WO2020038298A1 - 一种基于微囊的疫苗 - Google Patents

一种基于微囊的疫苗 Download PDF

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WO2020038298A1
WO2020038298A1 PCT/CN2019/101078 CN2019101078W WO2020038298A1 WO 2020038298 A1 WO2020038298 A1 WO 2020038298A1 CN 2019101078 W CN2019101078 W CN 2019101078W WO 2020038298 A1 WO2020038298 A1 WO 2020038298A1
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antigen
vaccine
tumor
microcapsules
microspheres
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PCT/CN2019/101078
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English (en)
French (fr)
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马光辉
魏炜
习晓博
叶通
那向明
卿爽
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中国科学院过程工程研究所
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Priority to US17/269,912 priority Critical patent/US20210308258A1/en
Priority to JP2021509850A priority patent/JP7257500B2/ja
Priority to EP19851585.0A priority patent/EP3842069A4/en
Publication of WO2020038298A1 publication Critical patent/WO2020038298A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
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    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to a microcapsule-based vaccine comprising an antigen and a matrix of a biodegradable polymer blend, such as an anti-tumor vaccine.
  • tumor vaccines have been able to mimic the way the body fights pathogens and restore the natural "recognition” and response to tumor cells, thereby specifically destroying tumor cells, and therefore have received more and more attention.
  • CN101601860A discloses a vaccine based on polymer particles, wherein the antigen composition is in the form of an antigen adsorbed on a biodegradable polymer particle and an unadsorbed antigen (free antigen).
  • the average particle diameter of the particles is 0.1-20 ⁇ m, prepared by the following methods: polymer particles are mixed with the hepatitis B surface antigen solution, and the suspension prepared by the adsorption of the polymer particles and the antigen solution is the vaccine product, and the vaccine of the present invention can be quickly induced in the body The immune response induces the body to produce higher levels of humoral and cellular immunity.
  • CN102489230A discloses a method for preparing biodegradable material microcapsules.
  • the method includes the preparation of open-cell microspheres, the filling of capsule core materials, and the sealing of open-cell microspheres.
  • the method is more gentle To avoid damage to biologically active substances; the remaining capsule core material in the solution after embedding can be recycled; more environmentally friendly, reaching the same particle size and pore level as previously reported, and a larger internal cavity volume, which is more conducive to capsules Loading of core material.
  • the biodegradable material microcapsules can be used for embedding small molecules and biological macromolecules, and can also be loaded with nano- and micron-sized particles.
  • an open-pored microsphere made of a biodegradable polymer is prepared by a double emulsion-solvent removal method, and then the open-pored microsphere is filled with a tumor antigen, and then the open-pored microsphere is made Sealed, made into a sealed microcapsule with a larger particle size that can be locally retained in the mouse body. Because the microcapsules are not easy to be phagocytosed and metabolized, they are more easily degraded, and during the degradation process, the body is stimulated to produce a continuous inflammatory response.
  • an antigen reservoir As an antigen reservoir, it can continuously recruit antigen-presenting cells such as dendritic cells (DC) to the injection site to engulf the antigen, and cooperate with the acidic microenvironment produced by the metabolism of the polymer matrix, which can significantly increase the number of cell recruitment and increase the cell's response to the antigen.
  • DC dendritic cells
  • Endocytosis stimulates the maturation and differentiation of DCs, eventually homing DCs to lymph nodes, stimulates the body to produce an efficient and durable immune response, promotes the continuous proliferation and differentiation of T cells, and produces specific and durable killings, which can significantly inhibit tumor growth Prolong the survival time of mice.
  • the present invention provides a vaccine comprising an antigen and a matrix of a biodegradable polymer blend, the polymer blend containing a hydrophobic polymer and an amphiphilic block copolymer, and the vaccine is in a microcapsule
  • the microcapsules contain a multi-chamber structure inside.
  • the average particle diameter of the microcapsules is preferably 10-100 ⁇ m, more preferably 30-60 ⁇ m.
  • the microcapsules are prepared by the following method: polymer blending
  • the open-pore microspheres are prepared, and then mixed with the antigen-containing solution, and then the open-pore microspheres loaded with the antigen solution are sealed to form sealed microcapsules loaded with the antigen.
  • the release of the antigen synergizes with the cell recruitment behavior during local retention in the vaccine, so that the released antigen is engulfed by the immature DCs recruited, Significantly improves the utilization of the antigen.
  • 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 a hepatitis B surface antigen.
  • chemokines such as granulocyte colony factor (GM-CSF), macrophage inflammatory protein 3 ⁇ (MIP-3 ⁇ ), and monocyte chemokines can also be loaded into the porous microsphere system.
  • GM-CSF granulocyte colony factor
  • MIP-3 ⁇ macrophage inflammatory protein 3 ⁇
  • monocyte chemokines can also be loaded into the porous microsphere system.
  • MCP-1 Protein 1 to enhance the recruitment of cells by microcapsules, further improve antigen utilization and enhance immune response.
  • Figure 3 Particle size distribution of microspheres prepared from PLA and PELA
  • FIG. 4 SEM image of the internal structure of microspheres prepared by PLA and PELA
  • FIG. 11 Recruitment behavior of inflammatory cells by microcapsules of the present invention: (a) Representative tissue section images of locally recruited cells containing sealed microcapsules (b) Quantitative analysis of the number of cells recruited per microcapsule
  • FIG. 12 Comparison of antigen utilization rates of different vaccine preparations: (a) number of OVA + cells, (b) antigen endocytosis of OVA + cells (expressed as mean fluorescence intensity), (c) antigen utilization rate
  • Fig. 13 Change curve of pH in local microenvironment during degradation of microcapsules of the present invention
  • the present invention relates to a vaccine for the prophylactic or therapeutic treatment of diseases such as tumors in mammalian subjects, said vaccine comprising an antigen and a matrix of a biodegradable polymer blend, said polymer
  • the blend contains a hydrophobic polymer and an amphiphilic block copolymer, the vaccine exists in the form of microcapsules, and the interior of the microcapsules contains a multi-chamber structure.
  • the average particle diameter of the microcapsules is preferably 10-100 ⁇ m, more preferably It is 30-60 ⁇ m, and the microcapsules are prepared by firstly preparing an open-pore microsphere having a through-hole channel from a polymer blend, and then mixing it with a solution containing an antigen, and then making an opening containing an antigen solution The microspheres are sealed to form sealed microcapsules loaded with antigen.
  • the vaccine according to the invention may also contain a pharmaceutically acceptable carrier, salt or diluent.
  • the vaccine is an anti-tumor vaccine.
  • the vaccine is simple to prepare, has the universality applicable to the prevention of recurrence, metastasis suppression and treatment of any kind of tumor, and has good antitumor effect.
  • the tumor antigen is first taken up and processed by Antigen Presenting Cells (APCs), and then turned into antigen peptides, and then the major histocompatibility complex on the cell surface (Major Histocompatibility Complex, MHC) -like molecules.
  • APC Antigen Presenting Cells
  • MHC Major histocompatibility Complex
  • CTL Cytotoxic T Lymphocytes
  • the antigen is a tumor antigen.
  • Tumor antigens play an important role in tumorigenesis, development, and induction of the body's anti-tumor immune effect, and are the target molecules for tumor immunotherapy. Tumor antigens are divided into two major categories according to their specificity: (1) tumor-associated antigens, which are antigens that are highly expressed in tumor tissues and that are also expressed to some extent in normal tissues; (2) tumor-specific antigens, which are expressed only Antigens in tumor tissues do not exist in normal tissues, so they have better safety as tumor antigens.
  • tumor-associated antigens which are antigens that are highly expressed in tumor tissues and that are also expressed to some extent in normal tissues
  • tumor-specific antigens which are expressed only Antigens in tumor tissues do not exist in normal tissues, so they have better safety as tumor antigens.
  • tumor-specific antigens which are expressed only Antigens in tumor tissues do not exist in normal tissues, so they have better safety as tumor antigens.
  • the anti-tumor antigen is obtained by collecting peptides of surface proteins of live tumor cells, and the peptides are obtained by periodically adding live tumor cells to primary cultures without causing cell death.
  • Protease obtained by processing. For example, live tumor cells previously eluted from the growth medium are subjected to primary culture, tumor cells are treated with protease without causing cell death, and released surface tumor antigens are collected. Then, after a period of time, the primary cultured live tumor cells are repeatedly treated with protease, and the interval of this period of time is sufficient to allow the surface tumor antigen activity to be restored by the cells.
  • the surface tumor antigen is accumulated (enriched) until a dose sufficient for vaccination is reached, and the components of the obtained surface tumor antigen are controlled.
  • the tumor antigen is, for example, mucin (MUC1), a tumor membrane antigen, a tumor whole cell antigen, or a tumor nascent antigen.
  • MUC1 mucin
  • a tumor membrane antigen for example, a tumor membrane antigen, a tumor whole cell antigen, or a tumor nascent antigen.
  • the anti-tumor vaccine of the present invention may contain one or more tumor antigens, the purpose is to generate one or more cytotoxic T lymphocyte clones, each of which recognizes a specific antigenic peptide to generate a more effective immune response.
  • the tumor antigens are preferably selected to induce an immune response against one type of tumor or tumor cell.
