Targeted protein degradation nano-vesicle as well as preparation method and application thereof

Abstract

The invention discloses a targeted protein degradation nano-vesicle as well as a preparation method and application thereof. The nano-vesicle comprises a shell and a targeted chimera wrapped in the shell, the shell is formed by fusing cell membranes and lipidosome; the shell carries a PD-1 protein and a targeting molecule. The targeted protein degradation nanovesicle provided by the invention can penetrate through a blood brain barrier to enter the intracranial, CAR protein specifically targets ligands on the surfaces of glioma cells, PD-1 protein interacts with mPD-L1 on the surfaces of the glioma to inhibit immune escape, PROTAC molecules efficiently degrade endogenous cPD-L1 of the glioma cells, and precise and efficient immunotherapy of the glioma is achieved.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a targeted protein degradation nanovesicle, its preparation method, and its application. Background Technology

[0002] Glioblastoma multiforme (GBM) is one of the most common primary malignant brain tumors, characterized by extremely poor prognosis, high invasiveness, and frequent recurrence. Even with maximal safe resection, radiotherapy, and standard chemotherapy, the median survival is less than two years. In recent years, immunotherapy has shown great potential in various solid tumors, particularly immune checkpoint inhibitors (ICBs) that block the programmed death-ligand-1 (PD-L1) / programmed death receptor-1 (PD-1) axis. PD-L1 on the surface of tumor cells can be considered an immune escape signal; its binding to PD-1 on cytotoxic T lymphocytes (CTLs) induces CTL dysfunction, exhaustion, and even apoptosis. In fact, anti-PD-1 and anti-PD-L1 antibodies have significantly improved the prognosis of patients with melanoma, lung cancer, and renal cell carcinoma. However, clinical trials targeting GBM have not observed equivalent efficacy, despite high PD-L1 expression in GBM and a significant positive correlation with poor prognosis. There are two main reasons for treatment resistance: First, CKLF-like MARVEL transmembrane domain protein 6 (CMTM6) mediates the recycling of membrane-type PD-L1 (mPD-L1), preventing it from entering the lysosomal degradation pathway; Second, GBM cells have extremely high levels of cytoplasmic PD-L1 (cPD-L1), which can activate the MAPK / ERK pathway to promote tumor survival and can serve as a "reserve pool" to continuously replenish mPD-L1, thereby continuously suppressing the immune system.

[0003] Targeted protein degradation technology, utilizing the ubiquitin-proteasome (UPS), endocytosis-lysosome, or macroautophagy pathways to selectively degrade pathogenic proteins, has become a highly promising therapeutic strategy. Among these, the proteolytic-targeting chimera (PROTAC) consists of a bifunctional ligand that binds to a target protein (POI) and an E3 ubiquitin ligase. This ternary complex enables the POI to be polyubiquitinated and ultimately degraded via UPS. Compared to traditional inhibitors and gene tools, PROTAC-mediated degradation has cyclical properties, can sustainably inhibit the target protein at lower doses, and holds promise for reduced toxicity. Currently, PROTAC has shown potential in the treatment of various oncogenic targets. However, PROTAC faces three major bottlenecks in the treatment of GBM: First, the blood-brain barrier (BBB) ​​prevents PROTAC from entering the brain, further exacerbating its low bioavailability; second, the lack of targeted systemic circulation means that higher doses are needed to achieve effective intracranial concentrations, which may lead to the risk of "targeted tumor detachment"; third, although PROTAC can degrade cPD-L1, it is difficult to prevent mPD-L1 from repositioning on the surface of tumor cells, and membrane-bound PD-L1 will still exert T-cell immunosuppressive effects. These bottlenecks greatly limit the application of PROTAC in the treatment of GBM. Summary of the Invention

[0004] This application aims to address at least one of the technical problems existing in the prior art. To this end, embodiments of this application propose a targeted protein degradation nanovesicle. This novel nanovesicle can penetrate the blood-brain barrier and enter the intracranial space. The CAR protein specifically targets the ligand on the surface of glioma cells, the PD-1 protein interacts with mPD-L1 on the glioma surface to inhibit immune escape, and the PROTAC molecule efficiently degrades endogenous cPD-L1 in glioma cells, thereby achieving precise and efficient immunotherapy for gliomas.

[0005] This application also proposes a preparation method.

[0006] This application also proposes an application.

[0007] According to a first aspect of this application, a targeted protein degradation nanovesicle is proposed, the targeted protein degradation nanovesicle comprising an outer shell and a targeting chimera encapsulated within the outer shell; the outer shell is formed by the fusion of a cell membrane and a liposome; the outer shell carries PD-1 protein and a targeting molecule.

[0008] In some embodiments of this application, the PD-1 protein and the targeting molecule are located on the surface of the shell.

[0009] In some embodiments of this application, the targeting chimera includes at least one of a proteolytic targeting chimera, a lysosomal targeting chimera, and an autophagy targeting chimera.

[0010] In some embodiments of this application, the protein hydrolysis targeting chimera includes an E3 ligase ligand and a PD-L1 binding ligand.

[0011] In some embodiments of this application, the E3 ligase ligand includes at least one of 4-fluorothalidomide, thalidomide, pomalidomide, lenalidomide, VH032 ligand, VH298 ligand, and RG7388.

[0012] In some embodiments of this application, the PD-L1 binding ligand includes at least one of BMS-1233, BMS-1166, BMS-202, and BMS-1198.

[0013] In some embodiments of this application, the cell membrane is derived from at least one of immune cells, tumor cells, and bacteria.

[0014] In some embodiments of this application, the immune cells include macrophages.

[0015] In some embodiments of this application, the raw material components of the pH-sensitive liposomes include pH-responsive lipids, cholesterol, and DPPC.

[0016] In some embodiments of this application, the pH-responsive lipids include DSPE-PEOz2000 and / or DOPE.

[0017] In some embodiments of this application, the targeting molecule includes a CAR molecule.

[0018] In some embodiments of this application, the CAR molecule includes an extracellular domain, a transmembrane domain, and an intracellular domain.

[0019] In some embodiments of this application, the target antigen of the extracellular domain is selected from at least one of IL-13Rα2, HER2, EGFRvIII, GD2, EphA2, C1QBP, CD133, CSPG4, and B7-H3.

[0020] In some embodiments of this application, the transmembrane domain is selected from at least one of CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD134, CD137, CD154, ICOS, OX40, DAP10, and DAP12.

[0021] In some embodiments of this application, the intracellular domain includes at least one of 4-1BB and CD3ζ.

[0022] In some embodiments of this application, the co-stimulatory domain is selected from at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1, CD2, CD7, LIGHT, NKG2C, B7-H3, DAP10, and DAP12.

[0023] In some embodiments of this application, the activation domain is selected from at least one of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

[0024] In some embodiments of this application, the mass ratio of the PD-1 protein to the CAR molecule is 1:1.

[0025] According to a second aspect of this application, a method for preparing the targeted protein degradation nanovesicles described in the first aspect of this application is proposed, the method comprising the following steps: S1. Provides targeted chimeras; S2. Preparation of complex liposomes: Complex liposomes were prepared using the targeting chimera and lipids obtained in step S1; S3. Preparation of PD-1 nanovesicles and targeting molecule nanovesicles: obtain cell membranes expressing PD-1 protein and cell membranes expressing targeting molecules, and then prepare PD-1 nanovesicles and targeting molecule nanovesicles respectively. S4. Preparation of nanovesicles: Targeted protein degradation nanovesicles were prepared using the composite liposomes obtained in step S2, the PD-1 nanovesicles obtained in step S3, and the targeted molecule nanovesicles.

[0026] In some embodiments of this application, step S1 includes obtaining a targeted chimera by condensing an E3 ligase ligand, a PD-L1 binding ligand, and a linker.

[0027] In some embodiments of this application, the linker in step S1 includes at least one of N-boc-1,4-butanediamine, N-boc-1,3-propanediamine, and N-boc-1,5-pentanediamine.

