Bispecific antibody modified tumor cell-derived drug-loaded vesicle and preparation

Abstract

The invention discloses a bispecific antibody modified tumor cell-derived drug-loaded vesicle and a preparation. The tumor cell-derived drug-loaded vesicles comprise vesicles released by apoptotic tumor cells and antitumor drugs wrapped in the vesicles and used as effective components; the vesicles are modified with a bispecific antibody through DSPE-PEG-NH2, and the bispecific antibody can achieve a synergistic effect with an anti-tumor drug wrapped by the vesicles at a targeting part of the vesicles; the bispecific antibody is a PD-L1 / TGF (Transforming Growth Factor)-beta bispecific antibody. The tumor cell-derived drug-loaded vesicle provided by the invention can target tumor tissues, responds to release a bispecific antibody in a tumor subacid environment, and reduces tumor matrix components by regulating and controlling tumor-related fibroblasts, so that the enrichment and deep penetration of the drug-loaded vesicle at a tumor part are increased.

Description

Technical Field

[0001] The present application belongs to the field of anti-tumor therapeutic technology, and more specifically, relates to a bispecific antibody-modified tumor cell-derived drug-loaded vesicle and preparation. Background Art

[0002] Chemotherapy is one of the key strategies for the treatment of advanced cancer. It can induce immunogenic cell death (ICD) of tumor cells and stimulate the body's anti-tumor immune response. However, the actual efficacy of traditional chemotherapy drugs is often limited by their insufficient accumulation in solid tumor tissues. This phenomenon is mainly attributed to the obstruction of complex physiological and pathological barriers. In addition, the immunosuppressive microenvironment of the tumor, such as the enhanced function of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), and the expression of PD-L1 in tumor cells induced by chemotherapy drugs, further weaken the anti-tumor immune response stimulated by chemotherapy, posing another important challenge in treatment. In view of this, the development of innovative combination therapy regimens aimed at enhancing the efficacy of chemotherapy drugs and overcoming the influence of the tumor immunosuppressive microenvironment is of vital significance for improving the treatment effect of patients with advanced cancer.

[0003] Clinical studies have shown that the extracellular matrix (ECM) composed of highly activated stromal cells and a large amount of accumulated matrix proteins in tumor tissues, high tumor tissue interstitial fluid pressure caused by abnormal structure and dysfunctional tumor blood vessels and highly squeezed and dysfunctional lymphatic vessels, as well as solid stress caused by rapid proliferation of tumor cells are the main causes of tumor tissue hardening. Abnormal mechanical microenvironment can also form a physical barrier to tumor therapeutic drugs including monoclonal antibodies and effector T cells, hindering their deep penetration into tumor tissues, and hardened tumor tissues will further compress tumor blood vessels, aggravate tumor tissue hypoxia, form a tumor immunosuppressive microenvironment, reduce the sensitivity of therapeutic drugs, and affect the effect of drug treatment. Therefore, regulating the tumor mechanical microenvironment will help the drug treatment of solid tumors.

[0004] Tumor-associated fibroblasts (CAFs) are a key type of cell in the abnormal mechanical microenvironment of tumors. Activated CAFs produce a large amount of ECM (including collagen, α-SMA, etc.). Therefore, inhibiting the activation of CAFs is expected to reshape the mechanical microenvironment and immunosuppressive microenvironment of liver cancer and improve the therapeutic effect of anti-tumor drugs. The activation of CAFs is related to an important cell transforming growth factor-β (TGF-β), which is a cytokine that is expressed in large quantities in the tumor microenvironment and has multiple functions. It plays an important role in the occurrence and development of cancer. TGF-β can regulate the growth and differentiation of various cells and promote the proliferation of tumor cells. TGF-β secreted by tumor cells can also induce fibroblast activation and differentiation into CAFs through the TGF-β-Smad signaling pathway, resulting in CAFs autosecreting a large amount of TGF-β, which further promotes the activation of fibroblasts. Existing studies have found that TGF-β not only activates fibroblasts, but also induces ECM protein expression and ECM reconstruction. Therefore, the activation of TGF-β signals in CAFs is considered to be an important cause of ECM deposition and reconstruction in tumor tissues. How to effectively block the TGF-β signal in tumor tissue is the key to inhibiting CAFs activation and regulating the tumor mechanical microenvironment. At present, some inhibitors or antibodies targeting the TGF-β signaling pathway have been clinically studied, but they all face problems such as low specificity and poor pharmacokinetics, and the treatment effect is not ideal. Summary of the invention

[0005] In order to inhibit transforming growth factor-β in a targeted manner and regulate the mechanical microenvironment of tumors, the present application discloses a bispecific antibody-modified tumor cell-derived drug-loaded vesicle, including vesicles released from apoptotic tumor cells and anti-tumor drugs encapsulated in the vesicles as active ingredients; The vesicle is modified with a bispecific antibody by DSPE-PEG-NH2, and the bispecific antibody can synergistically act with the anti-tumor drug encapsulated by the vesicle at the targeting site of the vesicle; the bispecific antibody is a PD-L1 / TGF-β bispecific antibody.

[0006] Preferably, the apoptotic tumor cells are liver cancer cells or breast cancer cells.

[0007] Preferably, the particle size of the tumor cell-derived drug-loaded vesicles is 200 nm to 400 nm.

[0008] Preferably, the anti-tumor drug is doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel or dextran.

[0009] The present application also discloses a preparation comprising the above-mentioned tumor cell-derived drug-loaded vesicles.

[0010] The present application also discloses a method for preparing the above tumor cell-derived drug-loaded vesicles, comprising the following steps: S1. The drug-loaded vesicles and DSPE-PEG-NH2 are mixed and fully incubated at a mass ratio of 1: (0.3-1), so that the DSPE-PEG-NH2 is modified on the surface of the phospholipid bilayer of the drug-loaded vesicles through its hydrophobic end to obtain amino-modified drug-loaded vesicles; the drug-loaded vesicles include vesicles released from apoptotic tumor cells and anti-tumor drugs as active ingredients encapsulated in the vesicles; S2. The amino-modified drug-loaded vesicles, glutaraldehyde and bispecific antibodies are mixed and fully incubated to modify the bispecific antibodies on the amino groups, thereby obtaining the tumor cell-derived drug-loaded vesicles.

[0011] Preferably, the incubation conditions in step S1 and / or step S2 are 0-4° C. for 6-24 hours.

[0012] Preferably, in step S2, the mass ratio of the amino-modified drug-loaded vesicles, glutaraldehyde and bispecific antibody is 1:(50-150):(0.2-0.5).

