Microcapsule-based vaccine

Abstract

The present invention relates to a vaccine comprising an antigen and a biodegradable polymer blend matrix, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer, the vaccine being present in the form of microcapsules containing a multi-chamber structure inside the microcapsules, the average particle size of the microcapsules preferably being 10-100 [mu] m, more preferably being 30-60 [mu] m, the preparation method of the microcapsule comprises the following steps: firstly, preparing an open-pore microsphere from a polymer blend, then mixing the open-pore microsphere with a solution containing an antigen, and then sealing the open-pore microsphere loaded with the antigen solution to form a sealed microcapsule loaded with the antigen.

Description

Technical Field

[0001] The present invention relates to a microcapsule-based vaccine, which comprises an antigen and a biodegradable polymer blend matrix, and the vaccine is, for example, an anti-tumor vaccine. Background Art

[0002] With the development of tumor biology and immunology, immunotherapy, which mainly features regulating the body's own immune system, has opened up a new way for tumor treatment. Among many immunotherapy strategies, tumor vaccines can mimic the way the body fights pathogens, restore the natural "recognition" and response to tumor cells, and thus specifically eliminate tumor cells, so they have received more and more attention.

[0003] One of the problems faced by anti-tumor vaccines in exerting an efficient effect is that simple tumor antigens are difficult to stimulate a long-lasting and effective immune response in the body. Using nanosized carriers to load tumor antigens can effectively improve the endocytosis and maturation of immune cells, thus stimulating a stronger immune response in the body. However, because of their small size, nanocarriers are easily cleared by the body, so it is difficult to produce a long-lasting anti-tumor effect. Encapsulating antigens by the double emulsion solution method to prepare micron-sized carriers effectively avoids the above problems. However, it also faces the dilemma that the antigen release is too slow to stimulate an effective immune response.

[0004] CN101601860A discloses a polymer particle-based vaccine, in which the antigen composition is in two forms: antigen adsorbed on biodegradable polymer particles and unadsorbed antigen (free antigen). The average particle size of the particles is 0.1-20 μm, and it is prepared by the following method: mixing polymer particles with hepatitis B surface antigen solution, and the suspension obtained by the adsorption of polymer particles and antigen solution is the vaccine product. The vaccine of the present invention can rapidly induce an immune response in the body and induce a high level of humoral immunity and cellular immunity in the body.

[0005] CN102489230A discloses a preparation method of biodegradable material microcapsules, including the preparation of open microspheres, the loading of core materials, and the sealing of open microspheres. Compared with the traditional double emulsion embedding method, this method is gentler, avoiding damage to bioactive substances; the remaining core materials in the solution after embedding can be recycled; it is more environmentally friendly, reaching the same particle size and pore size levels as previously reported, and having a larger internal cavity volume, which is more conducive to the loading of core materials. However, this patent document only mentions that the biodegradable material microcapsules can be used to embed small molecules, biological macromolecules, and can also load nanoparticles and micron-sized particles.

[0006] Despite the various studies mentioned above, the development of vaccines that can better stimulate the body to produce specific immunity, such as killing tumor cells through cytotoxic T cells, is of great significance for its clinical application. SUMMARY OF THE INVENTION

[0008] The inventors of the present application have discovered that an open-pored microsphere prepared from a biodegradable polymer is prepared by a double emulsion-solvent removal method, and then the open-pored microsphere is filled with tumor antigens, and then the open-pored microsphere is sealed to prepare a sealed microcapsule with a larger particle size that can be locally retained in mice. Since the microcapsule is not easily phagocytosed and metabolized, it is easier to degrade, and stimulates the body to produce a sustained inflammatory response during the degradation process. Therefore, the microcapsule can continuously recruit antigen-presenting cells such as dendritic cells (DC) to arrive at the injection site to phagocytize antigens, and the acidic microenvironment produced by the synergistic polymer matrix metabolism can significantly increase the number of cells recruited, increase the cell endocytosis of antigens, stimulate DC maturation and differentiation, and finally DC home to the lymph nodes, stimulating the body to produce an efficient and lasting immune response, promoting the continuous proliferation and differentiation of T cells, and producing specific and lasting killing, which can significantly inhibit tumor growth and prolong the survival time of mice.

[0009] Based on this, the present invention provides a vaccine, which comprises an antigen and a biodegradable polymer blend matrix, wherein the polymer blend contains a hydrophobic polymer and an amphiphilic block copolymer, and the vaccine exists in the form of a microcapsule, wherein the interior of the microcapsule contains a multi-chamber structure, and the average particle size of the microcapsule is preferably 10-100 μm, more preferably 30-60 μm. The microcapsule is prepared by the following method: first, open-pore microspheres are prepared from the polymer blend, and then the open-pore microspheres are mixed with a solution containing an antigen, and then the open-pore microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen.

[0010] Preferably, in the vaccine of the present invention, by optimizing the preparation process parameters, the release of the antigen and the cell recruitment behavior during the local retention of the vaccine in vivo produce a synergistic effect, so that the released antigen is phagocytosed by the recruited immature DCs, which significantly improves the utilization rate of the antigen.

[0011] Preferably, the vaccine is a therapeutic vaccine, such as an anti-tumor vaccine or a therapeutic hepatitis B vaccine, and the preferred antigen is a tumor antigen or hepatitis B surface antigen.

[0012] In the vaccine of the present invention, a certain amount of chemokines, such as granulocyte colony factor (GM-CSF), macrophage inflammatory protein 3α (MIP-3α) and monocyte chemoattractant protein 1 (MCP-1), can also be loaded in the porous microsphere system to enhance the recruitment of cells by the microcapsules, further improve the utilization rate of the antigen, and enhance the immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 SEM image of microspheres prepared from PLA

[0014] Figure 2 SEM image of microspheres prepared from PLA and PELA

[0015] Figure 3 Particle size distribution diagram of microspheres prepared from PLA and PELA

[0016] Figure 4 SEM image of the internal structure of microspheres prepared from PLA and PELA

[0017] Figure 5 Internal pore size distribution diagram of microspheres prepared from PLA and PELA

[0018] Figure 6 SEM image of the sealed microcapsules of the present invention

[0019] Figure 7 Internal structure of the sealed microcapsules of the present invention

[0020] Figure 8 Loading rate and embedding rate of the sealed microcapsules of the present invention against antigen OVA

[0021] Figure 9 Loading rate and embedding rate of the sealed microcapsules of the present invention against polypeptide MUC1

[0022] Figure 10 Quantitative diagram of antigen fluorescence intensity at the injection site of different vaccine preparations

[0023] Figure 11 Recruitment behavior of the microcapsules of the present invention to inflammatory cells: (a) Representative tissue section images of tissues locally containing sealed microcapsules recruiting cells (b) Quantitative analysis of the number of cells recruited by each microcapsule

[0024] Figure 12 Comparison of antigen utilization rates of different vaccine preparations: (a) OVA + Number of cells, (b) OVA + Antigen endocytosis amount of cells (expressed as average fluorescence intensity), (c) Antigen utilization rate

[0025] Figure 13 pH change curve in the local microenvironment during the degradation process of the microcapsules of the present invention

[0026] Figure 14 CD86 + Ratios of MHC-I and MHC-II in DCs

[0027] Figure 15 Tumor growth curve of E.G7 tumor-bearing mice after immunization with different vaccine preparations

[0028] Figure 16 Tumor growth curves of B16 tumor-bearing mice after immunization with different vaccine preparations

[0029] Figure 17 Quantitative analysis of metastatic foci in lung (a) and kidney (b) tissues after treatment with different vaccine preparations

[0030] Figure 18 Tumor growth curves of 4T1 tumor-bearing mice after immunization with different vaccine preparations

[0031] Figure 19 Tumor growth curves of recurrent tumors after surgery after treatment with different vaccine preparations

[0032] Figure 20 Bioluminescence imaging pictures and quantification of the postoperative recurrence model

[0033] Figure 21 H&E staining pictures of cells recruited by blank sealed microcapsules (a) and sealed microcapsules loaded with chemokine GM-CSF (b) in subcutaneous tissue Detailed description of the invention

[0035] Vaccine

[0036] The present invention relates to a vaccine for prophylactic or therapeutic treatment of diseases such as tumors in mammalian subjects. The vaccine comprises an antigen and a biodegradable polymer blend matrix, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer. The vaccine exists in the form of microcapsules with a multi-chamber structure inside. The average particle size of the microcapsules is preferably 10 - 100 μm, more preferably 30 - 60 μm. The microcapsules are prepared by the following method: first, open-pored microspheres with through pores are prepared from the polymer blend, then they are mixed with a solution containing the antigen, and then the open-pored microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen. The vaccine of the present invention may also contain a pharmaceutically acceptable carrier, salt or diluent.

[0037] In a preferred embodiment of the present invention, the vaccine is an anti-tumor vaccine. The vaccine is easy to prepare and has the versatility to be applicable to the prevention of recurrence, inhibition of metastasis and treatment of any type of tumor, and has a good anti-tumor effect. After the anti-tumor vaccine is administered, the tumor antigen is first taken up and processed by the antigen presenting cell (Antigen Presenting Cell, APC) to turn it into an antigenic peptide, which then binds to the major histocompatibility complex (Major Histocompatibility Complex, MHC) molecules on the cell surface. The APC further presents the peptide information to T cells, thereby generating a subsequent immune response process. In this process, most antigens are processed into peptides in the lysosome, which then bind to the MHC-II molecules and present them to CD4. + T cells; more importantly, a small number of antigens can be processed into peptides by the proteasome in the cytoplasm, bind to MHC-I molecules and present to CD8 + T cells are transformed into cytotoxic T lymphocytes (CTL). On the one hand, CTL directly binds to tumor cells to produce lytic killing function, and on the other hand, CD4 + T cells and CD8 + T cells jointly secrete cytokines to kill tumors, ultimately causing tumor apoptosis and lysis.