[0028] In some embodiments of this application, the lipids mentioned in step S2 include pH-responsive lipids, cholesterol, and DPPC.

[0029] In some embodiments of this application, the pH-responsive lipids include DSPE-PEOz2000 and / or DOPE.

[0030] In some embodiments of this application, the mixed weight ratio of the pH-responsive lipids, cholesterol, DPPC and PROTAC molecules is (2~6):(2~6):(10~15):1.

[0031] In some embodiments of this application, step S2 further includes mixing the targeting chimera and lipids using an organic solvent, including dichloromethane and / or DMSO.

[0032] In some embodiments of this application, the collection of cell membranes in step S3 includes three differential centrifugations: centrifuging at 3000-3500 g for 4-6 min, centrifuging at 19000-21000 g for 20-40 min, and centrifuging at 75000-85000 g for 1.5-3 h.

[0033] In some embodiments of this application, the mass ratio of the target chimera, the total protein content of PD-1 nanovesicles, and the total protein content of the target molecule nanovesicles in the composite liposomes in step S4 is 1:(2~4):(2~4).

[0034] In some embodiments of this application, step S4 includes mixing the components and then sequentially subjecting them to ultrasonic fusion and extrusion.

[0035] In some embodiments of this application, the power of the ultrasonic fusion is 60~80 W.

[0036] In some embodiments of this application, the ultrasound fusion time is 8-12 minutes, wherein the ultrasound is performed intermittently with a working time of 1-3 seconds and an interval of 3-5 seconds.

[0037] In some embodiments of this application, the mechanical extrusion includes 9 to 15 extrusion passes through polycarbonate films of 350-450 nm and 150-250 nm in sequence.

[0038] According to a third aspect of this application, the application of the targeted protein-degrading nanovesicles described in the first aspect of this application or the targeted protein-degrading nanovesicles prepared by the preparation method described in the second aspect of this application in the preparation of products for tumor immunotherapy is proposed.

[0039] In some embodiments of this application, the tumor includes at least one of solid tumors and non-solid tumors.

[0040] In some embodiments of this application, the solid tumor includes, but is not limited to, at least one of the following: glioma, melanoma, colorectal cancer, malignant tumor, glioma, mesothelioma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, prostate cancer, Burkitt lymphoma, head and neck cancer, colon cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small bowel cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine carcinoma, carcinoid tumor, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Kaposi's sarcoma, multicentric Castrmann's disease, AIDS-related primary exudative lymphoma, neuroectodermal tumor, rhabdomyosarcoma, and osteosarcoma.

[0041] In some embodiments of this application, the non-solid tumor includes, but is not limited to, tumors originating from the blood, lymphatic system, or bone marrow.

[0042] In some embodiments of this application, the non-solid tumor includes, but is not limited to, at least one of lymphoma, leukemia, and multiple myeloma.

[0043] This application has at least the following beneficial effects: This application provides a targeted protein degradation nanovesicle, an engineered cell membrane nanovesicle with triple functions of "drug synergy, precise targeting, and immune regulation." By efficiently overexpressing tumor-targeting CAR and PD-1 proteins on the surface of macrophages, cell membrane vesicles are extracted and fused with pH-responsive liposomes pre-encapsulated with PROTACs that efficiently degrade PD-L1 to prepare a biomimetic nanovesicle drug. This method not only overcomes the shortcomings of traditional antibodies, such as rapid metabolism, poor tumor targeting, and strong drug resistance, but also solves the problems of short in vivo circulation time, low bioavailability, and off-targeting of PROTAC drugs. Furthermore, macrophage cell membrane nanovesicles have the natural ability to penetrate the blood-brain barrier and stably overexpress dual-receptor proteins targeting glioma surface ligands on the macrophage membrane surface, preparing engineered cell membrane nanovesicles displaying specific antibodies and loading PROTAC-targeted degradation drug molecules for application in the immune regulation of gliomas.

[0044] PROTACs need to enter the cytoplasm or nucleus and rely on the ubiquitin-proteasome system (UPS) to degrade target proteins. PROTACs require simultaneous binding to the target protein and E3 ligase to form a ternary complex. If PROTACs are encapsulated within vesicles or embedded in a membrane, their dynamic conformation may be restricted, affecting intermolecular interactions. Macrophage membrane nanovesicles (such as exosomes or engineered membrane vesicles) typically deliver their contents into the cell via endocytosis / membrane fusion, but may remain in endosomes / lysosomes, making it difficult to release PROTACs into the cytoplasm, leading to functional failure. Therefore, even if nanovesicles successfully deliver PROTACs to cells, avoiding lysosomal retention and promoting cytoplasmic release is crucial. Therefore, this protocol pre-encapsulates PROTACs using pH-responsive liposomes, ensuring adequate PROTAC loading while avoiding prolonged accumulation in lysosomes. Secondly, this application utilizes macrophage membrane nanovesicles with high PD-1 expression in tandem with PROTAC to construct a "dual-mode degradation chimera," which synergistically degrades PD-L1 on the cell membrane surface and intracellularly—a function not possessed by single PROTAC or LYTAC. Furthermore, simultaneously achieving degradation via the endosome-lysosome and ubiquitin-proteasome pathways using a single molecule is impossible; the "dual-mode degradation chimera" constructed in this application is the first to fill this gap in the field. Attached Figure Description