[0013] According to another aspect of the present application, there is also provided the use of the above-mentioned tumor cell-derived drug-loaded vesicles in drugs for treating tumors. In general, compared with the prior art, the above technical solution conceived by the present application realizes the synergistic treatment of bispecific antibodies and anti-tumor drugs by coupling bispecific antibodies through the pH-sensitive imine bond on DSPE-PEG-NH2 on the basis of preparing tumor cell-derived drug-loaded vesicles loaded with anti-tumor drugs; The present application utilizes the biological targeting of tumor cell-derived drug-loaded vesicles to efficiently deliver bispecific antibodies and anti-tumor drugs to the tumor area, releases bispecific antibodies in response to the acidic conditions of the tumor, neutralizes TGF-β in the tumor tissue, inhibits the activation of tumor-associated fibroblasts, reduces the secretion of tumor matrix, reduces the solid stress of tumor tissue, improves the mechanical microenvironment of the tumor, promotes the normalization of blood vessels, and further enhances the enrichment and deep penetration of tumor cell-derived vesicles in the tumor tissue; After the tumor cell-derived drug-loaded vesicles of the present application release bispecific antibodies in tumor tissues, they can be efficiently taken up by tumor cells and inhibited; at the same time, they can induce ICD effect and activate T cell anti-tumor immunity; the tumor cell-derived drug-loaded vesicles can not only improve the tumor mechanical microenvironment, promote CD8 + The infiltration of T cells in tumor tissues can block the action of PD-L1, relieve the tumor immunosuppressive microenvironment, enhance T cell anti-tumor immunity, and exert a good synergistic effect between chemotherapy and immune drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The laser particle size analyzer test results of Example 1 and the comparative example are shown below: Figure 2 The drug release of DOX in Example 1 and the comparative example; Figure 3 The drug release of YM101 in Example 1 and the comparative example; Figure 4 The results of the deactivation effect test on tumor-associated fibroblasts of Example 1 and the comparative example; Figure 5 The results of Example 1 and the comparative example on the improvement of the subcutaneous tumor matrix microenvironment of mouse liver cancer; Figure 6 The distribution results of Example 1 and the comparative example in the tumor tissue of H22 tumor-bearing mice with poor matrix; Figure 7 The distribution results of Example 1 and the comparative example in the tumor tissue of H22 tumor-bearing mice with rich matrix; Figure 8 The results of uptake and killing of Example 1 and the comparative example in H22 cells; Fig. 9 The results of uptake and killing of Example 1 and the comparative example in 4T1 cells; Fig.10 The results of the detection of ATP and CRT released by tumor cells in vitro by Example 1 and the comparative example; Fig.11 The results of in vitro activated DCs in Example 1 and the comparative example are shown in FIG. Fig.12 The results of in vitro activated T cells in Example 1 and the comparative example; Fig.13 The combination of DOX@MPs and YM101 enhances CD8 + T cell activity; Fig.14 The improvement of the immune microenvironment of subcutaneous liver cancer tumors in mice by Example 1 and the comparative example; Fig.15 The tumor inhibition effects of Example 1 and the comparative example on a mouse liver cancer subcutaneous tumor model; Fig.16 The tumor inhibition effects of Example 1 and the comparative example on the mouse breast cancer 4T1 orthotopic tumor model; Fig.17 The tumor inhibition effect of MTX@MPs-YM101 in Example 2 and the comparative example on the mouse subcutaneous liver cancer tumor model; Fig.18 The tumor inhibition effect of 5-Fu@MPs-YM101 in Example 3 and the comparative example on the mouse liver cancer subcutaneous tumor model; Fig.19The effect of DOX@MPs-Y101D of Example 4 and the comparative example on inhibiting in vitro cultured human hepatocellular carcinoma tumors. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0016] Glossary: Bispecific antibody: Bispecific antibody (BsAb, abbreviated as bispecific antibody) is an artificial antibody that can specifically bind to two antigens or antigen epitopes at the same time.

[0017] YM101: anti-mouse PD-L1 and TGF-β bispecific antibody (anti-TGF-β / PD-L1 bispecificantibody). Y101D: anti-human PD-L1 and TGF-β bispecific antibody (anti-TGF-β / PD-L1 bispecific antibody). Microparticles (MPs): Bio-nanovesicles produced when cells are stimulated by relevant signals to trigger the cell membrane to bulge outward to encapsulate the cell contents and release them to the outside of the cell in the form of vesicles. The particle size is 100-1000nm. MPs of biological origin have attracted widespread attention as drug carriers because of their good biocompatibility, long circulation and specific cell targeting.

[0018] DSPE-PEG-NH2: An active phospholipid PEG reagent with an active primary amine group at one PEG terminus. DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) is a saturated 18C phospholipid commonly used in the synthesis of liposomes. Polyethylene glycol (PEG)-coupled DSPE is hydrophilic and can be used for drug delivery, gene transfection, and biomolecule modification. PEGylation of phospholipids significantly improves the blood circulation time and stability of encapsulated drugs.

[0019] Bispecific antibodies of TGF-β and PD-L1 provide a new strategy for cancer treatment. It can not only block the interaction between PD-L1 on tumor cells and PD-1 on T cells to prevent T cell exhaustion, but also promote T cell-mediated anti-tumor activity. At the same time, by destroying the TGF-β / Smad signaling pathway, TGF-β is inactivated, thereby inhibiting CAFs activation. This dual mechanism of action makes bispecific antibodies an effective drug for improving the efficacy of chemotherapy. However, similar to other antibodies, bispecific antibodies also face the problems of short half-life and insufficient enrichment in tumor tissues. Therefore, it is necessary to develop a method that can accurately deliver bispecific antibodies and chemotherapeutic drugs to tumor sites. Cells secrete extracellular vesicles ranging in size from 100 to 1000 nanometers under various internal and external stimuli. Due to their good biocompatibility, extremely low immunogenicity, excellent stability and inherent tumor targeting ability, tumor cell-derived MPs have significant potential as carriers for targeted drug delivery and can be used for tumor chemotherapy and immunotherapy, significantly improving the efficacy of tumor treatment.