[0038] In a preferred embodiment of the present invention, the antigen is a tumor antigen. Tumor antigens play an important role in tumor occurrence, development and inducing the body's anti-tumor immune effect, and are target molecules for tumor immunotherapy. Tumor antigens are divided into two categories according to their specificity: (1) tumor-associated antigens, which refer to antigens that are highly expressed in tumor tissues and also expressed to a certain extent in normal tissues; (2) tumor-specific antigens, which refer to antigens that are only expressed in tumor tissues but not in normal tissues, and therefore have better safety as tumor antigens. However, in the early stages of tumor vaccine development, due to technical limitations, it was difficult to discover and purify tumor-specific antigens, which in turn limited their wide application. In recent years, with the development of gene editing technology, researchers have successively discovered some specific antigens and new antigens as new targets for tumor vaccine immunity, thereby preparing personalized tumor antigens in a targeted manner to improve the immune effect of the vaccine, and ultimately solving the problem of antigen selection. However, suitable tumor antigens are still difficult to stimulate effective immune responses. The main reason is that simple tumor antigens have a short half-life and will be rapidly degraded and metabolized after injection, making it difficult for the body's immune system to be effectively activated.

[0039] In some embodiments of the present invention, the anti-tumor antigen is obtained by collecting peptides of the surface proteins of live tumor cells, and the peptides are obtained by periodically adding proteases that do not cause cell death to the live tumor cells in primary culture for treatment. For example, the live tumor cells pre-eluted from the growth medium are subjected to primary culture, and the tumor cells are treated with proteases without causing cell death, and the released surface tumor antigens are collected. Then, after a period of time, the live tumor cells in primary culture are repeatedly treated with proteases, and the interval of this period of time is sufficient for the activity of the surface tumor antigen to be restored by the cells. The surface tumor antigens are accumulated (enriched) until the dose required for vaccination is met, and the composition of the obtained surface tumor antigens is controlled.

[0040] In a preferred embodiment of the present invention, the tumor antigen is, for example, mucin (MUC1), tumor membrane antigen, tumor whole cell antigen, or tumor neoantigen.

[0041] The anti-tumor vaccine of the present invention may comprise one or more tumor antigens, aiming to generate one or more cytotoxic T lymphocyte clones, each of which recognizes specific antigenic peptides to produce a more effective immune response. When there are several tumor antigens, it is preferred to select the tumor antigens to induce an immune response against one type of tumor or tumor cells.

[0042] In some embodiments of the present invention, the anti-tumor vaccine of the present invention has a basic composition of a mixture of different antigens or a mixture of an antigen and different immune stimulants.

[0043] For tumor treatment, significantly activating CTL and secreting Th1-type cytokines can help the body attack and eliminate tumors. When the vaccine components are released, whether sufficient APCs are recruited to phagocytose the vaccine is also a crucial factor affecting whether an effective immune response can be stimulated. APCs are widely distributed in various tissues of the body, so they play a crucial role in the process of detecting pathogenic microorganisms. In the peripheral tissue skin, Langerhans cells, other types of DCs and macrophages can all detect danger signals and antigens, and after being activated, they home to lymphoid organs to present antigen information and activate the immune response. The migration of these immune cells in the body between peripheral tissues and lymphoid organs is complex, and their migration behavior is mainly regulated by numerous chemokines.

[0044] In a preferred embodiment of the present invention, the antigen is hepatitis B surface antigen, and the vaccine is a therapeutic hepatitis B vaccine.

[0045] Hepatitis B is a global infectious disease caused by the hepatitis B virus (HBV). So far, 2 billion people worldwide have been infected with the hepatitis B virus, of which about 350 million are chronic hepatitis B infected patients. There is still no effective treatment for hepatitis B. Currently, interferon and lamivudine commonly used in antiviral treatment can quickly inhibit virus replication, but they are prone to rebound after drug withdrawal and are also likely to lead to the emergence of mutant strains.

[0046] In the body of chronic HBV infected patients, due to the existence of immune tolerance, the specific T cell response is low. The purpose of therapeutic vaccines is to exert the cytolytic effect of the cell-mediated immune response that clears the virus, mainly the specific cytotoxic T lymphocytes (CTL). That is, CTL kills infected hepatocytes by releasing perforin and granzyme or induces apoptosis of infected hepatocytes through the Fas-mediated pathway. However, recent studies have shown that the non-cytolytic mechanism involving cytokines secreted by Th1 cells and CTL plays a dominant role in antiviral immunity. Cytokines such as IFN-α can inhibit virus replication and even clear viral DNA through a non-cytopathic manner. In particular, studies on IFN-γ in various animal models have shown that it has the effect of inhibiting the intermediate products of HBV replication and HBV-specific mRNA, and plays an important role in virus clearance. It promotes the secretion of cytokines mainly IFN-γ by stimulating its own cellular immune response to clear the virus.

[0047] Regarding hepatitis B surface antigen, there are also various antigen types to choose from. The hepatitis B surface antigen expressed by Saccharomyces cerevisiae and Hansenula polymorpha can be selected, and the CHO hepatitis B surface antigen expressed by the mammalian cell system can also be selected. However, the preferred antigen type is the hepatitis B surface antigen obtained through the Hansenula polymorpha expression system. HBsAgs from different sources have significant differences in structure and properties. Even if they have the same or similar gene coding, HBsAg particles expressed by different expression systems may still have different molecular sizes, molecular weights, and different subunit numbers, and thus exhibit different charge properties, hydrophobicity, and immunogenicity, etc.

[0048] In a preferred embodiment of the present invention, the vaccine of the present invention further contains chemokines. Chemokines are a class of polypeptides with a molecular weight of 10 KDa, which mainly act on the receptor family related to the connection of cell surface G glycoproteins. Chemokines and other chemotactic attraction-related molecules are all produced locally and then diffuse to form a soluble or solid concentration gradient. At this time, cells expressing chemokine receptors will reach the place where the chemotactic information comes from relying on the concentration gradient information.

[0049] A number of chemokines are involved in the migration of DCs and monocytes, including monocyte chemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β), regulated upon activation normal T cell expressed and secreted (RANTES) chemokine, complement C5a, β-defensins, and bacterial-derived formyl peptides, etc. Since under normal circumstances, the proportion of DCs in peripheral tissues and blood is 1% or even lower, if a vaccine can create a chemokine base camp locally, by continuously releasing chemokines, mimicking and enhancing the behavior of in vivo DC recruitment to the injection site, the immune response can be significantly enhanced.

[0050] In a preferred embodiment of the present invention, the chemokine is, for example, macrophage inflammatory protein 3α (MIP-3α), monocyte chemoattractant protein 1 (MCP-1), and GM-CSF, preferably GM-CSF. The chemokine is contained in the antigen-containing solution and is loaded into the sealed microcapsules by the opening microspheres. In a preferred embodiment of the present invention, the closed structure of the microcapsule chamber of the vaccine of the present invention can ensure that the molecules loaded in the sealed microcapsules are effectively encapsulated in the cavity, achieving a sustained and effective release. There is no significant burst release during the entire release process, no plateau phase, the metabolic rate is slow and effective, and the final effective metabolic rate is as high as 90%, and basically the antigen is completely released.

[0051] The excellent antigen retention ability of the microcapsules ensures a more sustained release of the antigen in vivo. Along with the release of the antigen, the in situ retention effect after microcapsule injection will cause an inflammatory response, and then recruit inflammation-related cells to phagocytose the carrier and the released antigen. The release kinetics of the antigen and the polymer matrix used as an adjuvant are important in controlling the immune response and will affect the final immune effect of the vaccine. When the release kinetics of the two are out of sync, that is, when the antigen is slowly released and the adjuvant is released too fast or too slow, it will affect the activation level of the local APCs that phagocytose the vaccine, making it difficult to stimulate an effective immune response.

[0052] Among numerous APCs, DC (Dentritic cells) are the most powerful professional APCs in the body. According to their state of maturation and differentiation, they are divided into: DC precursor cells, immature and mature DCs. DC precursor cells are cells without DC phenotype or function under normal physiological conditions. When the body is faced with pathogenic microorganism infection or inflammatory stimulation, they are rapidly mobilized, differentiated and developed into immature DCs; immature DCs are the state of most DCs under normal physiological conditions, which can efficiently uptake, process and present antigens and have strong migration ability; mature DCs have weakened antigen uptake ability due to being activated, but their ability to present antigen information and stimulate the activation of naive T cells is enhanced. A large amount of DC chemokines are locally produced in the sealing microcapsules of the present invention, which is beneficial to recruiting more DCs, enabling antigens to be processed and presented more effectively, and inducing a more effective immune response in the body.

[0053] In a preferred embodiment of the present invention, the locally generated inflammatory microenvironment continuously recruits inflammation-related cells and APCs to the injection site to phagocytose antigens. During this process, the secretion of chemokines plays a crucial role in the type and quantity of cell recruitment. The degradation rate of the polymer matrix as a carrier and the antigen release behavior will significantly affect the secretion amount of chemokines.

