Method and device for enhancing in situ tumor vaccine immunity based on cavitation induction

Abstract

Based on the method and device for enhancing the immune of tumor in situ vaccine induced by cavitation, the baseline state of the immune microenvironment of the target tumor is obtained, the induced cavitation pulse IAD is emitted, the immune activation state is monitored in real time, the immune activation state is evaluated and compared, the parameters of the induced cavitation pulse IAD are adjusted in real time, and the ablation cavitation pulse TD is emitted; compared with the prior art, by setting the induced cavitation pulse IAD and the ablation cavitation pulse TD which are emitted in sequence, the partial ablation of the tumor tissue is realized under the action of the induced cavitation pulse IAD, the immune activation of the tumor tissue is realized, the cavitation technology can establish the in situ vaccine platform on the tumor tissue, and the ablation boundary and ablation effect of the subsequent ablation cavitation pulse TD are laid.

Description

Technical Field

[0001] This invention relates to the field of tumor immunology technology, specifically to a method and device for enhancing in situ tumor vaccines based on cavitation induction. Background Technology

[0002] Tumor immunotherapy aims to activate or reshape a patient's own immune system to recognize and eliminate tumor cells. However, tumor immunotherapy depends on the presence of sufficient tumor-specific antigens in the tumor microenvironment and the effective activation of antigen-presenting cells. Many "cold tumors" (insufficient immune cell infiltration and weak antigenicity) have a response rate of less than 20% to existing immune checkpoint inhibitors (Immune Checkpoint).

[0003] Based on tumor immunity, a technical strategy is to induce tumor immune response by causing tumor cells to die in situ and release antigens through local treatment, thereby "transforming" tumor tissue into an autologous vaccine.

[0004] Existing in situ vaccine technologies typically include thermal ablation, cryoablation, irreversible electroporation, and ultrasonic cavitation ablation. Among these, ultrasonic cavitation ablation utilizes high-intensity focused ultrasound to generate inertial cavitation or boiling bubbles, mechanically pulverizing tissue into a cell-free homogenate. It does not rely on thermal effects and can achieve complete tissue liquefaction while fully preserving the natural conformation of tumor antigens.

[0005] While existing ultrasonic cavitation ablation technology has the potential for immune activation, the following problems exist in its practical application:

[0006] 1. Treatment parameters are geared towards ablation efficiency, neglecting the optimization of immune activation. Existing technologies select pulse parameters (pulse length, sound pressure amplitude, number of pulses, scanning interval, etc.) with the goal of maximizing tissue liquefaction rather than maximizing the anti-tumor immune response.

[0007] 2. The immune activation effect cannot be monitored and fed back in real time during the treatment process. Existing technologies cannot perceive changes in the immune status in the tumor microenvironment in real time during the treatment process, such as the release rate of tumor antigens, the recruitment of dendritic cells or macrophages, and the expression level of pro-inflammatory cytokines (such as IL-12 and TNF-α). These information are key indicators for determining whether the "in situ vaccine" has been effectively initiated, but existing technologies only rely on post-treatment sample analysis (results are usually obtained several hours to several days later). Existing technologies only assess the immune effect through sample analysis after the treatment is completed, and cannot perceive changes in the tumor microenvironment during the treatment process and make dynamic adjustments.

[0008] 3. The cavitation parameters “therapeutic dose” and “immunoadjuvant dose” are not separated. Existing technology attempts to use a single pulse sequence to accomplish two tasks simultaneously: physical ablation and immune activation. However, the two are often incompatible (high-energy pulses can ablate efficiently but may weaken immunogenicity due to excessive antigen destruction, while low-energy pulses can preserve antigens but have insufficient ablation efficiency).

[0009] 4. It cannot overcome the inhibition of immune activation by the cold tumor microenvironment. For cold tumors that are already insufficient in immunogenicity, simple cavitation antigen release may not be enough to activate effective anti-tumor immunity. Summary of the Invention

[0010] The present invention aims to overcome the deficiencies in the prior art and provide a cavitation-induced tumor in situ vaccine immune enhancement method and device that optimizes pulse parameters to have antigen release as the core objective, monitors and provides feedback on immune activation effects in real time, and separates and coordinates therapeutic dose pulses and immune adjuvant dose pulses.

[0011] To achieve the above-mentioned objectives, the present invention employs the following technical solution: a method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction, comprising the following steps:

[0012] Step S1: Obtain the baseline state of the immune microenvironment of the target tumor;

[0013] Step S2: Firing an induced cavitation pulse IAD, which is used to induce tumor antigen release and activate antigen-presenting cells, towards the focal region of the tumor tissue, and defining the parameters of the induced cavitation pulse IAD as the immune adjuvant dose, while continuously performing sparse scanning on the tumor tissue region.

[0014] Step S3: Monitor immune activation status in real time. At one or more time points after the emission of the induced cavitation pulse (IAD), collect the immune activation index (IAI) in the tumor microenvironment in real time.

[0015] Step S4: Assessment and comparison of immune activation status. Based on the antigen release status, one or more immune activation indices (IAI) collected in step S3 are classified into insufficient immune activation, optimal immune activation, and risk of immune depletion.

[0016] Step S5: Adjust the induced cavitation pulse IAD parameter in real time. Based on the evaluation results of step S4, adjust the induced cavitation pulse IAD parameter in real time until the immune activation index IAI is optimal for immune activation.

[0017] When the assessment result indicates insufficient immune activation, increase the release of tumor tissue antigens or activate the tumor immune microenvironment.

[0018] When the evaluation result indicates optimal immune activation, maintain the current IAD parameters for the induced cavitation pulse.

