A whole cell vaccine inactivation method and system directed to immunogenic cell death

Abstract

This invention belongs to the interdisciplinary field of biomedicine and nanotechnology, specifically relating to a method and system for inactivating whole-cell vaccines targeting immunogenic cell death. The method includes: contacting tumor cells with magnetic nanoparticles and internalizing them into the cell; placing the system to be treated in an alternating magnetic field, achieving heating from the inside out through in-situ intracellular heat generation; monitoring the temperature in real time with a fixed sampling period; dynamically adjusting the magnetic field strength and / or frequency based on the deviation between the temperature and the target temperature range, causing the temperature to fluctuate within the immunogenic cell death induction window; accumulating the effective time, and stopping the magnetic field after reaching the preset inactivation time. This invention, through the spatiotemporal coupling of magnetic nanoparticle internalization, closed-loop feedback control, and the accumulated effective time strategy, actively induces immunogenic cell death while ensuring 100% inactivation, obtaining a highly immunogenic whole-cell tumor vaccine. This solves the technical problems of thermal hysteresis, thermal overshoot, and inability to accurately induce immunogenic cell death in traditional thermal inactivation methods.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and nanotechnology, and specifically relates to a method and system for inactivating whole-cell vaccines targeting immunogenic cell death. Background Technology

[0002] Tumor immunotherapy has become an important treatment paradigm following surgery, radiotherapy, and chemotherapy. Among them, therapeutic tumor vaccines aim to activate the patient's own immune system to specifically recognize and attack tumor cells, and have the potential for persistence and high specificity. Whole-cell tumor vaccines, because they carry the complete antigen spectrum of tumor cells, including unknown tumor-associated antigens and neoantigens, can stimulate a broad polyclonal immune response and avoid immune escape caused by antigen selection bias.

[0003] Inactivation is a core step in the preparation of whole-cell vaccines to ensure biosafety and non-tumorigenicity. Ideal inactivation goes beyond simply killing cells; it transforms them into immune initiators capable of strongly activating adaptive immunity. The core of this transformation process is the induction of immunogenic cell death (ICD), a specific form of regulatory cell death characterized by the release or exposure of a series of danger signaling molecules from dying cells, including calreticulin (CRT) positivity, high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP), thereby efficiently activating dendritic cells and initiating antigen-specific T-cell immunity.

[0004] Existing inactivation methods mainly include physical and chemical methods. Physical methods include radiation inactivation, ultraviolet inactivation, traditional heat inactivation, and freeze-thaw / repeated freeze-thaw cycles. Radiation inactivation, often using 60-200 Gy gamma rays or X-rays, can cause protein oxidation, cross-linking, and DNA damage, potentially destroying key antigenic epitopes. Ultraviolet inactivation causes DNA damage, but has a relatively smaller impact on membrane protein antigens, although inactivation may be incomplete. Traditional heat inactivation, such as heating in a water bath or oven at 56°C for 30 minutes, leads to widespread protein denaturation and is not considered a preferred method for preparing vaccines that retain high immunogenicity; it is essentially a non-programmed destruction process. Freeze-thaw or repeated freeze-thaw cycles can cause cell lysis and release of contents, but the process is uncontrollable and antigens are severely damaged. Chemical methods commonly use cross-linking agents such as formaldehyde and glutaraldehyde, which non-specifically cross-link proteins, potentially masking antigenic epitopes and posing a risk of chemical residues.

[0005] More importantly, existing inactivation methods, especially physical heat inactivation, are limited by their inherent energy transfer and heat conduction mechanisms, making it difficult to meet the precise cellular stress control required for inducing ICD. Taking water bath heating as an example, the heat is conducted from the external heat source to the cell interior through the medium, which has the following limitations: First, the thermal response is severely delayed. When the external water temperature reaches the set point, the internal cell temperature takes a long time to reach equilibrium, making it difficult to accurately control the actual heat exposure dose. Second, there is a contradiction between temperature control precision and response speed. The large heat capacity of the system leads to large thermal inertia, resulting in temperature overshoot or slow drop after heating stops, making it impossible to achieve instantaneous start and stop of the heat source. Third, different thermal stress patterns may occur. Heating from the outside in may cause asynchronous heating of the cell membrane and internal organelles, making it impossible to simulate or trigger the optimal intracellular stress response pathway, and is severely affected by the heat conduction path and heat source uniformity.