[0054] In a preferred embodiment of the present invention, the antigen release and cell recruitment behavior synergistically exert the best effect, enabling the antigens released throughout the stage to be phagocytosed by the cells with the highest antigen presentation efficiency, that is, immature DC cells. These DC cells have the function of phagocytosing a large amount of antigens, further improving the utilization rate of antigens.

[0055] During the metabolism of the polymer matrix as a carrier, the locally generated inflammatory microenvironment continuously recruits inflammation-related cells and APCs to the injection site to phagocytose antigens. During this process, the secretion of chemokines plays a crucial role in the type and quantity of cell recruitment. The degradation rate of the carrier and the antigen release behavior will significantly affect the secretion amount of chemokines. In a preferred embodiment of the present invention, the acidic microenvironment generated by the metabolism of the polymer matrix as a carrier can significantly increase the number of cell recruitment. At the same time, it is found that the acidic microenvironment significantly enhances the endocytosis of antigens by cells. The acidic microenvironment constructed by microcapsules such as polylactic acid itself has strong immune adjuvant characteristics. This acidic microenvironment is also an important factor for improving the antigen utilization rate and can serve as a retention site for activated DCs, that is, without the need to add additional vaccine adjuvants and multiple immunizations, it can continuously provide activated APCs to home to lymph nodes. The acidic microenvironment can also significantly enhance antigen cross-presentation and the secretion of Th1-type cytokines.

[0056] In a preferred embodiment of the present invention, the vaccine of the present invention contains an immune stimulant enhancer, such as monophosphoryl lipid A (MPLA), cytosine-guanine oligodeoxynucleotide, and / or polyinosinic:polycytidylic acid.

[0057] Microcapsule Preparation

[0058] In a preferred embodiment of the present invention, the preparation of the microcapsule refers to the process of preparing a particulate composite by encapsulating or dispersing a functional material in a shell material through a certain method. Preferably, the microcapsule of the present invention has a multi-chamber structure inside, and the average particle size of the microcapsule is preferably 10 - 100 μm, more preferably 20 - 80 μm, and even more preferably 30 - 60 μm.

[0059] In a preferred embodiment of the present invention, the porosity of the open pore microspheres is at least above 40%, such as at least above 50%, preferably at least 60%, and more preferably above 70%, and has pore channels with a pore diameter of 1 - 5 μm. Preferably, the open pore microspheres are prepared by the double emulsion and solvent evaporation method. This method is convenient for using biodegradable polymers as the shell material and is suitable for the development of drug-loaded microcapsules. The open pore microspheres have a through-hole structure, a multi-chamber internal structure, and a porous shell. By controlling two dynamic processes of double emulsion evolution and double emulsion curing, the open pore structure and the number of open pores of the open pore microspheres can be easily controlled. Subsequently, the open pore microspheres are immersed in a solution containing an antigen, and the antigen diffuses into the interior of the microspheres. Three methods, namely solvent swelling method, irradiation method, and heating annealing method, can be used to seal the open pore microspheres.

[0060] In a preferred embodiment of the present invention, the preparation of the microcapsule mainly includes the preparation of open pore microspheres, the loading of antigens, and the sealing of the open pore microspheres. Preferably, the method specifically includes the following steps:

[0061] (1) Prepare an oil phase, which is a polymer matrix solution, and the solvent is an organic solvent; prepare an inner aqueous phase solution and an outer aqueous phase solution, and add a surfactant to the outer aqueous phase;

[0062] (2) Disperse the inner aqueous phase into the oil phase to form a water-in-oil primary emulsion; then disperse the primary emulsion into the outer aqueous phase to form a water-in-oil-in-water double emulsion;

[0063] (3) Use the solvent evaporation method to solidify the oil phase to obtain open pore microspheres with through-hole channels;

[0064] (4) Mix the open pore microspheres with a solution containing an antigen, and the antigen enters the internal cavity from the surface of the porous microspheres by diffusion mass transfer in the solution to obtain open pore microspheres loaded with an antigen;

[0065] (5) Seal the open pore microspheres to form sealed microcapsules loaded with an antigen.

[0066] In a preferred embodiment of the present invention, after the double emulsion is prepared, during the evolution process, the small droplets of the internal aqueous phase in the double emulsion will gradually fuse and become larger. At the same time, due to the salt concentration difference between the internal and external aqueous phases, the internal aqueous phase will escape. Therefore, during the evolution process, the fusion and escape of the internal aqueous phase occur simultaneously. The fusion of the internal aqueous phase will form a porous structure inside the microspheres, and the escape of the internal aqueous phase will form a porous morphology on the surface of the microspheres. In a preferred embodiment of the present invention, the polymer matrix is a biodegradable polymer blend matrix, and the polymer blend contains a hydrophobic polymer and an amphiphilic block copolymer.

[0067] In a preferred embodiment of the present invention, the hydrophobic polymer is a lactide polymer (commonly abbreviated as PLA), a glycolide polymer, a lactide-glycolide copolymer (commonly abbreviated as PELA), polycaprolactone, polyorthoester, and / or polyanhydride. Preferably, its weight-average molecular weight is 5,000 - 100,000 Daltons, and more preferably 10,000 - 50,000 Daltons.

[0068] In a preferred embodiment of the present invention, in the amphiphilic block copolymer, the hydrophilic block is a polyethylene glycol type, a polyacrylic acid type, a polyoxyethylene type, or a polyvinyl alcohol type, and the hydrophobic block is a lactone copolymer or homopolymer; preferably, the hydrophilic block is polyethylene glycol or monomethoxyethylene glycol, and the hydrophobic block is polylactic acid (PLA) or a copolymer of lactic acid and glycolic acid (PLGA); for the amphiphilic block copolymer, the hydrophilic block has a weight-average molecular weight of 500 - 30,000 Daltons, and the hydrophobic block has a weight-average molecular weight of 500 - 50,000 Daltons; preferably, the weight-average molecular weight of the hydrophilic block is 1,000 - 20,000 Daltons, such as 4,000 - 10,000 Daltons, and preferably the weight-average molecular weight of the hydrophobic block is 5,000 - 20,000 Daltons.

[0069] In a preferred embodiment of the present invention, the amphiphilic block copolymer is a copolymer of polyethylene glycol or monomethoxyethylene glycol and lactide and / or glycolide (commonly abbreviated as PELA). Preferably, its weight-average molecular weight is 5,000 - 10,000 Daltons, and more preferably 10,000 - 50,000 Daltons.

[0070] In a preferred embodiment of the present invention, the amphiphilic block copolymer can be used to stabilize the primary emulsion, so as to be able to prepare porous microspheres with a multi-chamber structure. The weight content ratio of the amphiphilic block copolymer in the polymer blend matrix of the microcapsule is at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40% or at least 50%. Due to its strong local inflammatory immune response, PLA is less used in the field of controlled drug release. As a material with better biocompatibility, PELA has been more widely used, and it can help to prepare open microspheres with higher porosity. By changing the ratio of the two, the hydrophobic properties and porosity of the microspheres can be improved, and at the same time, the inflammatory response of the carrier can be adjusted to improve the biocompatibility.

[0071] In a preferred embodiment of the present invention, the organic solvent in step (1) is a volatile organic solvent that is not miscible with water, such as n-butanol, methyl ethyl ketone, diethyl ether, chloroform, carbon tetrachloride, toluene, etc., including volatile organic solvents that are partially soluble in water, such as ethyl acetate, phenol, etc. Further preferably, it is one or at least a combination of two or more of alcohols, ketones, esters, ethers, alkylbenzenes, halogenated alkanes, and halogenated aromatic hydrocarbons that are not miscible with water. Typical but non-exhaustive examples of the combination include: a combination of alcohol and ketone, a combination of alcohol, ketone, ester, and ether, a combination of ester, alkylbenzene, and halogenated alkane, a combination of ester, alkylbenzene, halogenated alkane, and halogenated aromatic hydrocarbon, etc. Particularly preferably, it is one or at least a combination of two or more of alcohol, ester, alkylbenzene, chloroalkane, and chloroaromatic hydrocarbon.

[0072] In a preferred embodiment of the present invention, an immune stimulant enhancer, such as monophosphatide A, is added to the organic solvent described in step (1) during microcapsule preparation.

[0073] In a preferred embodiment of the present invention, an immune stimulant enhancer, such as cytosine-guanine oligodeoxynucleotide and / or polyinosinic acid, is further loaded during antigen loading.

[0074] In a preferred embodiment of the present invention, the solvent removal method in step (3) is solvent extraction, or the solvent can be removed by allowing it to evaporate by standing, or by stirring, or by other methods of removing the solvent.

[0075] In a preferred embodiment of the present invention, during the curing process in step (3), the inner aqueous phase and the outer aqueous phase merge to form through-holes.

[0076] In a preferred embodiment of the present invention, the open microspheres with internal and external through-hole channels described in step (3) have a porous structure.

[0077] In a preferred embodiment of the present invention, the open microspheres of the present invention are surface-porous microspheres.

[0078] In a preferred embodiment of the present invention, the porosity of the open-cell microspheres is at least above 40%, for example, at least above 50%, preferably at least 60%, more preferably above 70%, still more preferably above 80%, and preferably has pore channels with a pore diameter of 1-5 μm, for example. Preferably, the pore diameters inside the microspheres vary from 800 nm to 5 μm, and the average pore diameter is about 1 μm.