  • the basic composition of the antitumor vaccine of the present invention is a mixture of different antigens or a mixture of antigens and different immunostimulatory substances.
  • Th1-type cytokines For tumor treatment, significant activation of CTL and secretion of Th1-type cytokines can help the body attack and destroy tumors.
  • APCs are widely distributed in various tissues of the body, so they play a vital role in the discovery of pathogenic microorganisms.
  • danger signals and antigens can be detected by Langerhans cells, other types of DCs and macrophages, and after being activated, they return to lymphatic organs to present antigen information to activate the immune response.
  • the migration of these immune cells from peripheral tissues to lymphoid organs in the body is complex, and their migration behavior is mainly regulated by many chemokines.
  • the antigen is a hepatitis B surface antigen
  • the vaccine is a therapeutic hepatitis B vaccine.
  • Hepatitis B is a global infectious disease caused by hepatitis B virus (HBV). So far, 2 billion people worldwide have been infected with the hepatitis B virus, of which about 350 million people are chronic hepatitis B infection. There are no effective treatments for hepatitis B. Interferon and lamivudine commonly used in antiviral therapy currently can rapidly inhibit virus replication, but they can easily rebound after discontinuation of the drug, and can also lead to the generation of mutant strains.
  • HBV hepatitis B virus
  • the purpose of the therapeutic vaccine is to exert the cytolytic effect of the specific immune response of the cytotoxic T lymphocytes (CTLs), which is a virus-clearing cellular immune response, that is, CTL kills infected liver cells by releasing perforin, granzymes, or is mediated by Fas Pathways to induce apoptosis in infected hepatocytes.
  • CTLs cytotoxic T lymphocytes
  • Fas Pathways to induce apoptosis in infected hepatocytes.
  • Cytokines such as IFN- ⁇ can inhibit virus replication and even clear viral DNA in a non-cytopathic manner.
  • IFN- ⁇ In particular, studies of IFN- ⁇ in a variety of animal models have shown that it can inhibit HBV replication intermediates and HBV-specific mRNAs, and play an important role in virus clearance. It can stimulate IFN- ⁇ by stimulating its own cellular immune response. The secretion of major cytokines to clear the virus.
  • Hepatitis B surface antigen there are many types of antigens that can be selected. Hepatitis B surface antigen expressed by Saccharomyces cerevisiae and Hansenula yeast can be selected, and CHO hepatitis B surface antigen expressed by mammalian cell system can be selected, but the preferred antigen type Hepatitis B surface antigen obtained by Hansenula expression system.
  • HBsAg from different sources has large differences in structure and properties. Even if they have the same or similar gene codes, HBsAg particles expressed by different expression systems may still have different molecular sizes, molecular weights, and different numbers of subunits, and thus show different Charge, hydrophobicity, and immunogenicity.
  • the vaccine of the invention further contains a chemokine.
  • Chemokines are a class of peptides with a molecular weight of 10 KDa, which are mainly related to the family of G-glycoproteins linked to the cell surface. Chemokines and other molecules involved in chemotaxis are produced locally, and then diffuse to form a soluble or solid concentration gradient. At this time, cells expressing chemokine receptors will rely on the concentration gradient information to reach the source of chemotaxis information. local.
  • chemokines are involved in the migration of DCs and monocytes, including monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), and macrophage inflammatory protein 1 ⁇ (MIP- 1 ⁇ ), macrophage inflammatory protein 1 ⁇ (MIP-1 ⁇ ), regulated and activated normal T cell expression and secretion (Regulated activation, normal cell expressed and secreted, RANTES) chemokines, complement C5a, ⁇ -defensin ( ⁇ -defensins) and bacterial-derived formate peptides. Under normal circumstances, the proportion of DC in peripheral tissues and blood is 1% or lower. Therefore, if the vaccine can create a strong base of chemokines locally, through continuous release of chemokines, simulate and enhance DC injection in vivo Site-recruiting behavior can significantly enhance the immune response.
  • MCP-1 monocyte chemotactic protein 1
  • MIP-2 monocyte chemotactic protein 2
  • MIP- 1 ⁇
  • the chemokines are, for example, macrophage inflammatory protein 3 ⁇ (MIP-3 ⁇ ), monocyte chemotactic protein 1 (MCP-1), and GM-CSF, preferably GM-CSF
  • MIP-3 ⁇ macrophage inflammatory protein 3 ⁇
  • MCP-1 monocyte chemotactic protein 1
  • GM-CSF preferably GM-CSF
  • the antigen-containing solution contains a chemokine, and the chemokine is filled in a sealed microcapsule through an open-pore microsphere seal.
  • the closed structure of the microcapsule chamber of the vaccine of the present invention can ensure that the molecules contained in the sealed microcapsules are effectively encapsulated in the cavity to achieve a durable and effective release. There is no significant burst during the entire release process, there is no plateau phase, the metabolic rate is slow and effective, and the final effective metabolic rate is as high as 90%, and the antigen is basically completely released.
  • the excellent antigen retention ability of the microcapsules ensures a more durable release of the antigen in the body.
  • the in situ retention effect after the injection of the microcapsules will cause an inflammatory response, and then recruit inflammation-related cells to phagocytose the vector and release antigen.
  • the kinetics of the release of the antigen from the polymer matrix as an adjuvant plays an important role in controlling the immune response and will affect the final immune effect of the vaccine.
  • the release kinetics of the two are not synchronized, that is, when the antigen is slowly released, too fast or too slow release of the adjuvant will affect the level of APC activated by the local phagocytosis vaccine, and it is difficult to stimulate an effective immune response.
  • DCs Dentritic cells
  • immature and mature DCs are cells that have no DC phenotype or function under normal mechanisms.
  • Immature DCs are The state of most DCs under normal mechanism states can efficiently ingest, process, and present antigens, and has strong migration ability; mature DCs become weaker in their ability to take up antigens because they are activated, but present antigen information and stimulate The ability of initial T cells to activate is enhanced.
  • a large amount of DC chemokines are locally produced in the sealed microcapsules of the present invention, which is beneficial to recruit more DCs, enables the antigen to be processed and presented more efficiently, and induces the body to produce a more effective immune response.
  • the locally generated inflammatory microenvironment will continuously recruit inflammation-related cells and APCs to swallow antigen at the injection site.
  • the secretion of chemokines plays a role in the type and number of cell recruitment.
  • the crucial role of the polymer matrix as the carrier is the rate of degradation and antigen release behavior that can significantly affect the amount of chemokine secretion.
  • the synergistic effect of both the antigen release and the cell recruitment behavior exerts the best effect, so that the antigen released during the entire stage is phagocytosed by the cell with the highest antigen presentation efficiency, that is, immature DC cells
  • the DC cells have the function of phagocytosing a large amount of antigen, which further improves the utilization rate of the antigen.
  • the locally produced inflammatory microenvironment will continuously recruit inflammation-related cells and APCs to the injection site to engulf the antigen.
  • the secretion of chemokines plays a crucial role in the type and quantity of cell recruitment.
  • the speed of carrier degradation and the behavior of antigen release will significantly affect the secretion of chemokines.
  • the acidic microenvironment produced by the metabolism of the polymer matrix as a carrier can significantly increase the number of cells recruited, and it is found that the acidic microenvironment significantly improves the endocytosis of antigens by cells.
  • the acidic microenvironment itself has strong immune adjuvant characteristics.
  • This acidic microenvironment is also an important factor for improving the utilization rate of antigens. It can be used as a retention site for activated DCs, that is, without the need to add additional vaccine adjuvants and Under the premise of multiple immunizations, it can continue to provide activated APCs to homing to the lymph nodes, and the acidic microenvironment can also significantly improve antigen cross-presentation and Th1-type cytokine secretion.
  • the vaccine of the present invention contains an immunostimulatory enhancer, such as monophosphate A (MPLA), cytosine-guanine oligodeoxynucleotide, and / or polyinosine.
  • an immunostimulatory enhancer such as monophosphate A (MPLA), cytosine-guanine oligodeoxynucleotide, and / or polyinosine.
  • the preparation of microcapsules refers to a process in which a functional material is wrapped or dispersed in a shell material by a certain method to prepare a granular composite. Containing a multi-chamber structure, the average particle diameter of the microcapsules is preferably 10-100 ⁇ m, preferably 20-80 ⁇ m, and more preferably 30-60 ⁇ m.
  • the porosity of the open-pore microspheres is at least 40% or more, for example, at least 50% or more, preferably at least 60%, more preferably 70% or more, and having a pore size of 1-5 ⁇ m, for example. Kongdao.
  • the open-pore microspheres are prepared by a compound emulsion and solvent removal method, which is convenient for using a biodegradable polymer as a shell material and is suitable for the development of drug-loaded microcapsules.
  • the open-cell microspheres have a through-hole structure, a multi-cavity internal structure and a porous shell.
  • the preparation of microcapsules mainly includes the preparation of open-cell microspheres, the filling of antigens, and the sealing of open-cell microspheres.
  • the method specifically includes the following steps:
  • oil phase is a polymer matrix solution, wherein the solvent is an organic solvent; preparing an internal aqueous phase solution and an external aqueous phase solution, and adding a surfactant to the external aqueous phase;
  • the small droplets of the internal water phase in the multiple emulsion will gradually fuse and become larger during the evolution process.