[0045] The present application will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the preparation process of PROTAC in Example 1 of this application; Figure 2 This is an HPLC-MS result of the PROTAC molecule in Example 1 of this application; Figure 3 The above is the proton NMR spectrum of the PROTAC molecule in Example 1 of this application; Figure 4 This is a graph showing the encapsulation efficiency and stability test results of liposome TAC in Example 1 of this application; where a is the encapsulation efficiency and b is the stability. Figure 5 The image shows the CAR protein expression detection results of stable transfected Raw 264.7 cells with high CAR protein expression in Example 1 of this application; where a is the structure of CAR plasmid and PD-1 plasmid, b is the immunofluorescence result, the scale bar is 10 μm, and c is the flow cytometry result. Figure 6 This image shows the results of PD-1 protein expression detection in stable Raw 264.7 cells with high PD-1 protein expression in Example 1 of this application; where a is the result of flow cytometry, b is the result of western blot, c is the result of immunofluorescence, and the scale bar is 10 μm. Figure 7 This is a schematic diagram illustrating the preparation of BiTAC in Example 1 of this application; Figure 8 The image shows the verification results of BiTAC in Example 1 of this application; where a is a TEM image with scale bars of 50 nm (lower left corner) and 100 nm (lower right corner), b is a fluorescence image with scale bar of 1 μm, c is the signal colocalization of liposome TAC, PD-1 NVs and CAR NVs in BiTAC, d is the average particle size measured by NTA, e is the Zeta potential, f is the expression of PD-1 protein and CAR protein verified by western blot, and g is the SDS-PAGE result. Figure 9 The image shows the hIL-13Rα2 expression detection results in stably transfected GL-261 cells that highly express hIL-13Rα2 in the experimental examples of this application; where a is the structure of the hIL-13Rα2 plasmid, b is the immunofluorescence result, the scale bar is 20 μm, and c is the flow cytometry result. Figure 10 The figure shows the effect of PD-L1 monoclonal antibody and PD-1 NVs on PD-L1 in GL-261 cells in the experimental examples of this application; where a is the immunofluorescence result, the scale bar is 20 μm, and b is the flow cytometry result of mPD-L1. Figure 11 The figure shows the detection results of PD-L1 in GL-261 cells after PROTAC treatment in the experimental examples of this application; where a is the PD-L1 content of GL-261 cells after treatment with different concentrations of PROTAC for 48 hours, b is the PD-L1 content of GL-261 cells after treatment with 3.0 μM PROTAC for different times, c is the PD-L1 content of GL-261 cells after treatment with 3.0 μM PROTAC for 48 hours followed by PROTAC elution and detection at different time points, d is the effect of different degradation pathway inhibitors on the ability of PROTAC to degrade PD-L1, and e is the immunofluorescence result of PROTAC degrading PD-L1 in GL-261 cells. The scale bar is 20 μm. Figure 12Figure 1 shows the detection results of the degradation effect of BiTAC and its components on PD-L1 in GL-261 cells in the experimental examples of this application. Figure 2 shows the PD-L1 content in GL-261 cells after treatment with different concentrations of BiTAC for 48 hours; Figure 3 shows the degradation of PD-L1 after treatment with TAC-PD-1, CAR-NVs, and BiTAC; Figure 4 shows the fluorescence of lysosomal escape ability of liposome TAC and PD-1 NVs in GL-261 cells (scale bar: 20 μm); Figure 5 shows the fluorescence of lysosomal escape ability of BiTAC in GL-261 and B16F10 cells (scale bar: 20 μm); Figure 6 shows the colocalization statistics of BiTAC fluorescence signal and lysosomal fluorescence signal in Figure 6; Figure 7 shows the mPD-L1 and cPD-L1 content in GL-261 cells after treatment with PD-1 NVs, liposome TAC, and BiTAC for 48 hours; Figure 8 shows the effect of different degradation pathway inhibitors on PD-1... The effect of NVs on the ability of mPD-L1 degradation, where h represents the effect of different degradation pathway inhibitors on the ability of BiTAC to degrade total PD-L1; Figure 13 The figure shows the detection results of the degradation effect of BiTAC and its components on PD-L1 in GL-261 cells in the experimental examples of this application; where a and b are the immunofluorescence results of BiTAC and LipoTAC degrading PD-L1 in GL-261 cells and B16F10 cells, respectively. The scale bar of the three columns on the left is 20 μm, and the scale bar of the magnified field is 5 μm. c and d are the statistical results of a and b, respectively. Figure 14 The images show the detection results of the effects of BiTAC and its components on T cell anti-tumor immunity in the experimental examples of this application. Image a shows the immunofluorescence of BiTAC blocking PD-1 in tumor cells and binding to hIL-13Rα2 on the surface of GL-261 cells; the scale bar for the three columns on the left is 20 μm, and the scale bar for the magnified field of view is 5 μm. Image b shows a three-dimensional confocal image of TAC-PD-1, CAR-PD-1 hNVs, and BiTAC targeting and penetrating GL-261 tumor spheres; the 20–80 μm values ​​marked above the image correspond to different depths of the GL-261 tumor spheres, i.e., the Z-axis depth; the scale bar is 20 μm. Image c shows a three-dimensional confocal image of liposomal TAC and BiTAC promoting T cell infiltration into GL-261 tumor spheres; the scale bar is 20 μm. Image d shows αPD-L1 and PD-1... Flow cytometry analysis results of NVs, liposomal TAC and BiTAC enhancing the T cell killing ability against tumor spheres, G1~G5 correspond to control group, αPD-L1, PD-1 NVs, liposomal TAC and BiTAC respectively; Figure 15Figure 1 shows the therapeutic effects of BiTAC and its components on a mouse model of melanoma in the experimental examples of this application. Specifically, a) is a schematic diagram of the construction and treatment process of the melanoma mouse model; b) is the tumor growth curve of different treatment groups; c) is a representative graph of tumor size in different treatment groups; d) is the statistical results of tumor weight in different treatment groups; e) is the mPD-L1 flow cytometry analysis results of B16F10 tumor tissue in different treatment groups; and f) is the CD8+ expression in tumor tissue of different treatment groups. + T-cell proportional flow cytometry analysis results, g and h represent CD8+ in tumor tissue, respectively. + Flow cytometry analysis results of the expression ratios of T cell activation markers IFN-γ and GraB, i represents the flow cytometry analysis results of the proportion of M1 phenotype macrophages infiltrating tumor tissue in different treatment groups (CD11b). + F4 / 80 + CD80 + ), j represents the total PD-L1 expression level in tumor tissues of different treatment groups; Figure 16 The images show the results of the in vitro blood-brain barrier crossing ability detection of BiTAC and its components in the experimental examples of this application; where a is a schematic diagram of the in vitro blood-brain barrier crossing model, b is a confocal imaging image and statistical results of TAC-PD-1, CAR-NVs and BiTAC crossing into the lower chamber and being taken up by GL-261 cells, with a scale bar of 50 μm, c is a flow cytometry analysis result of the crossing efficiency verification of TAC-PD-1, CAR-NVs and BiTAC, d is the verification of the MCP-1 / CCR2 axis between M1 macrophage membrane vesicles and GL-261 cells, and e is a three-dimensional fluorescence imaging image and statistical results of the blood-brain barrier crossing mechanism of liposomal TAC, TAC-PD-1 and BiTAC, with a scale bar of 100 μm; Figure 17 The figures show the results of in vivo detection of the targeting ability of BiTAC and its components to glioma lesions in the experimental examples of this application; where a is the photoacoustic imaging of liposomal TAC, TAC-PD-1 and BiTAC targeting intracranial glioma lesions in mice, b is the flow cytometry analysis results of the mechanism of BiTAC targeting GL-261 cells, c is the brain fluorescence signal map and brain lesion signal distribution map after tail vein injection of TAC-PD-1 and BiTAC at different times, and d is the brain slice fluorescence map corresponding to 48 h in c, with a scale bar of 50 μm; Figure 18Figure 1 shows the therapeutic effect of BiTAC and its components in a mouse model of glioma in the experimental examples of this application; where a is a schematic diagram of the construction and treatment process of the mouse model of glioma, b is a freight elevator imaging map of glioma signals at different treatment times, c is the glioma growth curve of different treatment groups, d is the mouse survival curve of different treatment groups, e is the PD-L1 flow cytometry analysis results of GL-261 cells in different treatment groups, and f is the CD8+ in glioma tissue of different treatment groups. + T cell proportional flow cytometry analysis results, g and h are CD8+. + Flow cytometry analysis results of the expression ratios of T cell activation markers IFN-γ and Gra B, i represents the proportion of Treg cells in glioma tissues of different treatment groups, and j represents the flow cytometry analysis results of the proportion of M1 phenotype macrophages infiltrating tumor tissues of different treatment groups (CD11b). + F4 / 80 + CD80 + ), k represents the IL-2 content in the serum of mice in different treatment groups analyzed by ELISA. Detailed Implementation

[0046] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0047] Example 1: Preparation of BiTAC-targeted protein degradation nanovesicles This embodiment provides a BiTAC nanovesicle targeting protein degradation, and its preparation method is as follows: 1) Preparation of PROTAC A schematic diagram of the PROTAC preparation process is shown below. Figure 1 As shown. PROTAC was constructed by using 4-fluorothalidomide, a thalidomide derivative approved by the FDA, as the E3 ligase ligand, and BMS-1166 as the PD-L1 protein binding ligand. N-boc-1,4-butanediamine was selected as the linker.

[0048] ① 4-Fluoro-thalidomide (1.03 mM, soluble in DMF) was mixed with N-boc-1,4-diaminobutane (1.13 mM) and DIPEA (2.06 mM) and stirred at 90 °C for 12 hours. The product was diluted with ultrapure water and extracted with ethyl acetate. Purification was then performed by column chromatography using petroleum ether / ethyl acetate (10:1, v:v) as eluent to obtain pomalidomide-NH-C4-NH-Boc. ② Subsequently, a Boc deprotection reaction was carried out. Pomalidomide-NH-C4-NH-Boc was dissolved in 5 mL of dichloromethane, and 1 mL of a solution of 1,4-dioxane hydrochloride (HCl concentration 4 mol / L) was added dropwise. The mixture was stirred in an ice-water bath for 2 hours to obtain pomalidomide-C4-NH2·HCl. ③ To synthesize the PROTAC molecule, BMS-1166 (10 mg, dissolved in DMF) was mixed with HATU (1.2 equivalents), DIPEA (7.0 equivalents), and pomalidomide-C4-NH2·HCl (1.1 equivalents), and then stirred at room temperature for 12 hours. The PROTAC molecule was purified by column chromatography using dichloromethane / methanol (20:1, v:v) as the eluent. The final product was analyzed by LC-MS (TripleTOF® 6600+, SCIEX) and... 1 Characterized by 1H NMR spectroscopy (400MHz, Bruker).