[0079] In a preferred embodiment of the present invention, in step (3), after the oil phase is solidified, the residual surfactant is removed, and it is particularly preferred to remove the residual surfactant by sieving or centrifugal washing.

[0080] In a preferred embodiment of the present invention, preferably, the sealing process of the open-cell microspheres includes a solvent swelling method, an irradiation method, and a thermal annealing method. Those skilled in the art can also seal the open-cell microspheres according to the professional knowledge / new technologies they master.

[0081] In the present invention, those skilled in the art can understand that the "sealing" means embedding or fixing antigens and / or other substances such as chemokines in or retaining them inside the microcapsules. In the present invention, the sealed microcapsules do not mean that there are no openings on the surface of the microcapsules, but only mean that antigens and / or other substances such as chemokines can be partially embedded or fixed in or retained inside the microcapsules through the sealing process, and antigens and / or other substances such as chemokines can be released from the microcapsules, for example, through the degradation of the microcapsules.

[0082] In a preferred embodiment of the present invention, the thermal annealing method is an ideal sealing method. For example, the unique self-healing sealing property of biodegradable polymer blends such as polylactic acid can be utilized. It is a method of irradiating or heating to make the molecules on the surface of the microspheres absorb energy and rearrange to heal the pores on the surface. For example, by slowly heating the open-cell microspheres loaded with antigens to near the glass transition temperature of the microspheres, preferably, for example, 1-2 °C below the glass transition temperature of the microspheres, and after a period of time, slowly cooling, so as to seal the pores on the surface of the open-cell microspheres to prepare sealed porous microcapsules. At the same time, the antigens are effectively encapsulated in the microcapsules, and the loading rate and embedding rate are stable. The temperature required for the entire heating process is close to the glass transition temperature of the polylactic acid material. To achieve the healing and sealing of the microspheres, the heating conditions need to be controlled, and at the same time, it will not have too much impact on the morphology of the microspheres.

[0083] In other embodiments of the present invention, the porous microspheres can be prepared by phase separation of a polymer and a porogen. During the porogen process, the solvent is often referred to as the porogen, and usually a small oil-soluble molecule is selected. The phase separation of the polymer and the solvent can occur during the polymer chain growth process. Due to the growth of the polymer molecular weight or intermolecular cross-linking, the macromolecules gradually precipitate from the small molecule solvent to form a solid polymer. It can also occur in a polymer solution system. Due to changes in environmental conditions, such as temperature changes, removal of good solvents or addition of poor solvents, the solubility of the solvent in the polymer decreases, causing the polymer to precipitate into a solid phase.

[0084] One of the objects of the present invention also lies in a method for inducing a cytotoxic cell response against tumor cells or tumors in a patient. This method includes administering to the patient an effective amount of the vaccine of the present invention, preferably an anti-tumor vaccine, especially by intravenous injection or infusion, preferably by infusion. The object of this method is especially to induce the activation of dendritic cells and CD8 + cytotoxic cell response in the patient, and obtain specific CD4 + helper and CD8 + cytotoxic response.

[0085] The vaccine of the present invention can be used for the prevention and treatment of diseases such as cancer or hepatitis, such as hepatitis B, and more particularly for immunotherapy. The present invention provides a method for prophylactically or therapeutically treating cancer or hepatitis, such as hepatitis B, which comprises administering to a mammalian subject in need thereof a prophylactically or therapeutically effective amount of the vaccine.

[0086] In the present invention, the term "immunization" refers to active immunization, that is, the induction of a specific immune response due to administration, which is, for example, a small amount of antigen via subcutaneous, intradermal, intramuscular, oral or nasal routes, and the antigen is recognized as foreign by the immunized individual and is therefore immunogenic in a suitable formulation. Therefore, the antigen is used as a "trigger" for the immune system to establish a specific immune response against the antigen.

[0087] According to the present invention, immunization can be therapeutic or prophylactic. By way of example, it may be possible to achieve prophylactic protection against the occurrence of cancer diseases by immunizing individuals who do not have cancer. Examples of individuals who may be vaccinated prophylactically are individuals with an increased risk of developing cancer, although this application is not limited to such individuals. Patients at risk of cancer may already have tumors, either as primary tumors or metastases, or show a tendency to cancer.

[0088] In the present invention, the term "effective amount" means the amount of an antigenic / immunogenic composition that induces an immune response when administered to a human or animal. A person skilled in the art can easily determine the effective amount according to conventional procedures.

[0089] The vaccine or pharmaceutical composition provided by the present invention can be prepared in the form of a sterile powder. For example, the sterile powder contains a vaccine and mannitol, and can be prepared by the following method: Take the microcapsules, rinse them with water for injection, transfer them to a freeze-drying tray, add mannitol and an appropriate amount of water for injection, and place them in a freeze dryer for freeze-drying; the freeze-dried product is screened and mixed evenly, aseptically filled, and capped to obtain the sterile powder. Before administering to a patient, the sterile powder is suspended in an acceptable dispersion medium, which is composed of one or several of a suspending agent, a pH regulator, an isotonic regulator, a surfactant, and water for injection. The suspending agent can be one or more of sodium carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, and glycerol. The isotonic regulator can be one or more of sodium chloride, glucose, mannitol, and sorbitol, etc. The surfactant is a nonionic surfactant, such as the polysorbate series (such as polysorbate 80, polysorbate 60, etc.).

[0090] The pharmaceutical composition provided by the present invention, such as the vaccine microcapsules, can administer the vaccine of the present invention to a patient using methods well-known to those skilled in the art, such as intra-arterial, intravenous, percutaneous injection, and intranasal, transbronchial, intramuscular or oral administration. The dosage and administration method vary according to the weight and age of the patient and the administration method; and those skilled in the art can make selections as needed.

[0091] The liquid pharmaceutical composition is usually formulated to have a pH between about 3.0 - 9.0, more preferably between about 4.5 - 8.5 and still more preferably between about 5.0 - 8.0. The pH of the composition can be maintained by using buffers such as acetate, citrate, phosphate, succinate, Tris or histidine, usually in the range from about 1 mM - 50 mM. The pH of the composition can be additionally adjusted by using a physiologically acceptable acid or base.

[0092] The pharmaceutical composition is administered to an individual in a prophylactically effective amount or a therapeutically effective amount (as the case may be, although prophylaxis can be considered as treatment), which is sufficient to show benefit to the individual. Generally, this will elicit a therapeutically useful activity that is beneficial to the individual. The actual amount of the compound administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescribing such as dosage determination, etc. is within the responsibility of general practitioners and other doctors, and usually takes into account the condition being treated, the situation of the individual patient, the site of delivery, the administration method, and other factors known to the doctor.

[0093] The scope of the specific embodiments disclosed herein, these embodiments are only intended to illustrate several aspects of the present invention.

[0094] Comparative Example 1: Open-cell microspheres prepared from PLA

[0095] Dissolve 100 mg of racemic carboxyl-terminated polylactic acid (PLA, from Jinan Daigang Bioengineering Co., Ltd.) with a molecular weight of 20,000 Daltons in 2 mL of ethyl acetate (oil phase, O), add 0.5 mL of 0.05% aqueous sodium chloride solution (inner aqueous phase W1), and use an ultrasonic crusher to prepare a primary emulsion (water-in-oil, W1 / O). Then pour the primary emulsion into 15 mL of an aqueous solution containing PVA (purchased from Kuraray, Japan) (outer aqueous phase W2), and use a homogenizer to emulsify and prepare a double emulsion (water-in-oil-in-water, W1 / O / W2). After that, use a vertical suspension instrument to suspend for 25 min for double emulsion evolution. Subsequently, pour the double emulsion into 500 mL of ultrapure water, and use magnetic stirring at room temperature for 10 min (500 revolutions per minute) to solidify the microspheres. Finally, centrifuge multiple times (500 g, 5 min), discard the supernatant, collect the microspheres at the bottom, add 1 mL of ultrapure water to the microspheres again, and store at 4°C.

[0096] Absorb 50 μL of the PLA microsphere suspension, drop it on the tin foil, and leave it to dry at room temperature. Use conductive glue to paste the tin foil containing the sample on the sample preparation stage, sputter gold, and then observe the microsphere morphology with SEM. The prepared PLA microspheres are as Figure 1 shown: The microspheres prepared solely with PLA have no obvious porous structure and are not suitable for use as the porous microspheres of the present invention to further develop the sealing microspheres of the present invention.

[0097] Example 1: Preparation of open-cell microspheres from PLA and PELA

[0098] Dissolve 95 mg of racemic carboxyl-terminated polylactic acid (PLA, from Jinan Daigang Bioengineering Co., Ltd.) with a molecular weight of 20,000 Daltons and 5 mg of a copolymer of polyethylene glycol (mPEG, Mw: 2000)-racemic carboxyl-terminated polylactic acid (PLA, Mw: 36000) with a molecular weight of 38,000 Daltons (PELA, purchased from Jinan Daigang Bioengineering Co., Ltd.) in 2 mL of ethyl acetate (oil phase, O), add 0.5 mL of 0.05% aqueous sodium chloride solution (inner aqueous phase W1), and use an ultrasonic crusher to prepare a primary emulsion (water-in-oil, W1 / O). Then pour the primary emulsion into 15 mL of an aqueous solution containing PVA (outer aqueous phase W2), and use a homogenizer to emulsify and prepare a double emulsion (water-in-oil-in-water, W1 / O / W2). After that, use a vertical suspension instrument to suspend for 25 min for double emulsion evolution. Subsequently, pour the double emulsion into 500 mL of ultrapure water, and use magnetic stirring at room temperature for 10 min (500 revolutions per minute) to solidify the microspheres. Finally, centrifuge multiple times (500 g, 5 min), discard the supernatant, collect the microspheres at the bottom, add 1 mL of ultrapure water to the microspheres again, and store at 4°C.

