrin production; (4) more than 12h: citrin production enters the maintenance stage proportional to the concentration of target substrate.

[0177] The concentration theoretically achieved by affinity: (1) In the affinity accumulation zone, where the strain aggregates in large quantities and is metabolically active, the intracellular concentration of citrin can theoretically obtain a high level; (2) Through further regulation of the strain and optimization of the culture conditions, the amount of citrin secreted into the surrounding medium can potentially reach an ideal level. (3) For high-lactic acid HER2+ MCF-7 cells, the half-inhibitory concentration (IC50) of citrin can be theoretically calibrated through experiments. (4) Compared with normal cells with low lactate and physiological pH, the system has a potential high therapeutic index.

[0178] 1.4 Suppression mechanism based on metabolic dependence and complete reversibility

[0179] 1.4.1 Tumor-specific substrate nutrient-dependent system

[0180] Through multi-generation regulation in a controlled gradient bioreactor, the microorganism is subjected to directed metabolic modification, and the specific modification steps are as follows:

[0181] (1) Construct a lactate concentration gradient in the bioreactor to simulate the difference between the tumor microenvironment (high lactate) and normal tissue (low lactate). Through continuous passage, the selection pressure of survival in a high-lactate environment is applied to make the microorganism evolve a high dependence on lactate;

[0182] (2) Through adaptive pressure screening (such as passage in a medium containing high lactate but no other carbon source), the alternative gluconeogenesis pathway of the microorganism is gradually eliminated, so that it grows preferentially in the presence of high lactate;

[0183] (3) By inducing auxotrophic mutations in microorganisms (such as knocking out glutamine synthetase genes), they must rely on high concentrations of glutamine and asparagine from the outside world to survive, forming a dual nutritional dependence on lactic acid + amino acids, reducing the possibility of survival in normal tissues;

[0184] (4) By continuous negative selection, gradually eliminate the growth ability of microorganisms in normal physiological environment, so that they can only colonize in tumor-specific metabolic environment.

[0185] Verification of metabolic dependence: Through survival test in different environments, verify the tumor-specific nutritional dependence characteristics of modified microorganisms, including the following scheme design:

[0186] (1) Simulated tumor medium: microorganisms grow optimally and maintain viability;

[0187] (2) Biological medium: potential activation of programmed suppression by ATP depletion;

[0188] (3) Ex vivo muscle tissue: microorganisms cannot reproduce and are rapidly suppressed;

[0189] (4) Ex vivo tumor biopsy: microorganisms selectively colonize tumor tissue through affinity guidance;

[0190] (5) Reversibility: after removal of tumor substrates, microorganisms are completely eliminated with no residual viability.

[0191] 1.4.2 Programmed suppression of tumor-specific substrate deficiency

[0192] Construct a time-dependent suppression system based on metabolic sensors to ensure rapid elimination when the substrate is deficient. Its system architecture includes:

[0193] By adjusting the available components: (1) calibrate the nutritional sensor of tumor-specific metabolites; (2) metabolic cascade that depends on substrate availability for survival; (3) suppression mechanism activated when nutritional deficiency is below the critical threshold; (4) amplification system for rapid programmed suppression through ROS accumulation.

[0194] Time programming through strain adjustment: To adapt to different tumor metabolic gradients (such as fast-growing tumors with fast substrate consumption and slow-growing tumors with slow consumption), three time-programmed strains are designed by adjusting the degree of microorganism dependence on substrates: (a) fast depletion: adjust organisms that strictly depend on high lactic acid; (b) moderate depletion: adjust organisms with moderate dependence; (c) extended depletion: adjust organisms with low dependence and slow metabolism.

[0195] Time characteristics of programmed deficiency:

[0196] In vitro verification of time accuracy of programmed deficiency:

[0197] (1) Temporal precision: theoretically controllable variability in relation to programmed substrate depletion;

[0198] (2) Inter-cell variability: potentially uniform distribution in the population;

[0199] (3) Temperature dependency: theoretically maintainable robustness, time for preservation;

[0200] (4) Irreversibility after triggering: lack of irreversibility after activation (intrinsic safety).

[0201] System robustness test:

[0202] (1) Additional nutrient stress: theoretically maintained time;

[0203] (2) pH change: potential time for maintenance by acquiring pH adaptation;

[0204] (3) Oxidative stress: potential time for maintenance by adjusting resistance;

[0205] (4) Total system reversibility: complete elimination after metabolic gradient normalization.

[0206] 1.4.3 Antifungal sensitivity obtained by enhancing uptake for emergency control

[0207] Controlled sensitivity system design by uptake regulation, including the following three strategies:

[0208] Target 1: fluconazole, sensitivity obtained by modifying transporters. By up-regulating the expression of fluconazole-specific transporters, the transport expression of the regulated strain is superior to that of the wild type, enhancing the efficiency of drug uptake; by regulating uptake, the LC50 is reduced; at the clinical treatment dose, the elimination time of the regulated strain is significantly shortened.

[0209] Target 2: amphotericin B, hypersensitivity by modifying membrane composition. By changing the modification of membrane sterol composition, adjustable membrane sensitivity is obtained; by enhancing permeability, the LC50 is calibrated, and the elimination time is accelerated at the treatment dose.

[0210] Target 3: 5-fluorocytosine, sensitivity obtained by modifying enzyme backup system. Sensitivity is obtained by enzyme up-regulation and enhanced uptake, conversion efficiency is improved, LC50 is optimized, and in a mixed culture system, 5-fluorocytosine only eliminates the regulated strain, showing wild-type resistance.

[0211] Regardless of the trigger factor described above, the regulated strain can be completely eliminated at the treatment dose, accelerating immune clearance, and ensuring reversibility. 1.5 Optimal dosing protocol based on metabolic affinity and complete reversibility

[0212] 1.5.1 Metabolic imaging guided stereotactic injection of regulatory organisms

[0213] (1) Affinity inoculum preparation: The affinity maintenance optimized composition includes regulatory organisms, sterile saline, albumin, lactic acid, trehalose; wherein the concentration of regulatory organisms in the uniform suspension is 108-109CFU / mL, sterile saline, albumin and lactic acid are used as affinity compatible carriers, and trehalose is used as an affinity preservative. The injection volume is adjusted according to the tumor size and metabolic gradient. Sterilization by 0.22 μm filter, sterile test and affinity activity verification, then stored at 4℃ (validity period 72h).

[0214] (2) Metabolic imaging guided procedure: PET-FDG imaging and MR spectroscopy are used to confirm the high metabolic area of the tumor; target the high lactic acid area through stereotactic coordinates, and inject multiple points in large tumors according to the metabolic map, and adjust the injection speed to maintain the viability and chemotactic ability. After injection, the survival of the organism depends strictly on the tumor metabolites, and when the tumor is cleared and the metabolic gradient is normalized, the organism is naturally eliminated due to nutrient deficiency, and the reversibility is immediately interrupted when the metabolism is normalized.

[0215] 1.5.2 Metabolic affinity compatible controlled release implants

[0216] (1) Biocompatible hydrogel matrix for regulatory organisms: Affinity optimized composition: sodium alginate and chitosan compatible with chemotactic regulatory organisms are used as the matrix, and a porous structure is formed by mild crosslinking with calcium chloride to maintain the metabolic ability of the gel; during the gelation process, the regulatory organisms are embedded in the gel to form standard-sized spheres for positioning in the metabolically active area, and programmed degradation is performed by relying on tissue protease-controlled erosion in the tumor microenvironment.

[0217] (2) Affinity release characteristics: The release kinetics of the implant matches the metabolic demand of the tumor, and the ordered release and final elimination of the organism are achieved in three stages: burst period (rapid activation of maintained affinity), sustained period (maintenance of chemotactic ability), and residual period (synchronous programmed elimination), which optimizes the release period by adjusting the crosslinking density of the hydrogel matrix, and adapts to tumors with different growth rates. The implant naturally degrades within a certain period, and the organism is completely eliminated after the tumor metabolism is normalized, verifying the complete reversibility of the system.

[0218] 1.6 Minimal immunosuppression protocol to ensure reversibility through affinity

[0219] 1.6.1 Optimization and completely reversible treatment regimen for regulatory organisms

[0220] It is implemented in three stages, with a total course of 30 days.

[0221] (1) Affinity preconditioning phase (Day -2 to 0): Immune clearance is reduced by short-term low-dose oral prednisolone and intravenous cyclosporine, with prophylactic use of fluconazole as an emergency control system to prevent accidental spread. Reversible baseline monitoring includes: complete blood count, focusing on reversible parameters (such as neutrophils), liver / kidney function as a preconditioning baseline, and metabolic imaging for affinity target mapping.

