A functionalized CAR-M cell, its preparation method and application

Abstract

This invention relates to the field of biomedical technology, specifically to a functionalized CAR-M cell, its preparation method, and its applications. The preparation method involves synthesizing PVCL nanogel via polymerization precipitation, followed by amination and redox reaction to synthesize well-dispersible PVCL-MnO2. Simultaneously, CAR-M cells targeting the EphA2 antigen on the surface of GBM tumor cells are prepared. After treatment with PVCL-MnO2, the CAR-M cells exhibit enhanced phagocytic and killing abilities against tumor cells and can be visualized and tracked under MRI. Furthermore, this invention assists focused ultrasound technology in overcoming the physical barrier of glioblastoma multiforme, enabling more effective treatment with functionalized CAR-M cells, and has potential application value in the field of tumor immunotherapy.

Description

Related applications

[0001] This application claims priority to Chinese patent application filed on May 23, 2025, with application number 202510670216.7, entitled "A Functionalized CAR-M Cell and Its Preparation Method and Application". Technical Field

[0002] This invention relates to the field of technology, specifically to a functionalized CAR-M cell, its preparation method, and its application. Background Technology

[0003] Glioblastoma multiforme (GBM) is the most aggressive and recurrent primary malignant brain tumor. Currently, the standard treatment strategy includes surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy. However, even with standard treatment, the median survival for GBM patients is less than 15-18 months, and the 5-year survival rate is less than 5%. Treatment faces challenges such as the high heterogeneity of the tumor, the immunosuppressive microenvironment, and limitations imposed by the blood-brain barrier (BBB) ​​and blood-brain tumor barrier (BBTB). In recent years, immunotherapy has gradually become a new hot topic in GBM research to address these challenges. The core theoretical basis of this therapy lies in utilizing the body's immune system to recognize and eliminate tumor cells, restoring and enhancing the anti-tumor immune response. This strategy differs from traditional surgery, radiotherapy, and chemotherapy, emphasizing the elimination of tumors by regulating the host's immune system and establishing long-term immune memory to prevent recurrence. Currently, research in immunotherapy encompasses peptide vaccines, dendritic cell (DC) vaccines, chimeric antigen receptor T-cell (CAR-T) therapy, immune checkpoint inhibitors, and oncolytic viruses. The U.S. Food and Drug Administration (FDA) has approved several immunotherapy strategies for cancer treatment, including monoclonal antibody therapies targeting cytotoxic T-lymphocyte associated protein-4 (CTLA-4), programmed cell death protein 1 (PD-1), and its ligand PD-L1, as well as CAR-T immunotherapy. While immune checkpoint blockade (ICB) has shown significant efficacy in various solid tumors, it has not demonstrated the same therapeutic effect in GBM clinical trials. The main reason for this limited efficacy lies in the immunosuppressive microenvironment of GBM, where insufficient T-cell infiltration is one of the key factors affecting ICB efficacy. Cancer vaccines exert their anti-tumor effects by targeting tumor-associated antigens to activate the body's immune response. However, due to the limited specific antigens of GBM, relatively few vaccine therapies have been able to enter Phase III clinical trials.Furthermore, research results on CAR-T therapy in central nervous system tumors have not met expectations. Its main limitation lies in the potential activation of microglia and astrocytes by CAR-T cells, inducing anti-host immune responses and thus causing neurotoxicity. Tumor vaccines and CAR-T cells can also lead to the continuous loss of tumor antigens and an increase in the number of suppressor cells and factors in the tumor microenvironment (TME), resulting in drug resistance. Therefore, effectively improving the ability of therapeutic drugs or immune cells to penetrate the tumor blob and BBTB, restoring the immune response in the tumor microenvironment, and enhancing the specific killing effect of immune cells on GBM have become key points for overcoming the current bottleneck in GBM treatment.

[0004] Macrophages are highly plastic cells with multiple functions and can be activated into different phenotypes depending on their environment. They are generally classified into two types: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). M1 macrophages promote Th1 responses and enhance the recruitment of Th1 cells to sites of inflammation by secreting cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1 beta (IL1β), and interleukin-12 (IL-12), as well as CXCL9 / CXCL10 chemokines. They also upregulate genes involved in antigen processing and presentation, as well as co-stimulatory molecules, thereby enhancing T cell responses and exerting anti-tumor effects. M2 macrophages, on the other hand, suppress T cell function and secrete chemokines that recruit Treg cells, playing a crucial role in suppressing anti-tumor immunity. In the GBM microenvironment, microglia and macrophages (TAMs) are the most abundant immune cell populations, accounting for 30% to 50% of cells in the GBM microenvironment. They are crucial for promoting tumor progression and creating an immunosuppressive environment that hinders treatment. Most TAMs are M2 phenotyped, promoting GBM proliferation, angiogenesis, metastasis, and immunosuppression. Given their significant role in treatment resistance and GBM progression, TAMs have received considerable attention as targets for GBM therapy, broadly categorized into two types: reducing the number of TAMs or reprogramming TAMs in the tumor microenvironment. However, due to the complexity of the tumor immune microenvironment and the diversity of immunosuppressive cells, the polarization of TAMs in the tumor microenvironment is unstable and variable. It is difficult to completely alter the tumor immunosuppressive microenvironment and exert the killing effect of M1-type TAMs on tumor tissue. Furthermore, the addition of targeted receptors is needed to achieve more durable M1 polarization. Therefore, to address the aforementioned issues, researchers have developed a novel immunotherapy strategy: chimeric antigen receptor macrophages (CAR-M). This involves introducing specific CAR genes into macrophages, enabling them to target and engulf tumor cells. This approach not only maintains the macrophages in the M1 anti-tumor phenotype but also enhances their antigen-presenting capacity and promotes T-cell activation.