[0020] The present application provides a method for preparing drug-loaded vesicles derived from tumor cells, which specifically includes: S11. Tumor cells and anti-tumor drugs were mixed according to (5 7 ~15 7 The mixture is mixed at a ratio of 1:1 mg to cause apoptosis of tumor cells, and the drug-loaded vesicles released by the apoptotic tumor cells are collected; in some embodiments, the tumor cells are mouse liver cancer cells or breast cancer cells such as 4T1, H22, B16F10 or MC38 cells; in other embodiments, the anti-tumor drug is doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel or dextran; S12. The drug-loaded vesicles and DSPE-PEG-NH2 are mixed in a mass ratio of 1: (0.3-1) and fully incubated. Since DSPE has a hydrophobic end, it can be inserted into the phospholipid bilayer of the tumor cell-derived vesicles, so that DSPE-PEG-NH2 is modified on the surface of the tumor cell-derived vesicles to obtain amino-modified drug-loaded vesicles; S2. The amino-modified drug-loaded vesicles, glutaraldehyde and bispecific antibodies are mixed at a mass ratio of 1: (50-150): (0.2-0.5) and fully incubated so that the bispecific antibody is modified on the amino group; and then centrifugal extraction and other steps are performed to obtain the tumor cell-derived drug-loaded vesicles, wherein the particle size of the drug-loaded vesicles is 200nm-400nm. In some embodiments, the incubation conditions in step S1 and / or step S2 are incubation at 0-4° C. for 6-24 hours.

[0021] The drug-loaded vesicles prepared by the above method are modified with PD-L1 / TGF-β bispecific antibodies through DSPE-PEG-NH2; wherein the amino end of DSPE-PEG-NH2 is connected to the bispecific antibody through glutaraldehyde, and the hydrophobic end of one end of DSPE is bound to the phospholipid bilayer of the drug-loaded vesicles.

[0022] The bispecific antibody is modified on the surface of vesicles (MPs) loaded with anticancer drugs using an acidic pH-responsive imine bond. The bispecific antibody is modified on the surface of the vesicle and acts synergistically with the antitumor drug loaded by the vesicle. When the tumor cell-derived drug-loaded vesicles are administered through the tail vein and enter the tumor tissue through the body fluid circulation, the slightly acidic environment of the tumor induces the release of the bispecific antibody modified on the surface of the drug-loaded vesicles. The released bispecific antibody reprograms CAFs and reduces tumor ECM, promoting the accumulation and deep penetration of tumor cell-derived drug-loaded vesicles in the tumor. After the tumor cell-derived drug-loaded vesicles enter the tumor cells, they induce the immunogenic death (ICD) of the tumor cells and effectively activate CD8 + T cell-mediated immune response. In addition, ECM degradation can further promote CD8 + T cell infiltration in the tumor area. At the same time, the bispecific antibody can effectively block the function of PD-L1 overexpressed by tumor cells induced by tumor cell-derived drug-loaded vesicles, thereby restoring CD8 + The activity of T cells effectively inhibits tumor growth and produces long-term anti-tumor immune memory to inhibit tumor recurrence. Therefore, the tumor cell-derived drug-loaded vesicles can be used to prepare drug preparations for treating tumors and have the potential to be used in treating tumors.

[0023] Comparative Example 1 1. Experimental Materials and Reagents The mouse H22 liver cancer cells used were used, and the UV device was a conventional cell clean bench.

[0024] 2. Obtaining Tumor-loaded Vesicles (1) Take 10 7 H22 tumor cells were cultured in a sterile culture dish with a diameter of 10 cm. After overnight culture, the culture medium was discarded, washed with PBS, irradiated with ultraviolet light for 2 hours, and then doxorubicin (DOX) was added to make the final concentration of DOX in the culture medium 200 μg / mL and the final concentration of cells 500w / mL.

[0025] (2) 24 hours after the application of doxorubicin, the tumor was separated. First, the supernatant was taken for step-by-step separation. After centrifugation at 200g and 600g for 10 minutes each, it was centrifuged at 14000g for 1 minute to remove cells and debris. The supernatant after centrifugation was further centrifuged at 20000g for 1 hour to obtain drug-loaded vesicles DOX@MPs produced by apoptosis of H22 mouse liver cancer cells, that is, vesicles loaded with doxorubicin.

[0026] Comparative Example 2 Comparative Example 1 was repeated with the same steps, except that in step (1), after UV light irradiation, step (2) was directly performed to obtain non-drug-loaded vesicle MPs.

[0027] Comparative Example 3 S1. The vesicles obtained in Comparative Example 2 were mixed with DSPE-PEG 2K -NH2 (2K represents the molecular weight of polyethylene glycol) was incubated at a ratio of 1 mg protein: 500 μg at 4 °C for 12 h, and then the supernatant was removed by centrifugation at 20,000 g to obtain amino-modified vesicles; S2. Subsequently, the amino-modified vesicles, glutaraldehyde (GA) and YM101 were incubated at 4°C for 12 h in a ratio of 1 mg protein: 50 mg: 200 μg, and the supernatant was removed by centrifugation at 20,000 g to obtain MPs-YM101, i.e., vesicles modified with YM101 antibodies.

[0028] Comparative Example 4 Comparative Example 1 was repeated with the same steps, except that methotrexate (MTX) was used instead of DOX to obtain MTX@MPs drug-loaded vesicles.

[0029] Comparative Example 5 Comparative Example 1 was repeated with the same steps, except that 5-fluorouracil (5-Fu) was used instead of DOX to obtain 5-Fu@MPs drug-loaded vesicles.

[0030] Comparative Example 6 Comparative Example 3 was repeated with the same steps, except that Y101D was used instead of YM101 in step S2 to obtain MPs-Y101D, i.e., vesicles modified with Y101D antibody.

[0031] Example 1 S1. The drug-loaded vesicles of Comparative Example 1 were mixed with DSPE-PEG 2K -NH2 was incubated at 4°C for 12 h at a ratio of 1 mg protein: 500 μg, and then the supernatant was removed by centrifugation at 20,000 g to obtain amino-modified drug-loaded vesicles; S2. Subsequently, the amino-modified drug-loaded vesicles, glutaraldehyde (GA) and YM101 were incubated at 4°C for 12 h in a ratio of 1 mg protein: 50 mg: 200 μg, and the supernatant was removed by centrifugation at 20,000 g to obtain DOX@MPs-YM101, i.e., doxorubicin-loaded vesicles modified with YM101 antibodies.

[0032] Example 2 Example 1 was repeated with the same steps, except that the drug-loaded vesicles of Example 4 were used in step S2, and MTX@MPs-YM101, i.e., methotrexate-loaded vesicles modified with YM101 antibody, were finally obtained.

[0033] Example 3 Example 1 was repeated with the same steps, except that the drug-loaded vesicles of Example 5 were used in step S2, and 5-Fu@MPs-YM101, i.e., 5-fluorouracil-loaded vesicles modified with YM101 antibody, were finally obtained.

[0034] Example 4 Example 1 was repeated with the same steps, except that Y101D was used instead of YM101 in step S2, and finally DOX@MPs-Y101D, i.e., doxorubicin-loaded vesicles modified with Y101D antibody, were obtained.