[0019] When the assessment result indicates a risk of immune exhaustion, pause or reduce the IAD parameter of the induced cavitation pulse.

[0020] Step S6: Firing an ablation cavitation pulse TD, and continuously performing immune activation index (IAI) detection during and after the firing of the ablation cavitation pulse TD. The parameters of the ablation cavitation pulse TD are defined as the ablation dose, while continuously performing intensive scanning of the tumor tissue area.

[0021] Step S7: Repeat steps S2-S6 until the desired result is achieved.

[0022] As a preferred embodiment of the present invention, step S1 classifies tumors into three types based on the acquisition information: fully immune-infiltrated hot tumors, poorly immune-infiltrated cold tumors, and excluded tumors. The acquisition method for the baseline state of the immune microenvironment includes at least one of acoustic feature calibration, imaging calibration, and biomarker calibration.

[0023] As a preferred embodiment of the present invention, the parameters of the cavitation pulse IAD induced in step S2 include at least one of the following: shock scattering mode, low-intensity tissue damage mode, and boiling tissue damage low-energy variant mode.

[0024] As a preferred embodiment of the present invention, in step S2, during the sparse scanning of the tumor tissue region, the distance between two adjacent scanning focal points is greater than the damage diameter of a single pulse. While the tumor tissue region forms a local release of antigens, it retains the immune response initiation platform. The area of ​​tumor tissue liquefaction caused by the induced cavitation pulse IAD does not exceed 10%-40% of the total area covered by the induced cavitation pulse IAD.

[0025] As a preferred embodiment of the present invention, the method for collecting the immune activation index (IAI) in step S3 includes at least one of acoustic indicators, optical indicators, and biosensor indicators.

[0026] As a preferred embodiment of the present invention, the parameters of the ablation cavitation pulse TD in step S6 include either the shock scattering mode or the boiling tissue destruction low-energy variant mode.

[0027] In a preferred embodiment of the present invention, during the intensive scanning of the tumor tissue region in step S6, the spacing between scanning focal points is smaller than the damage diameter of a single pulse, the tumor tissue region is partially ablated, and 10%-30% of the tumor tissue is retained to maintain an antigen library for continuous antigen release.

[0028] As a preferred embodiment of the present invention, the requirements for step S7 include at least one of the following: imaging assessment showing complete regression of the target tumor, reaching the preset upper limit of the number of treatment cycles, the immune activation index IAI remaining in a state of insufficient immune activation and ineffective regulation, and receiving a termination command from the operator.

[0029] As a preferred embodiment of the present invention, during steps S1-S7, the immune microenvironment modulator is delivered to the tumor area or the whole body.

[0030] A cavitation-induced tumor in situ vaccine immune enhancement device and a cavitation-induced tumor in situ vaccine immune enhancement method include an ultrasonic cavitation therapy unit, an immune status real-time monitoring unit, a drug co-delivery unit, and a control unit; the ultrasonic cavitation therapy unit includes a focused ultrasound transducer that can switch between induced cavitation pulse (IAD) and ablation cavitation pulse (TD), a scanner capable of sparse and dense scanning, and a power amplifier with programmable output power.

[0031] Compared to existing technologies, by setting up sequentially emitted induced cavitation pulses (IAD) and ablation cavitation pulses (TD), the induced cavitation pulses (IAD) achieve partial ablation of tumor tissue and immune activation of tumor tissue, enabling cavitation technology to establish an in-situ vaccine platform on tumor tissue, laying the foundation for the ablation boundary and ablation effect of subsequent ablation cavitation pulses (TD).

[0032] During the induction of cavitation pulse IAD emission, the immune status of tumor tissue is monitored in real time. Based on the monitored immune activation index IAI, the parameters of the induction of cavitation pulse IAD are dynamically adjusted to achieve an adaptive induction effect.

[0033] By inducing the cavitation pulse IAD phase and the ablation cavitation pulse TD phase, the immune activation index IAI can be implemented alternately or in parallel according to the patient's timing. This ensures the integrity of the antigen structure to activate immunity and achieves efficient physical reduction of the tumor. This decoupled design allows the cavitation technology to independently optimize two objectives, breaking through the bottleneck that the traditional single-parameter mode cannot achieve.

[0034] For patients with cold tumors that are weakly immunogenic and do not respond to ICIs, inducing mechanical cell death through cavitation pulsed IADs to release intact antigens and DAMPs can transform the tumor microenvironment from an immunologically "cold" state to a "hot" state, increase the infiltration of CD8+ T cells and polarize TAMs to the M1 type. In synergy with immune checkpoint inhibitors, the tumor control rate of PD-1 / PD-L1 antibodies can be increased from about 20% to more than 50% by monotherapy, providing a new treatment option for patients with cold tumors.

[0035] By dynamically monitoring the immune activation index (IAI) (such as changes in backscatter spectrum, pro-inflammatory / anti-inflammatory cytokine ratio, etc.), the treatment process can be objectively quantified. When the immune activation index (IAI) remains in the optimal range and the tumor burden decreases significantly, the system can automatically determine that "immune memory protection has been established" without relying on the operator's subjective experience, which is conducive to the standardized promotion of this technology.

[0036] By establishing a personalized immune status baseline through pretreatment and real-time monitoring of the initial induced cavitation pulse IAD, the parameters of the induced cavitation pulse IAD and the treatment strategy can be dynamically adjusted to achieve "precise immune dose" delivery for each patient and each tumor. This individualized adaptive strategy makes the treatment results more consistent and reproducible, and can meet the immune response needs of different patients and different tumor types for cavitation stimulation. Attached Figure Description

[0037] Figure 1 This is a flowchart illustrating the present invention;

[0038] Figure 2 This is a schematic diagram comparing the parameter space of two pulse modes: immune adjuvant dose and ablation dose.