[0006] These physical limitations mean that even with high-precision temperature-measuring physical inactivation systems, there are bottlenecks in achieving rapid, uniform, and instantaneously responsive programmed thermal stimulation at the cellular level. This is precisely the key to efficient and repeatable induction of ICD, and there is a lack of inactivation technologies that can precisely match the biological requirements of ICD induction in terms of physical mechanisms.

[0007] Magnetic iron oxide nanoparticles generate a thermal effect under an alternating magnetic field, which has been extensively studied for local physical therapy of tumors. Current technologies primarily focus on their high-temperature ablation function above 50°C, aiming to directly induce coagulative necrosis of tumor tissue through high temperatures—a direct killing mechanism for therapeutic purposes. However, the use of the magnetocaloric effect as an in vitro, controllable means of inducing programmed cell stress, particularly for precisely inducing ICDs at mild temperatures for vaccine preparation, has not been systematically reported or demonstrated in existing technologies. Although some studies have compared the effects of different inactivation methods on T cell activation, they have not explored the fundamental differences in the impact of different heat conduction and control methods on this precise biological process of ICDs at the physical mechanism level.

[0008] Therefore, there is an urgent need for a novel inactivation method and system that can overcome the limitations of traditional heat conduction from a physical mechanism perspective, precisely control the cell death process, and actively and efficiently induce ICD while ensuring 100% inactivation, thereby transforming tumor cells into immunogenic vaccine raw materials. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a whole-cell vaccine inactivation method and system targeting immunogenic cell death. It departs from the existing approach of simply targeting inactivation rate, instead employing a cell programming strategy. Through precise magnetocaloric control technology mediated by magnetic nanoparticles capable of generating thermal effects under alternating magnetic fields, combined with a high-precision feedback and cumulative effective time control strategy, the inactivation process of tumor cells in an in vitro environment is directed into a controllable immunogenic cell death process.

[0010] By using magnetic nanoparticles that can generate a thermal effect under an alternating magnetic field, the heat source is directly located inside the tumor cells at the target site. Under the action of the alternating magnetic field, heat is generated in situ and instantaneously within the cell. The heat does not need to pass through an external medium and acts directly on key intracellular organelles. The start and stop of the magnetic field corresponds to the instantaneous start and stop of heat generation, which fundamentally eliminates the thermal hysteresis and inertia of traditional heat conduction. This inside-out heating method can activate and match the intracellular stress pathways related to ICD in time and space.

[0011] A method for inactivating whole-cell vaccines targeting immunogenic cell death, comprising the following steps: S1, bringing tumor cells into contact with magnetic nanoparticles that can generate a thermal effect under an alternating magnetic field, and internalizing the magnetic nanoparticles into the tumor cells to form a system to be treated; S2, The system to be treated is placed in an alternating magnetic field, and the magnetic nanoparticles that can generate a thermal effect under the alternating magnetic field generate heat in situ inside the tumor cells, thereby heating the tumor cells from the inside out. S3, with a fixed sampling period Real-time monitoring of the temperature of the system to be processed ; S4, based on the temperature and target temperature range To mitigate the deviation, the magnetic field strength and / or frequency of the alternating magnetic field are dynamically adjusted to control the temperature. Within the preset target temperature range Fluctuations within a period; S5, cumulative temperature Within the target temperature range Valid time within When the effective time Reaching the preset cumulative inactivation time When the alternating magnetic field is stopped, the target temperature range is the temperature range for inducing immunogenic cell death, and the cumulative inactivation time is the time sufficient to inactivate tumor cells 100% and simultaneously achieve immunogenic cell death phenotypic transformation.

[0012] Preferably, the dynamic adjustment of the magnetic field strength and / or magnetic field frequency of the alternating magnetic field in step S4 is achieved based on the following magnetothermal field control model: (1) in, For real-time temperature, The magnetic field strength, The magnetic field frequency, The particle size of a single magnetic nanoparticle capable of generating a thermal effect under an alternating magnetic field. This refers to the number of magnetic nanoparticles within a single tumor cell that can generate a thermal effect under an alternating magnetic field. The heat dissipation coefficient of the system to be treated is... For ambient temperature, a proportional-integral-derivative control law or a hysteresis control law is used, based on the real-time temperature and the target temperature range. The deviation is dynamically calculated and the adjustment amount of the magnetic field strength and / or magnetic field frequency is output.