[0049] The HPLC-MS chromatogram of the prepared PROTAC molecule is as follows: Figure 2 As shown, the measured molecular weight of PROTAC is 967.3959, which is consistent with the theoretically calculated molecular weight of 967.46. The proton NMR spectrum of PROTAC is shown below. Figure 3 This also confirmed the molecular structure of PROTAC prepared in this embodiment. PROTAC was lyophilized and then dissolved in DMSO solution for later use.

[0050] 2) Preparation of liposome TAC (LipoTAC) LipoTAC was prepared by thin-film dispersion. 12 mg DPPC, 4 mg cholesterol, 4 mg DSPE-PEOz2000, and 1 mg PROTAC obtained in step 1) were placed in a round-bottom flask, and an appropriate amount of dichloromethane was added to dissolve the mixture until clear. The dichloromethane was evaporated under low pressure using a rotary evaporator, resulting in a uniform pale green film at the bottom of the flask. The film was then hydrated with sterile PBS solution under ultrasonic assistance to form a clear suspension of liposomes-PROTAC. The ultrasonication process included: placing the sample tube in an ice-water bath for 10 min, with intermittent ultrasonication consisting of 2 s working and 4 s rest intervals; finally, the sample was extruded using an Avanti micro-extruder (0.22 μm) to obtain LipoTAC with uniform particle size.

[0051] The encapsulation efficiency of PROTAC in LipoTAC was evaluated using LC-MS, and the results are as follows: Figure 4 As shown in a; by Figure 4 As can be seen from 'a', the encapsulation efficiency of LipoTAC prepared in this embodiment is as high as 70.80±1.46%; and LipoTAC has good stability, remaining stable after one week at 4℃. Figure 4 In (b), the particle size did not change significantly.

[0052] 3) Constructing Raw 264.7 cells that stably display the IL-13Rα2 ligand CAR protein and the PD-1 extracellular domain. Mouse monocytes / macrophages (Raw 264.7) were transfected using lentivirus. First, the basic frameworks of a mouse-derived CAR plasmid based on IL-13Rα2-CD28-4-1BB-CD3ζ and a mouse-derived PD-1 plasmid were constructed. The structures of the CAR and PD-1 plasmids are shown below. Figure 5 As shown in a, the expression plasmid used was pLV2-CMV-EGFP-Puro (V01447). The plasmid containing the target gene and the packaging vector were co-transfected into 293T cells. The envelope plasmid was pMD2.G-K47Q / R354Q (Plasmid #207323), and the packaging plasmid was psPAX2 (Plasmid #12260). Forty-eight hours after transfection, the supernatant of 293T cells was collected, and lentivirus was extracted by ultracentrifugation. RAW 264.7 cells were infected with the lentivirus carrying the target plasmid. After 48 hours of infection, stable transfected monoclonal cell lines with GFP fluorescence were screened using flow cytometry based on the fluorescence signal carried by the plasmid vector. The expression levels of CAR and PD-1 proteins on the cell membrane surface of stably transfected RAW 264.7 cells were assessed using Western blot, immunofluorescence staining, and flow cytometry. The results are shown in the figure. Figure 5b and c in Figure 6 As shown. The amino acid sequence of the CAR protein targeting IL-13Rα2 is: (SEQ ID NO:1); the amino acid sequence of the PD-1 protein is: MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPL (SEQ ID NO:2).

[0053] Depend on Figure 5 It can be seen that CAR protein is stably and highly expressed on the surface of Raw 264.7 cells; Figure 6It can be seen that PD-1 protein is stably and highly expressed on the surface of Raw 264.7 cells, and its protein expression level is 4.95 times that of Raw 264.7 cells with the M1 phenotype.

[0054] 4) Preparation of PD-1 NVs and CAR NVs nanovesicles Raw 264.7 cells stably expressing PD-1 and CAR, obtained in step 3), were homogenized, treated with DNase and RNase (Invitrogen), and centrifuged (3200 g, 5 min). The supernatant was collected and further centrifuged (20000 g, 30 min). The precipitate was then discarded, and the supernatant was centrifuged again using an ultracentrifuge (Optima MAX-XP, Beckman Coulter) (80000 g, 2 h). The cells were washed with PBS containing protease inhibitor tablets and extruded 13 times using an Avanti microextruder (0.22 μm) to obtain nanovesicles PD-1 NVs and CAR NVs with high expression of PD-1 and CAR proteins on their membrane surfaces.

[0055] 5) Preparation of BiTAC A schematic diagram of the preparation process of the nanovesicle chimeric BiTAC is shown below. Figure 7 As shown.

[0056] The LipoTAC obtained in step 2) was mixed with the PD-1 NVs and CAR NVs obtained in step 4) at a ratio of 1:3:3 (mass of PROTAC molecules in LipoTAC: total protein content of PD-1 NVs: total protein content of CAR NVs). The mixture was then fully fused using a combination of ultrasonic fusion and mechanical extrusion. Specifically, the mixed sample was first placed in an ice bath and treated with intermittent sonication at 70 W (2 seconds on, 4 seconds off) for 10 minutes to achieve initial intercalation and recombination of the membrane material. Subsequently, a liposome extrusion device was used to repeatedly extrude the mixed sample through polycarbonate membranes with specific pore sizes (400 nm, 200 nm) (11 times) to obtain uniformly sized and structurally stable fused nanovesicles. These were then collected by centrifugation and stored at 4°C for subsequent experiments.

[0057] The shape, particle size, and zeta potential of the prepared membrane nanovesicles were determined using a nanoparticle size tracer and dynamic light scattering instrument. The morphology, size, and uniformity of the vesicles were observed using transmission electron microscopy. The results are shown below. Figure 8 As shown in a~e in the diagram.

[0058] Figure 8 The TEM images shown in Figure a demonstrate that PD-1 NVs, CAR NVs, and BiTAC have distinct cup-shaped structures. Figure 8 The confocal fluorescence pattern shown in Figure b and the colocalization results shown in Figure c indicate that PD-1 NVs, CAR NVs, and LipoTAC exhibit significant colocalization. Figure 8 The average particle size results shown in Figure d and the Zeta potential results shown in Figure e indicate that the NTA values ​​of LipoTAC, PD-1 NVs, CAR NVs, and BiTAC are 156.3±3.8 nm, 148.3±7.5 nm, 167.8±3.8 nm, and 180.3±2.5 nm, respectively, and the Zeta potentials are -24.28±0.19 mV, -23.65±0.75 mV, -20.16±1.80 mV, and -21.89±1.07 mV, respectively.

[0059] Finally, to further investigate the expression levels and integrity of PD-1 and CAR proteins on the membrane, Western blot and SDS-PAGE were used for validation, and the results are as follows: Figure 8 As shown in f and g in the figure, “CAR-PD-1 hNVs” refers to the fused nanovesicles of PD-1 NVs and CAR NVs; the results show that PD-1 and CAR proteins maintain their integrity on PD-1 NVs and CAR NVs, respectively, and the prepared BiTAC completely retains PD-1 and CAR proteins.

[0060] Test case This experimental example tested and evaluated multiple properties of the BiTAC targeting protein degradation nanovesicles provided in Example 1, including PD-L1 degradation efficiency, PD-L1 degradation mechanism verification, T cell anti-tumor immune activation efficiency, melanoma treatment effect, blood-brain barrier crossing ability, glioma targeting ability, and in vivo glioma treatment effect.

[0061] 1. Degradation efficiency test of PD-L1 In this experiment, the degradation efficacy of PD-L1 by PD-1 NVs, PROTAC molecules, and BiTAC nanovesicles prepared in Example 1 was tested using GL-261 cells.