[0005] CAR-M immunotherapy, as an emerging cancer immunotherapy, has demonstrated remarkable anti-tumor potential. Its unique advantage lies in the macrophages' natural tumor homing ability, enabling them to actively infiltrate solid tumors. Once inside the tumor microenvironment, CAR-M cells not only directly kill tumor cells positive for targeted antigens through phagocytosis but also release cytotoxic factors, further enhancing the anti-tumor effect. Given its M1 phenotype, CAR-M cells can secrete various pro-inflammatory cytokines and chemokines, thereby reshaping the tumor microenvironment, promoting inflammatory responses, and attracting T cells and other immune cells to infiltrate the tumor site, enhancing the effectiveness of immune attack. More importantly, CAR-M cells can act as antigen-presenting cells (APCs), further inducing and strengthening adaptive immune responses by enhancing the antigen presentation process, thus achieving a more durable anti-tumor immune effect. Because macrophages are widely distributed in various tissues, their natural physiological characteristics make CAR-M cells potentially less likely to induce cytokine release syndrome (CRS) and neurotoxicity compared to CAR-T cells, resulting in better safety. To further enhance the anti-tumor effects of CAR-M cells, researchers are exploring ways to optimize CAR-M cell function, such as pluripotent stem cell-derived macrophages and CARs with the intracellular toll / IL-1R (TIR) ​​domain of the toll-like receptor 4, thereby significantly enhancing the anti-tumor effects of first-generation CAR-Ms. Therefore, CAR-M cell therapy, by stably maintaining an anti-tumor phenotype and achieving tumor targeting, opens up new avenues for cancer treatment, especially in the face of the complex tumor microenvironment, where its advantages become increasingly apparent.

[0006] Due to the extensive migration capabilities of macrophages, exogenously injected CAR-M cells are often difficult to monitor accurately after passing through organs such as the lungs and liver. Furthermore, there is a lack of reliable tracking methods to assess the real-time enrichment of CAR-M in tumor tissues. Accurately tracking the distribution of exogenous macrophages in vivo is a key issue for clinical applications during CAR-M cell therapy. Nanomedicine and molecular imaging offer new solutions for the visualization and tracking of immunocellular therapy. Based on the unique physicochemical properties of nanoparticles, their application prospects in molecular imaging are very broad, especially in immune cell tracking and tumor-targeted imaging. Currently, molecular imaging techniques commonly used for visual monitoring of immune cells mainly include the following: 1) Positron emission tomography (PET): Using direct radiolabeling or reporter gene imaging methods, researchers can accurately analyze the survival, expansion, and persistence of CAR-T cells in the tumor microenvironment. 2) Optical imaging: By labeling immune cells with fluorescent dyes inside or on their surface, the cell migration pathway and therapeutic effect can be visualized and monitored. 3) Magnetic resonance imaging (MRI): Superparamagnetic iron oxide nanoparticles (SPIONs) or other magnetic nanoparticles are used to label immune cells, enabling them to be visualized and tracked under MRI monitoring, allowing for in vivo observation of their dynamic distribution and homing ability in vivo.

[0007] Molecular imaging techniques for tracing immune cells are playing an increasingly important role in elucidating immune response mechanisms and optimizing immunotherapy. MRI technology, due to its non-invasive nature, high soft tissue contrast, and multidimensional imaging capabilities, has become a crucial tool for immune cell tracking. However, gadolinium (Gd)-based contrast agents, widely used in clinical practice, have been shown to be associated with nephrogenic systemic fibrosis (NSF), and long-term use may lead to the accumulation of Gd ions in the brain and bones, increasing health risks. Therefore, finding safer MRI contrast agents has become a research hotspot. In recent years, manganese oxide nanoparticles (MnO2) have gradually attracted attention as an alternative contrast agent for T1-weighted magnetic resonance imaging. When CAR-M cells engulf MnO2 nanoparticles, the particles enter lysosomes (pH≈4.5-5.0), where the acidic environment accelerates the degradation of MnO2, releasing Mn... 2+ This significantly enhances the T1-weighted imaging signal, enabling precise tracking of CAR-M cells within tumor tissue. Furthermore, Mn 2+It can also promote macrophage polarization towards the M1 type, enhancing CAR-M anti-tumor activity. However, MnO2 nanoparticles have poor stability under physiological conditions, which limits their in vivo application. To overcome this challenge, researchers developed a loading system based on poly(N-vinylcaprolactam) (PVCL) nanogels. PVCL nanogels have good biocompatibility and degradability, which can effectively improve the stability of MnO2 nanoparticles and enhance their circulation time in vivo. Therefore, loading MnO2 onto PVCL nanogels can not only improve the visualization and tracking ability of CAR-M cells, but also enhance the anti-tumor immune effect by regulating the polarization state of macrophages, providing an innovative solution for CAR-M-based precision tumor immunotherapy. However, the efficacy in GBM is still limited by the dual barriers of the blood-brain barrier (BBB) ​​and the blood-brain tumor barrier (BBTB). To address the delivery limitations of the BBB / BBTB barrier, focused ultrasound (FUS) technology, through microbubble-mediated cavitation, provides a non-invasive and effective means to overcome the GBM delivery bottleneck. It is expected to enhance the enrichment of functionalized CAR-M within GBM and thereby improve the distribution of effector cells within the tumor, thus enhancing its immune killing efficacy.

[0008] However, there are currently no reports on the present invention regarding a diagnostic and therapeutic nanocomposite for glioblastoma multiforme, its preparation method, and its application. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a functionalized CAR-M cell, its preparation method, and its application.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a functionalized CAR-M cell, comprising the following steps:

[0012] (1) Preparation of carboxyl-containing polymer nanogels;

[0013] (2) An amino group is introduced into the surface of the polymer nanogel through a chemical coupling reaction to obtain an aminated nanogel;

[0014] (3) Loading metal oxide nanoparticles into aminated nanogels to form composite nanogels;

[0015] (4) Construct a chimeric antigen receptor (CAR) gene sequence targeting tumor antigens and transfect macrophages with a lentiviral vector to obtain CAR-M cells;

[0016] (5) The composite nanogel was co-cultured with CAR-M cells to obtain functionalized CAR-M cells.

[0017] Preferably, the tumor is glioblastoma multiforme.

[0018] Preferably, the tumor antigen is EphA2.

[0019] Preferably, the raw materials for the polymer nanogel in step (1) include polymeric monomers, crosslinking agents and surfactants, and are prepared by precipitation polymerization.

[0020] Preferably, the polymerizing monomer is N-vinylcaprolactam (VCL), the crosslinking agent is N,N'-methylenebisacrylamide (BAC), and the surfactant is sodium dodecyl sulfate (SDS).