[0013] Preferably, the expression for the proportional-integral-derivative control law is: (2) in, , The set temperature within the target temperature range. , , These are the proportional coefficient, integral coefficient, and differential coefficient, respectively.

[0014] Preferably, the feedback control in S3 is based on the deviation between the temperature and the upper and lower limits of the target temperature range. To adjust the magnetic field strength and / or magnetic field frequency, including: When the temperature is lower When this occurs, increase the magnetic field strength and / or magnetic field frequency; When the temperature is higher When this occurs, the magnetic field strength and / or magnetic field frequency are reduced; When the temperature is within the target temperature range During this period, the magnetic field strength is maintained or finely adjusted to keep the temperature within the specified range; The adjustment amount of the magnetic field parameter is positively correlated with the magnitude of the deviation, and the target temperature range...

[0015] Preferably, the fixed sampling period described in S3 Within one second, the accuracy of the real-time temperature monitoring of the system under treatment is no less than 0.1℃. Preferably, the effective time mentioned in S5 The accumulation method is as follows: in each sampling period, if the monitored temperature is within the target temperature range, then the sampling period is accumulated. Accumulated to the valid time A counter; otherwise, it is not incremented, satisfying the following condition: (3) Preferably, the magnetothermal field control model further includes feedforward compensation for intracellular iron content, wherein the average iron content within the tumor cells is measured before step S2. According to the average iron content The initial magnetic field strength is preset according to the following relationship. : (4) in, , This is an empirical coefficient, ensuring that, without feedback control, the equilibrium temperature of the system approaches the target temperature range after applying the initial magnetic field.

[0016] An inactivated tumor cell vaccine obtained by a whole-cell vaccine inactivation method targeting immunogenic cell death, the vaccine comprising 100% inactivated tumor cells cultured for 7 days without clone formation; the tumor cell culture supernatant has a CRT positivity rate and ATP concentration of not less than 50%; the tumor cell culture supernatant has an HMGB1 concentration of not less than 20 pg / mL; and the tumor cell culture supernatant has an ATP concentration of not less than 100 nM.

[0017] A system for implementing a whole-cell vaccine inactivation method targeting immunogenic cell death includes: a magnetic field generating unit for generating an alternating magnetic field with a frequency of 300 kHz to 500 kHz and adjustable magnetic field strength; a temperature monitoring unit for real-time monitoring of the temperature of the system to be treated at a sampling frequency of not less than 1 time / second and an accuracy of not less than 0.1 ℃; and a control unit communicatively connected to the magnetic field generating unit and the temperature monitoring unit.

[0018] Preferably, the control unit is configured to execute the following control process to obtain a preset target temperature range. and cumulative effective inactivation time target Based on the real-time temperature obtained from the temperature monitoring unit Data, accumulating the real-time temperature Within the target temperature range Valid time within According to real-time temperature and target temperature range Deviation between upper and lower limits The system dynamically calculates and outputs adjustment commands to the magnetic field generating unit to adjust the magnetic field strength and / or frequency of the alternating magnetic field; when the accumulated effective time... To achieve the When the time comes, an output command is sent to control the magnetic field generating unit to stop working.

[0019] Compared with the prior art, the method of this application has the following beneficial technical effects: 1. Lock the ICD immunoconversion temperature window. Within the optimized ICD temperature window of 42–47 ℃ and 15–25 min, stabilize the temperature fluctuation within the target temperature window through second-level closed-loop feedback and cumulative effective time control, with batch repeatability CV < 5%.

[0020] 2. Targeted programming of immunogenic cell death: By activating the endoplasmic reticulum stress pathway through intracellular heat stress and oxidative stress, the cell death mode is guided from the random necrosis / late apoptosis of traditional methods to active and controllable immunogenic cell death. The CRT positivity rate, HMGB1, ATP, IDC activation and in vivo protective effect of the product are all superior to the existing inactivation treatment technology, realizing the functional paradigm transformation of whole cell vaccine preparation from killing to programming.