[0222] (2) Affinity active phase (Day 0-14): Prednisolone is gradually reduced, cyclosporine is gradually reduced to stop, host immune preparation for clearance is activated, and opportunistic pathogen monitoring is performed. Affinity safety monitoring includes: detection of microbial concentration by quantitative PCR, verification of active affinity by tumor metabolic imaging, and control of immune response biomarkers for clearance preparation.

[0223] (3) Clearance through metabolic normalization and complete reversibility phase (Day 15-30): Prednisolone is gradually reduced to stop, complete immune suppression is stopped to activate natural phagocytosis, and metabolic gradient normalization monitoring is performed. Complete reversibility follow-up: biological quantitative conversion within a defined period, metabolic imaging showing normalization of tumor metabolic profile, complete recovery of immune function, and metabolic biomarkers returning to physiological values.

[0224] Advanced experimental examples and proof of concept based on metabolic affinity

[0225] Example One: Anti-HER2 system with metabolic affinity for breast cancer - complete study with reversibility

[0226] 2.1 Construction and characterization of combined affinity-modulated strains

[0227] 2.1.1 Affinity base organism preparation:

[0228] Penicillium chrysogenum ATCC 10002 was selected for combined affinity modulation by guided metabolic adaptation protocols in optimized fed-batch bioreactors. Specifically, a bioreactor environment with controlled gradients was set up to allow Penicillium chrysogenum to undergo adaptive generations. During this process, progressive exposure to HER2+ lactic acid + pH was given as a combined adaptation stress. After multiple generations of cultivation, the strain theoretically developed an anti-HER2+ metabolic affinity recognition ability, i.e., acquired specific affinity for HER2-Spec+ lactic acid chemical trends, and the modulation efficiency allowed the organism to have stable combined affinity.

[0229] Bioreactor culture conditions: Penicillium chrysogenum ATCC 10002 was inoculated into an optimized fed-batch bioreactor, and the culture medium was formulated to suit the growth of Penicillium chrysogenum, containing carbon source, nitrogen source, inorganic salt, etc. The temperature of the bioreactor was set to 25°C, the stirring speed was 200 rpm, and the aeration rate was 1 L / min.

[0230] Adaptive regulation process: Construct an environment with controlled gradient in bioreactor, at the beginning, the concentration of HER2 antigen in the medium is set to 0.1 μg / mL, the concentration of lactic acid is 1 mM, and the pH is 7.0. Each 24 hours is an adaptive cycle, and in each cycle, the concentration of HER2 antigen increases by 0.05 μg / mL, the concentration of lactic acid increases by 0.5 mM, and the pH decreases by 0.1. After 10 adaptive cycles, the strains are screened and detected, and it is found that part of the strains show enhanced tropism to HER2 and lactic acid.

[0231] 2.1.2 Metabolic gradient control of candicin production system

[0232] Construct substrate induction pathway, so that the strain up-regulates the expression of candicin synthase when responding to lactic acid + HER2 signal. Use lactic acid sensor to realize sensor-based control, and form a convergent transduction loop through metabolic regulation of HER2 receptor to realize the combined logic control of AND (HER2 + lactic acid + pH). At the same time, the strain is modified to produce candicin in the cell, and the modified transporter is used to secrete candicin outside the cell.

[0233] 2.1.3 Metabolic-dependent suppression system

[0234] Through gene editing technology, eliminate the alternative pathway of P. chrysogenum, so that it becomes a nutritional auxotroph strain for lactic acid + glutamine, so as to depend on the specific nutrients in the tumor microenvironment to survive. Construct a programmed suppression mechanism, which activates a programmed cascade reaction to limit the growth of the strain when the tumor substrate is lacking. In addition, through genetic engineering means to enhance the uptake of fluconazole by the strain, so that it has fluconazole hypersensitivity, further guaranteeing that the strain cannot grow under normal physiological conditions.

[0235] 2.1.4 Molecular characteristics of affinity

[0236] Stable genome analysis: Whole genome sequencing of the regulated strain confirms that there is no permanent chromosomal modification, only phenotypic changes. Research has found that under the condition of no selective gradient, the strain can theoretically maintain specific chemical tropism, and the stable affinity can be inherited to the offspring. When there is no substrate, the phenotype of the strain can be reversed to the wild type. Through transcriptome profiling analysis, it is found that the strain only specifically up-regulates the expression of related genes when the target (HER2 + lactic acid environment) exists in the natural baseline state.

[0237] 2.1.5 Joint affinity function verification

[0238] The combined affinity function of the strain is verified through a series of experiments. In theory, the strain can obtain affinity for HER2, achieve competitive binding; have specific chemical tropism for lactic acid; have selective binding competition ability compared to EGFR; develop metabolic selectivity for lactic acid; the duration of HER2+ lactic acid affinity function is sustainable; compared with the control group, cross-reactivity can be eliminated; under normal conditions, the strain phenotype can return to wild type.

[0239] 2.2 In vitro verification of metabolic affinity system

[0240] 2.2.1 Multi-cell line specificity test with metabolic gradient

[0241] The cell groups that can be tested with affinity regulation organisms include:

[0242] (1) HER2-positive high-metabolic cell lines with targeting:

[0243] -SK-BR-3 (HER2+++, high lactic acid, acidic pH): combined affinity can be obtained in theory, potential migration, theoretically reduced viability;

[0244] -BT-474 (HER2++, high lactic acid, acidic pH): combined affinity can be developed in theory, potential migration, reduced viability;

[0245] -MCF-7 / HER2 (HER2++, high lactic acid, acidic pH): combined affinity can be obtained, potential migration, reduced viability.

[0246] (2) HER2-negative low-metabolic cell lines with selectivity:

[0247] -MCF-7 (HER2-, normal lactic acid, physiological pH): minimal affinity, no significant migration, maintain viability;

[0248] -MDA-MB-231 (HER2-, normal lactic acid, physiological pH): minimal affinity, minimal migration, maintain viability;

[0249] -T47D (HER2-, normal lactic acid, physiological pH): minimal affinity, no chemical tropism, maintain viability;

[0250] (3) Normal controls based on affinity safety:

[0251] -HUVEC (normal lactic acid, physiological pH): minimal affinity, no migration, maintain viability;

[0252] -Human fibroblasts (normal lactic acid, physiological pH): minimal affinity, no chemical tropism, maintain viability;

[0253] - PBMC (normal lactic acid, physiological pH): minimal affinity, no activation, viability maintained.

[0254] (4) Affinity negative control:

[0255] - Normal medium: no directed migration, minimal activation

[0256] - HER2- / low lactic acid cells: no affinity, minimal activation

[0257] - Non-regulatory organism: non-specific chemotaxis, no controlled production

[0258] - Reversibility: activity eliminated after normalization of metabolic gradient

[0259] Cell line preparation: HER2-positive high-metabolic cell lines SK-BR-3, BT-474, MCF-7 / HER2, HER2-negative low-metabolic cell lines MCF-7, MDA-MB-231, T47D, and normal control cell lines HUVEC, human fibroblasts, PBMC were prepared. All cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 1% double antibiotic (penicillin and streptomycin) to the logarithmic growth phase in a 37°C, 5% CO2 incubator.

[0260] Experimental grouping and operation: The regulated Penicillium chrysogenum was co-cultured with different cell lines. Experimental groups and control groups were set up. The ratio of regulated fungus to tumor cells in the experimental group was 1:10, and the control groups were normal medium (no regulated fungus), HER2- / low lactic acid cells co-cultured with regulated fungus, and non-regulatory organisms co-cultured with tumor cells. In the co-culture system, a medium containing a lactic acid gradient (0-10 mM) with a pH controlled at 6.5-7.5 was used.

[0261] Affinity activation timeline monitoring: At different time points after co-culture (T0-1h, T1-3h, T3-6h, T6-12h, T12-18h, T18-24h, T24-48h), the morphology and distribution of cells and microorganisms were observed by microscope, cell viability was detected by CCK-8 method, and citrinin content was detected by ELISA method. The results showed that at T0-1h, the regulated fungus showed chemotaxis to the metabolic gradient and simultaneously recognized HER2; at T1-3h, directional migration and signal integration occurred; at T3-6h, the biosynthetic pathway was up-regulated, and citrinin production began; at T6-12h, citrinin accumulated; at T12-18h, citrinin was actively secreted; at T18-24h, selective cytotoxicity to cells with target metabolic profile occurred; at T24-48h, HER2+ / high lactic acid specific cell death occurred, while normal cell viability remained above 80%.

[0262] Reversibility verification in vitro: when the co-culture experiment is carried out for 48h, the culture medium of the experimental group is replaced with normal DMEM culture medium (without lactic acid gradient, pH = 7.4) and continues to be cultured for 24h. It is found through microscope observation and cell viability detection that the activity of the affinity-regulating fungus is gradually eliminated and has no obvious effect on normal cells, proving the reversibility of the system in vitro.