[0021] Preferably, in step (2), the carboxyl group is activated by EDC / NHS and reacted with ethylenediamine (EDA) to introduce an amino group.

[0022] Preferably, the metal oxide in step (3) is manganese dioxide (MnO2), which is loaded onto the surface of the aminated nanogel through a potassium permanganate (KMnO4) redox reaction.

[0023] Secondly, the present invention provides the application of functionalized CAR-M cells as described above in the treatment of glioblastoma multiforme.

[0024] Furthermore, the present invention provides the application of functionalized CAR-M cells as described above in the visualization and tracing of glioblastoma multiforme.

[0025] Furthermore, the present invention provides the application of functionalized CAR-M cell synergistic focused ultrasound as described above in the immunotherapy of glioblastoma multiforme.

[0026] The advantages of this invention are:

[0027] 1. This invention uses PVCL nanogel as a template to prepare PVCL-MnO2 nanoparticles, and the resulting product has good stability. PVCL-MnO2 can be used to functionalize CAR-M cells and visualize them under MRI, and in conjunction with focused ultrasound technology, to achieve immunotherapy for glioblastoma in situ. This invention relates to three basic principles: (1) PVCL-MnO2 can promote the polarization of CAR-M cells to the M1 type, enhancing the phagocytosis and killing of tumor cells by CAR-M cells. (2) PVCL-MnO2 is mainly located in the lysosomes of CAR-M cells, and MnO2 dissociates in the lysosomes. 2+ It can realize MRI visualization of CAR-M cells. (3) Focused ultrasound technology breaks through the physical barrier of glioblastoma multiforme, promotes the aggregation of multifunctional CAR-M cells in the tumor site, and effectively enhances the anti-tumor effect of CAR-M cells.

[0028] The preparation process of this invention is mild and simple.

[0029] 2. The PVCL-MnO2 nanoparticles prepared by the method of the present invention have good stability.

[0030] 3. The PVCL-MnO2 prepared by this invention has good anti-tumor and MRI imaging effects, providing new ideas for the diagnosis and treatment of tumors and cancer immunotherapy. Attached Figure Description

[0031] Appendix Figure 1 This is a flowchart of focused ultrasound-assisted functionalized CAR-M cell therapy for glioblastoma.

[0032] Appendix Figure 2 These are TEM images of the PVCL-NH2 and PVCL-MnO2 nanogels prepared in this invention.

[0033] Appendix Figure 3 These are the test results of the hydrated particle size and surface potential of the nanogel prepared in this invention.

[0034] Appendix Figure 4 This invention relates to the CCK method for detecting the toxic effects of PVCL-MnO2 on BMDM cells.

[0035] Appendix Figure 5 These are laser confocal microscope images of FITC-labeled PVCL-MnO2 prepared in this invention after treating BMDM for different times.

[0036] Appendix Figure 6 The present invention provides a flow cytometry analysis of the phenotypic changes of BMDM after PVCL-MnO2 treatment for 24 h.

[0037] Appendix Figure 7 The present invention provides laser confocal microscopy imaging of BMDM cells transfected with EphA2 CAR.

[0038] Appendix Figure 8 The present invention provides a flow cytometry analysis of the phenotypic changes of CAR-M after 24 h of PVCL-MnO2 treatment.

[0039] Appendix Figure 9 This invention provides a flow cytometry method for detecting the phagocytic function of macrophages.

[0040] Appendix Figure 10 This invention is used to detect the anti-tumor effect of the CAR-M prepared in this invention.

[0041] Appendix Figure 11 This is an MRI imaging of PVCL-MnO2-labeled macrophages prepared in this invention in an in situ GBM model.

[0042] Appendix Figure 12 This invention provides an evaluation of the effects of different treatments on the growth of in situ GBM.

[0043] Appendix Figure 13 This is TUNEL staining of in situ GBM tumor tissues from different treatment groups prepared according to the present invention.

[0044] Appendix Figure 14 This invention provides a flow cytometry method for detecting the phenotypes of macrophages and dendritic cells in the tumor microenvironment.

[0045] Appendix Figure 15 This invention provides a flow cytometry method for detecting the expression of T cells in tumors from different treatment groups.

[0046] Appendix Figure 16 The present invention prepares an evaluation of the effect of FUS-open BBB / BBTB on the aggregation of CAR-M cells at the tumor site.

[0047] Appendix Figure 17 This invention relates to the effect of focused ultrasound-assisted functionalized CAR-M cell therapy on the growth of glioblastoma in situ.

[0048] Appendix Figure 18 This invention provides a flow cytometry method for detecting immune checkpoints PD-1 / PD-L1 in tumors.

[0049] Appendix Figure 19 This invention provides a flow cytometry method for detecting the expression of inhibitory immune cells in the tumor immune microenvironment. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0051] The following explanation uses functionalized CAR-M cells for glioblastoma multiforme as an example.

[0052] The preparation methods for functionalized CAR-M cells for glioblastoma multiforme are roughly as follows:

[0053] (1) Dissolve VCL, SDS and BAC in 50 mL of water and transfer to a round-bottom reaction flask. Fill the reaction flask with nitrogen and stir in a water bath at 70 °C for half an hour. Add ACMA solution and add AAc aqueous solution after 5 min. Continue the reaction for 4 h. After the reaction is completed, cool to room temperature and dialyze for 3 days to obtain PVCL-COOH nanogel.

[0054] (2) EDC and NHS were added to PVCL-COOH nanogel one after another. After stirring for 2 h, EDA was added. After stirring for 3 days, the mixture was dialyzed for 3 days to obtain PVCL-NH2 nanogel.

[0055] (3) KMnO4 solution was added dropwise to PVCL-NH2 nanogel, stirred overnight, and dialyzed for 3 days to obtain PVCL-MnO2 nanogel;

[0056] (4) The EphA2 (scfv) target gene was constructed into the PGMLV-EF1a-sp (CD8A)-MCS-hinge™ (CD8A)-CD64 vector. After the vector plasmid was successfully constructed, HEK-293T cells were used to prepare lentiviruses containing the CAR-EphA2 sequence.

[0057] (5) CAR-M cells were obtained by transfecting BMDM with CAR-EphA2 lentivirus;

[0058] (6) Functionalized CAR-M cells were obtained by co-culturing CAR-M cells with PVCL-MnO2 nanogel.