[0039] Figure 3 It is a flowchart of adaptive parameter adjustment based on immune status feedback;

[0040] Figure 4 This is a schematic diagram of the treatment cycle timing for multimodal synergistic therapy;

[0041] Figure 5 This is a schematic diagram comparing the immune activation effect of the present invention with that of existing tissue ablation techniques;

[0042] Figure 6 This is a schematic diagram of the structure of the present invention;

[0043] Figure labels: 1. Ultrasonic cavitation therapy unit; 2. Real-time immune status monitoring unit; 3. Drug co-delivery unit; 4. Control unit. Detailed Implementation

[0044] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0045] like Figure 1-6 As shown, the cavitation-induced tumor in situ vaccine immune enhancement method includes the following steps:

[0046] Step S1: Obtain the baseline state of the immune microenvironment of the target tumor.

[0047] Based on the acquisition information, tumors are classified into three types: fully immune-infiltrated hot tumors, poorly immune-infiltrated cold tumors, and exclusionary tumors. The baseline status of the immune microenvironment is obtained through at least one of the following methods: acoustic feature calibration, imaging calibration, and biomarker calibration.

[0048] Among them, acoustic feature calibration involves emitting low-energy probe pulses (sound pressure far below the cavitation threshold), receiving and analyzing the spectral characteristics of ultrasound backscattered signals from tumor tissue, and extracting feature parameters related to tumor cell density, interstitial pressure, blood supply, etc., as acoustic proxy indicators of immune status.

[0049] Imaging mapping uses contrast-enhanced ultrasound, elastography, or optical imaging techniques to assess tumor blood perfusion, tissue stiffness, and the extent of immune cell infiltration.

[0050] Biomarker identification involves detecting immune-related markers in tumor tissue, such as PD-L1 expression level, CD8+ T cell infiltration density, and Treg / effector T cell ratio, through puncture biopsy or liquid biopsy.

[0051] Based on the baseline status of the immune microenvironment, tumors were classified into three types: fully infiltrated hot tumors, poorly infiltrated cold tumors, and exclusionary tumors. Fully infiltrated hot tumors are characterized by a large accumulation of various immune cells inside and around the tumor. Poorly infiltrated cold tumors are characterized by almost no cytotoxic T cells inside the tumor, with only a small number of immune cells at the tumor periphery, or even no immune cells invading the tumor mass at all. Exclusionary tumors are characterized by the presence of immune cells, but they are all blocked at the tumor periphery, accumulating in the stroma at the tumor periphery, and are completely unable to enter the core cancer cell nests and come into contact with cancer cells.

[0052] Step S2: Firing an induced cavitation pulse IAD. The induced cavitation pulse IAD is fired toward the focal region of the tumor tissue to induce the release of tumor antigens and activate antigen-presenting cells. The induced cavitation pulse IAD does not aim for complete liquefaction of the tumor tissue, and the parameters of the induced cavitation pulse IAD are defined as the immune adjuvant dose. At the same time, sparse scanning of the tumor tissue region is continuously performed.

[0053] The parameters of the induced cavitation pulse IAD include at least one of the following: shock scattering mode, low-intensity tissue destruction mode, and boiling tissue destruction low-energy variant mode.

[0054] The shock scattering mode consists of short pulses of 3-20 cycles with a peak negative pressure of 8-15 MPa; the low-intensity tissue destruction mode has a pulse length of 1-10 ms and a low duty cycle; and the boiling tissue destruction low-energy variant mode has a pulse length of 1-5 ms and a peak positive pressure of <50 MPa.

[0055] During sparse scanning of tumor tissue regions, the distance between two adjacent scanning focal points is greater than the damage diameter of a single pulse. The distance between two adjacent scanning focal points can be 2-3 times the damage diameter of a single pulse. This ensures that some tissue within the tumor region is treated while some untreated tumor tissue regions form local antigen release areas, thus preserving the immune response initiation platform.

[0056] The area of ​​tumor tissue liquefaction caused by induced cavitation pulse IAD does not exceed 10%-40% of the total area covered by induced cavitation pulse IAD. Specifically, the area of ​​tumor tissue liquefaction caused by induced cavitation pulse IAD does not exceed 20%-30% of the total area covered by induced cavitation pulse IAD. The main effect is concentrated on the release of tumor antigens (>50% of cells undergo immunogenic cell death) rather than complete lysis.

[0057] Step S3: Monitor immune activation status in real time. Collect the immune activation index (IAI) in the tumor microenvironment at one or more time points after the emission of the induced cavitation pulse (IAD) (including multiple time windows within 0-24 hours after the emission of the induced cavitation pulse (IAD)).

[0058] The immune activation index (IAI) can be collected using at least one of the following methods: acoustic, optical, or biosensor-based.

[0059] Among them, acoustic indicators detect the shift or change in the spectral width of the backscattered signal of tumor tissue after cavitation, serving as proxy indicators of changes in tumor cell density and inflammatory cell infiltration. They also detect the energy changes of broadband noise in passive cavitation noise, reflecting the intensity and range of bubble activity, and detect subharmonic components, reflecting the correlation between changes in tissue elastic modulus and the degree of immune cell infiltration.