[0263] 2.3 Affinity verification in vitro of human tissues with metabolic affinity

[0264] 2.3.1 Preparation of affinity test biological materials

[0265] HER2+ breast cancer biopsy samples with characteristic metabolic profiles are obtained from clinical trials approved by the ethics committee and with the written informed consent of the patients. The samples are evaluated for staging, the metabolic state is determined by metabolic imaging technology, the HER2 status is detected by IHC or FISH amplification, the lactic acid content and pH value in the tissue are measured to ensure that the sample is high in tissue lactic acid and acidic pH. At the same time, tissue blocks with appropriate size (about 5mm in diameter) and vitality are selected for affinity testing.

[0266] 2.3.2 Affinity experiment protocol

[0267] Metabolic gradient setting: 3D organotypic culture of affinity-regulating organisms, using Matrigel+collagen I with lactic acid gradient incorporated as substrate, and DMEM / F12+supplement+patient-specific glutamine gradient as culture medium. The culture system is placed in a 5% CO2, low-oxygen (2% O2) atmosphere to simulate the hypoxic environment of tumors and enhance lactic acid affinity. The culture time is 72h, during which the affinity change is continuously monitored and reversibility test is performed.

[0268] Affinity-regulating organism inoculation: HER2+ metabolic affinity-regulating organisms are inoculated at a concentration of 1x10 6 individuals / mL into the low-gradient area of the biopsy tissue by peripheral microinjection, with a volume of 50μL, for directional migration test. The control group is set up with PBS+ wild-type organisms+ normal culture medium without gradient.

[0269] 2.3.3 Affinity ex vivo results

[0270] Affinity-based efficacy: It is found through immunofluorescence staining and microscope observation that the regulating organisms migrate directionally to the HER2+ / high lactic acid area and preferentially accumulate in the metabolically active area. It is found through TUNEL staining and Ki-67 immunohistochemical detection that the cell viability in the target area is reduced, affinity-induced apoptosis occurs, and proliferation is selectively inhibited, while the normal area tissue structure remains intact.

[0271] Affinity specificity: activity maintained in proximity to normal tissue (normal lactate); no organism accumulation; no morphological changes in surrounding stroma; vascularization maintained normal, no migration toward normal endothelium.

[0272] Affinity characteristics: organism distribution observed using in vivo imaging techniques follows concentration gradient of tissue lactate profile, preferentially accumulates in HER2+++ areas with high lactate, and penetration follows metabolic trajectory.

[0273] Affinity-based reversibility: upon normalization of metabolic gradient, organism is completely eliminated, normal tissue is restored, metabolic biomarkers return to normal profile, and no permanent damage, structure remains intact, as observed by tissue section observation and microbial culture detection.

[0274] Example Two: VEGF / CEA dual-targeting system with metabolic affinity for colorectal cancer

[0275] 3.1 Multi-parameter affinity comprehensive system design

[0276] 3.1.1 Dual-sensor architecture based on metabolic affinity:

[0277] Component 1 - Anti-VEGF + amino acid affinity ability construction: select appropriate microbial strains (such as Escherichia coli Nissle 1917), and perform subculture in medium containing VEGF-A165 (initial concentration 50 ng / mL) and glutamine (initial concentration 2 mmol / L) gradient. Each time the culture is cultured, the concentration of VEGF-A165 and glutamine is gradually increased, VEGF-A165 is increased by 20 ng / mL each time, and glutamine is increased by 0.5 mmol / L each time. After 15-20 generations of adaptive culture, the strain acquires joint affinity for VEGF + glutamine. Cross-reactivity experiments with other growth factors (such as PDGF, FGF) and amino acids (such as asparagine, leucine) are performed to screen strains with low cross-reactivity.

[0278] Component 2 - Anti-CEA + acidic environment affinity ability construction: continue subculture of the above-mentioned strains preliminarily screened in medium containing CEA (initial concentration 100 ng / mL) and pH gradient (initial pH 7.0). Each time the culture is cultured, the concentration of CEA is increased by 50 ng / mL each time, and the pH value is decreased by 0.2 each time. After 10-15 generations of culture, the strain acquires joint affinity for CEA + pH sensitivity. Reaction experiments with other glycoproteins (such as AFP, CA125) under physiological pH conditions are performed to verify the specificity of the strain and ensure that it is not sensitive to other glycoproteins and physiological pH.

[0279] Activation logic based on multi-parameter affinity: By means of genetic engineering, the strain is modified so that it can only activate the production mechanism of therapeutic substances when VEGF, CEA, glutamine and acidic pH environment are simultaneously met; otherwise, the strain remains dormant. This activation logic is realized by constructing a corresponding gene regulatory circuit. When the above four conditions are met at the same time, the regulatory circuit is started, and the synthesis and secretion of therapeutic substances are activated.

[0280] 3.1.2 Affinity-controlled bifunctional therapeutic cocktail

[0281] Formulation 1 - Affinity-generated anti-angiogenic peptide: This anti-angiogenic peptide targets VEGF+ cells of tumor endothelium in glutamine-rich microenvironment, whose mechanism of action is to inhibit the activity of VEGFR2, while inducing cytotoxicity of tumor endothelial cells through metabolic gradient. The half-inhibitory concentration (IC50) of the anti-angiogenic peptide under different glutamine concentrations and VEGF expression levels is determined by experiment, and the affinity-dependent IC50 is optimized.

[0282] Formulation 2 - Affinity-generated auristatin to CEA+ acidic pH: Auristatin targets CEA+ tumor epithelial cells in acidic environment, whose mechanism of action is to enhance the inhibition of tumor cell protein synthesis under acidic pH conditions. By adjusting the culture conditions, the affinity-dependent IC50 of auristatin is optimized, and its selective killing effect on target cells is improved.

[0283] Synergistic effect based on combined affinity: This dual-targeting system achieves synergistic effect through metabolic co-localization, improving the therapeutic effect. Based on multiple metabolic gradients, this system has low toxicity to normal tissues and has a high therapeutic index. When the metabolic profile of tumor tissue returns to normal, the system can be completely eliminated, with good reversibility.

[0284] 3.2 Advanced 3D model verification with metabolic affinity

[0285] 3.2.1 Multi-cell tumor spheroid verification with metabolic gradient

[0286] Affinity spheroid composition and preparation: The multi-cell tumor spheroid is composed of HCT-116 tumor cells (70%), HUVEC endothelial cells (20%), and related fibroblasts (CAF, 10%). Among them, HCT-116 cells are lactic acid producers and glutamine consumers, HUVEC cells are VEGF-responsive cells in the intermediate environment, and CAF cells have a normal metabolic profile. The tumor spheroid is prepared by hanging drop method, and the three cells are mixed in proportion, and the cell concentration is adjusted to 1×10 5Cells were seeded at 5 x 105cells / mL, 50 μL per drop on the coverslip, and incubated upside down to allow the cells to naturally aggregate into spheroids. The spheroid size was controlled at 300-500 μm to form a metabolic gradient. The spheroids were incubated for 7-10 days to allow the spheroids to mature and form a stable metabolic gradient, with hypoxic and acidic environment in the center and oxygen-rich environment in the periphery.

[0287] Affinity-based therapeutic protocol: The dual-modulated VEGF / CEA+metabolic affinity strain was inoculated into the periphery of the tumor spheroid at a concentration of 5 x 105cells / mL. The directional migration of the strain to the metabolically active center was tracked by in vivo imaging technology. The differential viability of the spheroid, angiogenesis, invasion ability, and metabolic gradient clearance were detected at the end of the experiment. 4 5 Affinity-based results: The selective migration ability of the strain, the effect on the volume of the spheroid, the inhibition of VEGF-induced sprouting, the penetration ability of the core of the spheroid, the selectivity for normal fibroblasts, and the reversibility of the system were evaluated by relevant detection methods. Theoretically, the selective migration ability of the strain can reach the metabolically active center, the affinity volume is reduced, the inhibition of VEGF-induced sprouting is more obvious in the glutamine-rich area, the core penetration follows the metabolic gradient completely, has affinity selectivity, and the organism is eliminated after substrate elution, and the structure is partially restored.

[0288] 3.2.2 Intestinal organ-on-chip model with controlled affinity

[0289] Affinity platform construction: An intestinal organ-on-chip with incorporated metabolic gradient was constructed. The gradient epithelium of the chip was composed of Caco-2 cells (normal cells with normal lactic acid metabolism) and HCT-116 cells (tumor cells with high lactic acid metabolism) at a certain ratio; the vascular chamber contained HUVEC cells and a dynamic flow system carrying VEGF / CEA; the gradient matrix was collagen incorporated with a glutamine gradient; controlled perfusion was achieved by microfluidic technology to simulate interstitial flow with metabolic transport.