  • the internal water phase Escape will occur, so the internal water fusion and escape occur simultaneously during the evolution process.
  • the fusion of the internal water phase will form a porous structure inside the microsphere, and the escape of the internal water phase will form a porous morphology on the surface of the microsphere.
  • the polymer matrix is a biodegradable polymer blend matrix, the polymer blend comprising a hydrophobic polymer and an amphiphilic block copolymer.
  • the hydrophobic polymer is a lactide polymer (commonly referred to as PLA), a glycolide polymer, a lactide-glycolide copolymer (commonly referred to as PLGA), a polycaprolactone
  • PLA lactide polymer
  • glycolide polymer glycolide polymer
  • lactide-glycolide copolymer commonly referred to as PLGA
  • polycaprolactone preferably has a molecular weight, weight average molecular weight of 5,000 to 100,000 Daltons, and more preferably 10,000 to 50,000 Daltons.
  • the hydrophilic blocks in the amphiphilic block copolymer are polyethylene glycols, polyacrylics, polyoxyethylenes, polyvinyl alcohols, and the hydrophobic blocks are lactones.
  • 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); said 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 hydrophilic block has a weight average molecular weight of 1,000-20,000 Daltons Dayton is, for example, 4,000 to 10,000 Daltons, and preferably the hydrophobic block has a weight average molecular weight of 5,000 to 20,000 Daltons.
  • amphiphilic block copolymer is a copolymer of polyethylene glycol or monomethoxyethylene glycol and lactide and / or glycolide (commonly referred to as PLGA).
  • the molecular weight is preferably 5,000 to 10,000 Daltons, and more preferably 10,000 to 50,000 Daltons.
  • the amphiphilic block copolymer can be used to stabilize a primary emulsion, so that porous microspheres having a multi-chamber structure can be prepared, and the amphiphilic block copolymer occupies microcapsules.
  • the polymer blend matrix has a weight content ratio of at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50%. Because of its strong local inflammatory immune response, PLA has less application in the field of controlled release of drugs. PELA has been used more as a more biocompatible material, which can help to prepare a higher porosity onset. Porous microspheres. By changing the ratio of the two, the hydrophobic properties and porosity of the microspheres will be improved. At the same time, the inflammatory response of the carrier can be adjusted and the biocompatibility can be improved.
  • the organic solvent in step (1) is a volatile organic solvent that is incompatible with water, such as n-butanol, methyl ethyl ketone, ether, chloroform, tetrachloromethane, toluene, etc.
  • volatile and partially soluble organic solvents such as ethyl acetate, phenol, etc., and is more preferably one of alcohols, ketones, esters, ethers, alkylbenzenes, halogenated alkanes, and halogenated aromatics that are not miscible with water.
  • benzene, a halogenated alkane, and a halogenated aromatic hydrocarbon is particularly preferably one or a combination of at least two of alcohols, esters, alkylbenzenes, chloroalkanes, and chloroaromatic hydrocarbons.
  • an immune stimulating enhancer such as monophosphate A is added to the organic solvent described in step (1) during the preparation of the microcapsules.
  • an immunostimulatory enhancer is further loaded when the antigen is loaded, such as cytosine-guanine oligodeoxynucleotide and / or polyinosine.
  • the solvent removal method in step (3) is a solvent extraction method
  • it may also be a method of standing to volatilize the solvent, or stirring to volatilize the solvent or other methods of removing the solvent.
  • the internal water phase and the external water phase are fused to form a through hole.
  • the open-cell microspheres with internal and external through-holes described in step (3) have a porous structure.
  • the open-pore microspheres according to the present invention are superficially porous microspheres.
  • the porosity of the open-cell microspheres is at least 40% or more, such as at least 50% or more, preferably at least 60%, more preferably 70% or more, more preferably 80% or more, and preferably having, for example, Pore with a diameter of 1-5 ⁇ m.
  • the pore diameter inside the microspheres ranges from 800 nm to 5 ⁇ m, and the average pore diameter is about 1 ⁇ m.
  • step (3) after the oil phase is solidified, the residual surfactant is removed, and it is particularly preferable to remove the residual surfactant by sieving or centrifugal washing.
  • the sealing process of the open-cell microspheres includes a solvent swelling method, an irradiation method, and a temperature-annealing method. Perforated microspheres are sealed.
  • sealing means embedding or fixing or retaining antigens and / or other substances such as chemokines in the microcapsules.
  • sealing the microcapsules does not mean that there are no openings on the surface of the microcapsules, but only means that the antigen and / or other substances such as chemokines can be partially embedded or fixed or retained in the microcapsules through the sealing process. Internally, antigens and / or other substances such as chemokines can be released from the microcapsules, for example, by degradation of the microcapsules.
  • the heating annealing method is an ideal sealing method.
  • the unique self-healing sealing characteristics of a biodegradable polymer blend matrix such as polylactic acid can be used.
  • the method allows the molecules on the surface of the microspheres to rearrange and absorb the surface pores.
  • by slowly raising the temperature of the open-cell microspheres loaded with the antigen to close to the glass transition temperature of the microspheres it is preferably lower than the microsphere's glass transition temperature.
  • the glass transition temperature is 1 ⁇ 2 °C. After a period of time, the temperature is slowly reduced, so that the pores on the surface of the open microspheres are closed to prepare sealed porous microcapsules.
  • the antigen is effectively encapsulated in the microcapsules. Buried rate is stable.
  • the temperature required for the entire temperature increase is close to the glass transition temperature of the polylactic acid material. To achieve the healing sealing of the microspheres, the temperature increase conditions must be controlled, while the microsphere appearance will not be affected much.
  • the open-pored microspheres can be prepared by phase separation of a polymer and a porogen.
  • the solvent is often referred to as a porogen, and small oil-soluble molecules are usually selected.
  • the phase separation of polymer and solvent can occur during the growth of polymer chain. Due to the increase of the molecular weight of the polymer or the cross-linking between the molecules, the macromolecules gradually precipitate from the small-molecule solvent to form a solid polymer. It can also occur in polymer solution systems. Due to changes in environmental conditions, such as changes in temperature, removal of good solvents, or addition of poor solvents, the solvent's ability to dissolve the polymer is reduced, causing the polymer to precipitate into a solid phase.
  • One of the objects of the present invention is also a method for inducing a cytotoxic cellular response against a tumor cell or tumor in a patient.
  • This method comprises administering to the patient an effective amount of a vaccine of the invention, preferably an anti-tumor vaccine, in particular by intravenous, injection or infusion, preferably by infusion.
  • a vaccine of the invention preferably an anti-tumor vaccine
  • the purpose of this method is especially to induce the activation of dendritic cells and CD8 + cytotoxic cellular responses in patients, to obtain specific CD4 + helper and CD8 + cytotoxic responses.
  • 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 the prophylactic or therapeutic treatment of cancer or hepatitis, such as hepatitis B, which comprises administering a prophylactic or therapeutically effective amount of a vaccine to a mammalian subject in need thereof.
  • the term "immunization” means active immunization, that is, the induction of a specific immune response due to administration, such as a small amount of antigen via the subcutaneous, intradermal, intramuscular, oral or nasal route, This antigen is recognized as foreign by the vaccinated individual and is therefore immunogenic in a suitable formulation. The antigen is therefore used as a "trigger" for the immune system to establish a specific immune response against the antigen.
  • the immunization may be therapeutic or prophylactic.
  • Examples of individuals who may apply such prophylactic vaccination are individuals who have an increased risk of developing cancer, although this application is not limited to such individuals.
  • Patients at risk for cancer may have developed tumors, either as primary tumors or metastases, or show a tendency to cancer.
  • the term "effective amount” means the amount of an antigenic / immunogenic composition that induces an immune response when administered to a human or animal. Those skilled in the art can easily determine the effective amount according to conventional procedures.
  • the vaccine or pharmaceutical composition provided by the present invention can be prepared into a sterile powder form.
  • the sterile powder contains a vaccine and mannitol, and can be prepared by the following methods: taking microcapsules, rinsing with water for injection, and transferring to a lyophilized dish In the process, mannitol and an appropriate amount of water for injection are added, and the lyophilized product is lyophilized in a freeze dryer; the lyophilized product is sieved, sterilized and packed, and capped to obtain a sterile powder.
  • the sterile powder Prior to administration to a patient, the sterile powder is suspended in an acceptable dispersion vehicle consisting of one of a suspending agent, a pH adjuster, an isotonicity adjuster, a surfactant, or It is composed of several kinds and water for injection.
  • the suspending agent may be one or more of sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate, and glycerin.
  • the isotonicity adjusting agent may be One or more of sodium chloride, glucose, mannitol, sorbitol, etc.
  • the surfactant is a non-ionic surfactant, such as a polysorbate series (such as polysorbate 80, polysorbate 60, etc.) ).
  • the pharmaceutical composition provided by the present invention can be administered 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 method of administration vary depending on the weight and age of the patient and the method of administration; those skilled in the art can choose as needed.
  • Liquid pharmaceutical compositions are generally 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 through the use of buffers such as acetate, citrate, phosphate, succinate, Tris or histidine, and typically ranges from about 1 mM to 50 mM.