[0062] 1) Since GL-261 cells themselves do not express IL-13Rα2, this experiment constructed a stable GL-261 cell line displaying hIL-13Rα2 (human-derived) via lentiviral infection for all subsequent cell validation experiments. The hIL-13Rα2 plasmid structure is shown below. Figure 9As shown in 'a', the amino acid sequence of the IL-13Rα2 protein is: MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDH The expression level of hIL-13Rα2 on the cell membrane surface of stably transfected GL-261 cells was assessed using immunofluorescence staining and flow cytometry. The results are as follows: Figure 9 As shown in b and c in the figure; the results indicate that hIL-13Rα2 is stably and highly expressed on the surface of GL-261 cells.

[0063] 2) Degradation efficiency of PD-1 NVs on PD-L1 Using PD-L1 monoclonal antibody (αPD-L1) as a control, the degradation effect of PD-1 NVs obtained in step 4) of Example 1 on PD-L1 in GL-261 cells was evaluated by immunofluorescence and flow cytometry. The results are as follows: Figure 10 As shown. The dosage of PD-L1 monoclonal antibody was 20 μg / mL, and the dosage of PD-1 NVs was 100 μg / mL; the cell volume was 2 × 10⁶ cells / mL. 4 Each dish was incubated for 24 h and then incubated with PD-L1 monoclonal antibody and PD-1 NVs (0-48 h).

[0064] Depend on Figure 10 It can be seen that both PD-L1 monoclonal antibodies and PD-1 NVs can lead to a decrease in PD-L1 in GL-261 cells, but the PD-L1 degradation induced by PD-L1 monoclonal antibodies recovers after 24 hours. Figure 10 (a) While the action time of PD-1 NVs is longer, the membrane PD-L1 (mPD-L1) content of GL-261 cells still recovered to near the untreated level after 48 hours. Figure 10 (b) Considering that PD-L1 in GL-261 cells is mainly distributed in the cytoplasm, achieving efficient degradation of endogenous PD-L1 in GL-261 cells may be an effective strategy to overcome the poor efficacy of anti-PD-L1 antibodies against GL-261 cells. The reason why PD-1 NVs are more effective at degradation than PD-L1 monoclonal antibodies may be that after the PD-1 protein on PD-1 NVs binds to the PD-L1 protein on the tumor cell membrane, it is internalized into the lysosome. At this time, some PD-L1 will relocate to the membrane through CMTM6. Overexpression of PD-1 can prevent the internal circulation relocation of PD-L1 through multivalent binding and anchoring, thereby achieving degradation via the lysosomal pathway (Yang S, Shim MK, Song S, et al. Liposome-mediated PD-L1 multivalent binding promotes the lysosomal degradation of PD-L1 for T cell-mediated antitumor immunity. Biomaterials. 2022;290:121841. doi:10.1016 / j.biomaterials.2022.121841).

[0065] 3) Degradation efficiency of PROTAC for PD-L1 To evaluate the degradation efficacy of PROTAC on PD-L1 in GL-261 cells, the PD-L1 content in GL-261 cells before and after treatment with PROTAC (step 1) of Example 1 was quantified using a Western blot experiment. The results are as follows: Figure 11 As shown.

[0066] Depend on Figure 11 As shown in a, the optimal concentration of PROTAC required for the degradation of PD-L1 in GL-261 is 3.0 μM. At this concentration, the PD-L1 content of GL-261 is reduced to 23% of that in the control group.

[0067] Depend on Figure 11 As shown in b, the PD-L1 content in GL-261 reached its lowest level after 48 hours of treatment with 3 μM PROTAC.

[0068] Depend on Figure 11 As shown in 'c', after eluting and removing PROTAC, the PD-L1 content of GL-261 cells recovers in about 36 hours. This is consistent with the result that PROTAC does not affect the cell transcriptome level. If it does affect the transcriptome level, the result should be that it is difficult to recover or the recovery time is prolonged.

[0069] Depend on Figure 11 As can be seen from d, the 26S proteasome inhibitor MG-132 and the E3 ubiquitin ligand pomalidomide (CC-4047) can both inhibit the degradation of PD-L1 in GL-261 cells by PROTAC, indicating that the degradation of PD-L1 in GL-261 cells by PROTAC is achieved through the ubiquitin-proteasome pathway.

[0070] Depend on Figure 11 As shown in Figure 'e', ​​compared to the control group, the intracytoplasmic PD-L1 (cPD-L1) signal of GL-261 cells treated with PROTAC was almost invisible, but a clear PD-L1 signal was visible on the cell membrane surface of GL-261 cells. This indicates that PROTAC can significantly degrade cPD-L1 in GL-261 cells, but the mPD-L1 remaining on the GL-261 cell membrane surface can still maintain its inhibitory function on effector T cells. Therefore, integrating the advantages of PROTAC and PD-1 NVs to achieve simultaneous regulation of cPD-L1 and mPD-L1 in GL-261 cells may be an effective strategy to significantly improve the efficacy of GBM in ICB therapy based on anti-PD-L1 antibodies.

[0071] 4) Degradation efficiency of BiTAC for PD-L1 To evaluate the degradation ability of BiTAC on PD-L1 in GL-261 cells, the optimal concentration of BiTAC was first screened using Western blot experiments. The results are as follows: Figure 12 As shown in a; the results showed that the optimal concentration of BiTAC for PD-L1 degradation in GL-261 cells was 0.3 μM, which was 10-fold lower than that of free PROTAC. This is attributed to the superior cell internalization ability of BiTAC, which overcomes the inherent low bioavailability of PROTAC.

[0072] The contribution of each component of BiTAC to the degradation of PD-L1 in GL-261 cells was then analyzed.

[0073] Western blot was used to detect the degradation ability of PD-1 NVs and LipoTAC on PD-L1 in GL-261 cells. The results are as follows: Figure 12 As shown in b in the figure; the results showed that both PD-1 NVs and LipoTAC can induce the degradation of PD-L1, but when PD-1 NVs and LipoTAC are used in combination (BiTAC), PD-L1 in GL-261 cells is further reduced, suggesting that the downregulation of PD-L1 may be mediated through two mechanisms.

[0074] Therefore, the intracellular distribution of LipoTAC, PD-1 NVs, and BiTAC was further investigated. Immunofluorescence staining was used to detect the lysosomal escape ability of LipoTAC, PD-1 NVs, and BiTAC in GL-261 and B16F10 cells. The results are as follows: Figure 12 As shown in c~e. The results indicate that LipoTAC exhibits good lysosomal escape function due to its pH responsiveness; PD-1 NVs, on the other hand, primarily escape through internalization mediated by binding to surface mPD-L1, demonstrating a much weaker lysosomal escape ability. Figure 12 (c) Figure 12 Colocalization analysis of d and e in the figure shows that the colocalization coefficient between BiTAC and lysosomes gradually decreases with prolonged endowment time, confirming that BiTAC possesses the functions of both LipoTAC and PD-1 NVs. It is not only widely distributed in the cell periphery, but internalized BiTAC also has good lysosomal escape capabilities. Therefore, PD-1 NVs should be the main contributor to mPD-L1 downregulation, CAR-NVs have no effect on mPD-L1 degradation, while internalized PROTAC significantly degrades cPD-L1 in GL-261 cells. In the figure, TAC-PD1 refers to vesicles prepared by ultrasonic fusion of LipoTAC and PD-1 NVs at a ratio of 1:3 (drug mass: protein mass); TAC-CAR refers to vesicles prepared by ultrasonic fusion of LipoTAC and CAR NVs at a ratio of 1:3 (drug mass: protein mass).

[0075] Subsequently, the total PD-L1, mPD-L1, and cPD-L1 levels in GL-261 cells treated with PD-1 NVs, LipoTAC, and BiTAC for 48 hours were quantified using Western blot. The results are as follows: Figure 12 As shown in f; the results showed that PD-1 NVs led to downregulation of mPD-L1 in GL-261 cells, but had no effect on cPD-L1; LipoTAC significantly degraded cPD-L1 in GL-261 cells, but had a poor effect on mPD-L1. Surprisingly, BiTAC simultaneously induced the degradation of both mPD-L1 and cPD-L1 in GL-261 cells.