[0290] Affinity-based drug administration: The drug was administered by the lumen route. The dual-modulated strain preparation with maintained chemotactic ability was injected into the loading chamber of the chip. The strain concentration in the preparation was 1 x 105cells / mL, and the administration volume was determined according to the capacity of the loading chamber. The migration of the strain and the reversibility of the system were continuously monitored.

[0291] 5 Affinity-based drug administration: The drug was administered by the lumen route. The dual-modulated strain preparation with maintained chemotactic ability was injected into the loading chamber of the chip. The strain concentration in the preparation was 1 x 105cells / mL, and the administration volume was determined according to the capacity of the loading chamber. The migration of the strain and the reversibility of the system were continuously monitored.

[0292] Affinity organ chip results evaluation: detection of the selective targeting ability of the strain to the HCT-116 / high gradient zone, the influence on the intestinal barrier integrity (monitored by TEER value), the selective cytotoxicity to HCT-116 cells and Caco-2 cells, the clearance of the strain, the controlled inflammation, and the functional recovery of the chip after gradient normalization. The results show that the strain has strong selective targeting effect on the HCT-116 / high gradient zone, the TEER is maintained, there is no normal barrier damage, the selective cytotoxicity to HCT-116 cells is reduced, and the Caco-2 cells are minimal. With complete organisms in the absence of substrate and immune clearance, the cytokine level is within the normal range by detection, and the chip fully recovers its function after gradient normalization.

[0293] Example Three: Safety verification of affinity and complete reversibility suppression

[0294] 4.1 Specific affinity-based biological suppression test

[0295] Affinity stability and non-transferability experimental setup: co-culture the affinity-regulated strain with a target organism group under multiple conditions, and verify its specific suppression ability by monitoring the interspecies transfer of the affinity phenotype, genetic stability, and reversibility.

[0296] Affinity test target organisms include: Escherichia coli K12 (laboratory strain, different natural chemotaxis), Saccharomyces cerevisiae BY4741 (yeast, different metabolic systems), wild-type Penicillium chrysogenum (same organism, non-affinity-regulated), human intestinal microbiota (multiple species, normal metabolic profile).

[0297] Affinity protocol: set up multiple metabolic gradients (lactic acid 0-10 mM, glutamine 0-5 mM, pH 6.0-7.4, aerobic / anaerobic alternation), simulate the differences between tumor microenvironment and normal physiological environment, continuously pass in the presence / absence of gradients, regularly detect the affinity phenotype, and perform directional migration analysis through directional migration experiments.

[0298] Affinity specificity results: detect chemotaxis through directional migration experiments, in which the affinity phenotype is only maintained in the regulated strain; after continuous passage, there is no permanent mutation in the genome of the regulated strain, and the phenotype is genetically stable; based on specific metabolic dependence suppression, metabolic barrier stability and affinity barrier stability are maintained; compared with the wild type, the affinity adaptability cost is under optimal conditions; and in the absence of target gradient generations, the strain completely regresses to non-specific chemotaxis.

[0299] 4.2 In vivo suppression verification and reversibility based on metabolic deficiency

[0300] Animal model construction: 6-8 week old BALB / c nude mice (body weight 18-22 g), SPF level environment feeding, IACUC approved. Lactic acid pre-conditioning: subcutaneous injection of 0.5 mL lactic acid solution (100 mM) in the flank, once a day for 3 consecutive days, to establish a local high lactic acid environment (confirmed by microdialysis detection lactic acid concentration > 8 mM).

[0301] Affinity-regulated organism inoculation: subcutaneous injection of affinity-regulated strains in the lactic acid pre-conditioned area, affinity carrier PBS + 1% albumin + lactic acid. Another immunosuppressed group: part of the mice were injected with cyclophosphamide (50 mg / kg) intraperitoneally, once every 3 days, to suppress natural immunity.

[0302] Metabolic affinity monitoring: colony forming units were counted by biopsy and tissue metabolic analysis, and the affinity was quantified by chemical tropism phenotype analysis and selective gradient culture; metabolic imaging was performed by bioluminescence and DET-FDG, and the radioactivity uptake in the tumor area was analyzed; the change of metabolic gradient was evaluated by MR spectrum detection of tissue lactic acid and glutamine peak area, and the correlation between strain number change and tissue metabolic profile was tracked. Continuous monitoring until day 42 to verify complete elimination and metabolic recovery.

[0303] Elimination kinetics results based on metabolic deficiency:

[0304] Day 7: CFU / g tissue (active growth in the presence of substrate);

[0305] Day 10: CFU / g tissue (plateau of stable metabolic gradient);

[0306] Day 14: CFU / g tissue (lactic acid depletion begins to be suppressed);

[0307] Day 17: CFU / g tissue (rapid decline in glutamine deficiency);

[0308] Day 21: CFU / g tissue (auxotroph activation almost completely eliminated);

[0309] Day 28: not detectable (complete elimination of metabolic dependence);

[0310] Days 35-42: confirm complete elimination + normalization of tissue metabolism.

[0311] The following conclusions are drawn from the above experiments: (1) precision of suppression: through the programmed depletion of metabolites, precise control of the elimination time of organisms is achieved; (2) inter-animal variability: individual metabolic differences result in some variability in the results, but the overall trend is consistent; (3) in vivo reversibility: after the organism is completely eliminated, no residual detectable individuals are left, and the metabolic profile of the host target area completely returns to normal, verifying the reversibility and safety of the system.

[0312] 4.3 Affinity emergency control test with accelerated reversibility

[0313] 4.3.1 Pharmacological control validation in affinity tumor model:

[0314] Model construction: MCF-7 breast cancer cell xenograft nude mouse model is used, the key feature of which is that the tumor tissue lactate level is significantly elevated. On day 0, the regulatory organism is inoculated, and metabolic imaging is used to guide intratumoral injection; then on day 7, emergency control is started to simulate the scene that needs emergency intervention. The number of days after intervention and metabolic indicators are continuously monitored.

[0315] Affinity control protocol:

[0316] Intervention 1: fluconazole treatment, making the organism sensitive by enhancing its drug uptake;

[0317] Intervention 2: suspension of immunosuppression, natural immune clearance;

[0318] Intervention 3: physiological saline metabolic elution, forcibly creating substrate deficiency by diluting lactate and glutamine in the tumor microenvironment;

[0319] Control group: including vehicle control (only injecting drug vehicle), drug-only control (administering drugs alone without organisms), organism-only control (without intervention), metabolic control (monitoring natural metabolic changes of tumors).

[0320] Affinity multi-path elimination results:

[0321] (1) Fluconazole (sensitivity acquisition): At T+6h, the number of active organisms decreases (promoting uptake); at T+12h, the active organisms decrease (intracellular drug concentration reaches a lethal level); at T+24h, the active organisms decrease (rapid clearance is achieved through acquired drug sensitivity).

[0322] (2) Suspension of immunosuppression (natural immune clearance): At T+12h, the host's natural immune response is activated, and macrophage phagocytosis of the organism is enhanced; at T+24h, the organism is cleared (mainly relying on natural phagocytosis); at T+48h, immune clearance (increased neutrophil infiltration, complement system activation); at T+72h, complete elimination (natural immune response reaches a peak and is reversible).

[0323] (3) Programmed metabolic washout: At T+24h, lactic acid, glutamine concentration in tumor microenvironment significantly decreased due to saline washout, forced substrate deficiency initiated; At T+48h, metabolic deficiency containment effect emerged (organisms entered stationary phase due to nutrient deficiency, auxotrophic genes activated); At T+72h: nutrient dependency completely eliminated (organisms died due to ATP synthesis blocked, ROS (reactive oxygen species) accumulated, unable to survive).

[0324] (4) Maintenance of anti-tumor effect: Compared with the control group, the tumor volume of each intervention group continued to decrease after the organism was removed, and long-term follow-up showed that there was no tumor rebound after the organism was removed. The vital signs of all intervention group mice were normal, and there was no abnormality in hematological indicators, suggesting no significant systemic toxicity. After the intervention ended, the metabolic profile of the tumor and surrounding tissue (lactic acid, glutamine level, energy metabolism pathway) was completely normalized, with no significant difference from normal tissue without inoculation of organisms, verifying the reversibility of the metabolic system.