  • the pH of the composition can additionally be adjusted by using a physiologically acceptable acid or base.
  • the pharmaceutical composition is administered to a subject in a prophylactically effective or therapeutically effective amount (as the case may be, although prevention may be considered a treatment), which is sufficient to show an advantage to the individual. Generally, this will lead to a therapeutically useful activity that is beneficial to the individual.
  • the actual amount of compound administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated.
  • the prescription of treatments, such as dosage decisions, is within the responsibility of GPs and other physicians, and generally considers the condition being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.
  • PLA microsphere suspension Pipette 50uL of PLA microsphere suspension, drop it on the foil, and dry at room temperature.
  • the tin foil containing the sample was pasted on a sample preparation table with a conductive adhesive, and the microsphere appearance was observed by SEM after gold spraying.
  • the resulting PLA microspheres are shown in Figure 1:
  • the microspheres prepared solely with PLA have no obvious porous structure and are not suitable for use as porous microspheres of the present invention to further develop the sealed microspheres of the present invention.
  • Sodium hydroxide aqueous solution (internal water phase W1), prepare a primary emulsion (water-in-oil, W1 / O) with an ultrasonic crusher, and then pour the primary emulsion into 15mL of an aqueous solution containing PVA (external water phase W2), and use a homogenizer Emulsion to prepare multiple emulsions (water-in-oil-in-water, W1 / O / W2). After that, it was suspended in a vertical suspension apparatus for 25 min) to undergo re-emulsion evolution.
  • the multiple emulsion was poured into 500 mL of ultrapure water, and the microspheres were solidified by magnetic stirring at room temperature for 10 min (500 rpm). Centrifuge for several times (500g, 5min), discard the supernatant and collect the bottom microspheres. Add 1mL of ultrapure water to the microspheres again and store at 4 ° C.
  • the inside of the microsphere also has a structure that is internally porous and penetrates each other, and the internal pore diameter is about 1-5 ⁇ m.
  • the porosity and average pore size distribution of the porous microspheres are calculated.
  • the porosity of the porous microspheres is 82%.
  • the pore diameter inside the microspheres ranges from 800 nm to 5 ⁇ m, and the average pore diameter is about 1 ⁇ m.
  • the suspension speed is 100 rpm to ensure that the microspheres are heated uniformly throughout the heating process without sedimentation.
  • the reaction takes 2 hours.
  • the supernatant was removed by centrifugation (500g, 5min) to obtain sealed microcapsules loaded with protein and peptide molecules.
  • the sealed microcapsule suspension containing the antigen OVA was pipetted, dropped on a foil, and dried at room temperature.
  • the tin foil containing the sample was affixed to the sample preparation table with a conductive adhesive, and the surface morphology of the microcapsules was observed by SEM after gold spraying.
  • the surface is completely closed, forming a sealed microcapsule.
  • the dried microcapsules were shredded with an ultra-thin blade.
  • the sample was then adhered to the conductive glue and sprayed with gold, and observed by SEM.
  • FIG. 7 although the inside of the microcapsule still has a porous structure inside, and the pore diameter is about 1-5 ⁇ m, the porous structure penetrating inside becomes a closed and independent porous structure.
  • the loading rate and embedding rate of the antigen OVA or peptide MUC1 in the encapsulated microcapsules were further evaluated.
  • the method for determining the content of OVA protein and MUC1 polypeptide is as follows.
  • the experimental conditions are as follows:
  • Detection conditions 0.1% trifluoroacetic acid in deionized water (mobile phase A); 0.1% trifluoroacetic acid in high-performance liquid-grade acetonitrile (mobile phase B); elution gradient: mobile B: 0% -60%, 0- 25min; flow rate: 1.0mL / min; detection wavelength: 220nm.
  • the final microcapsule drug loading rate (%) is calculated by dividing the mass of the antigen (OVA / MUC1) in the sealed microcapsules by the mass of the microspheres (dry weight) and multiplying by 100%; the microcapsule embedding rate (% ) Is calculated by dividing the mass of the antigen (OVA / MUC1) in the sealed microcapsule by the mass (concentration times volume) of the antigen before loading (OVA / MUC1), and then multiplying by 100%.
  • the microcapsule loading rate increases linearly.
  • the antigen concentration is 100 mg / mL
  • the loading rate of the microcapsules is as high as 20%.
  • the embedding rate of the sealed microcapsules has always remained at a relatively stable level, indicating that the embedding rate of the microcapsules depends only on the cavity volume of the microcapsules. This linear increase in loading rate and relatively stable entrapment rate will help regulate the ratio between antigen and microcapsules to meet the needs of different loading systems.
  • Preparation of sealed microcapsules loaded with antigen and MPLA is as follows: Pipette 1mL of MPLA-loaded microsphere suspension into a 1.5mL centrifuge tube, centrifuge to remove the supernatant, and the remaining microsphere volume is 500 ⁇ L.
  • the injection doses of microspheres and antigen were 3mg and 60 ⁇ g, respectively.
  • the embedding group maintained a strong fluorescence intensity throughout the two weeks of the test. It can be observed in the quantitative map that there is no obvious burst effect in early metabolism, and the release within 24 hours is 10%, due to sealing.
  • the antigen can only be slowly released from the nanopores on the surface of the material, so only 20% of the antigen is metabolized in 3d.
  • the surface and internal structure of the carrier changes. The antigen in the capsule was released in large quantities, and about 50% of the antigen was released on the 7th day; then the metabolic rate was further slowed, and it still had a strong fluorescence intensity after 14 days of metabolism. As shown in Figure 10, 35% of the antigen still remained unmetabolized. .
  • H & E staining Subsequent paraffin embedding, sectioning, hematoxylin & eosin (H & E) staining, and mounting slides were used to prepare H & E stained samples. Finally, the pathological section panoramic scanning and analysis system (Vectra 3.0, purchased from PerkinElmer) was used to observe the inflammatory response at the injection site. Mice were sacrificed at different metabolic times. The local tissues subcutaneously wrapped with the carrier were removed, and H & E stained samples were prepared. Finally, the pathological section panoramic scanning and analysis system was used to observe the inflammatory response at the injection site.
  • Vectra 3.0 purchased from PerkinElmer
  • Cy5-OVA Cy5 dye-labeled OVA
  • the blended group of Cy5-OVA and porous microspheres were set.
  • n 4, purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • the mice were sacrificed, and the muscle tissue containing microspheres was taken out, cut into small pieces with scissors, ground into a suspension, resuspended in PBS at 4 ° C, and centrifuged to remove the supernatant.
  • the number of OVA + cells and the fluorescence intensity of intracellular OVA were weak, indicating that the local antigen was almost completely metabolized on the 5th day.
  • the retention effect of porous microspheres in the blend group significantly increased the utilization rate of antigens, the number of OVA + cells increased by about 10 times, the endocytosis of cells by OVA increased by twice, and the corresponding antigen utilization rate increased by about 20 times. ; Because of the excellent antigen retention effect and effective antigen release ability of the embedding group, the number of OVA + cells increased by about 50 times, the OVA endocytosis 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, so that they work together to further improve the utilization rate of antigens.
  • the formula for calculating the utilization rate of antigens OVA + number of cells ⁇ fluorescence intensity.
  • the antigen utilization of the OVA group was normalized. This synergy can not only promote more cells to phagocytose the antigen, but also increase the endocytosis of the antigen by the cells and maximize the utilization of the antigen.
  • Example 6 Effect of acidic microenvironment of sealed microcapsules loaded with antigen on cell recruitment.
  • SNARF R ⁇ -1 is a pH (monitoring range 6-9) sensitive probe that excites at 488nm and receives at two wavelengths of 640nm and 580nm.
  • the fluorescence intensity ratio I640 / I580 corresponds to the pH value in the system. The smaller the ratio, the lower the pH of the system. Therefore, by loading SNARF ⁇ R -1 in the microspheres, the laser confocal microscope can be used to monitor the change in local microenvironment pH of the lactic acid microcapsule degradation product lactic acid in real time.
  • the microsphere-encapsulated tissue was removed and placed under a laser scanning confocal microscope to observe local tissue pH changes. Observe the fluorescence image, calculate the fluorescence intensity ratio I640 / I580, and calculate the actual pH value according to the standard curve.
  • the local microenvironment changed from neutral to acidic microenvironment after 3 days of metabolism.
  • the local pH changed from a neutral 7.2 to a weakly acidic 6.5.
  • the continuous metabolism of the material no further acidification of the local acidic environment was found, but it was stably maintained at a pH of about 6.5, that is, no lactic acid accumulation caused by the gradual degradation of ordinary non-porous polylactic acid microspheres occurred.
  • ordinary non-porous polylactic acid microspheres are degraded internally and externally, the lactic acid produced by internal metabolism will accumulate due to mass transfer obstruction, so the internal pH will be lower than the external, resulting in local lactic acid accumulation, which is easy to produce. Possible sudden drop in pH or instability.
  • the sealed microcapsules of the present invention effectively avoid the above-mentioned problems of lactic acid accumulation and hindered mass transfer because of its unique internal porous penetration structure, and efficiently replace the lactic acid produced by the degradation of internal materials with the outside; when the body metabolizes lactic acid and polylactic acid
  • the lactic acid produced by material degradation achieves a dynamic equilibrium in the body, and the local pH will be maintained at a relatively stable level, thereby creating a relatively stable microenvironment for local cells to function and helping to analyze the impact of steady state on local cells. .