[0059] Preferably, in step (1), the concentration of VCL is 15.64 mg / mL, the concentration of SDS is 0.17 mg / mL, the concentration of BAC is 0.82 mg / mL, the concentration of AVMA is 0.58 mg / mL, and the aqueous solution of AAc is 42.8 μL.

[0060] Preferably, in step (2), the concentration of PVCL-COOH nanogel is 5.4 mg / mL, the concentration of EDC is 6.4 mg / mL, the concentration of NHS is 3.85 mg / mL, and the volume of EDA is 200.4 μL.

[0061] Preferably, in step (3), the mass ratio of PVCL-NH2 nanogel to KMnO4 is 1:0.25, and the concentration of KMnO4 is 5 mg / mL; the KMnO4 solution is added dropwise to the PVCL-NH2 nanogel at a rate of 0.1 mL / min using a syringe pump; the concentration of PVCL-NH2 nanogel is 5.6 mg / mL; and the concentration of manganese in the PVCL-MnO2 nanogel is 125 μg / mL.

[0062] Preferably, the titer of the lentivirus containing the CAR-EphA2 sequence in step (4) is 2.21E8 TU / mL.

[0063] Preferably, the lentivirus transfection condition in step (5) is MOI=75.

[0064] Preferably, the concentration of manganese in the PVCL-MnO2 nanogel added to BMDM in step (6) is 5 μg / mL.

[0065] Example 1

[0066] Weigh 0.782 g VCL, 8.33 mg SDS, and 40.94 mg crosslinking agent BAC, dissolve them in 50 mL of water, and transfer the solution to a round-bottom reaction flask. Fill the flask with nitrogen gas and stir in a 70 °C water bath for half an hour. Add 29.16 mg of ACMA solution (dissolved in 4.17 mL of water), and after 5 min, add AAc aqueous solution (42.8 μL AAc + 10.42 mL of water). Continue the reaction for 4 h. After the reaction is complete, cool to room temperature and dialyze for 3 days in a dialysis bag (8000-14000D) to obtain PVCL-COOH nanogel. Take 210 mg of PVCL-COOH nanogel, add 287.5 mg EDC and 172.6 mg NHS respectively, stir for 2 h, add 200.4 μL EDA, stir for 3 days, and then dialyze for 3 days to obtain PVCL-NH2 nanogel. PVCL-NH2 nanogels were reacted with KMnO4 at a mass ratio of 1:0.25 to prepare a 5 mg / mL KMnO4 solution, which was then added dropwise to the PVCL-NH2 nanogels. After stirring overnight, the mixture was dialyzed for 3 days to obtain PVCL-MnO2 nanogels. TEM test results are attached. Figure 2PVCL-NH2 and PVCL-MnO2 are spherical. Image J statistical analysis shows that the size of PVCL-NH2 is 103.5 nm and the size of PVCL-MnO2 is 76.88 nm. Hydrated particle size and surface potential test results are attached. Figure 3 The hydrated particle size of PVCL-NH2 is 205.3 ± 1.8 nm, with a positive surface charge and a potential of 17.1 ± 0.68 mV; the hydrated particle size of PVCL-MnO2 is 238.8 ± 12.1 nm, with a negative surface charge and a potential of -20.6 ± 0.96 mV. The change in potential from positive to negative proves the successful synthesis of MnO2.

[0067] Example 2

[0068] With 1*10 4 BMDM cells were seeded in 96-well plates and cultured overnight. The culture medium was then removed, and complete culture medium containing different concentrations of PVCL-MnO2 nanogels was added. The plates were cultured for another 24 hours. The CCK-8 assay was performed according to the instructions, and absorbance at 450 nm was measured using a microplate reader. Cells without nanogel treatment served as a blank control, and cell viability was recorded as 100%. CCK assay results are attached. Figure 4 The results showed that low concentrations of PVCL-MnO2 did not have a toxic effect on BMDM. At a Mn concentration of 20 µg / mL, the cell viability of BMDM was greater than 100%. Subsequent in vitro cell experiments used a concentration of 5 µg / mL.

[0069] Example 3

[0070] Synthesize FITC-labeled PVCL-MnO2 nanogels, using 5×10 5 BMDM cells were seeded in confocal culture dishes and cultured in a cell incubator for 24 hours. PVCL-MnO2-FITC was added at 2 h, 8 h, and 12 h. After cell collection, the cells were washed three times with PBS, and preheated (37 °C) 50 nM Lyso-Tracker Red staining working solution was added, followed by incubation at 37 °C for 60 min. The Lyso-Tracker Red staining working solution was removed, fresh cell culture medium was added, and then an appropriate amount of Hoechst live cell staining solution (100X) was added dropwise to a final concentration of 1X. After mixing, the cells were incubated at 37 °C for 10 min. The dye-containing culture medium was aspirated, and the cells were washed three times with PBS before observation under a laser scanning confocal microscope. Subsequently, BMDM cells incubated with PVCL-MnO2 for 12 h were collected for bioelectron microscopy analysis to observe the distribution of nanomaterials within the cells (see attached image). Figure 5The results showed that the green fluorescence gradually increased with the extension of PVCL-MnO2 incubation time, indicating an increase in BMDM uptake. To further clarify the localization of PVCL-MnO2 after BMDM uptake, we used a red lysosomal probe for localization and found that the PVCL-MnO2 nanogel co-cultured with BMDM was mainly localized in lysosomes in the cytoplasm.