[0060] Optical indicators use fluorescence imaging or Raman spectroscopy to detect reactive oxygen species (ROS) levels and pH changes in the tumor microenvironment, reflecting early biochemical signals of the immune response. Real-time tracking is performed using immune cells (such as CD8+ T cells and dendritic cells) labeled with near-infrared fluorescent probes, making it suitable for superficial tumors or deep tumors using fiber optic endoscopy.

[0061] Biosensor indicators are used to detect the concentration ratio of pro-inflammatory cytokines (such as IL-12, TNF-α, IFN-γ) and anti-inflammatory cytokines (such as IL-10, TGF-β) in tumor interstitial fluid through implanted microneedle sensors or multimodal probes, as well as the concentration of ATP (reflecting the degree of immunogenic cell death) and the level of DAMPs such as HMGB1 in the tumor microenvironment.

[0062] Step S4: Assessment and comparison of immune activation status. Based on the antigen release status, one or more immune activation indices (IAI) collected in step S3 are classified into insufficient immune activation, optimal immune activation, and risk of immune depletion.

[0063] Set a first threshold T1, a second threshold T2, and a third threshold T3 respectively. The first threshold T1 corresponds to the insufficient immune activation setting, the second threshold T2 corresponds to the optimal immune activation setting, and the third threshold T3 corresponds to the immune exhaustion risk setting.

[0064] When the assessment results indicate insufficient immune activation, i.e., insufficient antigen release, no significant recruitment of dendritic cells, and an excessively low ratio of pro-inflammatory to anti-inflammatory cytokines in the TME.

[0065] When the assessment result indicates optimal immune activation, it means that antigen release is sufficient and the TME inflammatory polarization direction is correct.

[0066] When the assessment result indicates a risk of immune exhaustion, it means that persistent strong inflammatory signals may lead to immune exhaustion or feedback recruitment of immunosuppressive cells (such as MDSCs and Tregs).

[0067] Step S5: Adjust the induced cavitation pulse IAD parameter in real time. Based on the evaluation results of step S4, adjust the induced cavitation pulse IAD parameter in real time until the immune activation index IAI is optimal for immune activation.

[0068] When the assessment result indicates insufficient immune activation, at least one of the following procedures can be performed to enhance the release of tumor tissue antigens or activate the tumor immune microenvironment:

[0069] 1. Increase the acoustic pressure amplitude of the induced cavitation pulse IAD (in increments of 0.5-2 MPa) or extend the length of the induced cavitation pulse IAD to enhance antigen release.

[0070] 2. Shorten the IAD spacing of the induced cavitation pulse (increase the scanning density), increase the coverage of the processed area, and generate more antigen.

[0071] 3. Switch to the boiling tissue destruction mode (millisecond-level long pulses, higher sound pressure) to enhance the release of damage-associated molecular patterns (DAMPs).

[0072] 4. Administer in combination with immune checkpoint inhibitors (such as anti-PD-1 / PD-L1 antibodies) or immune adjuvants (such as CpG, STING agonists).

[0073] When the evaluation result indicates optimal immune activation, the current induced cavitation pulse IAD parameters are maintained, indicating that the current induced cavitation pulse IAD parameters have triggered effective anti-tumor immunity. The induced cavitation pulse IAD parameters are maintained at the current level, while the immune status is continuously monitored to keep it within the optimal range.

[0074] When the assessment indicates a risk of immune exhaustion, pausing or reducing the induced cavitation pulse IAD parameter suggests that excessive inflammatory stimulation may lead to immune exhaustion. At least one of the following actions can be taken:

[0075] 1. At this point, it is necessary to reduce the sound pressure of the induced cavitation pulse (IAD) or shorten the pulse length.

[0076] 2. Extend the interval between induced cavitation pulses (IADs) to allow sufficient cooling-off period for the immune response.

[0077] 3. Pause the induced cavitation pulse IAD and perform only local therapeutic dose ablation or wait in a blank state.

[0078] 4. Administer anti-inflammatory modulators (such as low-dose IL-2 to maintain Treg / Teff balance).

[0079] Step S6: Firing an ablation cavitation pulse (TD) and continuously performing immune activation index (IAI) detection during and after the firing of the ablation cavitation pulse (TD). The parameters of the ablation cavitation pulse (TD) are defined as the ablation dose, while intensive scanning of the tumor tissue area is performed continuously.

[0080] Once immune activation has been confirmed to be fully initiated (i.e., the immune activation index IAI reaches the optimal level for immune activation), the ablation cavitation pulse TD can be selectively emitted in a portion of the tumor tissue to target the tumor tissue volume.

[0081] The parameters of the ablation cavitation pulse TD include either the shock scattering mode or the boiling tissue destruction low-energy variant mode. The shock scattering mode has 3-20 cycles and a peak negative pressure of 15-20 MPa. The boiling tissue destruction low-energy variant mode uses millisecond-level pulses and a peak positive pressure of 60-75 MPa.

[0082] During intensive scanning of the tumor tissue area, the focal distance between the scanning focal points is smaller than the damage diameter of a single pulse, completely covering the target tumor area. The tumor tissue area is partially ablated under the action of the ablation cavitation pulse TD, retaining 10%-30% of the tumor tissue to maintain the antigen pool for continuous antigen release, ensuring that the ablation process does not weaken the established immune response due to excessive destruction of the antigen structure.

[0083] Step S7: Repeat steps S2-S6 until the desired result is achieved.

[0084] The requirements for step S7 include at least one of the following: imaging assessment showing complete regression of the target tumor, reaching the preset maximum number of treatment cycles, the immune activation index (IAI) remaining in a state of insufficient immune activation and ineffective regulation, or receiving a termination command from the operator.