[0076] The mechanism of PD-L1 degradation by BiTAC was further investigated. Western blot was used to study the degradation effects of BiTAC and its components on PD-L1 under different inhibitor conditions. The results are as follows: Figure 12 As shown in gh. By Figure 12As can be seen from the g in the figure, PD-1 NVs-mediated PD-L1 degradation is inhibited by bafloxacin A1 (BAF), a lysosomal degradation pathway inhibitor, rather than the ubiquitin-proteasome inhibitor MG-132, confirming the lysosomal degradation pathway of mPD-L1. Figure 12 As can be seen from h, since the cPD-L1 content of GL-261 is significantly higher than that of mPD-L1, the degradation of BiTAC is more susceptible to the influence of MG-132.

[0077] Finally, immunofluorescence staining was used to detect the degradation effects of LipoTAC and BiTAC on PD-L1 in GL-261 and B16F10 cells. The results are as follows: Figure 13 As shown in the figure. The results showed that LipoTAC could degrade cPD-L1 in GL-261 and B16F10 cells, but there was still a significant mPD-L1 fluorescence signal in the cell periphery. BiTAC, on the other hand, reduced the content of both mPD-L1 and cPD-L1, showing superior PD-L1 degradation performance.

[0078] In summary, the above results indicate that BiTAC can downregulate total PD-L1 in tumor cells through the endosome-lysosome and ubiquitin-proteasome pathways, overcoming the shortcomings of existing PD-L1 monoclonal antibodies and PROTAC alone. Furthermore, due to cell membrane vesicle hybridization and liposome encapsulation, BiTAC can significantly improve the bioavailability of PROTAC, achieving the function of "drug reduction and efficacy enhancement," and is expected to promote the clinical translation of PROTAC.

[0079] 2. Activation effect of BiTAC on T cell anti-tumor immunity To evaluate the efficacy of BiTAC in activating T cells for anti-tumor immunity, the ability of BiTAC to bind to antigenic epitopes on GL-261 and B16F10 cells was first verified. The results are as follows: Figure 14As shown in a, BiTAC was used to treat cells for 2 hours at a concentration of 175 μg / mL. GL-261 cell surface antigens hIL-13Rα2 and PD-L1, as well as B16F10 cell surface antigen PD-L1, were labeled with antibodies (primary antibodies: anti-PD-L1 antibody (Abcam, 28-8, ab205921), anti-IL13Rα2 antibody (ABclonal, A19318); secondary antibody: goat anti-rabbit IgG H&L (FITC) secondary antibody (Abcam, ab6717)). The results showed that BiTAC could bind to PD-L1 and hIL-13Rα2 on the surface of GL-261 cells, and also to PD-L1 on B16F10 cells, indicating that BiTAC could block mPD-L1 before mediating its internalization and degradation, thus relieving the inhibition of effector T cells by tumor cell mPD-L1.

[0080] Subsequently, the function of BiTAC was further investigated in GL-261 tumor spheroids. IL-13Rα2 GL-261 cells (2 × 10⁶ cells per well) were used. 4 (Number of cells) were seeded into 24-well plates pre-coated with poly(hydroxyethyl methacrylate) for suspension culture. The culture medium was prepared using serum-free DMEM / F12 medium supplemented with 1× B27, 0.4% BSA, 20 ng / mL LEGF, 20 ng / mL FGF, and 5 μg / mL insulin. After 7 days of culture, the resulting tumor spheroids were collected for subsequent experiments. Staining was performed using Hoechst (nuclear label) and DiD (cell membrane label). After staining, PBS was gently added, and the cells were gently inverted to mix and wash. The cells were then collected by centrifugation at 150 g for 1 min. The collected tumor spheroids were gently transferred to a 35 mm confocal dish for imaging. The results are shown below. Figure 14 As shown in b~d in the figure. The CAR-PD-1 hNVs in the figure represent vesicles prepared by fusing CAR NVs and PD-1 NVs membranes.

[0081] Depend on Figure 14 As can be seen from b, BiTAC inherits well the intratumoral penetration and diffusion ability conferred by the pH responsiveness of LipoTAC and the tumor targeting ability of CAR-PD-1 hNVs.

[0082] Next, GL-261 tumor spheres were pre-incubated with LipoTAC (25 μg / mL) or BiTAC (175 μg / mL) for 24 hours, followed by co-culture with activated mouse spleen T cells (CD3 / CD28 activated for 48 h). The tumor cell to T cell ratio was 5:1. Observation was performed using a confocal microscope, and the results are as follows: Figure 14 As shown in c; the results showed that, compared with the LipoTAC treatment group, the BiTAC treatment group had more T cell infiltrations and were more distributed in the center of the tumor spheroid.

[0083] The ability of BiTAC to inhibit T cell exhaustion was further investigated. To obtain lymphocytes and dendritic cells, lymphocytes were obtained from the spleen and lymph nodes of C57BL / 6 mice inoculated with tumors. The cell suspension obtained by grinding and filtration was collected, centrifuged at 300 g for 5 minutes, and then erythrocyte lysis buffer was added to the cell pellet, incubated for 5 minutes to lyse the erythrocytes; the lysis reaction was terminated with PBS, and centrifuged again at 300 g for 5 minutes. Viable cells were identified by trypan blue staining, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 / mL for subsequent experiments. To obtain tumor-reactive T cells, dendritic cells (DCs) were pre-stimulated with lysates of IL-13Rα2 GL-261 cells irradiated at a dose of 40 Gy (using a RadSource RS2000pro irradiator). After 48 hours, isolated lymphocytes were added, and the cells were co-cultured with mature dendritic cells for 7 days, supplemented with 10 ng / mL IL-2 and 5 ng / mL IL-7 during culture. After sorting using magnetic beads coated with CD3-specific antibodies, tumor-reactive T cells were collected. Activated T cells were co-cultured with pretreated (48 h pretreated, unwashed) tumor spheroids at a 5:1 effector-target ratio for 5 days. After treatment, the diameter of the tumor spheroids was measured under a microscope, and the cells were washed twice with PBS buffer containing calcium ions, followed by antibody staining for flow cytometry analysis. The results are shown below. Figure 14 As shown in d.

[0084] The results showed that CD8 in the BiTAC-treated group + The proportion of T cells was the highest, the activation marker IFN-γ was significantly upregulated, while the number of Treg cells expressing FoxP3 was the lowest, indicating that BiTAC significantly inhibited CD8. + T cell depletion.

[0085] 3. The therapeutic effect of BiTAC on melanoma The effects of different treatment regimens on tumor growth and immune response were evaluated using a mouse model of melanoma. Figure 15Figure 'a' illustrates the construction and treatment process of the B16F10 mouse model. Mice were subcutaneously inoculated with B16F10 melanoma cells on day 7, and then administered different dosing regimens (200 μg per mouse) via tail vein injection between days 0 and 8. Tumor growth was monitored on days 0, 2, 4, 6, and 8 post-treatment, and analyzed after euthanasia on day 14. Changes in tumor volume and weight are shown in Figure 'a'. Figure 15 As shown in b~d; the flow cytometry results are as follows. Figure 15 As shown in e~i; the detection results of the Western blot are as follows Figure 15 As shown in j in the figure.

[0086] Depend on Figure 15 As shown in b~d, the TAC-PD-1 group showed the most significant inhibition of tumor growth, with the smallest tumor volume and weight.

[0087] Depend on Figure 15 As can be seen from e, there are differences in PD-L1 expression in B16F10 tumor tissues among different treatment groups. PD-L1 expression was significantly inhibited in tumors of the TAC-PD-1 and LipoTAC groups.

