Delivery system based on sensitized mast cells, preparation method therefor, and application thereof

Abstract

Disclosed are a delivery system based on sensitized mast cells, a preparation method therefor, and an application thereof, as well as a pharmaceutical composition and a drug administration device involving the delivery system of sensitized mast cells. The delivery system utilizes the specific recognition of tumor antigens by sensitized mast cells to targetedly deliver drugs to tumor cells, while protecting the drugs from the clearance of the circulating immune system. Sensitized mast cells rapidly degranulate under the activation of tumor antigens to release drugs, thereby enhancing infection, exerting an oncolytic effect, lysing tumor cells and releasing antigens, and activating an anti-tumor immune response. The delivery system can be used as a promising platform technology for personalized therapy.

Description

Sensitized mast cell-based delivery system and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of drug delivery, in particular to a sensitized mast cell-based delivery system and preparation method and application thereof. BACKGROUND

[0002] At present, the drugs widely studied in clinical research include oncolytic viruses, proteins, bioactive factors, nanomedicines and many other types. Many drugs have good in vitro experimental effects, but poor in vivo experimental effects, which is caused by the fact that many drugs cannot accurately target tumors after entering the living body. Therefore, it is necessary to use a delivery system to accurately and efficiently deliver drugs to tumor regions.

[0003] So far, various cell-based delivery strategies have been developed by taking advantage of the inherent biocompatibility and chemotaxis of cells. Drug loading in cell carriers such as stem cells, tumor cells and immune cells can not only avoid being cleared by the circulatory system, but also target the tumor site by hitchhiking on cells. After the cell carrier transports the drug to the lesion, the drug must be released to the target cells to take effect. However, most cell carriers are based on inflammatory chemotaxis to accumulate in the tumor microenvironment, and they do not recognize tumor cells themselves, so it is difficult to accurately target tumor cells. In addition, the existing cell carriers also have the problem of inaccurate regulation of drug release, which may cause the drug to be retained in the cell carrier and difficult to take effect. How to make the cell carrier release the drug when it reaches the target site is a key problem that needs to be solved. Therefore, it is of great research significance to develop a cell delivery strategy that recognizes tumor cells and intelligently releases drugs to improve drug efficacy.

[0004] Mast cells, as important effector cells in the immune system, have unique target recognition and rapid response mechanisms. In allergic reactions, antigen-specific immunoglobulin E (IgE) binds to sensitized mast cells through high-affinity FcεRI receptors, and mast cells rapidly release a variety of stored granules and inflammatory mediators such as histamine and TNF-α after contacting antigens. This process shows that sensitized mast cells not only can effectively recognize specific antigens, but also have the ability to release a large amount of effector molecules. Based on their unique antigen targeting and rapid release functions, sensitized mast cells can be developed as a new type of delivery system for targeted delivery of various therapeutic substances. Compared with other cell carriers such as stem cells, tumor cells and immune cells, sensitized mast cells not only can achieve targeted delivery, but also have a more accurate release mechanism. There is no research report on the development of sensitized mast cells as a drug delivery system.

[0005] Oncolytic viruses are genetically engineered viruses that selectively infect and kill tumor cells without damaging normal cells. Oncolytic viruses kill tumor cells mainly through two mechanisms: ① They selectively infect tumor cells by taking advantage of the inactivation or deficiency of tumor suppressor genes in target cells, replicate extensively in tumor cells, and eventually lyse tumor cells; ② They activate various immune cells such as antigen-presenting cells, NK cells, and T cells by releasing a large amount of antigens from lysed tumor cells, thereby activating the body's anti-tumor immune response. Unlike conventional therapies, oncolytic virus therapy exerts anti-tumor effects by directly lysing tumor cells and indirectly enhancing the host's anti-tumor immune response, and has the advantages of high specificity, high killing efficiency, and low risk of drug resistance, making it one of the most promising anti-tumor biological therapies.