[0021] 3. Reconstructing the thermal stress conduction path to achieve subcellular precision inside-out thermal programming: Magnetic nanoparticles capable of generating thermal effects under alternating magnetic fields must be internalized into tumor cells to generate heat in situ, making the heat flow direction completely opposite to external heating. This solves the technical problems of thermal hysteresis, thermal overshoot, and uneven population temperature in existing inactivation technologies, and improves the level of preferential activation of key ICD signaling organelles such as the endoplasmic reticulum. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This describes the process of Hepa1-6 cells taking up vortex magnetic nano-iron oxide over time in the treatment system of Example 1 of the present invention. Figure 2 This is for the verification of cell viability of Hepa1-6 cells after treatment in Example 1 of the present invention; Figure 3 This is to verify the cell clone formation of Hepa1-6 cells treated in Example 1 of the present invention after seeding into 6-well plates and culturing continuously for 7 days; Figure 4 The CRT positivity rate of Hepa1-6 cells after treatment in Example 1 of this invention; Figure 5The release of HMGB1 and ATP secretion in Hepa1-6 cells after treatment in Example 1 of this invention; Figure 6 This illustrates the effect of Hepa1-6 cells treated in Example 1 of the present invention on dendritic cell activation. Figure 7 The tumor vaccine treated in Example 1 of this invention has an inhibitory effect on homologous tumors; Figure 8 This is the CRT positivity rate of low-level endocytosis-induced magnetothermal tumor cells in Comparative Example 3 of this invention; Figure 9 The low-level endocytosis-induced release of HMGB1 tumor cells by magnetothermal activity in Comparative Example 3 of this invention; Figure 10 This invention provides a comparative example 3 showing the effect of low-level endocytosis-induced magnetocaloric induction on tumor cell activity. Figure 11 This invention provides a comparative example 4 showing the effect of high-temperature magnetothermal induction on the CRT positivity rate and HMGB1 release in tumor cells. Detailed Implementation The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The internalization described in this invention refers to the active uptake of magnetic nanoparticles capable of generating a thermal effect under an alternating magnetic field by tumor cells into the cell interior, including subcellular structures such as the cytoplasm, endosomes, or lysosomes. Active uptake mechanisms include, but are not limited to, clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, phagocytosis, and co-occultation. This allows the magnetic nanoparticles to generate heat in situ within the cell when an alternating magnetic field is applied, achieving a heating effect from the inside out. The degree of internalization can be quantified and characterized by intracellular iron content. When the average intracellular iron content is below 10 pg / cell, even with the same alternating magnetic field parameters and target temperature, complete inactivation of tumor cells and efficient induction of immunogenic cell death cannot be achieved. Therefore, to achieve the technical effects of this invention, it is preferable that the average intracellular iron content of tumor cells is not less than 10 pg / cell, more preferably 30-50 pg / cell. Those skilled in the art can obtain the desired intracellular iron content by adjusting conventional parameters such as the concentration of magnetic nanoparticles, incubation time, and incubation temperature.