[0088] Depend on Figure 15 From f~h, we can see that CD8 in the tumor tissue of the TAC-PD-1 treatment group + The significantly increased proportion of T cells indicates that the treatment enhanced immune infiltration at the tumor site; correspondingly, CD8... + The T cell activation markers IFN-γ and GraB were also significantly increased in the TAC-PD-1 group, further supporting the T cell activation effect of the TAC-PD-1 group.

[0089] Depend on Figure 15 As can be seen from i, the proportion of M1 macrophages in the TAC-PD-1 group was significantly higher than that in other groups.

[0090] Depend on Figure 15 As can be seen from j, the expression level of total PD-L1 in the tumor tissue of the TAC-PD-1 group was significantly lower than that of other groups, further verifying the immunomodulatory effect of this treatment strategy.

[0091] The experimental results above show that TAC-PD-1 treatment effectively inhibits melanoma growth by enhancing T cell activation, increasing the infiltration of CD8+ T cells and M1 macrophages, and regulating PD-L1 expression, providing a potential immunotherapy strategy.

[0092] 4. Validation of BiTAC's blood-brain barrier crossing and glioma targeting capabilities 1) Blood-brain barrier crossing ability To evaluate the effectiveness of different nanocarriers (TAC-PD-1, CAR NVs, and BiTAC) in crossing the blood-brain barrier (BBB) ​​and targeting gliomas, a BBB simulation system was first designed, as shown in the schematic diagram below. Figure 16 As shown in a, it is used to study the cellular uptake of different nanocarriers after crossing the blood-brain barrier.

[0093] bEnd.3 cells (mouse brain microvascular endothelial cells) monolayer (1×10⁶ cells per well) were prepared. 5 GL-261 cells (1 × 10⁶ cells per well) were seeded in the upper chamber of a 24-well Transwell cell culture plate (Corning, model 3470) and cultured in DMEM medium containing 10% FBS; GL-261 cells (1 × 10⁶ cells per well) were seeded in the upper chamber of the plate. 5 (Number of cells) were seeded in the lower chamber. Subsequent experiments were conducted when the transepithelial / endothelial resistance of the upper chamber exceeded 200 Ω·cm². To evaluate the penetration efficiency of nanovesicles in the in vitro blood-brain barrier model, DiD-labeled TAC-PD-1 (100 μg / mL, nanovesicles prepared by fusing LipoTAC and PD-1 NVs), CAR-NVs (100 μg / mL, cell membrane vesicles extracted from Raw264.7 macrophages overexpressing M1 CAR protein), and BiTAC (100 μg / mL, nanovesicles prepared by fusing LipoTAC, PD-1 NVs, and CAR-NVs) were diluted with fresh culture medium and added to the upper chamber, incubated for 24 hours. GL-261 cells stained with DAPI and iFluor 488 were imaged using a confocal laser scanning microscope (ZEISS LSM980). Simultaneously, based on DiD fluorescence, the internalization of the aforementioned nanovesicles by GL-261 cells was analyzed by flow cytometry. To investigate the mechanism of their crossing of the blood-brain barrier, DiD-labeled BiTAC was pretreated with 20 μg / mL anti-CCR2 antibody for 1 hour to block the flow, followed by incubation in the upper chamber for 24 hours. Three-dimensional imaging of Hoechst 33342-stained bEnd.3 cells and GL-261 cells was performed using the Z-axis tomography function of a confocal microscope. The results are shown below. Figure 16 As shown in b~d in the diagram.

[0094] Depend on Figure 16 As shown in b, confocal imaging revealed that TAC-PD-1, CAR NVs, and BiTAC were all successfully taken up by GL-261 cells. Among them, BiTAC's uptake effect benefited from inheriting the targeting ability of CAR-NVs, showing a 2.89-fold enhancement effect.

[0095] Depend on Figure 16As shown in 'c', the uptake of BiTAC within 24 hours was 4.10 times higher than that of TAC-PD-1, and comparable to that of CAR-NVs, indicating that the targeted modified nanocarrier can significantly improve the uptake efficiency of cells.

[0096] Figure 16 In the figure, 'd' represents the validation results of the chemotactic ability between GL-261 cells and M1 macrophages. The results above 'd' show GL-261 cells treated with TNF-α, which mimics the inflammatory stimulation secreted by immune cells in the glioma microenvironment. Western blot results show that TNF-α stimulates GL-261 cells to express MCP-1. The results below 'd' show the CCR2 expression in M1 and M2 macrophage membrane vesicles without any overexpression. The results show that M1 macrophages highly express CCR2 and possess inherent BBB bootie-piercing ability. These results confirm the existence of the MCP-1 / CCR2 signaling pathway between BiTAC and GL-261 cells, suggesting that this pathway may play an important role in the uptake of nanocarriers.

[0097] To further investigate the mechanism by which BiTAC crosses the BBB, three-dimensional fluorescence imaging was performed, and the results are as follows: Figure 16 As shown in e; the results indicate that BiTAC demonstrates stronger blood-brain barrier crossing ability than TAC-PD-1, supporting its potential as a more effective brain drug delivery system.

[0098] 2) Targeting ability for gliomas Establishment of a mouse glioma model: Female C57BL / 6J mice were deeply anesthetized (using tribromoethanol, 20 μL / g) and their heads were shaved before being fixed on a stereotaxic instrument (RWD Life Science). The injection site for IL-13Rα2GL-261-Luc cells was then determined, located 0.2 cm lateral and 0.1 cm posterior to the anterior fontanelle. After drilling a hole at the marked location using a skull drill, a microsyringe was vertically inserted into the brain to a depth of 2.5 mm. An IL-13Rα2GL-261-Luc cell suspension (3 × 10⁶ cells) was slowly injected. 5 The solution was dissolved in 5 μL of serum-free DMEM / F12 medium and injected at a constant rate of 20 nL / s. After injection, the needle was left in place for 5 minutes, followed by suturing the wound.

[0099] Administration: On day 12 post-inoculation, successful tumor formation in mice was confirmed using the IVIS® Lumina III in vivo imaging system (PerkinElmer). Then, mice with tumors were injected intravenously via the tail vein with free DiR dye, DiR-labeled TAC-PD-1, TAC-CAR, and BiTAC (100 μg / 100 μL, DiR concentration 1 mg / mL). In vivo imaging of the mice was performed using the IVIS® Lumina III imaging system at 3, 6, 12, 24, and 48 hours post-inoculation. After 48 hours of imaging, the mice were euthanized, and major organs (including brain, heart, liver, spleen, lungs, and kidneys) were immediately removed for ex vivo imaging. Simultaneously, frozen sections of brain tissue were prepared, stained with DAPI, and observed using a VS200 slide scanner (Olympus).

[0100] In a mouse glioma model, photoacoustic imaging was used to visualize ( Figure 17 In the study, TAC-PD-1 NVs and CAR NVs were able to effectively cross the blood-brain barrier, but the CAR NVs group showed a more significant effect on glioma accumulation.

[0101] To demonstrate targeting, IL-13Rα2-GL-261-Luc cells were cultured in vitro and incubated with 100 μg of BiTAC pre-labeled with DiD. Flow cytometry was then used to detect the fluorescence signal carried on the cell surface. The αPD-L1, αIL-13Rα2, and αPD-L1+aIL-13Rα2 groups were pre-treated with 20 μg / mL αPD-L1, 20 μg / mL αL-13Rα2, or 20 μg / mL αPD-L1+20 μg / mL αIL-13Rα2, respectively, to block the surface antigen of IL-13Rα2-GL-261-Luc cells. The blank group consisted of untreated cells, and the control group consisted of cells without blocking treatment. The uptake rate was the uptake rate of BiTAC by IL-13Rα2-GL-261-Luc cells. Flow cytometry was used to detect the fluorescence signal carried on the cell surface. Figure 17 (b) found that the targeting effect of BiTAC is mediated by both PD-1 and CAR protein, and that CAR protein has the most significant effect on enhancing the targeting ability.