[0006] Currently, there are five oncolytic virus products approved for marketing worldwide, all of which are administered by intratumoral injection for solid tumors. Clinical results show that the objective response rate and overall survival of tumor patients treated with intratumoral injection of oncolytic viruses have been significantly improved. Although intratumoral injection can ensure that oncolytic viruses directly reach the tumor lesion, it still has limitations. First, intratumoral injection usually requires the guidance of imaging and ultrasound technology, which is complex to operate. Second, intratumoral injection is difficult to access deep internal organ tumors or multiple metastatic tumor lesions. For metastatic tumor patients who are not suitable for intratumoral injection, intravenous injection is the preferred administration method, which can allow oncolytic viruses to infect metastatic lesions extensively. However, intravenous injection of oncolytic viruses has always been a major challenge in clinical application, which greatly limits the widespread application and therapeutic effect of oncolytic virus therapy. First, oncolytic viruses are mainly obtained by modifying common viruses such as herpes viruses, vaccinia viruses, and adenoviruses. Most patients have specific neutralizing antibodies against these viruses in their blood. After oncolytic viruses are injected intravenously into the body, they will be rapidly combined with neutralizing antibodies and lose their infectivity, and eventually be phagocytosed and eliminated by immune cells. In addition, oncolytic viruses have limited tumor targeting ability, and they are difficult to accumulate in tumor sites through blood circulation. Therefore, it is urgent to develop a tumor-targeted delivery strategy for oncolytic viruses to improve the accumulation of oncolytic viruses in metastatic tumors and solve the pain points of oncolytic virus clinical application.

[0007] SUMMARY

[0008] To solve the above technical problems, the present application provides a delivery system based on sensitized mast cells and its preparation method and application. The present application uses tumor antigen-specific IgE to sensitize mast cells and loads drugs into the sensitized mast cells. By taking advantage of the specific recognition of sensitized mast cells to tumor antigens, drugs can be targeted to tumor cells, and at the same time, they are protected from being cleared by the circulating immune system. Subsequently, sensitized mast cells rapidly degranulate to release drugs under the activation of tumor antigens, thereby enhancing infection. Finally, the drugs exert oncolytic effects, lyse tumor cells, and release antigens to activate anti-tumor immune responses.

[0009] Specifically, the present application is realized by the following technical solutions:

[0010] The first aspect of the present application provides a delivery system, comprising a mast cell; the mast cell is an antigen-specific sensitized mast cell.

[0011] In some embodiments, the mast cell is sensitive to a specific antigen.

[0012] In some embodiments, the antigen comprises OVA, HER2, EGFR, CD20, and folate receptor alpha.

[0013] In some specific embodiments, the antigen comprises OVA and HER2.

[0014] In some embodiments, the delivery system further comprises a drug.

[0015] In some specific embodiments, the drug comprises a virus, a small molecule drug, a polypeptide, a protein, and a gene drug.

[0016] In some specific embodiments, the drug is encapsulated with nanoparticles.

[0017] In some embodiments, the drug is an oncolytic virus.

[0018] In some specific embodiments, the oncolytic virus is selected from the group consisting of enterovirus, reovirus, paramyxovirus, rhabdovirus, togavirus, herpesvirus, parvovirus, adenovirus, poxvirus, and hybrid virus.

[0019] In some embodiments, the drug is a protein.

[0020] In some specific embodiments, the protein is a PD-1 protein.

[0021] In some embodiments, the delivery system is formulated for oral, nasal, intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intraperitoneal, intrapleural, intravaginal, intraurethral, intratumoral, intracranial, or intraspinal administration.

[0022] In some specific embodiments, the delivery system is formulated for intravenous administration.

[0023] In some embodiments, the mast cell is a mammalian mast cell.

[0024] In some embodiments, the mast cell is a primate mast cell.

[0025] In some specific embodiments, the mast cell is a human mast cell.

[0026] A second aspect of the present application provides a method of preparing the delivery system of the first aspect, the method comprising the step of (a) incubating an antigen-specific IgE with a mast cell, allowing the antigen-specific IgE to bind to the surface of the mast cell to obtain a sensitized mast cell.

[0027] In some specific embodiments, the method further comprises the step of (b) incubating a drug with the mast cell, allowing the drug to be loaded inside the mast cell or on the cell membrane.

[0028] In some specific embodiments, the step (b) further comprises using a penetration enhancer, the penetration enhancer comprising PEI, Polybrene and cationic liposome.