[0325] Example Four: Comprehensive preclinical pilot study with full reversibility of metabolic affinity

[0326] 5.1 Affinity non-human primate model

[0327] 5.1.1 Experimental design

[0328] The experimental animals were selected from cynomolgus monkeys, with a gender-balanced design, age and weight matched, and with characteristic baseline metabolic profiles. The experimental protocol was approved by the animal ethics committee, with a duration covering the treatment period and the metabolic reversibility follow-up period, with full monitoring of metabolic dynamics from intervention to complete recovery.

[0329] Tumor induction with metabolic profile: a human MCF-7 / HER2+ breast cancer cell xenograft model was used, and high metabolic activity of the tumor was confirmed by PET-FDG metabolic imaging. Tumor localization: inoculated in the subcutaneous breast area of cynomolgus monkeys, with synchronous monitoring of local lactic acid gradient distribution. Target size: the tumor was confirmed to reach a stable size with metabolic activity by imaging. Characteristic verification: combined with MRI imaging (structural observation), metabolic profile (lactic acid, glutamine quantification), and biopsy (histopathology and metabolic marker detection), the metabolic characteristics and activity of the tumor were fully confirmed.

[0330] 5.1.2 Primate affinity treatment protocol:

[0331] Group 1 - HER2+ metabolic affinity complete system: HER2+ lactic acid affinity modulated penicillin was used as a modulating organism. Through ultrasound-guided stereotactic technology, the organism was precisely injected into the tumor metabolic active area. Prednisolone was given, and the reversibility of immunosuppression was monitored.

[0332] Group 2 - No affinity organism control: Wild type P. chrysogenum strain with no affinity modulation (no HER2 targeting and no lactic acid tropism) is used, other operations (inoculation method, immunosuppression, monitoring frequency, etc.) are completely consistent with group 1, to verify the key role of affinity in treatment effect.

[0333] Group 3 - Metabolic monitoring vector control: Inoculate PBS + albumin vector without organisms as a baseline control, to evaluate the effect of simple vector and experimental operation on host metabolism, and to exclude non-specific interference.

[0334] 5.1.3 Affinity enhancement monitoring

[0335] PET-FDG imaging is used to regularly monitor tumor glucose uptake rate, MR spectrum is used to quantitatively analyze lactic acid, glutamine concentration and pH value of tumor and surrounding tissues, and regular hematological monitoring is carried out. Metabolic biochemical analysis: detection of lactic acid, glutamine, pH value and other metabolic markers in serum / plasma. Organism quantification: detection of organism load in blood, tumor and normal tissues by quantitative PCR (specific to organism-specific genes), to accurately track its colonization, proliferation and removal process. Affinity biopsy: needle biopsy of tumor and suspected metastatic sites, combined with tissue section migration analysis (to observe the distribution and tropism of organisms in tissues), to verify their specific aggregation to metabolic active areas. Metabolic reversibility evaluation: after immunosuppression is suspended, immune function markers and metabolic indicators are dynamically monitored to evaluate the recovery ability of the system.

[0336] 5.1.4 Affinity results in primates

[0337] Affinity-based treatment effect (group 1): After a certain number of days after treatment, the tumor volume is reduced, and the reduction rate is related to the active affinity. Quantitative analysis shows that the concentration of organisms in the metabolic active area of the tumor is higher than that in normal tissues. Complete response: partial animals achieve complete elimination by metabolic clearance; partial response: partial animals have reduced tumor volume and reduced affinity; no progression: all animals have no tumor progression, indicating that the affinity-based control strategy effectively inhibits tumor growth.

[0338] Affinity systemic safety: no animal died during the experiment, indicating that the affinity of the system is completely safe. The animal weight remains stable, and there is no sudden increase or decrease in body weight due to systemic metabolic interference. Except for a short-term, reversible leukopenia during immunosuppression (which returned to normal after drug withdrawal), other indicators (red blood cells, platelets, liver and kidney function, etc.) were within the normal range. There were no significant abnormalities in serum metabolites, and the animals' behavior, food intake, social interaction, etc. were normal, and MRI brain imaging ruled out the possibility of affected brain metabolism.

[0339] Bacterial clearance based on metabolic deprivation: Bacteria proliferate rapidly in the high-lactate region of the tumor and reach a peak, then decrease in number as the lactate substrate is progressively depleted. In synergy with the cessation of immunosuppression, the bacteria are completely cleared within a certain number of days. No bacterial dissemination to tissues outside the tumor is observed, confirming that their metabolic dependency (survival only in high-lactate environments) limits the risk of dissemination. The host produces transient anti-bacterial antibodies, and the clearance is ultimately completed by innate immune mechanisms, without long-term immune tolerance or allergic reactions.

[0340] Complete reversibility based on metabolic normalization: After bacterial clearance, metabolic indicators such as lactate and glutamine in the serum and tissues return to baseline levels before the experiment. From the metabolic profile of the tissues, the lactate concentration and pH in the residual tumor area and normal tissues return to normal physiological conditions. After discontinuation of prednisolone, the number and function of immune cells are completely restored, without permanent immune damage. In long-term follow-up, no detectable long-term metabolic effects are observed.

[0341] Metabolic analysis histology (end point): Histological sections show a large number of necrotic tumor cells, and the metabolic profile of the residual tissue has normalized. The structure and physiological metabolic function of normal tissues around and far from the tumor are intact. There are no abnormalities in the metabolic indicators and histological examination of important organs such as the heart, liver, brain, and kidneys. Only mild reactive hyperplasia is observed in the lymph nodes, and the metabolic state is normal.

[0342] 5.2 Affinity pharmacokinetics and toxicology analysis with reversibility

[0343] The complete PK study of metabolic affinity comprehensively investigated various affinity monitoring parameters. These include active bacteria with affinity (detected by CFU / g tissue and migration ability evaluation), cytotoxic metabolites (detected by LC-MS / MS and analyzed for their correlation with active affinity), chemotactic ability (measured by binding and migration assays), metabolic biomarkers (lactate, glutamine, tissue pH), and organ function and metabolic reversibility parameters.

[0344] The key PK findings of affinity are of great significance. Bacteria reach Cmax after inoculation in metabolically active areas; affinity distribution is limited to tissues with target metabolic profiles; orange mold can be detected after gradient activation, reflecting controlled metabolic characteristics; the lack of bacteria is related to substrate depletion; selective accumulation only occurs in the metabolic microenvironment of the tumor; when the metabolic gradient is normalized, PK shows complete clearance reversibility.

[0345] Affinity-based toxicology studies showed that at the tested therapeutic dose, affinity NOAEL (no observed adverse effect level) had no off-target effects; compared with the effective dose, affinity had an advantage in safety margin due to metabolic specificity; as affinity was limited to specific tumor microenvironment, no target organs were affected; all effects were reversible, reflecting the advantage of natural affinity; long-term safety follow-up found no permanent metabolic changes.

[0346] Specific application case based on metabolic affinity Example Five: Anti-HER2 system with metabolic affinity for metastatic breast cancer - with complete reversibility Simulated clinical 6.1 metabolic affinity clinical scenario:

[0347] A female patient, 52 years old, with HER2-positive breast cancer (IHC 3+), accompanied by multiple liver metastases (lesions 2-4 cm) and lung micro nodules. The patient failed to receive trastuzumab combined with chemotherapy in the past. The current ECOG physical status is 1, and the organ function is preserved. Through PET-FDG imaging and MR spectrum detection, the patient's tumor metabolic profile characteristics are determined, combined with multiple index evaluation, the patient is determined as an ideal candidate for metabolic affinity-based treatment, and the treatment system can ensure complete reversibility.

[0348] Optional metabolic pretreatment: About 72 hours before treatment, controlled feeding or very low carbon diet can be implemented under the supervision of a physician. The purpose is to reduce insulin / IGF-1 levels and increase ketone bodies, thereby enhancing the difference between tumor microenvironment and normal tissue in glucose / lactate utilization, and improving the recognition and triggering contrast of the system / composition.

[0349] The measured affinity target parameters are as follows:

[0350] Tissue HER2 level: The number of receptors per cell exceeds the recognition threshold, providing the basis for HER2 targeted therapy;

[0351] Tumor lactate (MR spectrum): The level is elevated and exceeds the affinity threshold, meeting the substrate requirement of the metabolic affinity system;

[0352] Tumor glutamine: The content is elevated and exceeds the chemotaxis threshold, which can guide the organism to migrate directionally to the tumor area;

[0353] Microenvironment pH: Acidic (< activation threshold), providing an acidic environmental signal for system activation;

[0354] Glucose uptake (PET-FDG): SUV max value is high and exceeds the affinity target, indicating active tumor metabolism;

[0355] Serum markers: Abnormal levels of CEA (carcinoembryonic antigen) and CA 15-3 (carbohydrate antigen 15-3) can be used as treatment effect monitoring indicators;

[0356] Metabolic profile: compatible with HER2+ metabolic affinity regulation system, ensuring the applicability of the treatment regimen.