[0085] For imaging assessment to show complete regression of the target tumor, the immune activation index (IAI) must be in the optimal range for immune activation for three consecutive time points (interval ≥ 7 days).

[0086] A persistently low IAI (Immune Activation Index) indicates insufficient immune activation and ineffective regulation, suggesting that the tumor is immune-rejecting and requires a switch to other treatment modalities.

[0087] After the required conditions are met, a challenge experiment can be conducted again to evaluate the immune memory effect. By inoculating homologous tumor cells onto the contralateral side and monitoring the tumor growth curve, the tumor rejection status of the untreated control group can be compared to determine the immune memory protection effect of the present invention.

[0088] During steps S1-S7, immune microenvironment modulators are delivered to the tumor area or the whole body. These agents include, but are not limited to, immune checkpoint inhibitors, immune adjuvants, and tumor microenvironment modulators.

[0089] Immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies; immune adjuvants include TLR agonists (such as CpG ODN and poly I:C), STING agonists (such as cGAMP), and cytokines (such as IL-2, IL-12, GM-CSF, and IFN-γ); and tumor microenvironment modulators include TGF-β inhibitors, CXCR2 antagonists, and arginase inhibitors.

[0090] Immune checkpoint inhibitors, immune adjuvants, and tumor microenvironment modulators can be delivered via intratumoral injection, intravenous injection, locally implanted sustained-release devices, or ultrasound-mediated targeted microbubble / nanobubble delivery. The timing, dosage, and site of drug administration can be dynamically adjusted based on the Immune Activation Index (IAI) assessment results from step S4.

[0091] A cavitation-induced tumor in situ vaccine immune enhancement device and a cavitation-induced tumor in situ vaccine immune enhancement method include an ultrasonic cavitation therapy unit 1, an immune status real-time monitoring unit 2, a drug co-delivery unit 3, and a control unit 4; the ultrasonic cavitation therapy unit 1 includes a focused ultrasound transducer that can switch between induced cavitation pulse (IAD) and ablation cavitation pulse (TD), a scanner capable of sparse and dense scanning, and a power amplifier with programmable output power.

[0092] The focused ultrasound transducer is a single-element or multi-element phased array focused transducer with an operating frequency of 0.5-2.5 MHz, and can switch between immune adjuvant dosing mode and ablation dosing mode.

[0093] The power amplifier has programmable output power and supports two operating modes: short pulse (microsecond level) and long pulse (millisecond level).

[0094] The scanner is a mechanical or electronic scanner that controls the focus, supporting both sparse and dense scanning.

[0095] The real-time immune status monitoring unit 2 includes at least one of the following monitoring devices: a passive cavitation detection probe, a backscatter analysis module, and a biosensor interface.

[0096] The passive cavitation detection probe is set independently or embedded in the focused ultrasonic transducer to collect broadband noise, subharmonics and other signals generated during cavitation, with a frequency response range of 0.5-10 MHz.

[0097] The backscatter analysis module is used to analyze the spectral characteristics of ultrasound backscatter signals in tumor tissue before and after treatment.

[0098] The photoelectric detection module includes a fiber optic spectrometer, a fluorescence excitation or detection system, or a Raman spectrometer, used for optical detection of immune-related biochemical indicators in superficial tumors or deep tumors via endoscopy.

[0099] The biosensor interface is used to connect implantable microneedle electrodes or multimodal probes to detect the concentration of cytokines in tumor interstitial fluid.

[0100] The drug delivery unit 3 includes a drug reservoir, a drug administration control module, and a delivery transducer. The drug reservoir contains one or more immunomodulators. The drug administration control module controls the drug administration method (intratumoral injection, intravenous infusion, ultrasound-mediated microbubble delivery, etc.), drug flow rate, drug administration timing, and dosage. The delivery transducer is shared with or independently set with the focused ultrasound transducer, with a driving frequency of 0.5-2.0 MHz. The delivery transducer uses ultrasound-targeted microbubble delivery of drugs.

[0101] The control unit 4 is telecommunicationly connected to the ultrasonic cavitation therapy unit 1, the real-time immune status monitoring unit 2, and the drug co-delivery unit 3.

[0102] The control unit 4 includes an immune activation status assessment module, an adaptive decision-making module, a parameter storage module, and a timing control module.

[0103] The immune activation status assessment module converts one or more immune activation indices (IAI) collected in step S3 into quantitative indicators and compares them with preset first threshold T1, second threshold T2 and third threshold T3.

[0104] The adaptive decision-making module dynamically determines the induction parameters, scanning mode, and drug administration strategy for the next stage based on the comparison results of the immune activation index (IAI) with the first threshold (T1), the second threshold (T2), or the third threshold (T3). This decision-making module is implemented based on deterministic rules (threshold comparison + lookup table pre-stored strategy).

[0105] The parameter storage module stores pre-optimized IAD (Induced Cavitation Pulse) and TD (Disease Cavitation Pulse) parameter libraries for different tumor types (thermal tumors, cold tumors, exclusionary tumors), as well as the optimal values ​​for each threshold.

[0106] The timing control module precisely controls the timing relationship between the induced cavitation pulse (IAD), the ablation cavitation pulse (TD), the immune status monitoring window, and the drug delivery window.

[0107] The tumor region is located by image guidance, and the type of immune microenvironment of the tumor is determined by backscatter analysis or biopsy information. Based on this, the pre-optimized induced cavitation pulse IAD parameters and initial immune activation threshold suitable for the tumor type are called.