[0029] In some specific embodiments, the penetration enhancer is Polybrene.

[0030] A third aspect of the present application provides a pharmaceutical composition comprising the delivery system of the first aspect.

[0031] In some specific embodiments, the pharmaceutical composition further comprises a combination therapeutic agent, the combination therapeutic agent comprising one or more of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent and a cytotoxic drug.

[0032] A fourth aspect of the present application provides a drug delivery device, characterized in that the drug delivery device comprises:

[0033] (1) an infusion module for administering the delivery system of the first aspect or the pharmaceutical composition of the third aspect to a subject in need thereof, and (2) optionally, a pharmacodynamic monitoring module.

[0034] A fifth aspect of the present application provides use of the delivery system of the first aspect in the manufacture of a medicament for treating and / or preventing a tumor.

[0035] In some embodiments, the tumor is a hematological tumor or a solid tumor.

[0036] In some specific embodiments, the tumor is an OVA-positive or HER2-positive tumor.

[0037] A sixth aspect of the present application provides the delivery system of the first aspect for use in treating and / or preventing a tumor.

[0038] In some embodiments, the tumor is a hematological tumor or a solid tumor.

[0039] In some specific embodiments, the tumor is an OVA-positive or HER2-positive tumor.

[0040] A seventh aspect of the present application provides a method for treating a tumor, comprising administering the delivery system of the first aspect to a patient in need thereof.

[0041] In some specific embodiments, the mast cells are autologous or allogeneic to the patient.

[0042] In some embodiments, the tumor is a hematological tumor or a solid tumor.

[0043] In some specific embodiments, the tumor is an OVA-positive or HER2-positive tumor.

[0044] An eighth aspect of the present application provides the use of the delivery system of the first aspect in the treatment and / or prevention of a tumor.

[0045] In some embodiments, the tumor is a hematological tumor or a solid tumor.

[0046] In some specific embodiments, the tumor is an OVA-positive or HER2-positive tumor.

[0047] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred embodiments of the present application.

[0048] The reagents and raw materials used in the present application are commercially available.

[0049] The positive progress effect of the present application is that:

[0050] 1. Better targeting: most cell carriers are enriched to the tumor microenvironment based on inflammatory chemotaxis, which do not recognize tumor cells themselves, so it is difficult to accurately target tumor cells. Considering that specific antigens exist on the surface of tumor cells, sensitized mast cells designed against antigens are a better choice for targeted delivery of oncolytic viruses.

[0051] 2. Biological response: sensitized mast cells induce the biological effects of allergic reactions in vivo, mimic the mechanism of allergic reactions, and activate sensitized mast cells to release oncolytic viruses using tumor cell surface antigens to achieve precise regulation of oncolytic virus release to enhance the infection efficiency of target cells, avoiding the situation of drug retention in carrier cells.

[0052] 3. Unique sensitization approach: Several engineered immune cell-based therapies have gained approval for clinical applications, including chimeric antigen receptor (CAR) T cells for the treatment of hematological malignancies. However, manipulating T cells to recognize tumor antigens requires complex genetic techniques and cumbersome manufacturing processes. In contrast, mast cells can be readily endowed with the ability to recognize antigens by simple incubation with antigen-specific IgE in vitro. It has been demonstrated that IgE capable of recognizing clinically validated cancer antigens such as HER2 / neu, EGFR, CD20, and folate receptor alpha can elicit potent effector functions. By customizing patient-specific IgE molecules, this MC-mediated delivery system can serve as a promising platform technology for personalized therapy. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is the characterization of sensitized mast cells in the embodiments of the present application. (a) is the flow cytometry analysis of the IgE expression level on sensitized mast cells. (b) is the SEM images of sensitized mast cells before and after activation. Scale bar: 2 μm.

[0054] Figure 2 is the result of adding Polybrene to improve the loading efficiency of oncolytic virus-loaded sensitized mast cells.

[0055] Figure 3 is the TEM images of oncolytic virus-loaded sensitized mast cells before and after activation in the embodiments of the present application. (a) is the TEM image of oncolytic virus-loaded sensitized mast cells. (b) is the TEM image of oncolytic virus released from sensitized mast cells after activation.