[0357] 6.2 Application of affinity protocol:

[0358] First 2 days: start the minimum dose of immunosuppressive treatment (prednisolone combined with cyclosporine), and this regimen does not interfere with the activity of the metabolic affinity system.

[0359] Day 0: stereotactic injection guided by metabolic imaging is used for the main liver lesions, and precise delivery of HER2+ lactic acid affinity regulation Eupenicillium sp. is performed; for lung metastases, systemic infusion is used to deliver affinity regulation organisms wrapped in autologous macrophages to the lesion site, and the targeting is enhanced by the tropism of macrophages.

[0360] Reversible metabolic baseline recording: all metabolic parameters (such as lactic acid, glutamine, pH, serum markers, etc.) are recorded in detail before treatment as a control standard for post-treatment efficacy evaluation and reversibility verification.

[0361] 6.3 Record the metabolic affinity activation timeline:

[0362] 0-4 hours (affinity chemical tropism and recognition phase): the regulation organism migrates to the high lactic acid area by sensing the metabolic gradient of the tumor microenvironment, and its migration speed is significantly higher than that in the control area (non-tumor tissue); at the same time, the organism specifically binds to target cells through the combined recognition mechanism of "HER2 (+) + lactic acid (+)"; in this stage, the metabolic sensors (pH-sensitive proteins, glutamine transporters, MCT lactic acid transporters) of the organism are activated, while normal tissues do not trigger system activation due to normal lactic acid levels and physiological pH, demonstrating strict tissue specificity.

[0363] 4-8 hours (affinity multi-parameter verification phase): the organism starts a four-input AND logic verification based on sensors, and only when "HER2 (+) ∩ lactic acid (+) ∩ glutamine (+) ∩ pH (+)" are met simultaneously, the treatment function is fully activated; metabolic signals are integrated through intracellular convergence transduction circuits, inducing substrate-dependent pathways to start, in which the expression of orsellinic acid synthase (a metabolic product synthesis enzyme with cytotoxicity) is up-regulated with the target metabolite concentration; at the same time, the gradient-controlled synthesis mechanism is started, and the production of intracellular active substances is proportional to the amount of available substrate in the tumor microenvironment.

[0364] 8-24 hours (affinity-guided active treatment phase): Affinity-related auristatins reach peak extracellular concentrations in the high metabolic gradient region (tumor core); organism distribution strictly follows the lactate / glutamine gradient, forming a therapeutic concentration within the effective radius; HER2-positive and metabolically active tumor cells preferentially uptake auristatin, while normal cells have limited uptake due to low lactate levels and normal pH, demonstrating metabolic profile selectivity.

[0365] Days 2-7 (gradient-controlled amplification phase): Organisms proportionally self-replicate in metabolically rich regions, with auristatin production correlating with lactate / glutamine availability; progressive necrosis occurs in the high-metabolic region of the tumor, with the tumor volume preferentially decreasing; normal tissues maintain their structure and function as the metabolic environment does not support organism survival, demonstrating selective affinity.

[0366] Days 7-14 (affinity consolidation phase): Under the guidance of the affinity system, metabolically active tumor lesions continue to decrease in volume; the degree of elimination of lung metastatic nodules is related to the baseline FDG uptake level; the number of organisms within the target region remains stable, relying on the sustained metabolic gradient to maintain CFU levels; systemic metabolic parameters remain stable, preparing for subsequent metabolic clearance.

[0367] Days 14-18 (programmed metabolic deficiency containment phase): As tumor cells die in large numbers, the tumor microenvironment's metabolic profile begins to normalize, with lactate and glutamine levels falling to normal ranges; organisms initiate a deficiency cascade due to substrate deficiency, with ATP / AMP sensors activated and energy reserves depleted; simultaneously, acquired auxotrophic mechanisms are triggered, leading to a rapid decrease in the number of organisms; MR spectroscopy shows that the metabolic gradient gradually normalizes, marking the beginning of metabolic reversibility.

[0368] Days 18-35 (final clearance and complete metabolic reversibility phase): Quantitative detection of organisms is negative, achieving complete deficiency clearance; the tumor microenvironment's metabolic profile is completely normalized, with lactate, pH, and glutamine returning to physiological values; the tumor response is consolidated, with complete volume reduction maintained after metabolic normalization; systemic metabolic parameters return to normal; long-term follow-up reveals no detectable permanent metabolic changes, confirming complete reversibility.

[0369] 6.4 Recorded Affinity Safety Control

[0370] Systemic toxicity is limited to the tumor site where metabolic activity is active, with metabolic function indicators (glucose, systemic lactate, blood pH) remaining normal, demonstrating the safety of local affinity. Due to the use of minimal immunosuppression, opportunistic infections do not occur. Organisms are limited to target metabolic profile tissues, with complete systemic metabolic reversibility, and no permanent changes in basal metabolic indicators.

[0371] 6.5 Comparison of Metabolic Affinity Efficacy

[0372] Objective response rate is significantly higher than standard chemotherapy, affinity selection is significantly better than non-selective systemic treatment, systemic toxicity is limited to the target site, and multi-organ toxicity of traditional treatment is significantly reduced; the duration of response is longer, as the affinity system has a self-amplification effect, and repeated treatment is not required; the quality of life of patients is significantly improved due to metabolic selective treatment.

[0373] 6.6 Affinity response biomarkers

[0374] Serum CEA and CA15-3 levels decreased to normal range; tissue lactic acid was confirmed to be completely metabolically normalized by MR spectroscopy detection; PET-FDG uptake showed that tumor glucose metabolism returned to normal; circulating tumor cell detection was not detectable, suggesting that the affinity system effectively eliminated tumor cells.

[0375] 6.7 Detailed metabolic reversibility monitoring

[0376] Glucose metabolism: blood glucose curve returned to normal, insulin sensitivity was normal; lipid metabolism: blood lipid profile (cholesterol, triglycerides) was within the normal range; acid-base balance: blood pH, bicarbonate, and whole-body lactic acid levels returned to physiological state; mitochondrial function: peripheral blood mononuclear cell cell respiration and ATP production capacity were normal; long-term metabolic follow-up found no permanent metabolic changes.

[0377] 6.8 Affinity vs. traditional treatment advantages

[0378] Selective targeting: precisely targeting specific tumor metabolic microenvironment, rather than non-specific systemic distribution of traditional treatment;

[0379] Self-amplification: treatment effect is proportional to tumor metabolic load, better than fixed-dose traditional treatment;

[0380] Natural containment: relies on metabolic dependence to limit organism spread, reducing the risk of dissemination of traditional treatment;

[0381] Metabolic reversibility: complete recovery of normal metabolic profile after treatment, avoiding permanent functional damage of traditional treatment;

[0382] Re-treatability: if tumor recurrence occurs with a new metabolic profile, the system can be re-adjusted for secondary treatment.

[0383] 6.9 Affinity cost-benefit analysis

[0384] Cost per quality-adjusted life year (QALY) is lower than traditional targeted therapy;

[0385] Due to good metabolic reversibility, the need for long-term metabolic monitoring is reduced;

[0386] Selectivity toxicity is small, the number of hospitalization days is significantly reduced;

[0387] Follow-up cost reduction (no long-term monitoring of metabolic sequelae), health return on investment (ROI) is better than standard care.

[0388] 6.10 Metabolic affinity case conclusion

[0389] Metabolic affinity-based systems show directional migration to the tumor microenvironment, selective targeting of metabolic profile changes, treatment amplification proportional to the nutritional gradient, programmed lack of suppression, and most importantly, complete metabolic reversibility, without permanent changes in systemic metabolism. The revolutionary feature of complete reversibility through metabolic gradient normalization confers safety features based on the natural mechanism of nutritional deficiency. Compared with traditional treatment, this system has significant advantages in selectivity, safety and cost-effectiveness, providing a new treatment idea for HER2-positive metastatic breast cancer (especially cases of traditional treatment failure).

[0390] Example six: diagnostic kit based on metabolic affinity

[0391] The present example also discloses a diagnostic kit based on metabolic affinity for detecting and verifying the presence of fungal and / or bacterial communities in tumor samples and characterizing the related metabolic microenvironment. Among them, sample pretreatment is specifically:

[0392] 7.1 Sample type: collect clinical tumor biopsy tissues (such as colorectal cancer, breast cancer), puncture fluid, peripheral blood and ex vivo cultured tumor cell line samples (n=50, including known positive and negative control samples).

[0393] 7.2 Sample pretreatment

[0394] For biopsy tissues and puncture fluids: use the DNA / RNA extraction kit in the kit to extract microbial and host genomic nucleic acids according to the instructions, while reserving part of the sample for metabolite detection.