[0071] Example 4

[0072] 2×10 5 BMDM cells were seeded in 6-well plates, reacted with PVCL-MnO2 nanogel for 24 h, and then collected. BMDM cells were digested with cell digestion buffer, washed once with FACs buffer at 1200 rpm / 5 min, blocked with Fc receptors for 10 min, and then incubated with surface fluorescent antibodies Percp Cy5.5-CD11b, BV421-F4 / 80, and APC-CD86 on ice for 30 min in the dark. At room temperature, 500 μL / tube of Fixation Buffer was added to fix the cells for 20 min in the dark, centrifuged at 1200 rpm / 5 min, and washed once with FACs buffer. 10× Intracelluar Staining Perm Wash Buffer was diluted to 1× with deionized water and used to wash the cells twice. The fixed / permeabilized cells were resuspended in Intracelluar Staining Perm Wash Buffer, PE-CD206 fluorescent antibody was added, and incubated for 30 min. The reaction volume was 100 μL. Add 2 mL of Intracelluar Staining Perm Wash Buffer, wash twice at 350 g / 5 min, and discard the supernatant. Resuspend the cells in 500 μL of FACs buffer and perform analysis (see attached image). Figure 6 Untreated BMDM cells expressed 37.5% CD206, which was downregulated to 4.8% by PVCL-MnO2. Untreated BMDM cells expressed 35.1% CD86, which was upregulated to 82.1% after PVCL-MnO2 treatment. These results indicate that PVCL-MnO2 can induce M1 polarization in BMDM cells and downregulate M2 macrophage markers.

[0073] Example 5

[0074] To ensure optimal transfection conditions, at 2×10 4BMDM cells were seeded in 96-well plates. After cell adhesion, different MOI values ​​were set, and BMDM cells were transfected with a control lentivirus (ZSGreen fluorescence). Observations were performed under an inverted fluorescence microscope from 72 to 96 hours. An MOI value of approximately 80% transfection efficiency was selected as the optimal transfection condition. Based on the microscopic observations, MOI=75 was chosen for subsequent experiments. Further, CAR-EphA2 lentivirus with eGFP fluorescence was constructed and transfected into BMDM cells at MOI=75. CAR-M cells were obtained after 72 hours, and CAR expression was observed using a laser scanning confocal microscope. The results are attached. Figure 7 As shown, macrophages successfully displayed green fluorescence (eGFP), indicating that CAR-EphA2 lentivirus was successfully transfected. Since eGFP fluorescence expression is mainly used to trace CAR, the above results also indicate that CAR-EphA2 is expressed in macrophages.

[0075] Example 6

[0076] Bone marrow-derived macrophages (BMDM) were extracted from 8-week-old mice and plated on day 6. The cells were divided into the following groups: 1) Control group; 2) CAR-M group; 3) CAR-M+PM group (CAR-M cells were reacted with PVCL-MnO2 for 24 h). After treatment, macrophages were digested with cell digestion solution, centrifuged, washed once with FACs buffer, blocked with Fc receptors for 10 min, and then incubated with surface fluorescent antibodies Percp Cy5.5-CD11b, BV421-F4 / 80, and APC-CD86 on ice for 30 min in the dark. At room temperature, 500 μL / tube of Fixation Buffer was added to the cells, fixed in the dark for 20 min, centrifuged at 1200 rpm for 5 min, and washed once with FACs buffer. Wash cells twice with 1× Intracelluar Staining Perm Wash Buffer, resuspend cells in 100 μL Intracelluar Staining Perm Wash Buffer, add PE-CD206 fluorescent antibody, and incubate for 30 min. Add 2 mL Intracelluar Staining Perm Wash Buffer, wash twice, 1200 rpm / 5 min, and discard the supernatant. Resuspend cells in 500 μL FACs buffer and perform instrumental analysis (see attached image). Figure 8 CAR-M can promote macrophage M1 polarization, with an M1 / M2 ratio of 0.27, which is higher than that of the control group (1.6). PVCL-MnO2 nanogel can promote macrophage M1 polarization more significantly, with an M1 / M2 ratio of 2.5.

[0077] Example 7

[0078] Bone marrow-derived macrophages (BMDM) were extracted from 8-week-old mice and plated on day 6. The cells were divided into three groups: 1) Control group; 2) CAR-M group; and 3) CAR-M+PM group. After BMDM adherence, EphA2-CAR lentivirus was transfected at MOI=75. The medium was changed after 24 h, and PVCL-MnO2 was added and incubated for another 24 h after 48 h. Macrophages were then plated with tumor cells exhibiting RFP red fluorescence at a 1:1 ratio. After 8 hours, the cells were collected, washed once with PBS, and 1 mL of cell detachment solution was added to each well. The cells were incubated in a sterile cell culture incubator at 37 ℃ for 8 minutes, then gently pipetted to collect the cells into 15 mL centrifuge tubes. The cells were resuspended in 100 μL of cell staining buffer at 1500 rpm for 5 min. After Fc receptor blocking for 5 min, the surface fluorescent antibody Percp Cy5.5-CD11b was added to the single-cell suspension. The cells were incubated on ice for 20 min in the dark, washed twice, and then incubated at 350g for 5 min. The supernatant was discarded, and the cells were resuspended in 0.5 mL Cell Staining Buffer for analysis. The phagocytosis ratio was calculated as RFP. + CD11b + Cell count / RPF + Cell count × 100% indicates the proportion of tumor cells phagocytosed by macrophages (see appendix). Figure 9 Untreated macrophages phagocytosed 0.85% of tumor cells within 8 hours. Since CAR-M targets EphA2, which is highly expressed on tumor cells, its phagocytic capacity was enhanced, with 1.24% of tumor cells being phagocytosed. PVCL-MnO2 promoted M1 polarization of CAR-M, upregulating its phagocytic capacity, with 2.38% of tumor cells being phagocytosed by CAR-M. Therefore, PVCL-MnO2 can enhance the phagocytic capacity of CAR-M.

[0079] Example 8

[0080] Bone marrow-derived macrophages (BMDM) were extracted and plated on day 6, and divided into the following groups: 1) Control group; 2) CAR-M group; 3) CAR-M+PM group. After BMDM adherence, EphA2-CAR lentivirus transfection was performed at MOI=75. The medium was changed after 24 h, and PVCL-MnO2 was added and incubated for 24 h after 48 h. Macrophages and fluorescent tumor cells were plated at a ratio of 4:1, and samples were collected after 48 h. Cell detachment solution was added to the wells of the plate, and the cells were incubated in a sterile cell culture incubator at 37 ℃ for 8 minutes. After that, the cells were collected by gently pipetting into 15 mL centrifuge tubes and centrifuged at 1500 rpm for 5 min. The cells were washed twice with PBS. Annexin V-FITC binding solution was added and the cells were gently resuspended. Annexin V-FITC and PI Staining Solution were then added, and the cells were gently mixed and incubated at room temperature in the dark for 15 minutes before being analyzed by microarray. Figure 10 The proportion of apoptotic cells in the Con-M group was 6.4%, the proportion of apoptotic cells in the CAR-M group was 9.6%, and the proportion of apoptotic cells in the CAR-M group treated with PVCL-MnO2 was 14.2%, indicating that PVCL-MnO2 can effectively enhance the killing effect of CAR-M on tumor cells.