[0056] Figure 4 is the fluorescent microscope images of oncolytic virus-loaded sensitized mast cells infecting target cells in the embodiments of the present application. Scale bar: 100 μm.

[0057] Figure 5 is the pharmacokinetic curve of oncolytic virus-loaded sensitized mast cells after tail vein administration in the embodiments of the present application.

[0058] Figure 6 is the data of oncolytic virus delivered by oncolytic virus-loaded sensitized mast cells targeting tumor sites in the embodiments of the present application. (a) is the representative small-animal fluorescence imaging image of sensitized mast cells targeting and accumulating in mouse tumors. (b) is the fluorescence image of oncolytic virus transfection of EGFP in tumor tissue frozen sections after intravenous injection of oncolytic virus-loaded sensitized mast cells. OVs are oncolytic virus treatment group, MCs are mast cell treatment group, IgE-MCs are sensitized mast cell treatment group, OV@MCs are oncolytic virus-loaded mast cell treatment group, and OV@IgE-MCs are oncolytic virus-loaded sensitized mast cell treatment group.

[0059] Figure 7 is the in vivo efficacy data of the oncolytic virus-loaded sensitized mast cells in the B16F10-OVA subcutaneous tumor model in the embodiments of the present application. OVs is the oncolytic virus treatment group, MCs is the mast cell treatment group, IgE-MCs is the sensitized mast cell treatment group, OV@MCs is the oncolytic virus-loaded mast cell treatment group, and OV@IgE-MCs is the oncolytic virus-loaded sensitized mast cell treatment group.

[0060] Figure 8 is the intratumoral T cell immunophenotyping analysis after treatment in the embodiments of the present application. G1 is the OVs treatment group, G1 is the MCs treatment group, G1 is the IgE-MCs treatment group, G1 is the OV@MCs treatment group, and G1 is the OV@IgE-MCs treatment group. G1-G6 are PBS, OVs, MCs, IgE-MCs, OV@MCs, and OV@IgE-MCs, respectively.

[0061] Figure 9 is the treatment effect of the oncolytic virus-loaded sensitized human mast cells against HER2 antigen on the humanized PDX tumor model in the embodiments of the present application. OVs is the oncolytic virus treatment group, hMCs is the human mast cell treatment group, IgE-hMCs is the sensitized human mast cell treatment group, OV@hMCs is the oncolytic virus-loaded human mast cell treatment group, and OV@IgE-hMCs is the oncolytic virus-loaded sensitized human mast cell treatment group.

[0062] Figure 10 is the intratumoral T cell immunophenotyping analysis after treatment in the humanized PDX tumor in the embodiments of the present application.

[0063] Figure 11 is the characterization of the sensitized mast cells genetically engineered to express PD-1 protein in the embodiments of the present application. (a) is the PD-1 expression level on the sensitized mast cells analyzed by laser confocal analysis. (b) is the PD-1 expression on the sensitized mast cells analyzed by Western blot.

[0064] Figure 12 is the fluorescence microscope image of the sensitized mast cells engineered to express PD-1 protein binding to target cells and releasing PD-1 in the embodiments of the present application.

[0065] Figure 13 is the treatment effect of the sensitized mast cells engineered to express PD-1 protein on the B16F10-OVA lung cancer model in the embodiments of the present application. PBS is the control group, MC is the mast cell treatment group, IgE-MC is the sensitized mast cell treatment group, MC-PD-1 is the mast cell engineered to express PD-1 protein treatment group, and IgE-MC-PD-1 is the sensitized mast cell engineered to express PD-1 protein treatment group. (a) and (b) are the growth of the tumor observed by the in vivo bioluminescence imaging system and the quantitative data. (c) is the intratumoral CD8 + T cell infiltration analysis after treatment in the embodiments of the present application.

[0066] Figure 14 is a confocal image of sensitized mast cells loaded with LNP fluorescent nanoparticles and PLGA fluorescent nanoparticles in an embodiment of the present application. DETAILED DESCRIPTION

[0067] The present application is further illustrated by the following examples without thereby limiting the present application to the scope of the examples.