[0108] The driven focused ultrasound transducer emits induced cavitation pulses (IADs), and a sparse scanning strategy is used to generate multiple dispersed "antigen release hotspots" within the tumor region. The energy of these induced cavitation pulses is just enough to induce immunogenic cell death in tumor cells, releasing intact TAAs and DAMPs, but not enough to completely liquefy the tissue. Due to the use of sparse scanning, a large amount of untreated residual tumor tissue is retained between the treated areas, forming an "antigen reservoir" that can continuously provide antigen stimulation during treatment.

[0109] At multiple time points following induced cavitation pulse IAD (e.g., 0.5 h, 2 h, 6 h, 12 h, and 24 h after treatment), passive cavitation detection probes and / or other monitoring devices collected immune status indicators of the tumor region. For example, an increase in the backscatter signal spectral width may reflect the infiltration of dendritic cells and macrophages, and changes in the subharmonic components of passive cavitation noise may reflect changes in the elasticity of the tumor mesotherapy (TME) and cell density, which are important indirect indicators of immune cell recruitment.

[0110] The immune activation status assessment module of the control unit compares the collected indicators with pre-stored thresholds. If insufficient immune activation is detected, the system automatically increases the energy of the induced cavitation pulse IAD or switches to a more aggressive cavitation mode, or triggers the drug co-delivery unit 3 to release the immune adjuvant. If excessive immune activation or immune exhaustion signals are detected, the system reduces the pulse energy or pauses treatment, giving the immune system an appropriate "cooling-off period".

[0111] Once immune activation has been fully established (the Immune Activation Index (IAI) stabilizes at the second threshold (T2), an ablation cavitation pulse (TD) is emitted to perform intensive scanning of the tumor mass for highly efficient physical ablation, achieving tumor reduction. Even during the ablation cavitation pulse (TD) ablation phase, immune status monitoring is maintained to ensure that the ablation does not weaken the initiated immune response due to excessive destruction of the antigen structure.

[0112] Throughout the process, if real-time monitoring detects TME immunosuppressive signals (such as elevated IL-10 or enhanced Treg marker expression), the drug co-delivery unit 3 can be triggered to release ICIs or adjuvants. For example, if antigen-loaded dendritic cells have migrated to draining lymph nodes 24 hours after induced cavitation pulse IAD, the system can trigger the administration of PD-1 antibodies to maximize the T cell killing effect.

[0113] When the tumor completely regresses and the immune activation index (IAI) remains within the optimal range for immune activation, the system determines that it has reached the state of "immune memory protection." The establishment of systemic immune memory can be verified by a contralateral rechallenge experiment after treatment.

[0114] Experimental Example 1: A 1.5 MHz single-element spherical cap focusing ultrasonic transducer with a radius of curvature of 100 mm and an aperture of 60 mm was used. A PVDF hydrophone with a center frequency of 5 MHz was installed at the center opening of the focusing ultrasonic transducer for passive cavitation detection and backscatter signal acquisition.

[0115] The control unit 4 uses an FPGA timing control board, which can generate cavitation excitation signals with adjustable pulse length (1-20 cycles).

[0116] Tumor-bearing mice (B16F10 melanoma model, tumor volume approximately 100 mm³) were placed in a degassed water tank, and the focus of the focused ultrasound transducer was aimed at the center of the tumor.

[0117] The induced cavitation pulse IAD parameters were set to impact scattering mode, with a pulse length of 5 acoustic cycles (corresponding to 1.5 MHz, each cycle 0.667 μs, total pulse length 3.33 μs), a focal peak negative pressure of 10 MPa, a pulse repetition frequency of 100 Hz, a sparse scanning strategy, and a spacing between two adjacent focal points of 1.5 mm (this spacing is approximately 1.67 times the single pulse damage diameter of 0.9 mm).

[0118] The tumor boundary is located using a B-mode ultrasound imaging system, and the scanning path is planned. The scanning path covers approximately 50% of the tumor area, leaving about 50% of the tumor tissue untreated.

[0119] Drive the focused ultrasound transducer to emit induced cavitation pulses (IAD) according to the above parameters. Emitters emit 30 pulses at each focal position. After processing one focal position, move the focused ultrasound transducer to the next focal position and repeat the operation until all planned scanning points are covered.

[0120] During the emission of the induced cavitation pulse IAD, backscattered signals are continuously collected by a central hydrophone for subsequent assessment of immune activation status.

[0121] Within a preset time window after the induced cavitation pulse IAD is emitted (e.g., 0.5 hours, 2 hours, 6 hours, 12 hours, 24 hours after treatment), a low-energy probe pulse (sound pressure much lower than the cavitation threshold, e.g., 1 MPa) is emitted to acquire backscattered radio frequency data from the tumor region.

[0122] At least 100 frames of RF data are acquired in each time window, with a sampling rate of 50 MHz. The acquired RF data is bandpass filtered (passband 1-15 MHz) to remove noise other than the fundamental frequency component. A Fast Fourier Transform (FFT) is performed on each A-scan signal to obtain the spectral distribution.

[0123] Calculate the following spectral characteristics:

[0124] High-frequency band energy percentage: Calculate the percentage of total energy within the 4-10 MHz frequency band;

[0125] -3dB bandwidth: The frequency width corresponding to a 3dB decrease in spectral amplitude;

[0126] Spectral center frequency shift: the amount of change in center frequency relative to the pre-treatment baseline;

[0127] Spectral entropy: The Shannon entropy of the spectral energy distribution, calculated by the formula H = -∑p(f)logp(f), where p(f) is the normalized power spectral density.

[0128] The above-mentioned features are input into the immune activation assessment module, which pre-stores offline calibrated mapping functions (such as linear regression models) to convert the spectral features into the "immune activation index" (IAI), with a value range of 0-1.