[0081] Example 9

[0082] BMDM cells were extracted in advance and plated on day 6. EphA2-CAR lentivirus was transfected into BMDM cells with an MOI of 75 to prepare CAR-M cells, followed by treatment with PVCL-MnO2 (60 μg / mL) for 12 hours. Cells were digested with cell digestion solution, centrifuged, washed once with PBS, resuspended in PBS, counted, and stored at 4 ℃. After successful construction of the orthotopic GBM model, 500,000 CAR-M macrophages were injected via tail vein. MRI scans were performed before and 1 h, 2 h, and 3 h after tail vein injection to observe signal changes in the tumor site. MRI parameters were: spin-echo T1-weighted phase (T1WI), repetition time (TR) of 500 ms, echo time (TE) of 15 ms, field of view (FOV) of 60 mm × 60 mm, and slice thickness of 1.5 mm. The signal-to-noise ratio at the point of highest signal intensity in the tumor region was further quantitatively analyzed and compared (see attached). Figure 11Two hours after tail vein injection, the MRI signal in the CAR-M group significantly increased, indicating that the cells had entered the tumor region. The MRI signal in the Con-M group also showed a slight increase, but the increase was not significant. Compared to Con-M, CAR-M expresses the EphA2 chimeric antigen receptor, which can bind to the EphA2 antigen on the surface of tumor cells. Therefore, the number of macrophages entering the tumor region significantly increased, resulting in a higher MRI signal at the tumor site. Statistical analysis of the T1 SNR two hours after tail vein injection revealed that the T1 SNR value in the CAR-M group was significantly higher than that in the Con-M group, indicating that the expression of the chimeric antigen receptor on CAR-M cells can achieve effective targeting at the tumor site.

[0083] Example 10

[0084] After tumor formation in mice, they were divided into four groups: 1) Control group; 2) Con-M group (untreated BMDM macrophages); 3) CAR-M group; 4) CAR-M+PM group. Each mouse received a tail vein injection of 500,000 cells. MRI scans were performed every 3 days after cell therapy to monitor tumor growth. Mice were sacrificed on day 6 post-treatment, and brain tissue was removed and fixed in 4% paraformaldehyde for subsequent TUNEL staining. Mice were sacrificed on day 7 post-treatment, and tumor tissue was dissected on ice into sterile centrifuge tubes and thoroughly minced to 1-2 mm using ophthalmic scissors. 3Add 1 mL of tissue digestion solution, place the centrifuge tube on a constant temperature shaker, and incubate at 37 ℃ / 180 rpm for 40 min to digest the tissue. After incubation, filter the tissue homogenate in the centrifuge tube through a 70 µm sterile filter, then rinse the sterile filter with complete cell culture medium (containing FBS), collect the filtrate in a 50 mL centrifuge tube, and centrifuge at 1200 rpm / 5 min. Add an appropriate amount of culture medium, filter at 40 µm, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend in PBS to obtain the tumor tissue single-cell suspension. Add live / dead staining solution Aqua (1:1000) to the tumor single-cell suspension, incubate at room temperature in the dark for 20 min, wash once with FACs buffer, and incubate at 350 g / 5 min. Fc blocking was performed at room temperature in the dark for 10 min. 10 μL of Brilliant Stain Buffer was added to each tube, followed by the addition of surface fluorescent antibodies (APC Cy7-CD45, FITC-CD3, Spark violet 538-CD4, APC / Fire 810-CD8, BB700-CD25, PE-CF594-PD-1, Pacific Blue-CD11b, Spark NIR 685-F4 / 80, Spark Plus B550-CD86, BV570-CD11c, R718-MHC II, PerCP / Fire 806-CD103, BV785-XCR1). The tubes were incubated on ice in the dark for 30 min. After washing once with 2 mL of FACsbuffer, the tubes were incubated at 350 g for 5 min. 1x Fix / Perm working solution and 1x Perm / Wash Buffer working solution were prepared for membrane perforation, followed by intracellular / nuclear staining. Add 0.5 mL of 1x Fix / Perm working solution to each tube and resuspend the cell particles by vortexing for approximately 3 seconds. Incubate on ice in the dark for 40 minutes. Add 1 mL of 1x Perm / Wash buffer directly to each tube and centrifuge at 350 g for 6 minutes at 4 °C. Add another 2 mL of 1x Perm / Wash buffer to each tube and centrifuge at 350 g for 6 minutes at 4 °C. Resuspend the cells in each tube with 100 µL of 1x Perm / Wash buffer, add intracellular / nuclear flow cytometry antibody (BV421-Foxp3, PE-Arg 1), vortex for 10 seconds, and incubate on ice in the dark for 40 minutes. Wash the cells twice with 2 mL of 1x Perm / Wash buffer to each tube and centrifuge at 350 g for 6 minutes at 4 °C. Resuspend the cells in 350 µL of FACS and analyze. Results are attached. Figure 12-15As shown, the tumors in the Control, Con-M, and CAR-M groups exhibited the largest tumor growth during treatment, while the CAR-M+PM group showed the smallest growth. Further comparison of volume measurements at the end of treatment (V6) and the beginning of treatment (V0), calculating the volume ratio (ΔV), revealed that the CAR-M+PM group showed the slowest volume growth, only doubling, while the Control group increased 3.7-fold, the Con-M group 3.3-fold, and the CAR-M group 2.4-fold. This indicates that CAR-M cells enhanced with PVCL-MnO2 nanogel have a stronger tumor-suppressive effect. TUNEL staining of tumor tissue showed that the CAR-M+PM group had a larger area of ​​apoptotic cells, reflecting the stronger tumor-killing ability of the CAR-M+PM group. During tumor proliferation monitoring, functionalized CAR-M cells significantly inhibited tumor growth. Flow cytometry was used to assess the immune activation status of the tumor microenvironment after different treatments. First, the phenotype of TAM in the tumor microenvironment was evaluated. After CAR-M cells entered the tumor microenvironment, the proportion of Arg-1-expressing M2 macrophages was not significantly reduced compared to the control group, at 50.9% (Con-M group), 54% (CAR-M group), and 54.1% (control group), respectively. However, CAR-M cells enhanced by PVCL-MnO2 treatment significantly downregulated the expression of M2 macrophages (28.3%) while upregulating the M1 / M2 ratio. This result suggests that functionalized CAR-M cells can promote TAM polarization towards the M1 type, achieving TAM reprogramming in the GBM microenvironment. In this study, we examined the expression of antigen-presenting cells (DCs) in the tumor microenvironment, paying particular attention to changes in cDCs. The proportion of cDCs in the CAR-M+PM group was significantly upregulated compared to the CAR-M group. Based on its potential for T cell activation, we also evaluated CD4+ expression in the tumor. + T cells and CD8 + T cell expression. In the control group, CD4... + T cell expression was 24.4%, and after macrophage treatment, CD4 expression was... + The proportion of T cells was significantly increased in all groups, with 46.8% in the Con-M group, 39.1% in the CAR-M group, and 40.9% in the CAR-M+PM group, while the proportion of CD8 cells was significantly increased. + T cell proliferation was most pronounced in the CAR-M+PM group (31.2%), compared to only 17.8% in the control group. This is mainly because the macrophages and dendritic cells in the CAR-M+PM group exhibited stronger anti-tumor functions, promoting CD4+ proliferation. + and CD8 +The proliferation and activation of T cells effectively inhibited the rapid growth of GBM. Therefore, functionalized CAR-M cell therapy effectively enhanced the anti-tumor immune response by regulating the tumor microenvironment, particularly by improving macrophage polarization, dendritic cell antigen presentation function, and T cell proliferation.