[0068] Example 1

[0069] Bone marrow cells were obtained from euthanized mice and cultured at a density of 10 6 cells / mL in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, 1% non-essential amino acids (NEAA), 50 mM 2-mercaptoethanol, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 ng / mL recombinant mouse interleukin-3 (IL-3), and 20 ng / mL recombinant mouse stem cell factor (SCF). After 4 weeks of culture, mast cells were obtained.

[0070] IgE-MCs were obtained by sensitizing mast cells (MCs) (10 6 cells / mL) with mouse anti-OVA IgE monoclonal antibody (E-C1, Chondrex) at a final concentration of 1 pg / mL for more than 8 hours. To activate the IgE-MCs, 20 pg / mL of OVA was added to the IgE-MCs suspension (10 6 cells / mL). The results are shown in Figure 1, where the IgE bound to the surface of the mast cells increased over time, and the sensitized mast cells degranulated upon stimulation with the antigen, releasing the intracellular granules.

[0071] Example 2

[0072] Oncolytic virus was loaded into the sensitized mast cells. The oncolytic virus was recombinant Ad5-hTERTp-E1A-EGFP oncolytic adenovirus. The oncolytic virus was propagated in HEK293 cells and purified by CsCl2 equilibrium gradient centrifugation.

[0073] OV@IgE-MCs were prepared by co-incubating IgE-MCs (10 6 cells / mL) with OVs at MOI = 250 for 4 hours with the addition of the infection enhancer Polybrene (8 pg / mL) to optimize the loading of OVs, and the results are shown in Figure 2. After the incubation, the cells were collected and washed twice with PBS for subsequent studies. To activate the IgE-MCs, 20 pg / mL of OVA was added to the IgE-MCs suspension (106 To evaluate the nature of OVs released by OV@IgE-MCs in response to antigen, 20 pg / mL of OVA was added to OV@IgE-MCs for 30 min stimulation. OV@IgE-MCs and antigen-activated OV@IgE-MCs were fixed with 2.5% glutaraldehyde and stained with 2% uranyl acetate. After staining, samples were washed twice with PBS buffer and dehydrated with gradient ethanol (50%, 70%, 90%, 95%, and 100%, 15 min each). The treated samples were embedded by immersion in Eponate 12 resin. Ultrathin sections (80 nm) were mounted on copper grids and stained with lead citrate and uranyl acetate. Sections were observed by transmission electron microscopy (Tecnai G2 Spirit; 120 kV; FEI). As shown in Figure 3, OVs were loaded inside sensitized mast cells. Upon stimulation of antigen, sensitized mast cells degranulated and oncosome particles were observed in these granules, indicating that sensitized mast cells can release oncolytic viruses in a degranulation manner in response to specific antigens.

[0074] Example 3

[0075] B16F10-OVA cell line stably expressing OVA protein on cell membrane surface was established by transfecting B16F10 cells with lentivirus constructed by fusing transmembrane signal peptide sequence encoding fusion membrane targeting integrin protein, epitope sequence of OVA protein and transmembrane peptide sequence.

[0076] B16F10-OVA cells were seeded in 24-well plates at a density of 50,000 cells per well, then infected with OVs, OV@MCs or OV@IgE-MCs and incubated for the indicated time. After incubation, virus infection was studied by analyzing EGFP positive cells by fluorescence microscopy and flow cytometry. As shown in Figure 4, sensitized mast cells actively adhered to tumor cells, resulting in the strongest fluorescence of virus-carrying genome EGFP in tumor cells. Compared with naked oncolytic viruses, oncolytic viruses released by sensitized mast cells in a degranulation manner were more easily taken up by tumor cells, thereby enhancing infection.

[0077] Example 4

[0078] To study the pharmacokinetics of OV@IgE-MCs, free OVs or OV@IgE-MCs were injected into each mouse at a dose of 1 x 10 8 VP, 2 x 10 6Doses of cells were administered to C57BL / 6 mice by intravenous injection. Viral concentration in blood was determined by qRT-PCR analysis at 0.125, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 hours. By detecting the oncolytic virus blood concentration, it was found that sensitized mast cells increased the circulation time of oncolytic virus in peripheral blood (Figure 5), indicating that the use of mast cell carrier delivery can protect OV from rapid clearance by the circulating immune system.