  • the acidic environment improves antigen utilization by increasing cell recruitment and stimulating cell endocytosis of the antigen. It also shows that only the synergy between the antigen release behavior and the cell recruitment behavior can improve the utilization of the antigen, such as the need for a stimulus
  • the beneficial microenvironment of immune cells phagocytosing antigens can further improve the utilization of antigens. Therefore, the combination of antigen release behavior, cell recruitment behavior, number and type of DCs, and acidic microenvironment to stimulate endocytosis is required to maximize antigen utilization.
  • Both the blending group and the embedding group loaded with NaHCO 3 demonstrate that any of the above factors are indispensable. Only the sealed microcapsules of the present invention can mobilize all of these factors at the same time, and play their respective functions in synergy, so that the antigen is most efficient. use.
  • antigens After DCs have swallowed antigens, the antigens are processed and processed to become antigenic peptides, which are expressed on the surface of APC in the form of antigenic peptide-major histocompatibility complex (MHC) for T cells to recognize.
  • MHC antigenic peptide-major histocompatibility complex
  • APC would not effectively express co-stimulatory molecules (CD80, CD86, CD40) to activate T cells.
  • CD80, CD86, CD40 co-stimulatory molecules
  • the processing and presentation of antigens is mainly through the lysosomal pathway. This process is restricted by MHC-II molecules and mainly mediates T-cell humoral immune-related reactions.
  • the exogenous antigen can be presented through the cytosol presentation pathway, that is, the MHC class I molecular pathway, which directly activates cytotoxic T cells (CTL), thereby killing target cells most directly and effectively. Therefore, for therapeutic tumor vaccines, more antigens need to be presented as MHC-I molecules in order to kill tumors more effectively.
  • CTL cytotoxic T cells
  • the acidic environment can significantly increase APC, especially DC's endocytosis of antigen, however, the activation of APC, the amount and way of antigen presentation need further investigation.
  • the specific operation is as follows: firstly dissolve OVA with 20 mM NaHCO 3 and PBS solution to prepare sealed microcapsules.
  • OVA mM NaHCO 3
  • PBS phosphate buffered bovine serum
  • For the mixing method and sealing method, refer to the loading of antigen. Injected into the thigh muscles of female C57BL / 6 mice (n 6, purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) for 6-8 weeks, and the tissues containing healing microcapsules were removed after 5 days of dissection.
  • DC-co-stimulatory molecules were labeled with PE-CD11c, APC-CD80, APC-Cy7-CD86, eFlour 450-MHC-I, and BV655-MHC-II. To detect the percentage of local DC activation.
  • E.G7 tumor-bearing mice purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • 1 * 10 6 E.G7 tumor cells purchased from ATCC
  • mice were randomly divided into 6 groups, and the tumor volume was vaccinated at 50-60 mm 3 on the 4th day.
  • the 25-day survival rate (SR) was only 17%; the blended group was in the microspheres.
  • the survival rate was increased to 50%, but the tumors grew faster in the early stage; the tumors in the embedded group were significantly inhibited at an early stage, the growth rate was slow, and the 25-day survival rate of the mice was as high as 100%.
  • Example 8 Experimental results of sealed microcapsules based on B16 melanoma solid tumors in animals
  • B16 tumor-bearing mice purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • 5 * 10 5 B16 melanoma cells provided by Jilin University
  • the tumor-bearing mice were randomly divided into 6 groups, and the tumor area was vaccinated when the tumor area was> 10 mm 2 on the 4th day.
  • Example 9 Experimental results of sealed microcapsules based on B16 melanoma metastases in animals
  • a B16 metastatic model mouse (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) was constructed. 2 * 10 5 B16 melanoma cells (provided by Jilin University) were injected into the tail of female C57BL / 6 mice for 6-8 weeks. Intravenous, mice were then randomly divided into groups of 6 and vaccinated on the 4th day. The specific immunization strategy is consistent with that in Example 8. On the 18th day after vaccination, 2 mice were sacrificed in each group, and the metastasis of lung and kidney melanoma in the organ was observed, and the metastases in the organ were counted and counted.
  • the selected tumor neonatal antigen is 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: The 4T1 tumor cell line and normal Balb / c mouse tissues (provided by the National Key Laboratory of Biochemical Engineering) were compared and sequenced to screen tumor mutant genes, and then a computer algorithm was used to predict the mutant peptides (submitted to Shenzhen Yuce Biotechnology) Company), followed by peptide synthesis (submitted to Kingsley Biotech).
  • the preparation method of the AS04 adjuvant group is as follows: firstly dissolve MPLA in a 0.5% triethanolamine solution, then heat to 65 ° C. and hold it for 5 minutes to promote dissolution, and then use an ultrasonic probe to sonicate three times at 60 W power for 1 min. After the dissolution is complete, adjust by HCl The pH is around 7.4. Subsequently, the MPLA aqueous solution is mixed with the aluminum adjuvant, and the ultrasound is added for 30 seconds, and the tumor neonatal antigen of the present invention is added and mixed to ensure that the amount of each component of the vaccine of each mouse is MPLA (3 ⁇ g), the aluminum adjuvant (100 ⁇ g) and the tumor neonatal antigen 200 ⁇ g. In 100 ⁇ L physiological saline solution.
  • Example 3 For the preparation method of the encapsulated tumor nascent antigen and the sealed microcapsule group loaded with MPLA, refer to the description in Example 3 of the present invention.
  • mice Construct a 4T1 orthotopic tumor model mouse (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) and inject 5 * 10 5 4T1 breast cancer cells (purchased from ATCC) into the lower right of female Balb / c mice for 6-8 weeks In the mammary fat pad, the mice were randomly divided into groups of 6 mice, and immunized with vaccine on the 4th day.
  • the set groups are as follows: PBS group, tumor neonatal antigen alone group (200 ⁇ g), tumor neonatal antigen AS04 adjuvant group (aluminum adjuvant 100 ⁇ g, tumor neonatal antigen 200 ⁇ g, MPLA 3 ⁇ g), tumor neonatal antigen embedded and MPLA loaded Sealed microcapsule group (microspheres 3 mg, tumor neoantigen 200 ⁇ g, MPLA 3 ⁇ g).
  • Example 11 Effect of Animal Experiments on Sealed Microcapsules Loaded with Tumor Neoantigen Based on 4T1 Breast Cancer Recurrence
  • 4T1-luc breast cancer recurrence model mice were purchased (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.), and 5 * 10 5 4T1-luc breast cancer cells (purchased from ATCC) were transfected with Luciferase (luciferase). ), Injected into the right lower mammary fat pad of female Balb / c mice for 6-8 weeks, after the tumor grows to 200mm 3 , the tumor is surgically resected, and the residual tumor tissue size is controlled by bioluminescence imaging, and then the small The rats were randomly divided into groups of 6 and immunized on the 2nd day.
  • Luciferase Luciferase
  • the residual volume of the tumor lesions after surgical resection was similar, and after different treatment strategies, the tumor recurrence showed a significant difference. No tumor lesion was observed in the sealed microsphere group by bioluminescence imaging on the 14th day, and tumor recurrence occurred in all or part of the other groups.
  • the sealed microcapsules were sacrificed after 5 days of in vivo metabolism. Dissect the area where the microcapsules were injected into the back of the mouse, use forceps to grab the local subcutaneous tissue surrounding the carrier, then cut the tissue with scissors, and place it in a 4% formalin solution for 24 hours.
  • H & E staining samples were prepared by paraffin embedding, sectioning, hematoxylin & eosin (H & E) staining and mounting.
  • pathological section panoramic scanning and analysis system Vectra 3.0, purchased from PerkinElmer was used to observe the inflammatory response at the injection site.