[0035] Verification Example 1 DOX@MPs-YM101 structure verification The vesicles obtained in Example 1 and Comparative Example 1 were dissolved in PBS, and the particle size and zeta potential distribution were measured using a nanoparticle size and zeta potential instrument with a 633 nm He-Ne laser. The specific setting conditions were: temperature of 25° C., and equilibrium time of 120 s.

[0036] The hydrated particle size of the nano-drug-loaded vesicles was measured using a laser particle size analyzer. Figure 1 As shown; it can be seen from the figure that the hydrated particle size of DOX@MPs-YM101 in Example 1 is about 328 nm, while the hydrated particle size of DOX@MPs in Comparative Example 1 is about 294 nm, indicating that YM101 is successfully modified onto the surface of DOX@MPs, and the nano-drug-loaded vesicles can be passively targeted to the tumor area through the EPR effect.

[0037] Verification Example 2 Drug release of DOX@MPs-YM101 drug-loaded vesicles Take 10 mL of PBS buffer and adjust the pH to 7.4 or 6.5 with HCl or NaOH respectively as the release solution. Add 1 mL of DOX drug solution (concentration 150 μg mL -1) was placed in a dialysis bag (300KDa), sealed, and the dialysis bag containing the drug solution was immersed in 10 mL of the release solution. Finally, it was placed in a shaker at 37°C and 250 rpm. 1 mL of the release solution was taken out at 5min, 10min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, and 50h, and then 1 mL of the release solution with the corresponding pH was added. The drug release experiment was carried out under light-proof conditions throughout the process. The drug concentration of the released solution was determined by fluorescence and the cumulative release amount was calculated.

[0038] Figure 2 This is the DOX release curve of Example 1, with an initial concentration of DOX: 150 μg / mL; Figure 3 This is the drug release curve of YM101 in Example 1. The initial concentration is YM101: 230 μg / mL. It can be seen from the figure that YM101 has a certain pH responsiveness after being connected by an imine bond. As the pH decreases, the release amount increases. The drug release amount at 12h is 70.2% under pH 6.5 conditions, which shows that DOX@MPs-YM101 can effectively release YM101 under pH 6.5 conditions. In addition, the release amount of DOX is also greatly increased under pH 4.5 conditions.

[0039] Verification Example 3 Deactivation effect of DOX@MPs-YM101 on cancer-associated fibroblasts (1) Primary fibroblasts were collected using the direct tissue block method. Under sterile conditions, the dorsal skin of BALB / c mice was removed and cut into small tissue blocks (0.5-1.0 mm 3 ), then inoculate at the bottom of the cell culture flask, add fresh culture medium containing 15% fetal bovine serum and 1% penicillin-streptomycin, and culture at 37°C and 5% CO2 for 48 hours, then replace the culture medium (every 48 hours). After 1 to 3 days, primary fibroblasts grow out of the tissue block.

[0040] (2) The grown primary fibroblasts were collected and 20 ng / mL TGF-β was added to them for stimulation and induction for 48 h to obtain myofibroblasts.

[0041] (3) 3×10 5Myofibroblasts were cultured in a cell culture plate. After 12 hours, the culture medium was removed and PBS, MPs, YM101, MPs-YM101, DOX@MPs, and DOX@MPs-YM101 solutions were added (the concentration was based on 1 μg / mL DOX, 20 μg / mL MPs protein concentration, or 1.6 μg / mL YM101). Lactic acid was used to adjust the pH to 6.5 or 7.4. After 48 hours of culture, myofibroblasts from each group were collected for immunofluorescence staining, and the expression of characteristic proteins of myofibroblasts was observed by confocal microscopy. DOX@MPs-YM101 was added to myofibroblasts as the experimental group. Among them, Figure 4 The experimental results of the expression of α-SMA and Fibronectin in the PBS, MPs, YM101, MPs-YM101, DOX@MPs, and DOX@MPs-YM101 treatment groups are shown in Table 1. It can be seen that the immunofluorescence intensity was not affected by the MPs in Comparative Example 2 and the DOX@MPs in Comparative Example 1, which shows that MPs and DOX@MPs have no effect on the activation state of myofibroblasts. Free YM101, MPs-YM101 in Comparative Example 3, and DOX@MPs-YM101 in Example 1 can down-regulate the expression of α-SMA and Fibronectin in myofibroblasts, but MPs-YM101 in Comparative Example 3 and DOX@MPs-YM101 in Example 11 down-regulate the expression of α-SMA and Fibronectin in myofibroblasts most significantly. The above results indicate that the YM101 antibody released by DOX@MPs-YM101 at pH 6.5 has the effect of deactivating myofibroblasts.

[0042] Verification Example 4: Improvement of the stromal microenvironment of subcutaneous tumors of mouse liver cancer by DOX@MPs-YM101 (1) When the mouse liver cancer H22 subcutaneous tumor grows to a volume of 100 mm 3 At the time of the study, tumor-bearing mice (18-22 g) were randomly divided into six groups, with 6 mice in each group. Each mouse was injected with PBS, MPs, YM101, MPs-YM01, DOX@MPs, and DOX@MPs-YM101 solutions (0.5 mg / kg, DOX; 0.8 mg / kg, YM101; 10 mg / kg, MPs) through the tail vein every two days (a total of 5 times). After 13 days of administration, the mice were killed by cervical stenosis and the tumor tissues were removed.

[0043] (2) Take part of the tumor tissue from each group and cut it into 2 mm 3The small pieces of about 100 μg / mL were digested with 0.8 mg / mL collagenase at 37 °C for 40 min, passed through a 200-mesh stainless steel sieve once and a 200-mesh nylon mesh twice, and then lysed with erythrocyte lysis buffer and washed with PBS to obtain a single-cell suspension. 6 Fluorescent dye-labeled antibodies were added to each cell and incubated at 4°C in the dark for 30 min. The cells were collected by centrifugation, resuspended in 500 μL PBS, and the proportion of cancer-associated fibroblasts (CAFs) in each group was detected using a CytoFLEX S flow cytometer.

[0044] like Figure 5 As shown in the figure, DOX@MPs-YM101 can significantly reduce the expression of (CAF), and its effect is significantly better than that of free YM101. The above results show that DOX@MPs-YM101 can effectively reduce the expression of tumor-associated fibroblasts in vivo.

[0045] Verification Example 5 Accumulation of DOX@MPs-YM101 in the tumor site of tumor-bearing mice (1) Establishment of mouse H22 liver cancer subcutaneous tumor model: Establishment of subcutaneous tumor model: 100 μL of H22 liver cancer + myofibroblasts cell suspension (cell number: H22: 2×10 6 ; myofibroblasts: 1×10 6 ), the mouse hepatocellular carcinoma H22 subcutaneous tumor model was established.