[0085] Example 11

[0086] An orthotopic GBM mouse model was constructed. After tumor formation, the mice were divided into two groups: 1) CAR-M+PM group; 2) CAR-M+P-M+FUS group. MRI images (Ingenia 3.0T CX, Philips Healthcare) were scanned at 0.5 h, 1 h, and 2 h after tail vein injection. MRI scan parameters were: repetition time (TR) 500 ms, echo time (TE) 15 ms, field of view (FOV) 60 mm × 60 mm, and slice thickness 2 mm. The signal-to-noise ratio (SNR) at the point of highest MRI signal was measured and compared between the two groups.

[0087] After orthotopic GBM modeling in mice, treatment with and without FUS-treated open BBTB was performed, followed by tail vein injection of CAR-M+PM (Mn: 2 µg). Mice were sacrificed 1 h after tail vein injection. Tumor tissue was dissected, and manganese content was determined by ICP. Results are attached. Figure 16 As shown, after tail vein injection of CAR-M cells, the number of cells in the tumor gradually increased, and an enhancement of the MRI signal could be observed. After FUS enhanced BBB / BBTB permeability, the signal intensity in the tumor was higher than that in the untreated group. Quantitative analysis of the signal intensity in the tumor at different time points showed that the signal-to-noise ratio at the highest signal intensity was also higher than that in the CAR-M+PM group. To clarify the Mn content in the tumor at this time, the tumor tissue was digested and subjected to ICP detection. The tissue content in the CAR-M+PM group was 0.46 μg / g. After FUS opened the BBB / BBTB, the tissue content increased to 0.7 μg / g, consistent with the MRI imaging results, indicating that FUS technology can effectively enhance the penetration efficiency of CAR-M cells.

[0088] Example 12

[0089] After tumor formation in mice, they were divided into two groups: 1) CAR-M+PM group; 2) CAR-M+P-M+FUS group. Each mouse received a tail vein injection of 500,000 cells. MRI scans were performed every 3 days after cell therapy to monitor tumor growth. Mice were sacrificed on day 10 post-treatment, and tumor tissue was dissected on ice and transferred to sterile centrifuge tubes. The tissue was then thoroughly minced to 1-2 mm using ophthalmic scissors. 3Add 1 mL of tissue digestion solution, place the centrifuge tube on a constant temperature shaker, and incubate at 37 ℃ / 180 rpm for 40 min to digest the tissue. After incubation, filter the tissue homogenate in the centrifuge tube through a 70 µm sterile filter, then rinse the sterile filter with complete cell culture medium (containing FBS), collect the filtrate in a 50 mL centrifuge tube, and centrifuge at 1200 rpm / 5 min. Add an appropriate amount of culture medium, filter at 40 µm, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend in PBS to obtain the tumor tissue single-cell suspension. Add live / dead staining solution Aqua (1:1000) to the tumor single-cell suspension, incubate at room temperature in the dark for 20 min, wash once with FACs buffer, and incubate at 350 g / 5 min. Fc blocking was performed at room temperature in the dark for 10 min. 10 μL of Lrilliant Stain Buffer was added to each tube, followed by the addition of surface fluorescent antibodies (APC Cy7-CD45, FITC-CD3, Sparkviolet 538-CD4, APC / Fire 810-CD8, BB700-CD25, PE-CF594-PD-1, Pacific Blue-CD11b, Spark NIR 685-F4 / 80, Spark Plus B550-CD86, BV570-CD11c, R718-MHC II, PerCP / Fire806-CD103, BV785-XCR1, Percp-Ly6C, BV650-Ly 6G, PE / Fire 640-PD-L1). The tubes were incubated on ice in the dark for 30 min. After washing once with 2 mL of FACs buffer, the tubes were incubated at 350 g for 5 min. Prepare 1x Fix / Perm working solution and 1x Perm / Wash Buffer working solution for cell permeabilization and subsequent intracellular / nuclear staining. Add 0.5 mL of 1x Fix / Perm working solution to each tube and resuspend the cell particles by vortexing for approximately 3 seconds. Incubate on ice in the dark for 40 minutes. Add 1 mL of 1x Perm / Wash buffer directly to each tube and centrifuge at 350 g for 6 minutes at 4 °C. Add another 2 mL of 1x Perm / Wash buffer to each tube and centrifuge at 350 g for 6 minutes at 4 °C. Resuspend each tube in 100 µL of 1x Perm / Wash buffer, add intracellular / nuclear flow cytometry antibody (BV421-Foxp3, PE-Arg 1), vortex for 10 seconds, and incubate on ice in the dark for 40 minutes. Wash the cells twice with 2 mL of 1x Perm / Wash buffer to each tube and centrifuge at 350 g for 6 minutes at 4 °C.Cells were resuspended in 350 µL FACS and analyzed. Results are attached. Figure 17-19 As shown, the tumor volume in the CAR-M+PM group gradually increased, while the tumor volume in the CAR-M+P-M+FUS group gradually decreased. The tumor volume on day 9 after treatment and the tumor volume before treatment were calculated for each group, and the volume ratio ΔV was calculated. The tumor in the CAR-M+PM group increased 1.6 times, while the tumor in the CAR-M+P-M+FUS group increased only 0.6 times. These results indicate that the dual effect of focused ultrasound opening the BBB / BBTB can effectively synergistically enhance functionalized CAR-M treatment and inhibit tumor proliferation. Flow cytometry was used to detect depleted CD8... + T cell expression was observed, and focused ultrasound-assisted functionalized CAR-M cell therapy significantly improved T cell function, with PD-1 expression in the CAR-M+PM group being higher. + CD8 + T cell expression was 32.8%, compared to 13.4% in the CAR-M+P-M+FUS group, and CD8 expression was [missing information]. + The immunosuppressive effect of T cells is relieved by the downregulation of PD-1, allowing cytotoxic T cell function to be restored and effectively kill tumor cells. In the tumor microenvironment, the expression of the inhibitory ligand PD-L1 can interact with CD8+. + PD-1 binding on T cells induces the depletion of infiltrating T lymphocytes, leading to their loss of immune surveillance function. PD-L1 is expressed not only on GBM tumor cells but also on immune cells such as MDSCs and TAMs. Subsequently, we also evaluated PD-L1 expression on immune cells. PD-L1 expression was downregulated on antigen-presenting dendritic cells (DCs), TAMs, and PMN-MDSCs, with the most significant downregulation observed in PMN-MDSCs, decreasing from 23.2% to 6.5%. Therefore, these results indicate that synergistic therapy with focused ultrasound can effectively relieve the inhibitory effect of PD-1 / PD-L1 on T cells, thereby activating them.