[0079] Example 5

[0080] To investigate the biodistribution of IgE-MCs, Cy5.5-labeled MCs or IgE-MCs were intravenously injected into B16F10-OVA tumor-bearing mice (subcutaneous tumor and lung metastasis model) at a dose of 2x10 6 cells per mouse. In vivo fluorescence imaging was performed using an IVIS Spectrum system (Perkin Elmer) with Cy5.5-specific excitation / emission filters and a 1-second exposure time. It was observed that sensitized mast cells were targeted to enrich at the tumor site, enhancing the infection level of oncolytic virus in the tumor (Figure 6). This indicates that sensitized mast cells improve the efficiency of oncolytic virus targeted delivery by targeting tumor cells.

[0081] Example 6

[0082] To investigate the therapeutic effect of OV@IgE-MCs, 5x105B16F10-OVA cells were subcutaneously inoculated into the right flank of C57BL / 6 mice. Six days later, the mice were randomly divided into six groups (n=6) and received intravenous injection of OV, MCs, IgE-MCs, OV@MCs and OV@IgE-MCs at a dose of 1x10 8 viral particles per mouse or 2x10 6 cells per mouse, once every other day for a total of four administrations, while a PBS control group was set up. Tumor size was measured and calculated by the following formula: width 2 x length x 0.5. When the animals showed signs of deteriorating health or the tumor size exceeded 1.5 cm 3 , they were euthanized.

[0083] The results are shown in Figure 7. Compared with PBS-treated mice, mice receiving OV and OV@MCs treatment showed only moderate anti-tumor effects, while OV@IgE-MCs treatment showed the most significant tumor control effect.

[0084] Example 7

[0085] Oncolytic viruses ultimately lead to tumor cell lysis by infection and replication in tumors, releasing large amounts of tumor antigens to activate tumor-specific T cell immune responses. We preliminarily evaluated the T cell immune responses elicited by sensitized mast cells loaded with oncolytic viruses. Tumors from different treated mice were collected and cut into small pieces, homogenized in cold staining buffer containing digestive enzymes to form a single-cell suspension. T cell phenotypes in tumor tissues after intravenous administration were detected by flow cytometry by flow antibody staining of T cell-related markers, and the results are shown in Figure 8. Sensitized mast cells loaded with oncolytic viruses promoted T cells, especially CD8 + T cell infiltration in tumors, indicating that sensitized mast cells potentiated the anti-tumor immune response of oncolytic viruses.

[0086] Example 8

[0087] To further explore the clinical potential of sensitized mast cells as a cancer adoptive cell therapy, human HER2-specific IgE was designed to sensitize human mast cells from peripheral blood progenitor cells. CD34+ precursor cells were isolated from PBMCs of a volunteer (using EasySep Human CD34+ Positive Selection Kit, STEMCELL Technologies). CD34+ cells were cultured in StemSpan medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng / mL; Peprotech), human IL-3 (10 ng / mL; Peprotech), and human SCF (50 ng / mL; Peprotech) for 4 weeks. At week 5, cells were cultured in IMDM Glutamax I medium supplemented with sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, insulin-transferrin selenium (all from Invitrogen), ciprofloxacin (10 mg / mL; Sigma-Aldrich), IL-6 (50 ng / mL), and human SCF (50 ng / mL). After 8 weeks, human mast cells were obtained.

[0088] Preparation of OV@IgE-hMCs was done by co-incubation of IgE-hMCs (10 6 cells / mL) with OVs at MOI = 250 for 4 hours, during which the infection-promoting agent Polybrene (8 pg / mL) was added to optimize the promotion of OV loading. After incubation, cells were collected and washed twice with PBS for subsequent studies.