[0129] The first threshold T1 = 0.3; a value below this indicates insufficient immune activation.

[0130] The second threshold T2 = 0.6, and values ​​above this indicate that immune activation has reached its optimal level;

[0131] The third threshold T3 = 0.9: A value higher than this indicates a potential risk of excessive inflammation or immune depletion.

[0132] Data is automatically collected at each monitoring time point, the immune activation index (IAI) is calculated, and the IAI is compared with the above threshold. The parameters of the subsequent induced cavitation pulse (IAD) are determined based on the comparison results.

[0133] Example 1,

[0134] If the immune activation index (IAI) is < 0.3, indicating insufficient immune activation, the peak negative pressure of the subsequent cavitation pulse IAD will be increased by 2 MPa (maximum not exceeding 15 MPa); simultaneously, the length of the cavitation pulse IAD will be increased from 5 cycles to 10 cycles. If the immune activation index remains < 0.3 after two consecutive adjustments, the scanning strategy will be switched from sparse scanning (1.5 mm spacing) to medium-density scanning (1.0 mm spacing).

[0135] Example 2,

[0136] If the immune activation index (IAI) is 0.3 ≤ 0.8, the immune activation is optimal. Maintain the current induced cavitation pulse (IAD) parameters unchanged and continue to fire the next set of induced cavitation pulses (IADs) as planned.

[0137] Example 3,

[0138] If the immune activation index (IAI) is > 0.8, indicating a risk of immune exhaustion, the peak negative pressure of subsequent induced cavitation pulse IADs will be reduced by 2 MPa (with a lower limit of no less than 6 MPa). At the same time, the repetition frequency of induced cavitation pulse IADs will be reduced from 100 Hz to 50 Hz to give the immune system a longer "cooling-off period". If the immune activation index continues to be > 0.8, the induced cavitation pulse IAD firing will be suspended for 24 hours, during which time only monitoring will be performed.

[0139] The immune activation index (IAI) is considered to be fully established when it reaches 0.6 or higher and less than 0.8 twice consecutively (with an interval of at least 6 hours).

[0140] Set the ablation cavitation pulse TD parameters, either shock scattering mode or boiling tissue ablation mode. If it is shock scattering mode, the pulse length is 10 cycles, the peak negative pressure is 18 MPa, and the pulse repetition frequency is 200 Hz. If it is boiling tissue ablation mode, the pulse length is 10 ms, the peak positive pressure is 70 MPa, and the pulse repetition frequency is 1 Hz. The scanning strategy is dense scanning, and the focal spacing is set to 0.6 mm (less than the single pulse damage diameter of 0.9 mm).

[0141] A grid scan is performed on the entire tumor area, with 100 pulses (impact scattering mode) or 30 pulses (boiling mode) emitted at each focal point to ensure that the entire tumor volume is densely covered.

[0142] During the ablation cavitation pulse TD emission, backscatter signals are continuously acquired to monitor changes in the immune activation index (IAI). If the IAI drops below 0.3 (indicating that excessive ablation may have destroyed the antigen structure), the ablation cavitation pulse TD emission is immediately paused, and the ablation cavitation pulse IAD emission is switched back to a small number (e.g., 2-3 focal points) of induced cavitation pulse IADs to supplement antigen release until the tumor is completely ablated (as assessed by imaging) and the IAI stabilizes in the range of 0.3-0.8.

[0143] During the induction of cavitation pulse IAD and ablation of cavitation pulse TD, an anti-PD-1 antibody solution (concentration 5 mg / mL) is pre-filled in the drug reservoir. The drug delivery control module uses a microfluidic pump with an adjustable flow rate (0.1-10 μL / min), and the drug delivery interface is connected to an intratumoral injection needle or an indwelling intravenous catheter.

[0144] If the immune activation index (IAI) before cavitation pulse IAD is < 0.2, no drug is administered.

[0145] Two hours after induced cavitation pulse IAD, the immune activation index (IAI) was 0.2-0.4. No drug was administered, and the patient waited for antigen presentation.

[0146] If the immune activation index (IAI) is ≥ 0.4 24 hours after induced cavitation pulse IAD, intravenous infusion of anti-PD-1 antibody (200 μg / mouse, infusion time 10 minutes) is triggered.

[0147] Following ablation cavitation pulsed TD ablation, an immune activation index (IAI) of 0.3-0.6 triggered a second anti-PD-1 antibody infusion (200 μg / mouse).

[0148] After treatment, the maintenance immune activation index (IAI) dropped to <0.3, triggering a third infusion (200 μg / mouse).

[0149] Experimental Example 2:

[0150] The baseline immune activation index (IAI) is calculated by backscatter signal analysis. If the IAI is < 0.15, the tumor is classified as a "cold" tumor.

[0151] Compared to Experiment 1, Experiment 2 used more aggressive initial cavitation pulse IAD parameters: 10 pulse lengths, 13 MPa peak negative pressure, and 1.2 mm focal distance (covering approximately 70% of the tumor area). Ten minutes before the cavitation pulse IAD, CpG ODN (TLR9 ​​agonist, 50 μg / tumor) was delivered via intratumoral injection.

[0152] Six hours after treatment, monitor the immune activation index (IAI). If the IAI is still <0.2, fire another set of induced cavitation pulses (IAD) (with unchanged parameters) until the IAI reaches 0.3 or above or the maximum pulse count limit is reached.

[0153] When the monitored immune activation index (IAI) reaches 0.6 or higher and less than 0.8 twice consecutively (with an interval of at least 6 hours), it indicates that immune activation has been fully established.