[0090] Furthermore, in the GBM tumor immune microenvironment, Tregs, MDSCs, and M2-type TAMs constitute the main suppressive immune cell populations and are key factors in immune escape. Using flow cytometry analysis, we further evaluated whether FUS synergistic functionalized CAR-M cell therapy could reverse the immunosuppressive microenvironment. The expression of Tregs was downregulated in the FUS+CAR-M+PM group compared to the CAR-M+PM group (11.7% vs. 21.1% in the CAR-M+PM group). MDSCs were further classified into PMN-MDSCs (Ly6C-MDSCs) using Ly6C and Ly6G flow cytometry staining. - Ly6G + ) and M-MDSC (Ly6C + Ly6G -The main difference in tumor microenvironment between the two treatment groups was the expression of M-MDSCs, with 14.4% expression in the CAR-M+PM group and a downregulated expression of 8.3% in the FUS+CAR-M+PM group. Simultaneously, FUS combined with multifunctional CAR-M therapy could also significantly downregulate Arg. + Macrophages reduce the proportion of M2 type TAM. Therefore, FUS intervention can effectively enhance anti-tumor immunity. Combined with functionalized CAR-M cell therapy, it enhances T cell activation through pathways such as reducing immune escape mechanisms, decreasing immunosuppressive cells, and restoring T cell function, thus promoting a stronger anti-tumor immune response to inhibit GBM progression.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A functionalized CAR-M cell, characterized in that, The method for preparing the functionalized CAR-M cells includes the following steps: (1) Preparation of carboxyl-containing polymer nanogels; (2) An amino group is introduced into the surface of the polymer nanogel through a chemical coupling reaction to obtain an aminated nanogel; (3) Loading metal oxide nanoparticles into aminated nanogels to form composite nanogels; (4) Construct a chimeric antigen receptor gene sequence targeting tumor antigens and transfect macrophages with a lentiviral vector to obtain CAR-M cells; (5) The composite nanogel was co-cultured with CAR-M cells to obtain functionalized CAR-M cells.

2. The functionalized CAR-M cells according to claim 1, characterized in that, The tumor is glioblastoma multiforme.

3. The functionalized CAR-M cells according to claim 2, characterized in that, The tumor antigen is EphA2.

4. The functionalized CAR-M cells according to claim 1, characterized in that, The polymer nanogels mentioned in step (1) are made from VCL, SDS, BAC, ACMA and AAc, and are prepared by precipitation polymerization.

5. The functionalized CAR-M cells according to claim 4, characterized in that, In step (2), the carboxyl group is activated by EDC / NHS and reacted with EDA to introduce an amino group.

6. The functionalized CAR-M cells according to claim 4, characterized in that, The metal oxide mentioned in step (3) is MnO2, which is loaded onto the surface of the aminated nanogel through a KMnO4 redox reaction.

7. The functionalized CAR-M cells according to claim 5, characterized in that, In step (1), the concentration of VCL was 15.64 mg / mL, the concentration of SDS was 0.17 mg / mL, the concentration of BAC was 0.82 mg / mL, the concentration of AVMA was 0.58 mg / mL, and the aqueous solution of AAc was 42.8 μL.

8. The functionalized CAR-M cells according to claim 5, characterized in that, In step (2), the concentration of polymer nanogel is 5.4 mg / mL, the concentration of EDC is 6.4 mg / mL, the concentration of NHS is 3.85 mg / mL, and the volume of EDA is 200.4 μL.

9. The functionalized CAR-M cells according to claim 6, characterized in that: In step (3), the mass ratio of aminated nanogel to KMnO4 is 1:0.25, the concentration of KMnO4 is 5 mg / mL, the concentration of aminated nanogel is 5.6 mg / mL, and the concentration of manganese in the composite nanogel is 125 μg / mL.

10. The use of the functionalized CAR-M cells according to claim 1 in a medicament for treating glioblastoma multiforme.

11. The application of the functionalized CAR-M cells of claim 1 in the visualization and tracing of glioblastoma multiforme.

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