[0099] Absorb 50 μL of the above microsphere suspension, drop it on the tin foil, and leave it to dry at room temperature. Use conductive glue to paste the tin foil containing the sample on the sample preparation stage. After sputtering with gold, observe the surface morphology of the microspheres by SEM. As Figure 2 shown, the surface pore diameter is 1 - 2 μm, with a structure having surface openings. Its particle size distribution is as Figure 3 shown, and the average particle size of the microspheres is 60 μm. At the same time, in order to observe the internal structure of the microspheres, cut the dried microspheres into pieces with an ultra-thin blade. Then adhere the sample to the conductive glue, sputter with gold, and observe by SEM. As Figure 4 shown, the microspheres also have an internally porous and interconnected structure inside, and the internal pore diameter is about 1 - 5 μm.

[0100] Take 30 mg of the freeze-dried porous microspheres, measure them with a mercury porosimeter, measure in parallel three times, and calculate the porosity and average pore size distribution of the porous microspheres. The porosity of the porous microspheres is 82%. As Figure 5 shown, the pore diameters inside the microspheres vary from 800 nm to 5 μm, and the average pore diameter is about 1 μm.

[0101] Example 2: Preparation of sealed microcapsules loaded with antigens

[0102] Absorb 1 mL of the open microsphere suspension with a dry weight of 30 mg prepared in Example 1 into a 1.5 mL centrifuge tube, centrifuge to remove the supernatant, add 500 μL of 50 mg / mL or 10 mg / mL antigen OVA (purchased from Sigma) or 500 μL of 8 mg / mL MUC1 peptide segment (purchased from Gil Biochemical Shanghai Co., Ltd.) and mix with the microspheres. Place it on a vertical suspension instrument and suspend for 4 h (300 rpm) to allow the protein or peptide segment to fully enter the internal cavity of the microspheres through the penetrating pores of the microspheres. Then place the vertical suspension instrument and the microspheres in an incubator at 39 °C to heat and seal. During the process, the suspension speed is 100 rpm to ensure that the microspheres are heated evenly throughout the heating process and do not settle. After reacting for 2 h, the sealing is completed. Centrifuge (500 g, 5 min) to remove the supernatant to obtain sealed microcapsules loaded with protein and polypeptide molecules.

[0103] Absorb 50 μL of the sealed microcapsule suspension containing antigen OVA, drop it on the tin foil, and leave it to dry at room temperature. Use conductive glue to paste the tin foil containing the sample on the sample preparation stage. After sputtering with gold, observe the surface morphology of the microcapsules by SEM. As Figure 6 shown, the surface pores are completely sealed to form sealed microcapsules. At the same time, in order to observe the internal structure of the sealed microcapsules, cut the dried microcapsules into pieces with an ultra-thin blade. Then adhere the sample to the conductive glue, sputter with gold, and observe by SEM. As Figure 7 shown, although the microcapsules still have an internally porous structure inside and the pore diameter is about 1 - 5 μm, the internally penetrating porous structure has become a closed and independent porous structure.

[0104] After the microspheres were sealed, the loading rate and encapsulation rate of antigen OVA or peptide MUC1 in the sealed microcapsules were further evaluated. The methods for determining the contents of OVA protein and MUC1 polypeptide are as follows.

[0105] Extraction of protein OVA from the sealed microcapsules: Weigh 5 mg of freeze-dried microspheres loaded with OVA (n = 3), add 1 mL of 0.1 M sodium hydroxide aqueous solution, and react overnight at 4 °C for 12 h. After the microcapsules were completely degraded, centrifuge to obtain the supernatant, and titrate with 0.1 M hydrochloric acid to restore the pH of the solution to neutral. Pipette 100 μL of the supernatant and detect the protein content using a BCA kit.

[0106] Extraction of polypeptide MUC1 from the sealed microcapsules: Weigh 5 mg of freeze-dried microcapsules loaded with MUC1 polypeptide (n = 3). First, add 300 μL of acetonitrile to completely dissolve the material. After the system becomes clear, add 1.7 mL of 0.015 M HCl solution. Finally, use a 0.45 μm filter membrane to remove the insoluble substances and impurities in the system, and finally determine the concentration of MUC1 peptide by RP-HPLC. The experimental conditions are as follows:

[0107] Detection conditions: Deionized water with 0.1% trifluoroacetic acid (mobile phase A); HPLC-grade acetonitrile with 0.1% trifluoroacetic acid (mobile phase B); Elution gradient: Mobile phase B: 0% - 60%, 0 - 25 min; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm.

[0108] The calculation method for the final drug loading rate (%) of the microcapsules is the mass of the antigen (OVA / MUC1) in the sealed microcapsules divided by the mass (dry weight) of the microspheres, and then multiplied by 100%; the calculation method for the encapsulation rate (%) of the microcapsules is the mass of the antigen (OVA / MUC1) in the sealed microcapsules divided by the mass of the antigen (OVA / MUC1) before loading (concentration multiplied by volume), and then multiplied by 100%.

[0109] As Figure 8 and Figure 9 shown, as the concentrations of the initial antigens OVA protein and MUC1 peptide increased, the loading rate of the microcapsules showed a linear increasing trend. When the antigen concentration was 100 mg / mL, the loading rate of the microcapsules was as high as 20%. At the same time, as the antigen concentration increased, the encapsulation rate of the sealed microcapsules always remained at a relatively stable level, indicating that the encapsulation rate of the microcapsules only depends on the cavity volume of the microcapsules. This characteristic of linear increase in the loading rate and relative stability of the encapsulation rate will be beneficial for regulating the ratio between the antigen and the microcapsules, so as to meet the requirements of different loading systems.

[0110] Example 3. Preparation of sealed microcapsules loaded with antigen and MPLA

[0111] First, dissolve 1 mg of MPLA (purchased from Sigma) in 500 μL of chloroform (CHCl 3 ), then add 100 μL of chloroform to 1.9 mL of the oil phase ethyl acetate. Next, dissolve 95 mg of racemic carboxyl-terminated polylactic acid (PLA, with a molecular weight of 20,000 Daltons, purchased from Jinan Daigang Bioengineering Co., Ltd.) and 5 mg of the copolymer of polyethylene glycol (mPEG, Mw: 2000)-racemic carboxyl-terminated polylactic acid (PLA, Mw: 36,000) (PELA, purchased from Jinan Daigang Bioengineering Co., Ltd.) in 2 mL of the above oil phase containing chloroform and MPLA. Add 0.5 mL of 0.05% sodium chloride aqueous solution (inner aqueous phase W1), and use an ultrasonic disruptor to prepare a primary emulsion (water-in-oil, W1 / O). Then pour the primary emulsion into 15 mL of an aqueous solution containing PVA (outer aqueous phase W2), and use a homogenizer to emulsify and prepare a double emulsion (water-in-oil-in-water, W1 / O / W2). After that, use a vertical shaker to shake for 25 min for double emulsion evolution. Subsequently, pour the double emulsion into 500 mL of ultrapure water, and use magnetic stirring (500 rpm) at room temperature to solidify the microspheres. Finally, centrifuge multiple times (500 g, 5 min), discard the supernatant, collect the microspheres at the bottom, add 1 mL of ultrapure water to the microspheres again, and store them at 4°C.

[0112] The preparation process of the sealed microcapsules loaded with antigen and MPLA is as follows: Pipette 1 mL of the microsphere suspension loaded with MPLA into a 1.5 mL centrifuge tube, centrifuge to remove the supernatant, and the remaining microsphere volume is 500 μL. Then add 500 μL of 10 mg / mL antigen OVA (purchased from Sigma) or 500 μL of 8 mg / mL MUC1 peptide (purchased from Gil Biochemicals Shanghai Co., Ltd.) and mix with the microspheres. Place them on a vertical shaker and shake for 4 h (300 rpm) to allow the peptide to fully enter the internal cavity of the microspheres through the pores of the microspheres. Then place them in an incubator at 39°C to heat-seal. After 2 h, the sealing is completed. Centrifuge (500 g, 5 min) to remove the supernatant to obtain the sealed microcapsules loaded with antigen and MPLA. After calculation, the actual loading rate of OVA in the microcapsules is 2%, the actual loading rate of the peptide MUC1 is 1.7%, and the actual loading rate of MPLA is 1‰.

[0113] Example 4: Preparation of Sealed Microcapsules Loaded with Chemokines

[0114] Absorb 500 μL of the open microsphere suspension prepared in Example 2 with a dry weight of 30 mg into a 1.5 mL centrifuge tube. Centrifuge to remove the supernatant. The remaining microsphere volume is 500 μL. Add 500 μL of a 1 mg / mL GM-CSF chemokine (purchased from Peprotech) solution and mix it with the microspheres. Place it on a vertical suspension instrument and suspend for 4 h (300 revolutions per minute) to allow the chemokine to fully enter the interior of the microsphere cavity. Then place it in an incubator at 39 °C for temperature healing. After reacting for two hours, seal it to complete. Centrifuge to remove the supernatant to obtain the sealed microcapsules of the chemokine. After calculation, the actual loading rate of GM-CSF in the microcapsules is 2.4‰.