[0046] (2) When the mouse liver cancer H22 subcutaneous tumor grows to a volume of 300-400 mm 3 At the same time, tumor-bearing mice (18-22 g) were randomly divided into 3 groups, with 4 mice in each group. DOX, DOX@MPs, and DOX@MPs-YM101 (0.75 mg / kg, DOX) were injected into the tail vein of each mouse at a dose of 300 μL.

[0047] (3) 48 hours after caudal vein administration, the mice in each group were killed, and the tumors and tissues were removed and prepared into tissue homogenates, and the drug content was detected by an ELISA instrument.

[0048] In the H22 tumor model with poor matrix, the accumulation of DOX@MPs-YM101 in Example 1 and DOX@MPs in Comparative Example 1 at the tumor site was similar, with no significant difference. Figure 6 As shown, in the H22 tumor model with abundant tumor stroma, the accumulation of DOX@MPs-YM101 in the tumor site was significantly higher than that of DOX@MPs, as shown in Figure 7As shown, it indicates that the matrix at the tumor site will hinder the accumulation of nanocarriers at the tumor site, and DOX@MPs-YM101 can effectively degrade the matrix, thereby increasing the aggregation of nanocarriers at the lesion site, thereby achieving a better tumor inhibition effect.

[0049] Verification Example 6 Uptake and killing of H22 cells by H22 cell-derived drug-loaded vesicles DOX@MPs-YM101 (1) H22 cells were plated into a 12-well plate at a cell density of 2.0 × 10 cells per well. 4 After culturing overnight, the pH of the culture medium was adjusted to 7.5 or 6.5 using lactic acid, and then the modified drug-loaded vesicles (DOX@MPs-YM101) coupled with antibodies were added as the experimental group. Free doxorubicin (Free Dox) was obtained in a similar manner, and the drug-loaded vesicles (DOX@MPs) without antibody coupling were used as the control. The final concentration of DOX was 1μg / mL. After continuing to incubate at 37℃ for 4 hours, the cells were collected and the content of drug uptake in the cells was detected by flow cytometry. The results are shown in Figure 8 As shown in content a, it can be seen that the uptake of DOX@MPs of comparative example 1 and DOX@MPs-YM101 of example 1 by H22 mouse liver cancer cells was significantly higher than that of the free DOX group.

[0050] (2) H22 cells were cultured at 8×10 3 The cells were seeded at a density of 100 μg / mL in a 96-well plate. After overnight incubation, the serum-free medium was replaced. The pH of the medium was adjusted to 7.5 or 6.5 with lactic acid. Then, the modified drug-loaded vesicles (DOX@MPs-YM101) coupled with antibodies were added as the experimental group, where the final concentration of DOX was 1 μg / mL. PBS buffer, 20 μg / mL of MPs, 1.5 μg / mL of YM101, 20 μg / mL of MPs-YM101 (concentration of MPs: 20 μg / mL) and 20 μg / mL of DOX@MPs (concentration of MPs: 20 μg / mL) were used as the control group. Finally, after incubation for 24 hours at 37°C and 5% CO2, 10 μL of CCK-8 solution was added to each well and incubated for 4 hours. The absorbance of each well at 450 nm was detected using an enzyme reader. The results are shown in Figure 2. Figure 8 As shown in content b, it can be seen that the DOX@MPs of comparative example 1 and the DOX@MPs-YM101 of example 1 have stronger ability to kill tumor cells.

[0051] Verification Example 7 Uptake and killing of 4T1 cell-derived drug-loaded vesicles DOX@MPs-YM101 in 4T1 cells (1) 4T1 cells were plated into a 12-well plate at a cell density of 2.0 × 10 cells per well. 4After culturing overnight, the pH of the culture medium was adjusted to 7.5 or 6.5 using lactic acid, and then the modified drug-loaded vesicles (DOX@MPs-YM101) coupled with antibodies were added as the experimental group. Free doxorubicin (Free Dox) was obtained in a similar manner, and the drug-loaded vesicles (DOX@MPs) without antibody coupling were used as the control. The final concentration of DOX was 1 μg / mL. After continuing to incubate at 37°C for 4 h, the cells were digested and collected, and the content of drug uptake in the cells was detected by flow cytometry. The results are shown in Fig. 9 As shown in content a, it can be seen that the uptake of DOX@MPs of Comparative Example 1 and DOX@MPs-YM101 of Example 1 by 4T1 mouse breast cancer cells is significantly higher than that of the free DOX group.

[0052] (2) 4T1 cells were cultured at 8×10 3 The cells were seeded at a density of 1 μg / mL in a 96-well plate, and the serum-free medium was replaced after overnight incubation. The pH of the medium was adjusted to 7.5 or 6.5 with lactic acid, and then the modified drug-loaded vesicles (DOX@MPs-YM101) coupled with antibodies were added as the experimental group, in which the final concentration of DOX was 1 μg / mL. PBS buffer, 20 μg / mL of MPs, 1.5 μg / mL of YM101, 20 μg / mL of MPs-YM101 (concentrations of MPs: 20 μg / mL, YM101: 1.6 μg / mL) and 20 μg / mL of DOX@MPs (concentrations of MPs: 20 μg / mL, YM101: 1.6 μg / mL) were used as the control group. Finally, after incubation at 37°C and 5% CO2 for 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 4 h. The absorbance of each well at 450 nm was measured using an ELISA reader. The results are shown in Table 1. Fig. 9 As shown in content b, it can be seen that the DOX@MPs of comparative example 1 and the DOX@MPs-YM101 of example 1 have stronger ability to kill tumor cells.

[0053] Verification Example 8 Detection of ATP and CRT release from tumor cells induced by DOX@MPs-YM101 in vitro (1) H22 cells were cultured at 2×10 4 Cells / dish were seeded into confocal dishes or at 3 × 10 5Cells / well were inoculated into 6-well plates. After 12 h of culture, PBS, MPs, YM101, MPs-YM01, DOX@MPs, and DOX@MPs-YM101 (with a DOX concentration of 1 μg / mL, an MPs protein concentration of 20 μg / mL, or a YM101 concentration of 1.6 μg / mL) were added to the dish or wells, respectively. HCl was used to adjust the pH to 7.4 or 6.5. The final concentration of DOX was 2 μg / mL, and the cells were incubated for 6 h. After the incubation, the culture supernatant in the well was taken, and the amount of ATP in the supernatant was detected using an ATP detection kit. The results are shown in Figure 2. Fig.10 As shown in the left figure. The results showed that under pH 6.5 conditions, the concentration of ATP in H22 cells treated with DOX@MPs-YM101 increased significantly, which was 2.7 and 3.0 times that of the PBS and YM101 treatment groups, respectively.