[0024] Example 1 Combination Figures 1-7In one specific embodiment of the present invention, S1, tumor cells are brought into contact with magnetic nanoparticles capable of generating a thermal effect under an alternating magnetic field, and the magnetic nanoparticles capable of generating a thermal effect under an alternating magnetic field are internalized into the tumor cells to form a treatment system. Specifically, mouse hepatocellular carcinoma cell line (Hepatoma 1-6, Hepa1-6) is cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum. Vortex magnetic iron oxide nanoparticles with an average particle size of 50 nm are added to the cell culture medium at a concentration of 75 μg Fe / mL and incubated for 12 hours. Inductively coupled plasma mass spectrometry confirms that the average intracellular iron content of the treatment system of Hepa1-6 internalized with the vortex magnetic iron oxide nanoparticles is 35 pg / cell, which is within the preferred range of intracellular iron content. S2, the system to be treated is placed in a spiral coil alternating magnetic field, and the tumor cells are heated from the inside out by the in-situ heat generation inside Hepa1-6 cells through vortex magnetic nano iron oxide nanoparticles; S3, with a fixed sampling period The unit is S, and the temperature of the system to be processed is monitored in real time. Real-time monitoring is achieved through a fiber optic temperature probe with an accuracy of 0.1℃, which can be inserted directly into the system to be treated. S4, based on the temperature and target temperature range The deviation, with temperature measured in °C, involves dynamically adjusting the magnetic field strength and / or frequency of the alternating magnetic field to adjust the temperature. Within the preset target temperature range Fluctuations within a given time interval, wherein the dynamically adjusted magnetic field strength satisfies: (5) in, For real-time temperature, The magnetic field strength, The magnetic field frequency, The particle size of a single vortex magnetic nano-iron oxide is [value missing]. The unit is pg / cell, which represents the number of magnetic nanoparticles within a single tumor cell capable of generating a thermal effect under an alternating magnetic field. The heat dissipation coefficient is expressed in units of... , ℃ represents the ambient temperature. In this embodiment, a hysteresis control law is used as the feedback strategy. The control unit collects temperature data in real time, and when the temperature... At that time, the magnetic field strength was increased in increments of 0.5 kA / m, and when the temperature... The magnetic field strength is decreased in increments of 0.3 kA / m, with an initial magnetic field strength of 30 kA / m and a maximum magnetic field strength of 35 kA / m. The adjustment amount of the magnetic field parameters is positively correlated with the magnitude of the deviation. A larger deviation requires a larger step size to achieve rapid approach, while a smaller deviation requires a smaller step size to avoid overshoot. S5, cumulative temperature Within the target temperature range Valid time within When the effective time Reaching the preset cumulative inactivation time When the alternating magnetic field is stopped, the target temperature range is the induction window temperature range for immunogenic cell death, and the cumulative inactivation time is the time sufficient to inactivate tumor cells 100% and simultaneously achieve immunogenic cell death phenotypic transformation. The effective inactivation time is accumulated by using a fixed sampling period. The system monitors the temperature in real time. At the end of each sampling period, if the real-time temperature is within the target temperature range [42.7℃, 43.3℃], then... Accumulated to the valid time counter Otherwise, they are not accumulated. That is: (6) when When the preset 20 minutes are reached, the control unit automatically stops the magnetic field output. By controlling the cumulative effective time, the influence of temperature fluctuation on the heat dose is eliminated, ensuring the consistency of the treatment effect between batches. In this embodiment, the temperature first enters the target temperature range at 180 seconds, and then fluctuates within the range of 42.8~43.2℃. The magnetic field is automatically stopped when the cumulative effective time reaches 20 minutes, and the total processing time is 23.5 minutes.

[0025] like Figure 2 As shown, the cell viability of Hepa1-6 cells treated in Example 1 of the invention was verified. The cell viability of Hepa1-6 cells was verified using a cell counting kit-8, and the cell viability was 0%. Figure 3 As shown, Hepa1-6 cells treated in Example 1 of this invention were seeded into 6-well plates and cultured continuously for 7 days. No cell proliferation clones were observed, confirming 100% inactivation and non-tumorigenicity. Figure 4 As shown, the CRT positivity rate of Hepa1-6 cells after treatment in Example 1 of this invention was 80% after half an hour of treatment, stained with Alexa Fluor® 488-labeled anti-CRT antibody, and detected by laser confocal microscopy. Figure 5As shown in Example 1 of this invention, the release of HMGB1 and ATP secretion in Hepa1-6 cells after treatment were as follows: 24 hours after treatment, HMGB1 in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA) and was 29.45 ng / mL; 4 hours after treatment, ATP in the supernatant was detected by luciferase assay and was 2750 nM.

[0026] like Figure 6 As shown in Example 1 of this invention, the effect of Hepa1-6 cells treated with this method on dendritic cell activation was investigated. The treated Hepa1-6 cells were co-cultured with bone marrow-derived dendritic cells at a ratio of 1:2 for 24 hours. Flow cytometry was used to detect surface markers on dendritic cells, including differentiation cluster 80 and CD80. + Differentiation cluster 86 CD86 + The positive rate was 43.6%, the positive rate for Major Histocompatibility Complex (MHC) I was 36.8%, and the positive rate for MHC II was 37.8%.

[0027] like Figure 7 As shown, the inhibitory effect of tumor cells treated in Example 1 of this invention on homologous tumors was demonstrated by subcutaneous injection of 2×10⁻⁶ cells into the right ventral side of two groups of C57BL / 6 mice. 6 In Example 1 of this invention, Hepa1-6 cells were treated, and Hepa1-6 cells were inactivated by conventional water bath treatment at 43°C for 20 min. Another group of C57BL / 6 mice were not injected. Seven days later, 5 × 10⁶ cells were injected into the left ventral side of each of the three groups of C57BL / 6 mice. 5 Live Hepa1-6 cells were used, and tumor growth curves showed that no tumor growth was observed in C57BL / 6 mice injected with Hepa1-6 cells treated in Example 1 of this invention, with a tumor-free rate of 100%. The tumor-free rate of C57BL / 6 mice injected with Hepa1-6 cells after conventional water bath inactivation treatment at 43°C for 20 min was 16.6%, while the tumor-free rate of C57BL / 6 mice that were not injected was 0%.