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Abstract

一种疫苗，其包含抗原以及可生物降解的聚合物共混物基质，所述聚合物共混物含有疏水聚合物和两亲嵌段共聚物，所述疫苗以微囊的形式存在，微囊内部含有多腔室结构，所述微囊的平均粒径优选是10-100μm，更优选是30-60μm，所述微囊由以下方法制备：先由聚合物共混物制备开孔微球，然后使其与含抗原的溶液混合，接着使装载有抗原溶液的开孔微球封口，形成装载有抗原的封口微囊。

Description

一种基于微囊的疫苗 技术领域
本发明涉及一种基于微囊的疫苗，其包含抗原以及可生物降解的聚合物共混物基质，该疫苗例如是抗肿瘤疫苗。
技术背景
随着肿瘤生物学以及免疫学的发展，以调节自身免疫系统为主要特性的免疫疗法为肿瘤治疗开辟了一条崭新的思路。在众多的免疫治疗策略中，肿瘤疫苗能够模拟机体对抗病原体的方式，恢复对肿瘤细胞的天然“识别”和应答，从而特异性地消灭肿瘤细胞，因此受到了越来越多的关注。
抗肿瘤疫苗发挥高效作用面临的难题之一就是单纯的肿瘤抗原很难激发机体持久有效的免疫反应。利用纳米尺寸的载体来装载肿瘤抗原，可以有效地提高免疫细胞的内吞和成熟，从而激发机体较强烈的免疫反应，但是纳米载体因为尺寸较小，易被机体清除，所以很难产生持久的抗肿瘤效果。通过复乳溶液法包埋抗原制备微米尺寸的载体有效避免上述难题，然而又面临抗原释放过于缓慢而不能激发有效免疫应答的困境。
CN101601860A公开了一种基于聚合物颗粒的疫苗，其中的抗原组成为吸附于可生物降解聚合物颗粒上的抗原与未吸附的抗原(游离抗原)两种形式，所述的颗粒的平均粒径为0.1～20μm，通过以下方法制备：将聚合物颗粒与乙肝表面抗原溶液进行混合，通过聚合物颗粒与抗原溶液的吸附作用制得的悬液即为疫苗产品，本发明疫苗能够在机体内迅速诱导免疫应答，诱导机体产生较高水平的体液免疫和细胞免疫。
CN102489230A公开了一种生物可降解材料微囊的制备方法，含开孔微球的制备、囊芯材料的装填和开孔微球的封口，相比传统的复乳包埋方法，该方法更加温和，避免生物活性物质受损；包埋后溶液中剩余的囊芯材料可回收利用；更加环境友好，达到了与之前报道相同的粒径、孔径水平，且内腔容积更大，更有利于囊芯材料的装载。但是该专利文献仅仅提及该生物可 降解材料微囊可用于包埋小分子、生物大分子物质，还可以装载纳米、微米级尺寸的颗粒。
尽管有上述各种研究，但研究开发出能更好激发机体产生特异性免疫的疫苗，例如通过细胞毒性T细胞来杀伤肿瘤细胞，对其临床应用具有重要意义。
发明概述
本申请的发明人发现，利用复乳-溶剂去除法制备出一种由可生物降解聚合物制备的开孔微球，之后在该开孔微球中装填肿瘤抗原，然后使该开孔微球封口，制成粒径较大的能够在小鼠体内局部滞留的封口微囊，由于微囊不易被吞噬代谢，较易降解，且在降解的过程中刺激机体产生持续的炎症反应，从而微囊作为抗原储库可以不断地募集抗原递呈细胞如树突状细胞(DC)到达注射部位吞噬抗原，协同聚合物基质代谢产生的酸性微环境，能显著提高细胞募集的数目，提高细胞对抗原的内吞，刺激DC成熟分化，最终DC归巢至淋巴结，刺激机体产生高效持久的免疫反应，促使T细胞持续的增殖与分化，并产生了特异且持久的杀伤，能够显著地抑制了肿瘤的生长、延长了小鼠的生存时间。
基于此，本发明提供一种疫苗，其包含抗原以及可生物降解的聚合物共混物基质，所述聚合物共混物含有疏水聚合物和两亲嵌段共聚物，所述疫苗以微囊的形式存在，微囊内部含有多腔室结构，所述微囊的平均粒径优选是是10-100μm，更优选是30-60μm，所述微囊由以下方法制备：先由聚合物共混物制备开孔微球，然后使其与含抗原的溶液混合，接着使装载有抗原溶液的开孔微球封口，形成装载有抗原的封口微囊。
优选的是，在本发明疫苗中，通过制备的工艺参数优化，抗原的释放与疫苗体内局部滞留时细胞募集行为的产生协同作用，使在释放出来的抗原被募集过来的非成熟的DC吞噬，显著提高了抗原的利用率。
优选的是，所述疫苗是治疗性疫苗，例如是抗肿瘤疫苗或治疗性乙肝疫苗，优选的抗原是肿瘤抗原或乙肝表面抗原。
在本发明的疫苗中，还可以在多孔微球体系中装载一定量的趋化因子，如粒细胞集落因子(GM-CSF)、巨噬细胞炎症蛋白3α(MIP-3α)和单核细胞趋化蛋白1(MCP-1)，来增强微囊对细胞的募集，进一步提高抗原利用率， 增强免疫应答。
附图说明
图1由PLA制备的微球的SEM图
图2由PLA与PELA制备的微球的SEM图
图3由PLA与PELA制备的微球的粒径分布图
图4由PLA与PELA制备的微球内部结构的SEM图
图5由PLA与PELA制备的微球的内部孔径分布图
图6本发明封口微囊的SEM图
图7本发明封口微囊的内部结构
图8本发明封口微囊对抗原OVA的装载率和包埋率
图9本发明封口微囊对多肽MUC1的装载率和包埋率
图10不同疫苗制剂在注射部位的抗原荧光强度定量图
图11本发明微囊对炎症细胞的募集行为：(a)局部含有封口微囊的组织募集细胞的代表性组织切片图像(b)定量分析每个微囊募集的细胞数目
图12不同疫苗制剂抗原利用率的比较：(a)OVA +细胞数目，(b)OVA +细胞的抗原内吞量(以平均荧光强度表示)，(c)抗原利用率
图13本发明微囊降解过程中局部微环境中pH变化曲线
图14酸性和中性微环境下CD86 +的DC中MHC-I和MHC-II的比例
图15不同疫苗制剂免疫后E.G7荷瘤小鼠的肿瘤生长曲线
图16不同疫苗制剂免疫后B16荷瘤小鼠的肿瘤生长曲线
图17不同疫苗制剂治疗后肺部(a)与肾脏(b)组织中的转移灶定量分析
图18不同疫苗制剂免疫后4T1荷瘤小鼠的肿瘤生长曲线
[根据细则91更正 05.11.2019]　
[根据细则91更正 05.11.2019]　
图19不同疫苗制剂治疗后术后复发肿瘤生长曲线
[根据细则91更正 05.11.2019]　
d
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图20术后复发模型生物发光成像图片及定量
[根据细则91更正 05.11.2019]　
图21空白封口微囊(a)和装载趋化因子GM-CSF的封口微囊(b)在皮下组织募集细胞的H&E染色图
发明详述
疫苗
本发明涉及一种疫苗，其用于对哺乳动物受试者中的肿瘤等疾病的预防性或治疗性处理，所述疫苗包含抗原以及可生物降解的聚合物共混物基质，所述聚合物共混物含有疏水聚合物和两亲嵌段共聚物，所述疫苗以微囊的形式存在，微囊内部含有多腔室结构，所述微囊的平均粒径优选是10-100μm，更优选是30-60μm，所述微囊由以下方法制备：先由聚合物共混物制备具有贯穿孔道的开孔微球，然后使其与含抗原的溶液混合，接着使装载有抗原溶液的开孔微球封口，形成装载有抗原的封口微囊。本发明所述疫苗还可含有药学可接受的载体、盐或稀释剂。
在本发明优选的实施方式中，所述疫苗是抗肿瘤疫苗。所述疫苗制备简便，同时具有不论哪种肿瘤的复发预防、转移抑制及治疗均可适用的通用性，且抗肿瘤效果佳。当接种抗肿瘤疫苗后，首先抗原递呈细胞(Antigen Presenting Cell，APC)摄取和加工肿瘤抗原，使其变为抗原肽段，然后与细胞表面的主要组织相容性复合体(Major Histocompatibility Complex，MHC)类分子结合。APC进一步将肽段信息呈递给T细胞，进而产生后续的免疫应答过程。在此过程中，多数抗原在溶酶体内被加工成肽段后，会与MHC-II类分子结合并呈递给CD4 +T细胞；更重要的是，少部分抗原还可以通过胞质内的蛋白酶体加工成肽段，与MHC-I类分子结合并呈递给CD8 +T细胞，使之转化为细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte，CTL)。一方面CTL直接与肿瘤细胞结合产生裂解杀伤功能，另一方面CD4 +T细胞与CD8 +T细胞共同分泌细胞因子来杀伤肿瘤，最终使肿瘤凋亡裂解。