[0054] (2) The treated cells were washed 3 times with pre-cooled PBS, fixed with 4% PFA for 15 min, and then washed 5 times with PBS. After fixation, PBS containing 5% FBS was added for blocking at room temperature for 30 min. After blocking, anti-CRT rabbit polyclonal antibody prepared with blocking solution was added and incubated at room temperature for 30 min. After the primary antibody incubation, the cells were washed 5 times with PBS, and the cells were incubated with FITC-labeled goat anti-rabbit secondary antibody for another 30 min. After the secondary antibody incubation, the cells were washed 2 times with PBS, and the cells were resuspended with PBS. The proportion of CRT-positive tumor cells was detected by flow cytometry. The results are shown in the figure. Fig.10 As shown in the right figure, it can be seen that DOX@MPs-YM101 was also detected by flow cytometry in the pH 6.5 treatment group. + The cell ratio is also the highest. The above results show that under pH 6.5, DOX@MPs-YM101 of Example 1 can induce the release of ATP and exposure of CRT in tumor cells after treating tumor cells, thereby inducing immunogenic death of tumor cells.

[0055] Verification Example 9 In vitro activation of DCs and T cells (1) 1×10 6 Immature dendritic cells (DCs) were seeded into 12-well plates at a density of 1×10 / well. 5PBS, MPs, YM101, MPs-YM01, DOX@MPs, DOX@MPs-YM101 (with a concentration of 1 μg / mL DOX, 20 μg / mL MPs protein or 1.6 μg / mL YM101) were added to the dish or wells respectively, and the pH was adjusted to 7.4 or 6.5 with HCl. H22 tumor cells treated according to the above method, together with the culture medium supernatant of the killer cells, were added to the wells and incubated with DCs for 12 hours. Another well was set up to add lipopolysaccharide (LPS) to a final concentration of 4 μg / mL as a positive control. After the incubation, the cells were collected and stained with anti-CD80-PE and anti-CD86-FITC antibodies. The staining method was referred to the antibody instruction manual. After the staining, the cells were washed once with PBS, and the cells were resuspended in PBS and detected by flow cytometry. The results are shown as follows Fig.11 As shown, it can be seen that after incubating DCs with the supernatant of tumor cells after different treatments, the proportion of DCs mature positive cells in the DOX@MPs-YM101 and DOX@MPs treatment groups was significantly higher than that in the other treatment groups, indicating that the tumor cells treated with DOX@MPs-YM101 in Example 1 and DOX@MPs in Comparative Example 1 can significantly induce DCs maturation after incubation with DCs.

[0056] (2) 8×10 5 The isolated and cultured T cells were inoculated into a 12-well plate at a cell density of 1:10, and the DCs treated by the above method were added into the wells at a ratio of 1:10. The culture was continued for 5 days. After the culture was completed, the cells were collected and stained with anti-IFN-γ-APC, anti-Granzyme B-Bv421, and anti-IL-2-PE antibodies. The staining method was referred to the antibody manual. After the staining, the cells were washed once with PBS, and the cells were resuspended with PBS buffer and detected by flow cytometry. The results were as follows: Fig.12 As shown in the contents ac, it can be seen that after incubating T cells with DCs activated by H22 cells with different treatments, the IL-2, IFN-γ and Granzyme B positive cell ratios of T cells in the DOX@MPs-YM101 and DOX@MPs treatment groups were higher than those in the other treatment groups. The above results show that after DCs are induced to mature by tumor cells treated with DOX@MPs-YM101 in Example 1 and DOX@MPs in Comparative Example 1, they can activate more T cells, cause T cells to secrete multiple cytokines, and play an immunomodulatory role.

[0057] Verification Example 10 DOX@MPs combined with YM101 enhances the activity of CD8+T cells by blocking tumor cell PD-L1.

[0058] (1) Isolation and culture of T cells. Healthy BALB / c mice were killed by cervical dislocation, soaked in 75% alcohol for 5 min, and transferred to a clean bench. The mouse spleen tissue was removed and placed in a 6-well plate. 2 mL of PBS was added and the spleen was ground with a syringe piston until there was no obvious block tissue. The cell suspension was filtered through a 200-mesh nylon mesh, filtered twice, centrifuged at 300 g for 5 min, and the supernatant was discarded. 5 mL of red blood cell lysis buffer was added for 5 min. After lysis, the cells were washed with PBS three times. Then, the Mojosort Mouse CD3 Nanomagnetic Bead Negative Selection Kit and Mojosort Magnetic Pole were used to sort T lymphocytes. 2×10 6 Cells were added to a 6-well plate at a cell density of 100 cells / well and cultured with RPMI 1640 complete medium containing 20 ng / mL IL-2. The next day, half of the medium in the well was replaced with fresh RPMI 1640 complete medium containing 5 μg / mL anti-CD3, 5 μg / mL anti-CD 28 and 20 ng / mL IL-2, and the cells were collected for subsequent experiments after further culture for 3 days.

[0059] (2) H22 cells were treated with DOX@MPs (1 μg / mL, DOX) in Comparative Example 1 for 12 h, washed with PBS, centrifuged, and then treated with PBS, 1.2 μg / mL anti-TGF-β, 1.2 μg / mL anti-PD-L1, and 1.5 μg / mL YM101 for 4 h. 4 / well were inoculated in a 96-well plate, and then the T cells treated by the above method were added to the wells at a ratio of T cells: tumor cells of 10:1, and co-cultured for 48 h. In addition, tumor cells, blank culture medium and T lymphocyte wells were set as controls. After the culture was completed, lysis solution was added to the control tumor cell wells to lyse the cells for 10 minutes. After the lysis was completed, the tumor cell lysate was used as a positive control well. The supernatant of the culture medium in each well was aspirated and transferred to a new 96-well plate, and then the LDH concentration in the supernatant was detected using the LDH kit to calculate the killing effect of T cells on tumor cells.

[0060] from Fig.13 It can be seen that the activated CD8 + T cells had a stronger killing effect on H22 cells treated with YM101 and anti-PD-L1, indicating that YM101 can enhance CD8 + T cell killing effect.