[0089] A 72-year-old male gastric cancer patient with HER2 / neu positivity was obtained from resected primary tumor sample, subcutaneously transplanted into the axillary of NSG mice to establish PDX model. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. For humanized PDX model, 7 days after PDX tumor transplantation, PBMCs isolated from healthy human peripheral blood donors (5 x 10 6 cells per mouse) were injected intravenously. The reconstitution of human immune system was evaluated at designated time points by monitoring the reconstitution of T cells (hCD45+hCD3+). The therapeutic effect of oncolytic virus-loaded IgE-human mast cells (OV@IgE-hMCs) was evaluated in HER2-positive PDX mouse model with reconstituted human immune system. The results are shown in Figure 9. Tumor growth was significantly inhibited in mice treated with sensitized oncolytic virus-loaded human mast cells compared to mice treated with oncolytic virus and oncolytic virus-loaded human mast cells.

[0090] Example 9

[0091] Tumors from different treated mice were collected and cut into small pieces, homogenized in cold staining buffer containing digestive enzymes to form a single cell suspension. T cell phenotypes in humanized PDX tumors were detected by flow cytometry by flow antibody staining of T cell related markers. The results are shown in Figure 10. Sensitized oncolytic virus-loaded human mast cells significantly increased CD4+ and CD8+ T cell infiltration in tumors and induced activation of CD8+ T cells.

[0092] Example 10

[0093] A membrane expression PD-1 protein plasmid was constructed by fusing a transmembrane signal peptide sequence encoding a fusion membrane targeting integrin protein to the PD-1-EGFP protein sequence and a transmembrane peptide sequence. The membrane expression PD-1 protein plasmid (10 μg) was introduced into MCs (10 6 cells / mL) by setting the Lonza electroporator at the DS130 program to obtain MC-PD-1, and then the MC-PD-1 (10 6 cells / mL) was sensitized with mouse anti-OVA IgE monoclonal antibody (E-C1, Chondrex) at a final concentration of 1 μg / mL for more than 8 hours to obtain IgE-MC-PD-1. After incubation, the cells were collected and washed twice with PBS for subsequent studies. The results are shown in Figure 11. PD-1 protein was observed on the membrane of mast cells under confocal observation, and western blot experiments were performed on IgE-MC-PD-1 extracted protein, and PD-1 protein bands were observed, indicating that the production of PD-1 protein by mast cells through genetic engineering technology was successful.

[0094] To evaluate the ability of IgE-MC-PD-1 to release PD-1 protein in response to antigen, B16F10-OVA cells were seeded in confocal dishes and incubated overnight, followed by co-culture with MC-PD-1 and IgE-MC-PD-1. Cell-cell interactions were observed using fluorescence microscopy. The results, shown in Figure 12, indicate that IgE-MC-PD-1 actively adheres to tumor cells and releases PD-1 onto the tumor cell surface, while this was not observed in tumor cells treated with MC-PD-1. This suggests that IgE-MC-PD-1 can release drugs onto tumor cells in response to antigen stimulation.

[0095] Example 11

[0096] Figure 13 shows the therapeutic effect of engineered PD-1 protein-sensitized mast cells on the B16F10-OVA lung cancer model in this embodiment of the invention. PBS is the control group, MC is the mast cell treatment group, IgE-MC is the sensitized mast cell treatment group, MC-PD-1 is the engineered PD-1 protein-sensitized mast cell treatment group, and IgE-MC-PD-1 is the engineered PD-1 protein-sensitized mast cell treatment group. (a) and (b) show the tumor growth and quantitative data observed using an in vivo bioluminescence imaging system. (c) shows the CD8+ in the tumor after treatment in this embodiment of the invention. + T-cell infiltration analysis.

[0097] To evaluate the effects of IgE-MC-PD-1 on lung tumors, C57BL / 6 mice were intravenously injected with 5 × 10⁻⁶ IgE-MC-PD-1. 5 Five days after tumor cell inoculation, mice were randomly divided into 6 groups (n=5). Each mouse was intravenously injected with OV, MC, IgE-MC, MC-PD-1, and IgE-MC-PD-1 at a dose of 2×10⁻⁶ B16F10-OVA-luc cells. 6 Each mouse was treated four times every other day. On day 20, mouse lung tissue was dissected, perfused with d-fluorescein (Thermo Scientific Pierce), and tumor growth was observed using an in vivo bioluminescence imaging system. Lung regions were quantified to mean radiation using Living Image software. Mouse lung tumors were homogenized in a cold staining buffer containing digestive enzymes to form a single-cell suspension. T-cell infiltration in the tumor was detected by flow cytometry using antibody staining for T-cell-related markers. As shown in Figure 13, compared with other treated mice, tumor growth was significantly inhibited in IgE-MC-PD-1 treated mice. IgE-MC-PD-1 significantly increased CD8+ T-cell infiltration in the tumor.