[0102] Further tail vein injection imaging ( Figure 17 As shown in c), both TAC-PD-1 and BiTAC successfully accumulated in the brain at different time points, with BiTAC exhibiting the strongest fluorescence signal in the brain. Finally, fluorescence imaging of brain tissue sections (…) Figure 17 (d) further validates that these nanocarriers can effectively target glioma lesions, demonstrating good brain-directed delivery capabilities.

[0103] The above experimental results demonstrate that BiTAC has great potential in improving drug delivery for brain tumors, effectively crossing the blood-brain barrier and targeting gliomas, thus significantly improving treatment outcomes.

[0104] 5. The therapeutic effect of BiTAC on gliomas in vivo To evaluate the in vivo therapeutic effect of BiTAC on glioma, a mouse model of glioma was constructed. The schematic diagram of its construction process is shown below. Figure 18 As shown in a. Specifically, C57BL / 6 mice were inoculated with GL-261 glioma cells and then administered different treatment regimens (G1: PBS, G2: CAR-PD-1 hNVs, G3: TAC-CAR, G4: BiTAC) via tail vein injection at different time points. The dosage was 100 μg per mouse, administered every five days. Tumor volume was measured every five days using the IVIS® Lumina III in vivo imaging system during the treatment process, and survival was assessed on day 20. Final observation was performed on day 50. To monitor bioluminescence signals, each mouse was intraperitoneally injected with D-fluorescein potassium (3 mg), and fluorescence signals were captured 10 minutes after injection. The results are shown in Figure 1. Figure 18 As shown in b~d in the diagram.

[0105] Depend on Figure 18 As shown in b, by monitoring the changes in glioma signal at different time points in different treatment groups through in vivo imaging, the BiTAC group had the lowest glioma signal after 20 days of treatment, indicating that the glioma regressed significantly after treatment.

[0106] Depend on Figure 18 As shown in 'c', the tumor growth curve indicates that the BiTAC group can significantly inhibit tumor growth, and the inhibitory effect is the most obvious compared with other groups.

[0107] Depend on Figure 18 As shown in d, the survival time of BiTAC mice was significantly prolonged, and the survival time was significantly better than that of other groups, indicating that BiTAC treatment can effectively delay the progression of glioma and improve the survival rate of mice.

[0108] Next, flow cytometry was used to detect immune infiltration and immune cell activation in the glioma tissue of glioma mice. The results are as follows: Figure 18 As shown in e~j in the diagram.

[0109] Depend on Figure 18 As can be seen from 'e', ​​PD-L1 in glioma cells varied across different treatment groups. + There were significant differences in the proportion of cells, with the BiTAC group showing higher PD-L1 levels. + The proportion of cells was significantly lower than in other groups, indicating that PD-L1 expression was significantly inhibited.

[0110] Depend on Figure 18 From f~h, we can see that the CD8 of the BiTAC group... + The proportion of T cells increased significantly, indicating that BiTAC treatment can enhance T cell infiltration; in CD8 + The BiTAC group showed significantly increased expression levels of T cell activation markers IFN-γ and GraB, indicating that these treatments can effectively activate CD8. + The anti-tumor function of T cells.

[0111] Depend on Figure 18 As can be seen from i, the proportion of Treg cells in the BiTAC group was significantly reduced, indicating that these treatments can promote anti-tumor immune responses by reducing the proportion of immunosuppressive Treg cells.

[0112] Depend on Figure 18 As can be seen from j in the data, the BiTAC group M1 macrophages (CD80) + The significant increase in the proportion of M1 indicates that the treatment can effectively induce the immune response in the tumor microenvironment to transform into the more tumor-suppressive M1 type.

[0113] The levels of IL-2 in mouse serum were analyzed by ELISA, and the results are as follows: Figure 18 As shown in k in the figure; the results showed that the serum IL-2 level in the BiTAC group was significantly higher than that in other groups, further supporting the idea that BiTAC treatment enhances the anti-tumor immune response by activating the function of T cells and other immune cells.

[0114] In summary, BiTAC treatment works by activating CD8. + Immunotherapy reduced T cells, increased M1 macrophage infiltration, and effectively inhibited glioma growth and prolonged survival in mice by increasing IL-2 levels. These results provide strong support for the application of immunotherapy in glioma treatment.

[0115] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

Claims

1. A type of targeted protein degradation nanovesicle, characterized in that, The targeted protein degradation nanovesicles include an outer shell and a targeted chimera encapsulated within the shell; the outer shell is formed by the fusion of a cell membrane and a liposome; the outer shell carries the PD-1 protein and the targeting molecule.

2. The targeted protein degradation nanovesicles according to claim 1, characterized in that, The targeting chimera includes at least one of the following: a proteolysis targeting chimera, a lysosome targeting chimera, and an autophagy targeting chimera; The proteolytic targeting chimera includes an E3 ligase ligand and a PD-L1 binding ligand; The E3 ligase ligand includes at least one of 4-fluorothalidomide, thalidomide, pomalidomide, lenalidomide, VH032 ligand, VH298 ligand, and RG7388. The PD-L1 binding ligand includes at least one of BMS-1233, BMS-1166, BMS-202, and BMS-1198.

3. The targeted protein degradation nanovesicles according to claim 1, characterized in that, The cell membrane can be derived from at least one of immune cells, tumor cells, and bacteria; The immune cells include macrophages.

4. The targeted protein degradation nanovesicles according to claim 1, characterized in that, The raw material components of the liposomes include pH-responsive lipids, cholesterol, and DPPC; The pH-responsive lipids include DSPE-PEOz2000 and / or DOPE.

5. The targeted protein degradation nanovesicles according to claim 1, characterized in that, The targeted molecules include CAR molecules; The CAR molecule includes an extracellular domain, a transmembrane domain, and an intracellular domain. The target antigen of the extracellular domain is selected from at least one of IL-13Rα2, HER2, EGFRvIII, GD2, EphA2, C1QBP, CD133, CSPG4, and B7-H3. The transmembrane domain is selected from at least one of CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD134, CD137, CD154, ICOS, OX40, DAP10, and DAP12; The intracellular domains include co-stimulatory domains and activation domains; The co-stimulatory domain is selected from at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1, CD2, CD7, LIGHT, NKG2C, B7-H3, DAP10, and DAP12. The activation domain is selected from at least one of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

6. The method for preparing the targeted protein degradation nanovesicles according to any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: S1. Provides targeted chimeras; S2. Preparation of complex liposomes: Complex liposomes were prepared using the targeting chimera and lipids obtained in step S1; S3. Preparation of PD-1 nanovesicles and targeting molecule nanovesicles: obtain cell membranes expressing PD-1 protein and cell membranes expressing targeting molecules, and then prepare PD-1 nanovesicles and targeting molecule nanovesicles respectively. S4. Preparation of nanovesicles: Targeted protein degradation nanovesicles were prepared using the composite liposomes obtained in step S2, the PD-1 nanovesicles obtained in step S3, and the targeted molecule nanovesicles.

7. The preparation method according to claim 6, characterized in that, The lipids mentioned in step S2 include pH-responsive lipids, cholesterol, and DPPC; The pH-responsive lipids include DSPE-PEOz2000 and / or DOPE; The mixed weight ratio of the pH-responsive lipids, cholesterol, DPPC and PROTAC molecules is (2~6):(2~6):(10~15):

1.

8. The preparation method according to claim 6, characterized in that, In step S4, the mass ratio of the target chimera, the total protein content of PD-1 nanovesicles, and the total protein content of the target molecule nanovesicles in the composite liposome is 1:(2~4):(2~4).

9. The preparation method according to claim 6, characterized in that, Step S4 includes mixing the components and then sequentially subjecting them to ultrasonic fusion and extrusion; The power of the ultrasonic fusion is 60~80 W; The ultrasound fusion time is 8-12 minutes.

10. The use of the targeted protein-degrading nanovesicles as described in any one of claims 1 to 5 or the targeted protein-degrading nanovesicles prepared by the preparation method described in any one of claims 6 to 9 in the preparation of products for tumor immunotherapy.

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