[0061] Verification Example 11 DOX@MPs-YM101 improves the immune microenvironment of subcutaneous liver cancer in mice (1) After the drug administration in Verification Example 10, the mice were killed by cervical dislocation, and the tumor tissue was removed, washed and separated to obtain a single cell suspension of the tumor tissue. 6 Each cell was added with fluorescent dye-labeled antibodies (concentrations were: CD45, 0.2 mg / ml; CD3, 0.2 mg / ml; CD8, 0.2 mg / ml; CD69, 0.5 mg / ml), and incubated at 4°C in the dark for 30 min. The cells were collected by centrifugation, resuspended in 500 μL PBS, and the CD8 expressed in the tumor tissue was detected using a CytoFLEX S flow cytometer. + T cells (such as Fig.14 Content a) and activated CD8 + The number of T cells (eg Fig.14 Content b), the ratio of PMN MDSCs and Tregs (as shown in Fig.14 It can be seen that the adverse effects of the DOX@MPs-YM101 group were confirmed to significantly increase the CD8 + T cells and activated CD8 + The number of T cells was significantly decreased, and the proportion of PMN MDSC and Treg in tumor tissues was significantly downregulated.

[0062] Verification Example 12 Antitumor effect of DOX@MPs-YM101 on subcutaneous liver cancer model in mice (1) The construction of mouse liver cancer H22 subcutaneous tumor model was the same as step (1) of Validation Example 5; (2) When the mouse liver cancer H22 subcutaneous tumor grows to a volume of 100 mm 3 At the time of the experiment, tumor-bearing mice (18-22 g) were randomly divided into six groups, with 6 mice in each group. Each mouse was injected with 200 μl of PBS, MPs, YM101, MPs-YM01, DOX@MPs, and DOX@MPs-YM101 solutions (the dosage was 0.5 mg / kg based on DOX, 0.8 mg / kg based on YM101, or 10 mg / kg based on MPs) through the tail vein. The drugs were given every two days (5 times in total). Subsequently, the tumor size was measured at a fixed time every day. The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V=L×W was calculated. 2 / 2. like Fig.15 As shown, DOX@MPs-YM101 of Example 1 can significantly inhibit tumor growth in the early stage, and its tumor inhibition effect is better than that of MPs-YM101 of Comparative Example 3 and DOX@MPs of Comparative Example 1, indicating that under the synergistic effect of drugs DOX and YM101, it has a better therapeutic effect. Verification Example 13 Inhibitory effect of 4T1 cell-derived drug-loaded vesicles DOX@MPs-YM101 on 4T1 in situ tumors 1. Experimental Materials and Reagents Mouse 4T1 breast cancer cells and BALB / C mice were purchased from Alva (Wuhan) Biotechnology Co., Ltd. 2. Experimental steps (1) Establishment of mouse liver cancer 4T1 orthotopic tumor model: 50 μL of 4T1+myofibroblasts cell suspension (cell number: H22: 5×10 5 ; myofibroblasts: 2.5×10 5 ), the mouse 4T1 breast orthotopic tumor model was established.

[0063] (2) When the mouse tumor volume reaches 100mm 3 At the same time, tumor-bearing mice were randomly divided into 6 groups, with 6 mice in each group. Each mouse was injected with 200 μl of PBS, MPs, YM101, MPs-YM01, DOX@MPs, and DOX@MPs-YM101 (0.5 mg / kg, DOX; 0.8 mg / kg, YM101; 10 mg / kg, MPs) through the tail vein every two days (5 times in total). On the 13th day after the first administration, the longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L×W was calculated. 2 / 2. By Fig.16 It can be seen from the data that 4T1 cell-derived drug-loaded vesicles DOX@MPs-YM101 have a good inhibitory effect on 4T1 in situ tumors.

[0064] Verification Example 14: Antitumor effect of MTX@MPs-YM101 on subcutaneous liver cancer model in mice (1) The construction of mouse liver cancer H22 subcutaneous tumor model was the same as step (1) of Validation Example 5; (2) When the mouse liver cancer H22 subcutaneous tumor grows to a volume of 100 mm 3 At the time of the study, tumor-bearing mice (18-22 g) were randomly divided into six groups, with 6 mice in each group. Each mouse was injected with 200 μl of PBS, MPs, YM101, MPs-YM01, MTX@MPs, and MTX@MPs-YM101 (0.5 mg / kg, DOX; 0.8 mg / kg, YM101; 10 mg / kg, MPs) through the tail vein every two days (5 times in total). Subsequently, the tumor size was measured at a fixed time every day. The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L×W was calculated. 2 / 2. By Fig.17It can be seen from the data that the H22 cell-derived drug-loaded vesicles MTX@MPs-YM101 of Example 2 have a good inhibitory effect on H22 tumors.

[0065] Verification Example 15: Antitumor effect of 5-Fu@MPs-YM101 on subcutaneous liver cancer model in mice (1) The construction of mouse liver cancer H22 subcutaneous tumor model was the same as step (1) of Validation Example 5; (2) When the mouse liver cancer H22 subcutaneous tumor grows to a volume of 100 mm 3 At the time of the study, tumor-bearing mice (18-22 g) were randomly divided into six groups, with 6 mice in each group. Each mouse was injected with 200 μl of PBS, MPs, YM101, MPs-YM01, 5-Fu@MPs, and 5-Fu@MPs-YM101 (0.5 mg / kg, DOX; 0.8 mg / kg, YM101; 10 mg / kg, MPs) through the tail vein every two days (5 times in total). Subsequently, the tumor size was measured at a fixed time every day. The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L×W was calculated. 2 / 2. By Fig.18 It can be seen from the data that the H22 cell-derived drug-loaded vesicles 5-Fu@MPs-YM101 of Example 3 have a good inhibitory effect on H22 tumors.

[0066] Verification Example 16 DOX@MPs-Y101D inhibits human hepatocellular carcinoma tumors cultured in vitro (1) Tumor tissue was removed from the liver of a patient diagnosed with hepatocellular carcinoma by a clinician. In a laboratory biosafety cabinet, the patient's tumor tissue was cut into 5×5×5 mm slices and randomly divided into 12 groups, and PBS, MPs, Y101D, MPs-Y101D, DOX@MPs, and the DOX@MPs-Y101D group of Example 4 were added, respectively, in which the concentrations of doxorubicin and Y101D were 1 μg / mL and 1.6 μg / mL, respectively, and the concentration of MPs was 20 mg / kg. Acetic acid was used to adjust the pH to 7.4 or 6.5. Three samples were placed in each group and cultured in 6 mL RPMI complete medium. Peripheral blood mononuclear cells were isolated from the peripheral blood of the same patient. 1×10 6cells / mL was added to the above culture medium. After incubation at 37 °C for 48 h, the tumor sections were washed 3 times with PBS, then cut into pieces, and digested at 37 °C for 1 h with serum-free RPMI 1640 medium containing 0.8 mg / mL type I collagenase and 5 μg / mL DNase I. The cells were filtered once with a 200-mesh stainless steel sieve and centrifuged at 300 g for 5 min to obtain a cell suspension. After lysing the red blood cells with red blood cell lysis buffer, the red blood cells were washed and resuspended with PBS, and then filtered twice with a 40 μm cell nylon filter to obtain a single cell suspension of tumor tissue. Finally, the cell suspension was divided into multiple portions for detection.