在本发明优选的实施方式中，所述抗原是肿瘤抗原。肿瘤抗原在肿瘤发生、发展及诱导机体抗肿瘤免疫效应中发挥重要作用，是肿瘤免疫治疗的靶分子。肿瘤抗原根据其特异性分为两大类：(1)肿瘤相关抗原，指在肿瘤组织中高表达，在正常组织中也会有一定程度表达的抗原；(2)肿瘤特异性抗原，指仅表达在肿瘤组织却不存在于正常组织中的抗原，因此作为肿瘤抗原具有更好的安全性。然而在肿瘤疫苗发展的早期，由于技术的局限性，难以发现和纯化出肿瘤特异性的抗原，进而限制了其广泛的应用。近年来，随着基因编辑技术的发展，研究人员陆续发现了一些特异性的抗原和新抗原作为肿瘤疫苗免疫的新靶点，从而针对性的制备个性化的肿瘤抗原来提高疫苗的 免疫效应，最终解决了抗原选择的问题。然而合适的肿瘤抗原依然难以激发有效地免疫应答，主要原因是单纯的肿瘤抗原因其半衰期短，注射后会被快速地降解代谢，从而机体免疫系统难以被有效地激活。
在本发明一些实施方式中，所述抗肿瘤抗原是通过收集活的肿瘤细胞表面蛋白的肽类而得到的，所述肽类是通过向原代培养的活的肿瘤细胞周期性加入不使细胞死亡的蛋白酶进行处理而得到的。例如，将预先从生长培养基上洗脱的活的肿瘤细胞进行原代培养，用蛋白酶对肿瘤细胞进行不使细胞死亡的处理，收集释放的表面肿瘤抗原。接着，经过一段时间，用蛋白酶对原代培养的活的肿瘤细胞进行重复处理，该段时间的间隔足以使得表面肿瘤抗原的活性被细胞恢复。积聚(富集)表面肿瘤抗原直至达到满足疫苗接种的剂量，控制得到的表面肿瘤抗原的组分。
在本发明优选的实施方式中，所述肿瘤抗原例如是黏蛋白(MUC1)、肿瘤膜抗原、肿瘤全细胞抗原，或者肿瘤新生抗原。
本发明的抗肿瘤疫苗可包含一种或多种肿瘤抗原，目的是产生一个或多个细胞毒性T淋巴细胞克隆，其各自识别特异性抗原性肽，以产生更有效的免疫应答。在存在数种肿瘤抗原时，优选选择所述肿瘤抗原以诱导针对一种类型的肿瘤或肿瘤细胞的免疫应答。
在本发明一些实施方式中，本发明的抗肿瘤疫苗，其基本组成是不同抗原的混合物或者抗原与不同免疫刺激物质的混合物。
对于肿瘤治疗而言，显著地激活CTL和分泌Th1型细胞因子才能帮助机体攻击和消灭肿瘤。在疫苗组分释放时，是否募集到充足的APC来吞噬疫苗也是影响其能否激发有效免疫应答的至关重要的因素。APC广泛分布在机体的各个组织中，因此它们在发现病原微生物的过程中发挥着至关重要的作用。在外周组织皮肤中，朗格汉斯细胞，其他类型的DC和巨噬细胞均可以检测到危险信号和抗原，被激活后归巢到淋巴器官递呈抗原信息而激活免疫响应。机体内这些免疫细胞在外周组织到淋巴器官之间的迁移是复杂的，其迁移行为主要受到众多趋化因子的调控。
在本发明优选的实施方式中，所述抗原是乙肝表面抗原，该疫苗是治疗性乙肝疫苗。
乙型肝炎(Hepatitis B)是由乙肝病毒(hepatitis B virus,HBV)引起的全球性传染病，迄今为止全球有20亿人感染过乙型肝炎病毒，其中约3.5亿人为慢 性乙肝感染者，对于乙型肝炎尚无有效的治疗药物，目前抗病毒治疗上常用的干扰素和拉米呋啶，虽然能迅速抑制病毒复制，但是停药后易反跳，也易导致变异株的产生。
在慢性HBV感染者体内由于免疫耐受的存在，特异性T细胞反应低下。治疗性疫苗的目的是发挥清除病毒作用的细胞免疫反应主要特异性细胞毒性T淋巴细胞(CTL)的溶细胞作用，即CTL通过释放穿孔素、颗粒酶杀伤感染的肝细胞或通过Fas介导的途径来诱导感染的肝细胞凋亡。但是近年来研究显示，Thl类细胞和CTL分泌的细胞因子参与的非溶细胞机制在抗病毒免疫中起着主导作用。IFN-α等细胞因子都可以通过非细胞病变的方式来抑制病毒复制，甚至清除病毒DNA。尤其是IFN-γ在多种动物模型中的研究表明其具有抑制HBV复制中间产物和HBV特异mRNA的作用，在病毒清除中发挥着重要作用，通过激发自身的细胞免疫应答促进以IFN-γ为主的细胞因子的分泌来清除病毒。
就乙肝表面抗原而言，可做选择的抗原类型也有多种，可选择酿酒酵母和汉逊酵母表达的乙肝表面抗原，也可选择哺乳动物细胞系统表达的CHO乙肝表面抗原，但优选的抗原类型为通过汉逊酵母表达体系得到的乙肝表面抗原。来源不同的HBsAg在结构和性质上有较大差别，即使具有相同或相似的基因编码，由不同表达系统表达的HBsAg颗粒仍可能具有不同的分子大小、分子量以及不同的亚基数，进而表现出不同的电荷性、疏水性以及免疫原性等。
在本发明优选的实施方式中，本发明的疫苗进一步含有趋化因子。趋化因子是一类分子量在10KDa的多肽，主要与细胞表面G糖蛋白连接的相关受体家族作用。趋化因子和其他趋化吸引相关的分子均是在局部产生，然后扩散形成可溶或者固态的浓度梯度，此时表达趋化因子受体的细胞就会依靠浓度梯度信息到达趋化信息来源的地方。
许多的趋化因子与DC和单核细胞的迁移有关，包括单核趋化蛋白1(MCP-1)、单核细胞趋化蛋白2(MCP-2)、巨噬细胞炎症蛋白1α(MIP-1α)、巨噬细胞炎症蛋白1β(MIP-1β)、调节活化正常T细胞表达和分泌的(Regulated upon activation normal T cell expressed and secreted，RANTES)趋化因子、补体C5a、β-防御素(β-defensins)和细菌衍生的甲酸基肽等。由于正常情况下，DC在外周组织及血液中的比例为1％甚至更低，因此如果 疫苗可以在局部创造一个趋化因子的大本营，通过不断释放的趋化因子，模拟并增强体内DC向注射部位募集的行为，从而可以显著地增强免疫应答。
在本发明优选的实施方式中，所述趋化因子例如是巨噬细胞炎症蛋白3α(MIP-3α)、单核细胞趋化蛋白1(MCP-1)和GM-CSF，优选GM-CSF，在所述含抗原的溶液中含有趋化因子，该趋化因子经开孔微球封口被装填在封口微囊中。在本发明优选的实施方式中，本发明疫苗的微囊腔室闭合的结构可以保证封口后的微囊所装载的分子被有效的封装在空腔中，实现持久有效的释放。整个释放过程中没有显著的突释，没有平台期，代谢速率缓慢且有效，最终有效代谢率高达90％，基本上抗原被完全释放。
微囊出色的抗原滞留能力保证了抗原在体内更为持久的释放，伴随着抗原的释放，微囊注射后因原位滞留效应会导致炎症应答，进而募集炎症相关的细胞吞噬载体及释放出来的抗原。抗原与作为佐剂的聚合物基质释放的动力学在控制免疫反应时起重要性，会影响疫苗最终的免疫效应。当二者释放动力学不同步时，即当抗原缓慢释放，佐剂过快或者过慢释放均会影响局部吞噬疫苗的APC被激活的水平，从而难以激发有效的免疫应答。
在众多的APC中，DC(Dentritic cells)是机体中功能最强大的专职APC，根据其成熟分化的状态分为：DC前体细胞、未成熟和成熟DC。DC前体细胞为正常机理状态下尚无DC表型或功能的细胞，当机体面临病原微生物感染或炎症刺激的作用下迅速地被动员、分化和发育为未成熟的DC；未成熟的DC是正常机理状态下大部分DC的状态，可以高效地摄取、加工和递呈抗原，具备较强的迁移能力；成熟的DC因其被激活，所以摄取抗原能力变弱，但递呈抗原信息、刺激初始T细胞活化的能力增强。本发明封口微囊的局部产生了大量的DC趋化因子，有利于募集更多的DC，使抗原被更有效地加工和递呈，诱导机体产生更有效的免疫应答。
在本发明优选的实施方式中，局部产生的炎症微环境会不断地募集炎症相关的细胞及APC到注射部位吞噬抗原，在此过程中，趋化因子的分泌对细胞募集的种类与数量发挥了至关重要的作用，作为载体的聚合物基质降解的速度和抗原释放行为会显著影响趋化因子的分泌量。
在本发明优选的实施方式中，抗原释放与细胞募集行为两者协同作用发挥最佳的效果，使在整个阶段释放出来的抗原都被抗原递呈效率最高的细 胞，即非成熟的DC细胞吞噬，该DC细胞具备大量吞噬抗原的功能，进一步提高了抗原的利用率。
作为载体的聚合物基质代谢的过程中，局部产生的炎症微环境会不断地募集炎症相关的细胞及APC到注射部位吞噬抗原。在此过程中，趋化因子的分泌对细胞募集的种类与数量发挥了至关重要的作用，载体降解的速度和抗原释放行为会显著影响趋化因子的分泌量。在本发明优选的实施方式中，作为载体的聚合物基质代谢产生的酸性微环境能显著提高细胞募集的数目，同时发现酸性微环境显著提高细胞对抗原的内吞，聚乳酸等微囊构建的酸性微环境自身就具备很强的免疫佐剂特性，该酸性微环境是也是提高抗原利用率的重要的因素，能够作为被激活DC的滞留位点，即在不需要添加额外的疫苗佐剂及多次免疫的前提下，就可以持续的提供被激活的APC归巢至淋巴结，酸性微环境还能够显著提升抗原交叉递呈及Th1型细胞因子分泌。