[0154] Co-administration of ablation cavitation pulsed TD emission and drug reservoir was performed until imaging assessment (B-ultrasound or MRI) showed complete disappearance of the target tumor or only scar tissue, or the immune activation index (IAI) values ​​monitored for three consecutive times (7 days apart) were all in the range of 0.3-0.8 and showed a stable or slow decreasing trend (indicating that immune memory has been established and no continuous stimulation is required).

[0155] After each ablation cavitation pulse TD is completed, the immune activation index (IAI) after the current ablation cavitation pulse TD is recorded. After reaching the preset maximum number of cycles (e.g., 6 cycles), the endpoint assessment mode is entered. If imaging shows that there is still residual tumor (volume > 5 mm³), the next cycle of treatment is continued. If imaging shows that there is no residual tumor, and the immune activation index (IAI) in the most recent three cycles is ≥ 0.3 and ≤ 0.8, and the change rate of immune activation index (IAI) between two adjacent cycles is < 10%, it is determined that "immune memory protection has been established".

[0156] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention; therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0157] Although this document frequently uses reference numerals in the figures, such as "ultrasonic cavitation therapy unit 1," "real-time immune status monitoring unit 2," "drug co-delivery unit 3," and "control unit 4," the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of the invention; interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for enhancing the immunity of tumor in situ vaccines based on cavitation induction, characterized in that, Includes the following steps: Step S1: Obtain the baseline state of the immune microenvironment of the target tumor; Step S2: Firing an induced cavitation pulse IAD, which is used to induce tumor antigen release and activate antigen-presenting cells, towards the focal region of the tumor tissue, and defining the parameters of the induced cavitation pulse IAD as the immune adjuvant dose, while continuously performing sparse scanning on the tumor tissue region. Step S3: Monitor immune activation status in real time. At one or more time points after the emission of the induced cavitation pulse (IAD), collect the immune activation index (IAI) in the tumor microenvironment in real time. Step S4: Assessment and comparison of immune activation status. Based on the antigen release status, one or more immune activation indices (IAI) collected in step S3 are classified into insufficient immune activation, optimal immune activation, and risk of immune exhaustion. Step S5: Adjust the induced cavitation pulse IAD parameter in real time. Based on the evaluation results of step S4, adjust the induced cavitation pulse IAD parameter in real time until the immune activation index IAI is optimal for immune activation. When the assessment result indicates insufficient immune activation, increase the release of tumor tissue antigens or activate the tumor immune microenvironment. When the evaluation result indicates optimal immune activation, maintain the current IAD parameters for the induced cavitation pulse. When the assessment result indicates a risk of immune exhaustion, pause or reduce the IAD parameter of the induced cavitation pulse. Step S6: Firing an ablation cavitation pulse TD, and continuously performing immune activation index (IAI) detection during and after the firing of the ablation cavitation pulse TD. The parameters of the ablation cavitation pulse TD are defined as the ablation dose, while continuously performing intensive scanning of the tumor tissue area. Step S7: Repeat steps S2-S6 until the desired result is achieved.

2. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, Step S1 classifies tumors into three types based on the acquired information: fully immune-infiltrated hot tumors, poorly immune-infiltrated cold tumors, and excluded tumors. The methods for acquiring the baseline state of the immune microenvironment include at least one of acoustic feature calibration, imaging calibration, and biomarker calibration.

3. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, The parameters of the cavitation pulse IAD induced in step S2 include at least one of the following: shock scattering mode, low-intensity tissue damage mode, and boiling tissue damage low-energy variant mode.

4. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, In step S2, during the sparse scanning of the tumor tissue region, the distance between two adjacent scanning focal points is greater than the damage diameter of a single pulse. While the tumor tissue region forms a local release of antigens, it retains the immune response initiation platform. The area of ​​tumor tissue liquefaction caused by the induced cavitation pulse IAD does not exceed 10%-40% of the total area covered by the induced cavitation pulse IAD.

5. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, The method for collecting the immune activation index (IAI) in step S3 includes at least one of acoustic indicators, optical indicators, and biosensor indicators.

6. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, The parameters for the ablation cavitation pulse TD in step S6 include either the shock scattering mode or the boiling tissue destruction low-energy variant mode.

7. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, In step S6, during the intensive scanning of the tumor tissue area, the spacing between scanning focal points is smaller than the damage diameter of a single pulse, resulting in partial ablation of the tumor tissue area while retaining 10%-30% of the tumor tissue to maintain the antigen pool for continuous antigen release.

8. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, The requirements for step S7 include at least one of the following: imaging assessment showing complete regression of the target tumor, reaching the preset upper limit of the number of treatment cycles, the immune activation index (IAI) remaining in a state of insufficient immune activation and ineffective regulation, and receiving a termination command from the operator.

9. The method for enhancing the immune response of an in situ tumor vaccine based on cavitation induction according to claim 1, characterized in that, During steps S1-S7, immune microenvironment modulators are delivered to the tumor area or the whole body.

10. A device for enhancing the immune response of an in situ tumor vaccine based on cavitation, based on the method for enhancing the immune response of an in situ tumor vaccine based on cavitation as described in any one of claims 1-9, comprising an ultrasonic cavitation therapy unit (1), an immune status real-time monitoring unit (2), a drug co-delivery unit (3), and a control unit (4); wherein the ultrasonic cavitation therapy unit (1) comprises a focused ultrasound transducer that can switch between induced cavitation pulse (IAD) and ablation cavitation pulse (TD), a scanner capable of sparse scanning and dense scanning, and a power amplifier with programmable output power.

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