[0067] (2) After 48 hours of incubation, the researchers rinsed the tumor sections thoroughly with PBS, minced them, and digested them in RPMI 1640 medium containing 0.8 μg / mL type I collagenase and 5 μg / mL deoxyribonuclease I (DNaseI) at 37°C for 1 hour. The digested cell mixture was filtered twice through a 40-μm cell sieve and rinsed with PBS. 1×10 6 cells (100 μL), add appropriate amount of fluorescent-labeled antibodies according to the instructions, incubate at 4°C in the dark for 30 min, centrifuge at 300 g for 5 min, resuspend the cells in 400 μL PBS, and use CytoFlex S flow cytometer to detect the proportion of positive cells. Use FITC-anti-CD45 to circle tumor cells (CD45-), and then use Annexin V-PI apoptosis detection kit to detect tumor cell apoptosis. Use APC-CD11c, PE / Cy7-anti-CD80 or PE-anti-CD86 to detect the maturation of DCs. Use FITC-anti-CD45, PE / Cy7-anti-CD3, PE / Cy5.5-anti-CD8 to mark CD8 + T lymphocytes; APC-anti-CD69, APC-anti-IFN-γ, and APC-anti-GzmB were used to mark activated CD8 + T lymphocytes.

[0068] Fig.19 The above-mentioned embodiments and comparative examples inhibit the effect of in vitro culture of human hepatocellular carcinoma tumors; wherein, Fig.19 Content a is the percentage of CRT-positive cells. It can be seen that DOX@MPs-Y101D effectively induced the expression of CRT in tumor cells and improved its immunogenicity. Fig.19 Content b is the percentage of CD80 in CD11c. It can be seen that the antigens released by tumor cells promote the maturation of DCs. Fig.19Content c is the percentage of CD69 in CD11c, Fig.19 Content d is the percentage of GzmB in CD8, Fig.19 Content e is the percentage of IFN-γ in CD8, Fig.19 Content f is the percentage of apoptotic tumor cells. The above results show that under pH 6.5 conditions, DOX@MPs-Y101D significantly activated CD8 + T cells promoted the expression of GzmB and IFN-γ, and had a good ability to kill tumor cells. These data show that under the action of the acidic microenvironment of the tumor, DOX@MPs-Y101D has a synergistic effect of chemotherapy and immunotherapy and has a good anti-tumor therapeutic effect.

[0069] In summary, the system of this embodiment is based on the preparation of MPs loaded with anti-tumor drugs, and the bispecific antibody is coupled to the vesicle loaded with anti-tumor drugs through a pH-sensitive imine bond, so as to achieve the synergy between the bispecific antibody and the vesicle loaded with anti-tumor drugs, which is used for the treatment of tumor chemotherapy and combined immunotherapy. The biological targeting of MPs is utilized to enable efficient delivery of bispecific antibodies and anti-tumor drugs to the tumor area. In response to the acidic conditions of the tumor, the bispecific antibody is released, TGF-β is neutralized, the activation of CAFs is inhibited, the secretion of tumor matrix is ​​reduced, the solid stress of tumor tissue is reduced, the mechanical microenvironment of the tumor is improved, and the enrichment and deep penetration of the drug-loaded vesicle preparation in the tumor tissue are further enhanced; after the drug-loaded vesicle preparation releases the bispecific antibody in the tumor tissue, the drug-loaded vesicle preparation is taken up by the tumor cells, effectively killing cancer cells, especially cells and inducing the ICD effect, activating T cell anti-tumor immunity; in addition to improving the mechanical microenvironment of the tumor and promoting CD8 + In addition to infiltrating tumors, T cells can block the action of PD-L1, relieve the tumor immunosuppressive microenvironment, increase the number and deep infiltration of cytotoxic T cells in tumor tissues, improve the tumor immunosuppressive microenvironment, enhance the killing of tumor cells, inhibit the growth of tumor tissues, and have a good tumor immunotherapy effect.

[0070] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A bispecific antibody-modified tumor cell-derived drug-loaded vesicle, characterized in that: The invention comprises vesicles released from apoptotic tumor cells and anti-tumor drugs encapsulated in the vesicles as effective ingredients; The vesicle is modified with a bispecific antibody by DSPE-PEG-NH2, and the bispecific antibody can synergistically act with the anti-tumor drug encapsulated by the vesicle at the targeting site of the vesicle; the bispecific antibody is a PD-L1 / TGF-β bispecific antibody.

2. The tumor cell-derived vesicle according to claim 1, characterized in that The apoptotic tumor cells are liver cancer cells or breast cancer cells.

3. The tumor cell-derived drug-loaded vesicle according to claim 1, characterized in that: The particle size of the tumor cell-derived drug-loaded vesicles is 200nm-400nm.

4. The tumor cell-derived drug-loaded vesicle according to claim 1, characterized in that: The anti-tumor drug is doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel or dextran.

5. A preparation for treating tumors, characterized in that: It comprises the tumor cell-derived drug-loaded vesicles as described in any one of claims 1-4.

6. The method for preparing the tumor cell-derived drug-loaded vesicles according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. The drug-loaded vesicles and DSPE-PEG-NH2 are mixed and fully incubated at a mass ratio of 1: (0.3-1), so that the DSPE-PEG-NH2 is modified on the surface of the phospholipid bilayer of the drug-loaded vesicles through its hydrophobic end to obtain amino-modified drug-loaded vesicles; the drug-loaded vesicles include vesicles released from apoptotic tumor cells and anti-tumor drugs as active ingredients encapsulated in the vesicles; S2. The amino-modified drug-loaded vesicles, glutaraldehyde and bispecific antibodies are mixed and fully incubated to modify the bispecific antibodies on the amino groups, thereby obtaining the tumor cell-derived drug-loaded vesicles.

7. The preparation method according to claim 6, characterized in that: The incubation conditions in step S1 and / or step S2 are 0-4° C. for 6-24 hours.

8. The preparation method according to claim 6, characterized in that: In step S2, the mass ratio of the amino-modified drug-loaded vesicles, glutaraldehyde and bispecific antibody is 1:(50-150):(0.2-0.5).

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