[0395] For peripheral blood: use density gradient centrifugation to separate plasma for metabolite detection; Buffy coat layer for nucleic acid extraction.

[0396] For ex vivo cultured samples: collect cell culture supernatant for metabolite detection, and cell precipitate for nucleic acid extraction.

[0397] Stabilizers (trehalose, albumin) are added during the processing of all samples to prevent nucleic acid degradation and metabolite loss.

[0398] 7.3 The kit includes the following standardized components:

[0399] (1) Molecular detection module: specific PCR primers and probes for amplification and identification of characteristic sequences of fungi (e.g. Candida, Aspergillus, Malassezia, etc.) or reference bacterial strains, as well as genes related to metabolic response (e.g. MCT transporters, GlnPQ transport system, lactate metabolism-related enzymes). Specifically:

[0400] qPCR amplification: configure the reaction system in a 96-well plate, containing extracted nucleic acid template, specific primers, probes, PCR Mix.

[0401] Reaction conditions: 95°C pre-denaturation for 10 min, 95°C denaturation for 15 s, 60°C annealing and extension for 1 min, a total of 40 cycles, using a real-time fluorescence quantitative PCR instrument to detect the fluorescence signal.

[0402] Result interpretation: based on the standard curve of the reference strain, calculate the relative copy number of the target sequence in the sample, Ct value ≤ 35 and exponential growth of amplification curve, then determine as positive.

[0403] (2) Metabolic detection module: a system based on enzymatic color development or fluorescence reaction for quantitative detection of lactic acid (operation range 2-15 mM), glutamine (1-8 mM) and acidic pH (6.2-7.4). This module can define the metabolic characteristics of the tumor microenvironment. Specifically,

[0404] Lactic acid detection: take 50 μL of sample supernatant, add enzymatic color development reagent (lactate dehydrogenase, NAD+, etc.), incubate at 37°C for 30 min, then detect the absorbance at 450 nm by a microplate reader, and calculate the lactic acid concentration in the sample according to the standard curve drawn by the lactic acid standard solution (2-15 mM).

[0405] Glutamine detection: using fluorescence method, glutamine is generated after sample and glutaminase reaction, and further reacts with fluorescence substrate, the fluorescence intensity is detected at excitation light 485 nm and emission light 535 nm, and the concentration is calculated by comparing with 1-8 mM standard solution.

[0406] pH detection: use a pH meter to directly measure the pH value of the sample supernatant, and record the value in the range of 6.2-7.4.

[0407] (3) Control and standard components: including reference fungi / bacterial strains with characterized metabolic affinity, and standardized metabolite solutions (lactic acid, glutamine) for calibration and control during detection.

[0408] (4) Auxiliary solutions: sterile buffer, stabilizers (such as trehalose, albumin) and DNA / RNA extraction kit to ensure the stability of the sample and the repeatability of the results.

[0409] (5) Optional functional verification module: microfluidic or chemotaxis chamber for cell migration experiment, used to detect the chemotaxis response of reference strains under in vitro simulation of metabolic gradient conditions. This module is mainly used for scientific research experiments and is not a necessary part of clinical routine diagnosis. Among them:

[0410] Microfluidic experiment: inoculate the reference fungal / bacterial strain on one side of the microfluidic chip, and add gradient concentrations of lactic acid (2-15 mM) or glutamine (1-8 mM) solution on the other side. After incubation at 37°C for 4h, observe the migration distance and number of microorganisms to the high concentration metabolite area by inverted microscope.

[0411] Chemotaxis chamber: use Boyden chamber, add metabolite solution to the lower chamber and microbial suspension to the upper chamber, count the number of migrated colonies in the lower chamber after incubation, and calculate the chemotaxis index (migrated colony number / control group colony number).

[0412] 7.4 Method of use: The kit can be applied to tumor biopsy, puncture fluid, peripheral blood or in vitro culture samples. The detection is divided into three stages: (i) molecular level detection of microorganisms / metabolism related markers by qPCR; (ii) quantitative analysis of lactic acid, glutamine and pH and other metabolic characteristics; (iii) optional chemotaxis function verification under scientific research conditions.

[0413] 7.5 Expected results: The kit can generate a comprehensive detection report, including the presence or absence of microorganisms with potential tumor affinity, the strength of the metabolic gradient, and the verification results of in vitro chemotaxis reaction (if performed), which can visually display the metabolic affinity characteristics of tumor microecology.

[0414] This embodiment helps to reduce false positive results, improve standardization level across laboratories, and provide support for individualized treatment strategies through multi-module collaborative detection combined with molecular, metabolic and functional three-level analysis, while being compatible with existing clinical detection methods (PCR, enzymatic detection, GMP standard process).

[0415] The above specific embodiments are further illustrations of the technical solutions and beneficial effects of the present application, and are not limitations of the embodiments. Any obvious substitutions for those skilled in the art without departing from the concept of the present application are within the scope of protection of the present application.

Claims

1. A biological system based on metabolic affinity, characterized in that: Comprise: (a) Metabolic response module: a living organism consisting of endogenous filamentous fungi and / or exogenous filamentous fungi isolated from tumor tissue, and non-pathogenic commensal bacteria, said living organism comprising the following features: (i) selective response capability to tumor metabolites obtained by adaptive cultivation in a gradient-controlled bioreactor; (ii) multi-threshold metabolic sensors integrated into adaptive transduction circuits, requiring simultaneous satisfaction of at least two of the following tumor metabolic features to activate the system through convergent pathways: said tumor metabolic features include lactic acid concentration greater than 10 mM, glutamine concentration greater than 5 mM, pH less than 6.8, hypoxia less than 3% O2; (iii) biosynthetic pathways induced by tumor-specific substrates for the production of secondary metabolites, bioactive peptides, or extracellular lytic enzymes. (b) Biological containment module: comprising synergistic redundant mechanisms: (i) nutritional dependency on conditions of lactic acid concentration greater than 8 mM and glutamine concentration greater than 3 mM; (ii) protease-mediated intracellular apoptosis pathway triggered by ATP depletion and reactive oxygen species accumulation; (iii) inducible pharmacological sensitivity to fluconazole, amphotericin B, or 5-fluorocytosine; (c) Controlled drug delivery system: comprising image-guided stereotactic injection, surgical implantation of biocompatible hydrogel formulations, or combined immunosuppression.

2. The metabolic affinity-based biological system according to claim 1, characterized in that: The living organism is obtained by gradient bioreactor metabolic adaptation protocol, optimizing affinity to tumor metabolites under controlled gradients of 2-15 mM lactic acid, 1-8 mM glutamine, and pH 6.2-7.4; acquisition of normal physiological substrate specificity is achieved by negative selection pressure exerted through alternating growth / selection cycles in normal vs. tumor media; maintenance under stable selection in the absence of gradients to verify stable acquisition; chemical tropism functional half-life greater than 24 h; resistance to affinity loss is obtained by integrating the chemical tropism pathway into essential metabolic circuits.

3. The metabolic affinity-based biological system according to claim 1, wherein: The biological logic of the multi-threshold metabolic sensors is activated for bioactivity production through specific transduction circuits integrated by three input signals, including: (i) surface receptors preferentially bound to selection adaptation detecting tumor protein markers greater than 5 ng / ml, including but not limited to HER2, PSA, CEA, VEGF; (ii) lactic acid sensors enhanced by modifiable metabolic enzymes and similar upregulatable MCT transporters, threshold greater than 10 mM; (iii) acidic pH sensors through pH-sensitive proteins and modifiable ion channels, threshold less than 6.

8.

4. The metabolic affinity-based biological system according to claim 1, wherein: The biosynthetic pathways enhance production of multiple bioactive metabolites through enzyme upregulation selection including: (i) optimization of potency of secondary metabolites through biosynthetic enzymes and type I polyketide synthases overexpression by high productivity selection in presence of tumor substrates; (ii) calibration of activity of bioactive compounds through specific enzyme expression control and optimizable controlled release biosynthetic pathways; (iii) bioactive cyclic peptides reaching effective concentrations through upregulatable non-ribosomal peptide synthetases and substrate dependent allosteric control; (iv) lysing enzymes with obtainable specificity for tumor extracellular matrix components through directed evolution of catalytic active sites. Multiple bioactive metabolites are produced through lactic acid / glutamine dependent positive feedback loop control with enhanced secretion release through modifiable ABC transporters.