[0115] Example 5, Synergistic Effect of Antigen Release and Cell Recruitment of Sealed Microcapsules Loaded with Antigen

[0116] First, different vaccine preparations were subcutaneously injected into the back of female C57BL / 6 mice at 6 - 8 weeks old (n = 4, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). A simple fluorescent antigen (antigen OVA labeled with dye Cy5, OVA-Cy5) group, a blend group of fluorescent antigen and porous microspheres, and an embedding group of fluorescent antigen encapsulated in sealed microcapsules were set up respectively. The injection doses of microspheres and antigen in all groups were 3 mg and 60 μg respectively. At different stages of metabolism (1 h, 2 h, 4 h, 8 h, 0.5 d, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 17 d, 21 d, 25 d, and 30 d), then a small animal in vivo imaging system was used to observe the different metabolic behaviors of the antigen in the porous microspheres and sealed microspheres.

[0117] As Figure 10 shown, the local fluorescence intensity of the simple OVA antigen almost completely disappeared at the 3rd day, indicating that the antigen metabolism rate is very fast and it is basically metabolized completely. The burst release of the blend group within 24 h was 50%. Because in the early stage of metabolism, the antigen free outside the microspheres will be rapidly metabolized by tissues, so the metabolism rate is relatively fast; then the antigen in the porous microspheres will be further released. However, there was still a strong fluorescence intensity at the 5th day. It can be observed from the figure that 10% of the antigen still remained unmetabolized. The reason is the local retention of the microspheres and the retardation effect of the pores, and the antigen metabolism was significantly delayed, and the total metabolism time was extended to about 7 d. In contrast, the embedding group maintained a strong fluorescence intensity throughout the two weeks of detection. It can be observed in the quantitative figure that there was no obvious burst release effect in the early metabolism, and the release within 24 h was 10%. The reason is the closure of the pores on the surface of the sealed microcapsules, and the antigen can only be slowly released from the nanopores on the material surface temporarily. So only 20% of the antigen was metabolized at the 3rd day; as the metabolism time extended, the surface and internal structures of the carrier changed, and a large amount of the antigen in the microcapsules was released. About 50% of the antigen was released at the 7th day; subsequently, the metabolism rate further slowed down, and there was still a very strong fluorescence intensity after 14 d of metabolism. AsFigure 10 As shown, 35% of the antigen still remains unmetabolized.

[0118] Along with the release of the antigen, the in-situ retention effect after microcapsule injection can lead to an inflammatory response, which in turn recruits inflammation-related cells to phagocytize the carrier and the released antigen. To further observe the phenomenon of cell recruitment, blank-sealed microcapsules from Example 2 were injected into the back of female 6-8-week-old C57BL / 6 mice (n = 4, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The mice were sacrificed at different metabolic times in vivo. The area of the back of the mice where the microcapsules were injected was dissected, and the local subcutaneous tissue wrapping the carrier was taken out with forceps. Then the tissue was cut with scissors and immersed in 4% formalin solution for 24 h. Subsequently, it was paraffin-embedded, sectioned, stained with hematoxylin & eosin (H&E), and mounted to prepare H&E-stained samples. Finally, the inflammatory response at the injection site was observed using a panoramic scanning and analysis system for pathological sections (Vectra 3.0, purchased from PerkinElmer). The mice were sacrificed at different metabolic times. The local tissue wrapping the carrier under the skin was taken out and H&E-stained samples were prepared. Finally, the inflammatory response at the injection site was observed using a panoramic scanning and analysis system for pathological sections.

[0119] As Figure 11 shown in a, at the 3rd day, cells (dark dots) were gradually recruited around the microcapsules (light circles); as the degradation time extended, the number of cells around the microspheres increased continuously, the inflammatory response enhanced, and even infiltration of inflammatory cells was observed inside some microcapsules. The Inform software was used to count the number of dots and the number of microcapsules, and the number of cells recruited by each microcapsule locally was calculated quantitatively. Through analysis, as Figure 11 shown in b, about 20 cells could be recruited by each microsphere on average at 14 days. Therefore, during the entire metabolic process, although the local inflammatory response gradually increased due to the accumulation of degradation products. At the same time, no phenomena such as local tissue swelling, granulation, and suppuration were found during the dissection of the mice, indicating that the inflammatory response generated by the sealed microcapsules is safe.

[0120] A simple antigen group labeled with Cy5 dye-labeled OVA (Cy5-OVA), a blend group of Cy5-OVA and porous microspheres, and an embedding group with Cy5-OVA loaded in sealed microcapsules were respectively set up. They were respectively injected into the thigh muscles of female C57BL / 6 mice at 6 - 8 weeks old (n = 4, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 5 days, the mice were sacrificed, and the local muscle tissue containing microspheres was taken out, cut into small pieces with scissors, ground into a suspension, resuspended in PBS at 4°C, and the supernatant was removed by centrifugation. Then 1 mL of a mixed enzyme solution (1 mg / mL collagenase D and 100 U / mL recombinant DNase I dissolved in RPMI 1640 medium) was added, and the reaction was carried out at 37°C for 30 min. After that, it was washed with PBS again, and the impurities were filtered through a 20-μm cell sieve to obtain a single-cell suspension. According to the operation steps of flow cytometry sample staining, fluorescent antibodies against mouse FITC-CD11c and eFlour450-F4 / 80 were respectively added to the samples to label DC and macrophages. Further, OVA + cells, CD11c + OVA + cells and F4 / 80 + OVA + cells were detected, and the data were analyzed and processed with Flowjo software.

[0121] As Figure 12 shown, in the simple antigen group (control group), the number of OVA + cells and the fluorescence intensity of intracellular OVA were both very weak, indicating that the local antigen was almost completely metabolized at the 5th day. In the blend group, the retention effect of the porous microspheres significantly improved the antigen utilization rate. The number of OVA + cells increased by about 10 times, the endocytosis of OVA by cells increased by two times, and the corresponding antigen utilization rate increased by about 20 times; in the embedding group, due to its excellent antigen retention effect and antigen effective release ability, the number of OVA + cells increased by about 50 times, the endocytosis of OVA increased by more than 4 times, and the antigen utilization rate finally increased by more than 200 times. The above results show that the sealed microcapsules can more effectively regulate the antigen release and cell recruitment behavior in the microcapsule system, making the two work synergistically to further improve the antigen utilization rate. The antigen utilization rate calculation formula: OVA + cell number × fluorescence intensity, and it was normalized with the antigen utilization rate of the single OVA group. This synergistic effect can not only promote more cells to phagocytose antigens, but also increase the endocytosis of antigens by cells, maximizing the antigen utilization rate.

[0122] Example 6. Influence of the acidic microenvironment of the sealed microcapsules loaded with antigens on cell recruitment.

[0123] The lactic acid, which is the degradation product of polylactic acid microcapsules, can lead to the acidification of the local microenvironment. The degree, change and duration of acidification may all have an impact on the later immune effects. Therefore, it is necessary to precisely monitor the acidification process and changes of the local microenvironment. SNARF ○R -1 is a pH (monitoring range 6 - 9) sensitive probe, which is excited at 488 nm and received at two wavelengths of 640 nm and 580 nm. The fluorescence intensity ratio I640 / I580 corresponds to the pH value in the system. The smaller this ratio, the lower the pH of the system. Therefore, by loading SNARF ○R -1 into the microspheres, the change of the pH of the local microenvironment caused by the lactic acid, which is the degradation product of polylactic acid microcapsules, can be monitored in real time by using a laser confocal microscope.

[0124] The specific operation is as follows: Sealed microcapsules loaded with SNARF ○R -1 are subcutaneously injected into the back of female C57 / BL6 mice (n = 6). The mice are sacrificed on the 3rd day, 5th day, 7th day and 14th day respectively. The tissues subcutaneously wrapping the microspheres are taken out and placed under a laser scanning confocal microscope to observe the change of the local tissue pH. Observe its fluorescence image, calculate the fluorescence intensity ratio I640 / I580, and calculate the actual pH value according to the standard curve.

[0125] The experiment found that the local microenvironment changed from neutral to acidic after 3 days of metabolism. As Figure 13 shown, the local pH changed from neutral 7.2 to weakly acidic 6.5. Along with the continuous metabolism of the material, no further acidification of the local acidic environment was found, but it was stably maintained at about pH 6.5, that is, there was no problem of lactic acid accumulation caused by the gradual degradation of ordinary non-porous polylactic acid microspheres. When the inside and outside of the material of ordinary non-porous polylactic acid microspheres degrade simultaneously, the lactic acid produced by internal metabolism will accumulate due to mass transfer obstruction. As a result, the pH inside will be lower than that outside, leading to local lactic acid accumulation, and it is easy to produce possible sudden decrease or instability of pH. The sealed microcapsule of the present invention effectively avoids the above problems of lactic acid accumulation and mass transfer obstruction because of its unique internal porous through structure, enabling the lactic acid produced by the degradation of the internal material to be efficiently replaced with the outside; when the metabolism of lactic acid by the body and the lactic acid produced by the degradation of polylactic acid material achieve a dynamic balance in the body, the local pH will be maintained at a relatively stable level, thus creating a relatively stable microenvironment for local cells to function, and at the same time contributing to the analysis of the influence of homeostasis on local cells.