[0028] Comparative Example 1 Combination Figure 4 , 5 Hepa1-6 cells without magnetic nanoparticles that can generate a thermal effect under an alternating magnetic field were placed in a precision water bath for water bath treatment. The temperature was set to 43.0±0.3 ℃. An optical fiber probe was placed below the liquid surface of the Eppendorf tube in the same precision water bath to monitor the temperature in real time. The precision water bath adopted proportional-integral-derivative temperature control with a nominal accuracy of ±0.1℃.

[0029] It takes about 140 seconds for the sample to rise from room temperature to 43.0℃. When the precision water bath shows 43.0℃, the actual temperature fluctuation range of the Eppendorf tube is 42.5–43.6℃, ​​with the overshoot reaching a maximum of 43.9℃. In order to achieve a cumulative effective time of 20 minutes, the actual total processing time needs to be extended to 38 minutes.

[0030] like Figure 4 As shown, the CRT positivity rate of Hepa1-6 cells after treatment in Comparative Example 1 was 10.6% after staining with Alexa Fluor® 488-labeled anti-CRT antibody and laser confocal microscopy detection half an hour after treatment. Figure 5 As shown, after treatment with Comparative Example 1, the release of HMGB1 and ATP secretion in Hepa1-6 cells were measured. After 24 hours of treatment, HMGB1 in the supernatant was 1.78 ng / mL by enzyme-linked immunosorbent assay. After 4 hours of treatment, ATP in the supernatant was 103 nM by luciferase assay.

[0031] Even if the temperature control target and method are as close as possible to Example 1, the external heating source cannot fit the endoplasmic reticulum preferential stress pattern caused by intracellular heat production. The difference in immunogenicity comes from the spatial distribution of thermal stress rather than the temperature integral dose. At the same time, based on the difference in thermodynamics, Example 1 is superior to Comparative Example 1 in terms of control difficulty and effective temperature control time.

[0032] Comparative Example 2 Hepa1-6 cell suspensions without internalized nanoparticles were irradiated from above the liquid surface with an 808 nm infrared laser with a spot diameter of 8 mm. The fiber optic temperature measurement was maintained within the range of 43.0 ± 0.3 °C by adjusting the laser power.

[0033] Temperature distribution. Infrared thermal imaging shows that the surface temperature of the liquid can reach 45℃, while it is only 41℃ 2 mm below the surface. Cells settle at the bottom of the tube about 8 mm below the surface, indicating that the actual heating is severely uneven.

[0034] The inactivation results showed that after a cumulative irradiation of 30 minutes, the cells continued to proliferate and formed clones after 7 days, indicating that the inactivation was incomplete.

[0035] Surface heat sources cannot achieve a uniform heat dose across the entire population, and cannot simultaneously achieve 100% inactivation.

[0036] Example 2 Using the same treatment system as in Example 1, different cumulative times were set, and the results of the orthogonal experiment are shown in Table 1. Below the window, inactivation was incomplete; above the window, cell necrosis occurred, the release of damage-related molecular patterns decreased, and immunogenicity declined.

[0037] Table 1

[0038] When the effective time was extended to 35 min, the CRT positivity rate actually decreased to 16.4%, indicating that excessive heat stress had caused cells to enter the late necrosis or secondary necrosis stage, resulting in the degradation or loss of molecules such as CRT, highlighting the importance of precise control of the cumulative time.

[0039] Comparative Example 3 Combination Figures 8-10 In terms of treatment, Hepa1-6 cells were co-incubated with magnetic nanoparticles that could generate a thermal effect under an alternating magnetic field at 4°C for 2 hours, and the intracellular iron content was 9.46 pg / cell.