5. The metabolic affinity-based biological system according to claim 1, wherein: The intracellular apoptosis pathway includes: (i) acquisition of dependence on lactic acid concentrations greater than 8 mM and glutamine concentrations greater than 3 mM through deletion of gluconeogenesis and amino acid de novo synthesis by selection in media lacking; (ii) calibratable sensitivity to substrate availability through metabolic sensors monitoring intracellular pools of essential metabolites; (iii) activation of starvation-induced cell death cascade through ATP depletion and ROS accumulation triggering the apoptosis pathway; (iv) time precision of 8% relative to substrate depletion through metabolic consumption rate and kinetics of endogenous reserves control; (v) programmable duration of 7, 14, or 21 days through selection of variant strains with different metabolic rates and reserve storage capacities.

6. The metabolic affinity-based biological system according to claim 1, wherein: The inducible pharmacological sensitivity is obtained through: (i) fluconazole hypersensitivity through upregulation of MDR transporters and reduction of LC50 by selection for enhanced uptake; (ii) enhanced amphotericin B sensitivity through modification of membrane sterol composition and reduction of efflux pump activity; (iii) 5-fluorocytosine hypersensitivity through cytosine deaminase overexpression and nucleoside transporter facilitated uptake. Prodrugs are converted to intracellular accumulated active cytotoxic metabolites through metabolic cascades with elimination times less than 12 hours post administration.

7. The metabolic affinity-based biological system according to claim 1, wherein: The active organisms include bacterial variants including: (i) Escherichia coli Nissle 1917: tumor affinity through modification of Tar receptor and enhanced chemotaxis to lactic acid through Che pathway selection; (ii) Bifidobacterium longum: adaptation to grow in hypoxic environments of 0.5-3% O2 through upregulation of alternative oxidases and anaerobic fermentation pathways; (iii) Lactobacillus plantarum: optimized secretion system through signal peptidase and ABC transporter overexpression for efficient release of bioactive metabolites. Bacteria possess the same functional chemotactic abilities as fungi through species-specific adaptation of signal transduction pathways.

8. The metabolic affinity-based biological system according to claim 1, wherein: The surgically implantable biocompatible hydrogel formulation comprises: (i) a biocompatible hydrogel matrix incorporating living organisms by gentle mixing during gelation to maintain cell integrity and metabolic activity, maintaining greater than 95% viability and chemotactic ability of the living organisms cross-linked with 2% sodium alginate and 0.5% chitosan by calcium chloride; (ii) 10 6 -10 7 CFU / organism and uniformly distributed; (iii) release kinetics controlled by matrix porosity in three phases: 20% in burst phase at 0-2 hours, 60% in sustained phase at 2 hours-10 days, and 20% in residual phase at 10-21 days, releasing progressively with hydrogel erosion; (iv) complete degradation within 14-21 days by hydrolysis with endogenous alginate lyase, compatible with natural clearance, with no permanent residue.

9. The metabolic affinity-based biological system according to claim 1, wherein: The combined immunosuppression comprises: (i) minimal preconditioning: prednisolone 0.3 mg / kg / day and cyclosporine 2 mg / kg / day orally for 48 hours, preventing acute rejection while maintaining immune surveillance against pathogens; (ii) active phase: maintained for 14 days and tapered with a 25% reduction every 3 days, reducing the impact of long-term suppression; (iii) clearance phase: complete drug withdrawal within 21 days, activating natural immune clearance and phagocytosis of residual organisms; (iv) monitoring: active organisms and immune biomarkers are detected every 48 hours to verify safety; (v) total duration of 14-21 days, compared to 6-12 months of traditional systemic therapy, with more than 80% reduction in immunosuppression exposure.

10. Use of a metabolic affinity-based biological system according to any one of claims 1 to 9 for the preparation of an antitumor drug, characterized in that: The preparation of the antitumor drug comprises: (i) organisms based on metabolic affinity modulation according to any one of claims 1-9, in a homogeneous suspension with a defined concentration; (ii) a biocompatible drug carrier, including sterile buffered physiological saline, human blood albumin as a stabilizer, and trehalose as a cryoprotectant, to maintain organism viability and sustain modulated chemotactic ability; (iii) a low-temperature preservation system for stability, with controlled freezing and thawing protocols to maintain cell integrity and metabolic function; (iv) sterile primary packaging meeting GMP standards, with sterility testing and endotoxin control; (v) a diagnostic kit equipped with diagnostic reagents for identifying target protein markers of tumor metabolic profiles comprising different lactate, glutamine, pH compositions and modulated affinity recognition.

11. Use of the biological system based on metabolic affinity according to claim 10 for the preparation of antitumor drugs, characterized by the fact that: The types of formulations, divided by administration form, include: (i) isotonic injection suspensions for stereotactic administration, with optimized injection viscosity to maintain cell viability; (ii) biocompatible hydrogel preformed implants for surgical positioning, with release kinetics controlled by programmed degradation; (iii) mucosal adhesive formulations for transdermal / mucosal application by modulating epithelial permeability organisms; (iv) liposome formulations for systemic transport protection, with passive targeting and metabolic capacity maintenance functions. Different types of formulations have controlled microbial stability and maintain affinity bioactivity during storage.

12. The use of the biological system based on metabolic affinity according to claim 10 in the preparation of antitumor drugs, characterized by the fact that it is: The antitumor drug is suitable for: (i) HER2-positive breast cancer, with a metabolic adaptation protocol that potentially adapts the dual affinity against HER+ metabolism in a high-lactate environment; (ii) prostate adenocarcinoma, with an anaerobic bioreactor modulation protocol that adapts to hypoxic microenvironments and environments where the PSA marker is present; (iii) colorectal cancer, with a VEGF / CEA+ metabolic affinity dual-targeting system through a multiple adaptation protocol for low-glucose environments; (iv) brain tumors, with the potential to cross the blood-brain barrier through carrier cells loaded with adaptive neurotrophic organisms. It includes: (i) specific primers for quantitative PCR detection of modulated organisms; 13. Use of a metabolic affinity-based biological system according to any one of claims 1 to 9 for the preparation of a test kit, characterized in that: (ii) biosensor components for metabolic gradient verification; (iii) metabolic detection reagents for lactate, glutamine, pH detection; (iv) migration detection systems for affinity function verification. The combined immunosuppression comprises: (i) minimal preconditioning: prednisolone 0.3 mg / kg / day and cyclosporine 2 mg / kg / day orally for 48 hours, preventing acute rejection while maintaining immune surveillance against pathogens; (ii) active phase: maintained for 14 days and tapered with a 25% reduction every 3 days, reducing the impact of long-term suppression; (iii) clearance phase: complete drug withdrawal within 21 days, activating natural immune clearance and phagocytosis of residual organisms; (iv) monitoring: active organisms and immune biomarkers are detected every 48 hours to verify safety; (v) total duration of 14-21 days, compared to 6-12 months of traditional systemic therapy, with more than 80% reduction in immunosuppression exposure. The preparation of the antitumor drug comprises: (i) organisms based on metabolic affinity modulation according to any one of claims 1-9, in a homogeneous suspension with a defined concentration; (ii) a biocompatible drug carrier, including sterile buffered physiological saline, human blood albumin as a stabilizer, and trehalose as a cryoprotectant, to maintain organism viability and sustain modulated chemotactic ability; (iii) a low-temperature preservation system for stability, with controlled freezing and thawing protocols to maintain cell integrity and metabolic function; (iv) sterile primary packaging meeting GMP standards, with sterility testing and endotoxin control; (v) a diagnostic kit equipped with diagnostic reagents for identifying target protein markers of tumor metabolic profiles comprising different lactate, glutamine, pH compositions and modulated affinity recognition. The types of formulations, divided by administration form, include: (i) isotonic injection suspensions for stereotactic administration, with optimized injection viscosity to maintain cell viability; (ii) biocompatible hydrogel preformed implants for surgical positioning, with release kinetics controlled by programmed degradation; (iii) mucosal adhesive formulations for transdermal / mucosal application by modulating epithelial permeability organisms; (iv) liposome formulations for systemic transport protection, with passive targeting and metabolic capacity maintenance functions. Different types of formulations have controlled microbial stability and maintain affinity bioactivity during storage. The antitumor drug is suitable for: (i) HER2-positive breast cancer, with a metabolic adaptation protocol that potentially adapts the dual affinity against HER+ metabolism in a high-lactate environment; (ii) prostate adenocarcinoma, with an anaerobic bioreactor modulation protocol that adapts to hypoxic microenvironments and environments where the PSA marker is present; (iii) colorectal cancer, with a VEGF / CEA+ metabolic affinity dual-targeting system through a multiple adaptation protocol for low-glucose environments; (iv) brain tumors, with the potential to cross the blood-brain barrier through carrier cells loaded with adaptive neurotrophic organisms. It includes: (i) specific primers for quantitative PCR detection of modulated organisms; (ii) biosensor components for metabolic gradient verification; (iii) metabolic detection reagents for lactate, glutamine, pH detection; (iv) migration detection systems for affinity function verification.

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