[0126] In many physiological inflammatory conditions, a large number of cells are recruited to the inflammatory site, and the inflammatory site is accompanied by acidification. Therefore, there should be a certain correlation between acidity and cell recruitment behavior. Therefore, the focus is on investigating the effects of the neutral microenvironment (NM) and acidic microenvironment (AM) on cell recruitment. By loading the weak base salt NaHCO in the polylactic acid microcapsule 3 , the acid-base neutralization reaction between it and the lactic acid, the degradation product of the microcapsule, is used to eliminate the local acidity and construct a neutral microenvironment locally to compare with the acidic microenvironment of the polylactic acid microcapsule.

[0127] The acidic environment improves the antigen utilization rate by increasing cell recruitment and stimulating cells to endocytose antigens. At the same time, it also shows that the improvement of antigen utilization rate can be achieved by the coordinated cooperation of only antigen release behavior and cell recruitment behavior. For example, a beneficial microenvironment that stimulates immune cells to phagocytose antigens in large amounts is needed to further improve the antigen utilization rate. Therefore, the combined action of multiple factors such as antigen release behavior, cell recruitment behavior, DC number and type, and acidic microenvironment that stimulates endocytosis is required to maximize the antigen utilization rate. Both the blending group and the embedding group loaded with NaHCO 3 illustrate that any of the above factors is indispensable. Only the sealed microcapsules of the present invention can mobilize all these factors simultaneously and synergistically play their respective functions to make the antigen be utilized most efficiently.

[0128] After DC phagocytoses antigens, the antigens are processed and become antigen peptides, which are expressed on the surface of APCs in the form of antigen peptide-major histocompatibility complex (MHC) for T cell recognition. However, when APCs only phagocytose antigens and lack external stimulators, APCs will not effectively express co-stimulatory molecules (CD80, CD86, CD40) to activate T cells. At the same time, in most cases, after DCs phagocytose foreign antigens, their processing and presentation of antigens mainly occur through the lysosomal pathway, which is restricted by MHC-II molecules and mainly mediates reactions related to T cell humoral immunity; in some special cases, exogenous antigens can be presented through the cytosolic pathway, that is, the MHC class I molecule pathway, so as to directly activate cytotoxic T cells (CTL), and then most directly and effectively kill target cells. Therefore, for therapeutic tumor vaccines, more antigens need to be presented in the form of MHC-I molecules to more effectively kill tumors.

[0129] The acidic environment can significantly improve the endocytosis of antigens by APCs, especially DCs. However, the activation of APCs, the quantity and pathway of antigen presentation still need to be further investigated. The specific operation is as follows: First, use 20 mM NaHCO respectively 3OVA was dissolved in PBS solution to prepare sealed microcapsules. The mixing method and sealing method are referred to the loading of antigens. They were respectively injected into the thigh muscles of female C57BL / 6 mice aged 6 - 8 weeks (n = 6, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After dissection on the 5th day, the tissues containing healed microcapsules were taken out. They were cut into pieces with scissors, and then 1 mL of mixed enzyme solution (1 mg / mL collagenase D and 100 U / mL recombinant DNase I dissolved in RPMI 1640 medium) was added, and reacted at 37 °C for 30 min. Then they were homogenized in a cell homogenizer to make a single-cell suspension, the supernatant was removed by centrifugation, the cells were collected and filtered through a 20-μm cell sieve to remove impurities, and the cells were counted with a hand-held cell counter. Then they were stained and labeled with flow antibodies, and the co-stimulatory molecules and major histocompatibility complex markers on the surface of DC were labeled with PE-CD11c, APC-CD80, APC-Cy7-CD86, eFlour 450-MHC-I, and BV 655-MHC-II respectively to detect the proportion of locally activated DC.

[0130] As Figure 14 shown, in an acidic environment, DC was significantly activated. CD86 + The expression level of MHC-I molecules in DC was more than 40 times that of MHC-II, indicating that DC presented antigens more in the form of MHC-I, thus indicating that the microcapsules have the ability to efficiently promote antigen cross-presentation. However, in a neutral environment, CD86 + The proportion of MHC-I in DC decreased by more than twice, so it shows that the environment plays a very important role in the microcapsules promoting antigen cross-presentation.

[0131] Example 7. Animal experiment effect of sealed microcapsules based on E.G7 lymphoma solid tumor

[0132] First, E.G7 tumor-bearing mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were constructed. 1×10 6 E.G7 tumor cells (purchased from ATCC) were injected into the armpits of female C57BL / 6 mice aged 6 - 8 weeks. Then the tumor-bearing mice were randomly divided into groups of 6 each. On the 4th day, the tumor volume was 50 - 60 mm 3Vaccination was carried out at that time. The vaccine groups were set as follows: PBS, low-dose multiple OVA (20 μg, boosted immunization once at 7 days and 14 days respectively), high-dose single OVA (60 μg), porous microspheres blended with OVA (3 mg microspheres, 60 μg OVA), sealed microcapsules loaded with OVA (the preparation method is shown in Example 2) (3 mg microspheres, 60 μg OVA), and sealed microspheres embedded with OVA and loaded with MPLA (the preparation method is shown in Example 3) (3 mg microspheres, 60 μg OVA, 3 μg MPLA). Since the day of tumor model construction, the hair, body weight and survival of mice were observed every other day, and the length and width of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 1 / 2 × L × W 2 (L: length; W: width).

[0133] As Figure 15 shown by the tumor growth curve, in the simple antigen group, it was difficult to inhibit tumor growth in both the low-dose multiple group and the high-dose single group, and the survival rate (SR) at 25 days was only 17%; in the blending group, the survival rate was increased to 50% under the adjuvant effect of the microspheres, but the tumor grew faster in the early stage; the tumors in the embedding group could be significantly inhibited in the early stage, with a slow growth rate, and the survival rate of mice at 25 days was as high as 100%. It shows that the microcapsule system can achieve good therapeutic effects by making full use of tumor antigens without adding additional molecular adjuvants. When a small amount of adjuvant MPLA (3 μg) was loaded into the material of the healing microcapsules, not only the survival rate was as high as 100%, but also the survival rate of tumor-bearing mice was more significantly inhibited. At 26 days, the tumor volumes of 50% of the mice were basically the same as those at the initial stage of treatment, and more than 60% of the mice survived for more than 40 days, further confirming that the compatibility of the microsphere system and the MPLA system can further improve the tumor inhibition effect.

[0134] Example 8, Animal experiment effect of sealed microcapsules on B16 melanoma solid tumors

[0135] First, B16 tumor-bearing mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were constructed. 5×10 5 B16 melanoma cells (provided by Jilin University) were injected into the armpits of 6-8 week-old female C57BL / 6 mice. Then the tumor-bearing mice were randomly divided into groups of 6 each. When the tumor area > 10 mm 2Vaccination at that time. The groups were set as follows: PBS group, MUC1 peptide group (50 μg), embedding group of sealed microcapsules loaded with MUC1 peptide (3 mg of microspheres, 50 μg of peptide), and group of sealed microcapsules embedding MUC1 and loaded with MPLA (3 mg of microspheres, 60 μg of peptide, 3 μg of MPLA). Since the day of tumor model construction, the hair, body weight and survival of mice were observed every other day, and the length and width of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 1 / 2 × L × W 2 (L: length; W: width).

[0136] As Figure 16 shown by the tumor growth curve, neither PBS nor the high-dose single group could effectively inhibit the growth of melanoma, while the sealed microspheres could significantly inhibit the growth of tumors. The tumor volume of 33% of the mice was less than 500 mm 3 , and the survival rate within 22 days of monitoring was 100%. The sealed microspheres loaded with MPLA showed the best anti-tumor effect. At 22 days, the tumors hardly grew in 50% of the mice, and the melanoma even disappeared in some individual mice.

[0137] Example 9. Animal experiment effect of sealed microcapsules based on B16 melanoma metastatic tumors

[0138] First, B16 metastatic model mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were constructed. 2×10 5 B16 melanoma cells (provided by Jilin University) were injected into the tail veins of 6-8-week-old female C57BL / 6 mice. Then the mice were randomly divided into groups of 6 each, and vaccination was carried out on the 4th day. The specific immunization strategy was the same as that in Example 8. At 18 days after vaccination, 2 mice in each group were sacrificed, and the metastatic conditions of melanoma in the lungs and kidneys of the organs were observed, and the metastatic foci in the organs were counted and statistically analyzed.

[0139] As Figure 17 shown, a large number of melanoma metastases appeared in the lungs of the PBS group. It was found by counting that about 100 metastatic foci were distributed in the lungs of each mouse. At the same time, metastatic foci with a total volume of 100 mm 3 grew in the kidney tissue, confirming the successful construction of the melanoma metastasis model. 18 days after vaccination with the vaccine, when the mice in the sealed microsphere group were dissected, it was found that compared with the PBS group, the number of lung metastases decreased from 100 to 15 - 20, and the kidney tumor volume decreased from 100 mm 3 to 10 mm 3 . After adding MPLA to the sealed microspheres, the anti-tumor metastasis effect was further improved. There were hardly any obvious metastatic foci in the lungs, and no tumors were generated in the kidneys. Therefore, the combination of the microcapsule system and the TLR receptor agonist produced the best anti-tumor metastasis effect.

[0140] Example 10: Animal experiment effect of sealed microcapsules loaded with tumor neoantigens based on 4T1 breast cancer

[0141] The selected tumor neoantigens are a combination of 8 polypeptides in equal proportions (the amino acid sequences of the 8 polypeptides are: SPNRSWVSL, HPMYLFLSM, VAVKVNFYVI, KAPHNFQFV, YHYVLNSMV, EYSAMTTRGTI, GSPPRFFYM, and CPQTHAVVL). The preparation process of the polypeptide is as follows: Tumor mutant genes are screened by comparative sequencing of 4T1 tumor cell line and normal Balb / c mouse tissues (provided by the National Key Laboratory of Biochemical Engineering), and then mutant polypeptides are predicted by computer algorithms (submitted to Shenzhen Yuce Biotechnology Co., Ltd.), and then polypeptide synthesis is carried out (submitted to GenScript Biotechnology Co., Ltd.).