[0040] Magnetic field treatment was performed at the same target temperature as in Example 1, 43.0 ± 0.3 °C, with a cumulative time of 20 min. To achieve the required magnetic field strength to reach the target temperature, the initial alternating magnetic field strength was increased to 40 kA / m. Because the main heat conduction was from the membrane surface to the cell interior, the conduction efficiency was low, resulting in a CRT positivity rate of 15.6% and an HMGB1 concentration of 14.8 ng / mL. Trypan blue staining showed that some cells remained viable, and after 48 hours of culture, some cells resumed adhesion. Clonal formation was observed after 7 days.

[0041] Comparative Example 4 Combination Figure 11 3 nm of manganese ferrite was infused into the hepa1-6 cells to form the treatment system. Inductively coupled plasma mass spectrometry confirmed that the average intracellular iron content of the treatment system was 39 pg / cell. A constant alternating magnetic field of 35 kA / m was applied for 20 min without real-time temperature feedback. The magnetic field was rapidly increased to 46.8℃ from 0 to 3 min, and fluctuated between 46.0 and 47.5℃ from 3 to 20 min. The high temperature caused denaturation or shedding of calreticulin, resulting in a CRT positivity rate of 16.7% and an HMGB1 concentration of 8.46 pg / mL. Microscopic observation of cell morphology showed extensive swelling and rupture of cells, exhibiting necrosis characteristics.

[0042] The conclusions of Comparative Examples 3 and 4 together demonstrate that the combination of magnetic nanoparticles and the known method of alternating magnetic fields is insufficient to achieve the immunogenic cell death induction effect described in this invention.

[0043] Specifically, in Comparative Example 3, although the same alternating magnetic field parameters as in Example 1 were used, with a target temperature of 43.0 ± 0.3 °C, a cumulative effective time of 20 min, and a feedback control strategy, the intracellular iron content was only 9.46 pg / cell, indicating low-level endocytosis. The heat mainly originated from nanoparticles near the cell membrane surface, failing to generate efficient intracellular thermal stress from the inside out. This resulted in a CRT positivity rate of only 15.6% and HMGB1 release of only 14.8 ng / mL. Furthermore, some cells resumed proliferation after 48 hours, failing to achieve complete inactivation. This indicates that simply having feedback control and a temperature window accumulation time, without sufficient intracellular internalization—the spatial condition of intracellular magnetothermal—is insufficient to achieve the effects of this invention.

[0044] In Comparative Example 4, although full internalization of nanoparticles was achieved and the intracellular iron content was comparable to that of Example 1, the actual temperature rapidly rose to 46.0-47.5℃ under a constant-intensity alternating magnetic field of 35 kA / m for 20 min without feedback control, falling within the high-temperature ablation range. The results showed a CRT positivity rate of only 16.7% and HMGB1 release of only 8.46 pg / mL, with cells exhibiting necrosis characteristics, failing to induce immunogenic cell death. This indicates that even with intracellular magnetothermal conditions, without precise feedback control and a real-time climate control strategy based on cumulative effective time, only high-temperature necrosis can be achieved, and the cell death mechanism cannot be directed towards immunogenic cell death.

[0045] In summary, Comparative Examples 3 and 4 demonstrate from both positive and negative perspectives that the technical effect of this invention does not originate from the known combination of magnetic nanoparticles and / or alternating magnetic fields, but rather relies on the spatiotemporal coupling among four factors: sufficient intracellular internalization, precise feedback control, cumulative effective time, and the immunogenic cell death temperature window. Without any one of these elements, the functional paradigm shift from random necrosis or high-temperature ablation to programmed immunogenic cell death cannot be achieved.

[0046] The method described in this invention is well-suited for the internalization of magnetic nanoparticles capable of generating thermal effects under alternating magnetic fields. Under typical internalization conditions, effective temperature control and immunogenic cell death induction can be achieved through the described feedback control strategy. Those skilled in the art will understand that internalization can be optimized using conventional techniques such as nanoparticle surface engineering, but this does not affect the implementation of the core solution of this invention.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0048] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for inactivating whole-cell vaccines targeting immunogenic cell death, characterized in that, Includes the following steps: S1, bringing tumor cells into contact with magnetic nanoparticles that can generate a thermal effect under an alternating magnetic field, and internalizing the magnetic nanoparticles into the tumor cells to form a system to be treated; S2, the system to be treated is placed in an alternating magnetic field, and the magnetic nanoparticles that can generate a thermal effect under the alternating magnetic field generate heat in situ inside the tumor cells, thereby heating the tumor cells from the inside out. S3, with a fixed sampling period Real-time monitoring of the temperature of the system to be processed ; S4, based on the temperature and target temperature range To mitigate the deviation, the magnetic field strength and / or frequency of the alternating magnetic field are dynamically adjusted to control the temperature. Within the preset target temperature range Fluctuations within a period; S5, cumulative temperature Within the target temperature range Valid time within When the effective time Reaching the preset cumulative inactivation time When the alternating magnetic field is applied, stop applying it. The target temperature range is the temperature range within the induction window for immunogenic cell death, and the cumulative inactivation time is the time sufficient to inactivate tumor cells 100% and simultaneously achieve immunogenic cell death phenotypic transformation.