[0098] Example 12

[0099] The fluorescent substance-loaded lipid nanoparticles (Fitc-LNP) and the fluorescent substance-loaded polymer nanoparticles (Cy5-PLGA) were separately incubated with the IgE-MC to obtain the sensitized mast cells loaded with the nanoparticles. After the incubation, the loading of the fluorescent nanoparticles was observed under a microscope. As shown in FIG. 14, the fluorescent substance-loaded lipid nanoparticles and the polymer nanoparticles were loaded into the interior of the sensitized mast cells.

[0100] Although the specific embodiments of the present application are described above, it should be understood by those skilled in the art that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and the essence of the present application. Therefore, the scope of protection of the present application is defined by the appended claims.

Claims

1. A delivery system characterized by, The delivery system comprises mast cells; the mast cells are antigen-specific sensitized mast cells.

2. The delivery system of claim 1, wherein, The mast cells are sensitive to specific antigens, which preferably include OVA, HER2, EGFR, CD20, and folate receptor alpha.

3. The delivery system of claim 1 or 2, wherein, The delivery system further comprises a drug, which preferably includes viral, small molecule drugs, polypeptides, proteins, and genetic drugs; preferably the drug is encapsulated with nanoparticles.

4. The delivery system of claim 3, wherein, The drug is an oncolytic virus, which is preferably selected from the group consisting of enterovirus, reovirus, paramyxovirus, rhabdovirus, togavirus, herpesvirus, parvovirus, adenovirus, poxvirus, and hybrid virus; and / or, the drug is a protein, which is preferably a PD-1 protein.

5. The delivery system of any of claims 1-4, wherein, The delivery system is formulated for oral, nasal, intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intraperitoneal, intrapleural, intravaginal, intraurethral, intratumoral, intracranial, or intraspinal administration.

6. The delivery system of any of claims 1-5, wherein, The mast cells are mammalian mast cells; preferably, the mast cells are primate mast cells; more preferably, the mast cells are human mast cells.

7. A method of preparing a delivery system according to any one of claims 1-6, characterized in that, The method comprises step (a): co-incubating antigen-specific IgE with mast cells, allowing the antigen-specific IgE to bind to the surface of the mast cells to obtain sensitized mast cells.

8. The method of claim 7, wherein, The method further comprises step (b): incubating a drug with the mast cells, allowing the drug to be loaded inside the mast cells or on the cell membrane; Preferably, step (b) further comprises using a co-infection agent, which includes PEI, Polybrene, and cationic liposomes, preferably Polybrene.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the delivery system of any one of claims 1-6; Preferably, the pharmaceutical composition further comprises a combination therapeutic agent, which includes one or more of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, and a cytotoxic drug.

10. A drug delivery device characterized in that, The administration device comprises: (1) an infusion module for administering the delivery system of any one of claims 1-6 or the pharmaceutical composition of claim 9 to a subject in need thereof, and (2) an optional pharmacodynamic monitoring module.

11. Use of the delivery system of any one of claims 1-6 in the manufacture of a medicament for treating and / or preventing a tumor; preferably, the tumor is a hematological tumor or a solid tumor; more preferably, the tumor is OVA-positive or HER2-positive.

12. Use of the delivery system of any one of claims 1-6 in treating and / or preventing a tumor; preferably, the tumor is a hematological tumor or a solid tumor; more preferably, the tumor is OVA-positive or HER2-positive.

13. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-12. The method comprises administering the delivery system of any one of claims 1-6 to a patient in need thereof; preferably, The mast cells are autologous or allogeneic to the patient; and / or, the tumor is a hematological tumor or a solid tumor; more preferably, the tumor is OVA-positive or HER2-positive.

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