[0142] The preparation method of the AS04 adjuvant group is as follows: First, MPLA is dissolved in a 0.5% triethanolamine solution, then heated to 65 °C and maintained for 5 min to promote dissolution, and then sonicated three times with a sonication probe at a power of 60 W for 1 min. After complete dissolution, the pH is adjusted to about 7.4 with HCl. Subsequently, the MPLA aqueous solution is mixed with aluminum adjuvant, sonicated for 30 s, and the tumor neoantigens of the present invention are added and mixed evenly to ensure that the dosage of each component of the vaccine for each mouse is MPLA (3 μg), aluminum adjuvant (100 μg), and tumor neoantigen 200 μg in 100 μL of physiological saline solution.

[0143] Among them, the preparation method of the sealed microcapsules embedding tumor neoantigens and loading MPLA can be referred to the description of Example 3 of the present invention.

[0144] 4T1 orthotopic tumor model mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) are constructed, and 5×10 5 4T1 breast cancer cells (purchased from ATCC) are injected into the right lower mammary fat pad of 6-8 week-old female Balb / c mice, and then the mice are randomly divided into groups of 6 each, and vaccinated on the 4th day.

[0145] The groups are set as follows: PBS group, simple tumor neoantigen group (200 μg), AS04 adjuvant group loaded with tumor neoantigens (aluminum adjuvant 100 μg, tumor neoantigen 200 μg, MPLA 3 μg), sealed microcapsule group embedding tumor neoantigens and loading MPLA (microspheres 3 mg, tumor neoantigen 200 μg, MPLA 3 μg).

[0146] Since the construction date of the tumor model, the hair, body weight and survival of the mice are observed every other day, and the length and width of the tumor are measured with a vernier caliper. The tumor volume is calculated according to the following formula: V = 1 / 2 × L × W2 (L: Length; W: Width).

[0147] As Figure 18 shown by the tumor growth curve, neither PBS nor the simple tumor neoantigen group could effectively inhibit the growth of breast cancer. The AS04 adjuvant group had a certain inhibitory effect on tumor growth, showing a relatively obvious delayed tumor growth. In contrast, the sealed microcapsule group showed the best anti-tumor effect, and the tumor volume was less than 500 mm in 83% of the mice at 28 d 3 =.

[0148] Example 11: Animal experiment effect of sealed microcapsules loaded with tumor neoantigens based on the postoperative recurrence of 4T1 breast cancer

[0149] A mouse model of postoperative recurrence of 4T1-luc breast cancer (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was constructed. 5×10 5 4T1-luc breast cancer cells transfected with Luciferase (purchased from ATCC) were injected into the right lower mammary fat pad of 6-8-week-old female Balb / c mice. When the tumor grew to 200 mm 3 , tumor resection was performed on the mice. The size of the residual tumor tissue was controlled by bioluminescence imaging to be similar. Then the mice were randomly divided into groups of 6 each, and vaccine immunization was carried out on the 2nd day.

[0150] The specific immunization strategy was the same as that in Example 10. Since the construction of the tumor model, the hair, body weight and survival of the mice were observed every other day. The recurrence of the tumor was monitored by bioluminescence imaging, and the length and width of the tumor were measured with vernier calipers. The tumor volume was calculated according to the following formula: V = 1 / 2×L×W 2 (L: Length; W: Width).

[0151] As Figure 19 shown by the tumor growth curve, neither PBS nor the simple tumor neoantigen group could effectively inhibit the postoperative recurrence of breast cancer. Tumor recurrence occurred in all mice, and the tumor progressed rapidly and the mice died quickly. The AS04 adjuvant group prevented tumor recurrence in some mice, and 13% of the mice still had no visible tumor growth at the 22nd day, but still died during long-term monitoring. The sealed microsphere group showed the best anti-recurrence effect after surgery. Only one mouse had tumor recurrence at the 22nd day, and 83% of the mice had no tumor recurrence. As Figure 20 shown by the bioluminescence imaging pictures and statistical charts, after surgical resection, the residual volume of the tumor lesions was similar, and after subsequent different treatment strategies, there were obvious differences in tumor recurrence. No tumor lesions were observed by bioluminescence imaging in the sealed microsphere group at the 14th day, and tumor recurrence occurred in all or part of the other groups.

[0152] Example 12. Comparison of the effect of chemokine-loaded sealed microcapsules on cell recruitment

[0153] In female C57BL / 6 mice aged 6 - 8 weeks (n = 4, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), 3 mg of blank sealed microspheres and sealed microcapsules loaded with 7 μg of chemokine GM-CSF were injected into the back respectively. After 5 days of in vivo metabolism, the mice were sacrificed. The back of the mice at the injection site of the microcapsules was dissected, and the local subcutaneous tissue wrapping the carrier was picked up with forceps, then the tissue was cut off with scissors and immersed in 4% formalin solution for 24 h. Subsequently, paraffin embedding, sectioning, hematoxylin & eosin (H&E) staining and coverslipping were performed to prepare H&E staining samples. Finally, the inflammatory reaction at the injection site was observed with a panoramic scanning and analysis system for pathological sections (Vectra 3.0, purchased from PerkinElmer).

[0154] As Figure 21 shown, after 5 days of in vivo metabolism, only a small number of inflammatory cells (black dots) were recruited around the blank microspheres (light-colored round objects). However, significantly more cells could be recruited around the microcapsules loaded with chemokine GM-CSF, indicating that the antigen released from the microcapsules loaded with chemokine GM-CSF could be phagocytosed by more cells, thereby enhancing the efficiency of antigen presentation and the final immune effect.

Claims

1. A vaccine comprising an antigen and a biodegradable polymer blend matrix, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer, the vaccine existing in the form of microcapsules with a multi-chamber structure inside, the average particle size of the microcapsules preferably being 10 - 100 μm, more preferably 30 - 60 μm, and the microcapsules being prepared by the following method: first, open-pored microspheres are prepared from the polymer blend, then they are mixed with a solution containing the antigen, and then the open-pored microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen.

2. The vaccine according to claim 1, wherein the vaccine is used for prophylactic or therapeutic treatment of tumors or hepatitis in mammalian subjects, the vaccine being, for example, a therapeutic vaccine, preferably an anti-tumor vaccine or a therapeutic hepatitis B vaccine, and the preferred antigen being a tumor antigen or hepatitis B surface antigen.

3. The vaccine according to claim 1, which contains chemokines, such as granulocyte colony-stimulating factor (GM-CSF), macrophage inflammatory protein 3α (MIP-3α), and monocyte chemoattractant protein 1 (MCP-1), and preferably, the chemokines are contained in the solution containing the antigen and are loaded into the sealed microcapsules through the sealing of the open-pored microspheres.

4. The vaccine according to claim 1, wherein the hydrophobic polymer is a lactide polymer, a glycolide polymer, a lactide-glycolide copolymer, polycaprolactone, polyorthoester, and / or polyanhydride, preferably having a weight-average molecular weight of 5,000 - 100,000 daltons, more preferably 10,000 - 50,000 daltons.

5. The vaccine according to claim 1, wherein the amphiphilic block copolymer is a copolymer of polyethylene glycol or monomethoxy polyethylene glycol with lactide and / or glycolide, preferably having a weight-average molecular weight of 5,000 - 10,000 daltons, more preferably 10,000 - 50,000 daltons.

6. The vaccine according to claim 1, wherein the weight content ratio of the amphiphilic block copolymer in the polymer blend matrix of the microcapsules is at least 5%, preferably at least 10%, at least 15% or at least 20%.

7. The vaccine according to claim 1, which contains immune stimulatory enhancers, such as monophosphoryl lipid A, cytosine-guanine oligodeoxynucleotides, and / or polyinosinic:polycytidylic acid.

8. The vaccine according to claim 1, wherein the open-pored microspheres loaded with the antigen are sealed by heating them to a temperature close to the glass transition temperature of the microspheres, preferably, for example, 1 - 2 °C below the glass transition temperature of the microspheres.

9. The vaccine according to claim 1, wherein the open-pored microspheres are prepared by the double emulsion solvent evaporation method.

10. The vaccine according to claim 1, wherein the porosity of the open-pored microspheres is at least 40% or more, for example, at least 50% or more, preferably at least 60%, more preferably 70% or more, and preferably has pore channels with a pore diameter of 800 nm - 5 μm.

11. A pharmaceutical composition containing any one of the above vaccines or any one of the above sealed microcapsules, and a pharmaceutically acceptable carrier.

12. Use of any of the above vaccines, any of the above open microspheres or any of the above sealed microcapsules in the preparation of anti-tumor vaccines or therapeutic vaccines.

13. Use of any of the above vaccines or any of the above sealed microcapsules in the preparation of anti-tumor drugs, drugs for treating hepatitis such as hepatitis B, or drugs for inducing cytotoxic cell responses against tumor cells or tumors.

14. A method for inducing a cytotoxic cell response against tumor cells or tumors in a patient, comprising administering to the patient an effective amount of a vaccine, preferably an anti-tumor vaccine.

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