2. The method according to claim 1, characterized in that, The feedback control in S3 is based on the deviation between the temperature and the upper and lower limits of the target temperature range. To adjust the magnetic field strength and / or magnetic field frequency, including: When the temperature is lower When this occurs, increase the magnetic field strength and / or magnetic field frequency; When the temperature is higher When this occurs, the magnetic field strength and / or magnetic field frequency are reduced; When the temperature is within the target temperature range During this period, the magnetic field strength is maintained or finely adjusted to keep the temperature within the specified range; The adjustment amount of the magnetic field parameter is positively correlated with the magnitude of the deviation, and the target temperature range... .

3. The method according to claim 1, characterized in that, The fixed sampling period mentioned in S3 shall not exceed 1 second, and the accuracy of real-time monitoring of the temperature of the system to be processed shall not be less than 0.1℃.

4. The method according to claim 1, characterized in that, In step S4, the magnetic field strength and / or magnetic field frequency of the alternating magnetic field are dynamically adjusted based on the following magnetothermal field control model: （1） in, For real-time temperature, The magnetic field strength, The magnetic field frequency, The particle size of a single magnetic nanoparticle capable of generating a thermal effect under an alternating magnetic field. This refers to the number of magnetic nanoparticles within a single tumor cell that can generate a thermal effect under an alternating magnetic field. The heat dissipation coefficient of the system to be treated is... For ambient temperature, a proportional-integral-derivative control law or a hysteresis control law is used, based on the real-time temperature and the target temperature range. The deviation is dynamically calculated and the adjustment amount of the magnetic field strength and / or magnetic field frequency is output.

5. The method according to claim 1, characterized in that, The effective time in step S5 The accumulation method is as follows: in each sampling period, if the monitored temperature is within the target temperature range, then the sampling period is accumulated. Accumulated to the valid time The counter is used unless otherwise incremented, satisfying the following condition: (2)。 6. The method according to claim 1, characterized in that, The target temperature range is 43-45℃, and the cumulative inactivation time is 15-25 minutes.

7. The method according to claim 1, characterized in that, The internalization process results in an average intracellular iron content of the tumor cells of not less than 10 pg / cell, preferably 30-50 pg / cell.

8. The method according to claim 4, characterized in that, The expression for the proportional-integral-derivative control law is: （3） in, , The set temperature within the target temperature range. , , These are the proportional coefficient, integral coefficient, and differential coefficient, respectively.

9. An inactivated tumor cell vaccine prepared by the whole-cell vaccine inactivation method targeting immunogenic cell death as described in any one of claims 1-8, characterized in that, The vaccine contains 100% inactivated tumor cells that have not formed clones after 7 days of culture; the culture supernatant of the tumor cells has a CRT positivity rate and ATP concentration of not less than 50%; the culture supernatant of the tumor cells has an HMGB1 concentration of not less than 20 pg / mL; and the culture supernatant of the tumor cells has an ATP concentration of not less than 100 nM.

10. A system for implementing a whole-cell vaccine inactivation method targeting immunogenic cell death as described in any one of claims 1-8, comprising: The magnetic field generating unit is used to generate an alternating magnetic field with a frequency of 300 kHz to 500 kHz and an adjustable magnetic field strength. A temperature monitoring unit is used to monitor the temperature of the system to be processed in real time at a sampling frequency of not less than 1 time / second and an accuracy of not less than 0.1 ℃; a control unit is communicatively connected to the magnetic field generating unit and the temperature monitoring unit respectively, and is configured to execute steps S3-S5.

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