在本发明优选的实施方式中，本发明疫苗其含有免疫刺激增强剂，例如单磷酸脂A(MPLA)、胞嘧啶-鸟嘌呤寡聚脱氧核苷酸和/或聚肌胞苷酸。
微囊制备
在本发明优选的实施方式中，微囊的制备，指的是通过一定的方法使功能材料被包裹或分散在壳层材料中，从而制备颗粒状复合物的过程，优选的本发明微囊内部含有多腔室结构，所述微囊的平均粒径优选是10-100μm，优选20-80μm，更优选是30-60μm。
在本发明优选的实施方式中，所述开孔微球的孔隙率至少在40％以上，例如在至少50％以上，优选在至少60％，更优选70％以上，具有例如孔径1-5μm的孔道。优选的是，开孔微球由复乳与溶剂去除法制备，该方法便于采用生物降解的高分子为壳层材料，适用于载药微囊的开发。所述开孔微球具有贯穿孔结构，具有多腔的内部结构和多孔的壳层，通过复乳演变和复乳固化两个动态过程的控制，可容易控制开孔球的开孔结构与开孔数量，随后将开孔球浸入到含有抗原的溶液中，抗原通过扩散进入微球内部，可选用溶剂溶胀法、照射法和升温退火法三种对开孔微球进行封口。
在本发明优选的实施方式中，微囊的制备主要包括开孔微球的制备、抗原的装填，以及开孔微球的封口，优选所述方法具体包括以下步骤：
(1)配制油相，所述油相为聚合物基质溶液，其中溶剂为有机溶剂；配制内水相溶液和外水相溶液，外水相添加表面活性剂；
(2)将内水相分散到油相当中，形成油包水初乳液；再将初乳液分散到外水相中，形成水包油包水复乳液；
(3)利用溶剂去除法，使油相固化，得到具有贯穿孔道的开孔微球；
(4)将开孔微球与含抗原的溶液混合，抗原在溶液中通过扩散传质的方式从多孔微球的表面进入内部的空腔中，得到装载有抗原的开孔微球；
(5)开孔微球的封口，形成装载有抗原的封口微囊。
在本发明优选的实施方式中，在制备出复乳液后，演变的过程中复乳液中的内水相小液滴会逐渐融合而变大，同时由于内外水相的盐浓度差，内水相会发生逃逸，所以在演变的过程中内水相融合和逃逸在同时进行。内水相的融合会形成微球内部多孔的结构，内水相的逃逸会形成微球表面多孔的形貌。在本发明优选的实施方式中，所述聚合物基质是可生物降解的聚合物共混物基质，所述聚合物共混物含有疏水聚合物和两亲嵌段共聚物。
在本发明优选的实施方式中，其中所述疏水聚合物是丙交酯聚合物(通常简称PLA)、乙交酯聚合物、丙交酯-乙交酯共聚物(通常简称PLGA)、聚己内酯、聚原酸酯和/或聚酸酐，优选是其分子量重均分子量为5,000-100,000道尔顿，更优选是10,000-50,000道尔顿。
在本发明优选的实施方式中，其中所述两亲嵌段共聚物中亲水嵌段为聚乙二醇类、聚丙烯酸类、聚氧乙烯类、聚乙烯醇类，疏水嵌段为内酯类共聚物或均聚物；优选亲水嵌段是聚乙二醇或单甲氧基乙二醇，疏水嵌段是聚乳酸(PLA)或乳酸和乙醇酸的共聚物(PLGA)；所述两亲嵌段共聚物，亲水嵌段具有500-30,000道尔顿的重均分子量，疏水嵌段具有500-50,000道尔顿的重均分子量；优选亲水嵌段重均分子量是1,000-20,000道尔顿，例如是4,000-10,000道尔顿，优选疏水嵌段重均分子量是5,000-20,000道尔顿。
在本发明优选的实施方式中，其中所述两亲嵌段共聚物是聚乙二醇或单甲氧基乙二醇与丙交酯和/或乙交酯的共聚物(通常简称PLGA)，优选是其分子量重均分子量为5,000-10,000道尔顿，更优选是10,000-50,000道尔顿。
在本发明优选的实施方式中，其中所述两亲嵌段共聚物可以用于稳定初乳液，从而能够制备具有多腔室结构的多孔微球，所述两亲嵌段共聚物占微囊中聚合物共混物基质的重量含量比至少是5％，优选至少是10％，至少是 15％、至少是20％、至少是30％、至少是40％或至少是50％。PLA因为其较强的局部炎症免疫应答，在药物可控释放领域应用较少，PELA作为一种生物相容性更好的材料得到了更多的应用，能够帮助制备得到更高孔隙率的开孔微球，通过改变两者的配比，微球的疏水特性和孔隙率就会得到改善，同时可以调节载体的炎症应答，改善生物相容度。
在本发明优选的实施方式中，其中步骤(1)中所述有机溶剂为挥发性且不与水互溶的有机溶剂，例如：正丁醇、甲乙酮、乙醚、氯仿、四氯甲烷、甲苯等，包括挥发性且可部分溶于水的有机溶剂，例如乙酸乙酯、苯酚等，进一步优选为不与水互溶的醇、酮、酯、醚、烷基苯、卤代烷烃、卤代芳烃中的1种或至少2种的组合，所述组合典型但非穷尽的实例有：醇、酮的组合，醇、酮、酯、醚的组合，酯、烷基苯、卤代烷烃的组合，酯、烷基苯、卤代烷烃、卤代芳烃的组合等，特别优选为醇、酯、烷基苯、氯代烷烃、氯代芳烃中的1种或至少2种的组合。
在本发明优选的实施方式中，在微囊制备时在步骤(1)中所述的有机溶剂中添加有免疫刺激增强剂，例如单磷酸脂A。
在本发明优选的实施方式中，在装填抗原时进一步装填免疫刺激增强剂，例如是胞嘧啶-鸟嘌呤寡聚脱氧核苷酸和/或聚肌胞苷酸。
在本发明优选的实施方式中，其中步骤(3)中所述溶剂去除法为溶剂萃取法，也可以是静置使溶剂挥发，或搅拌使溶剂挥发或其他去除溶剂的方法。
在本发明优选的实施方式中，其中步骤(3)中所述固化过程中，内水相与外水相融合，形成贯穿孔。
在本发明优选的实施方式中，步骤(3)所述的具有内外贯穿孔道的开孔微球，为多孔结构。
在本发明优选的实施方式中，本发明所述开孔微球为表面多孔微球。
在本发明优选的实施方式中，开孔微球的孔隙率至少在40％以上，例如在至少50％以上，优选在至少60％，更优选70％以上，更优选80％以上，优选具有例如孔径1-5μm的孔道。优选，微球内部的孔径从800nm-5μm不等，平均孔径在1μm左右。
在本发明优选的实施方式中，其中步骤(3)中，使油相固化后，除去残留的表面活性剂，特别优选通过过筛或离心洗涤除去残留的表面活性剂。
在本发明优选的实施方式中，优选地，所述开孔微球的封口工艺，包括溶剂溶胀法、照射法和升温退火法，所属领域技术人员也可根据其掌握的专业知识/新技术对开孔微球进行封口。
在本发明中，所属领域技术人员可以理解所述“封口”意味着将抗原和/或其他物质例如趋化因子包埋或者固定在或者滞留在微囊内部。在本发明中，封口微囊不意味着微囊表面没有任何开孔，而仅仅意味抗原和/或其他物质例如趋化因子，通过封口的工艺可以部分地包埋或者固定在或者滞留在微囊内部，抗原和/或其他物质例如趋化因子例如需通过微囊的降解可从微囊中释放出来。
在本发明优选的实施方式中，升温退火法是较为理想的封口方法，例如，可以利用聚乳酸等可生物降解的聚合物共混物基质独特的自愈合封口特性，它是采用照射或加热的方法使微球表面的分子吸收能量发生重排使表面的孔发生愈合封口，例如通过将载有抗原的开孔微球缓慢升温至接近微球的玻璃化温度，优选例如低于微球的玻璃化温度的1～2℃，一段时间后，缓慢降温，从而使开孔微球表面的孔封闭制备出封口的多孔微囊，同时抗原被有效的封装在微囊当中，且装载率和包埋率稳定。整个升温需要的温度接近聚乳酸材料的玻璃化温度，要实现微球的愈合封口，就要控制升温的条件，同时对微球形貌不会产生太大的影响。
在本发明的其它实施方式中，开孔微球能够通过聚合物和致孔剂的相分离来制备。在致孔过程中，溶剂常被称为致孔剂，通常选择油溶性小分子。聚合物与溶剂的相分离能够发生在聚合物链增长过程中，由于聚合物分子量的增长或分子间的交联，大分子逐渐从小分子溶剂中沉淀出来，形成固态高分子。也能够发生在高分子溶液体系中，由于环境条件改变，如温度的变化、良溶剂的去除或不良溶剂的添加，造成溶剂对高分子的溶解能力下降，使高分子沉淀成固相。
本发明的目的之一还在于在患者体内诱导针对肿瘤细胞或肿瘤的细胞毒性细胞应答的方法。此方法包括向该患者施用有效量的本发明疫苗优选抗肿瘤疫苗，特别是通过静脉内、注射或输注，优选通过输注施用。该方法的目的尤其在于诱导患者树突细胞的活化和CD8 +细胞毒性细胞应答，获得特异性CD4 +辅助和CD8 +细胞毒性应答。
本发明的疫苗，可以用于疾病如癌症或肝炎例如乙型肝炎的预防和处理，且更特殊地用于免疫疗法。本发明提供一品预防性或治疗性处理癌症或肝炎例如乙型肝炎的方法，其包含对有此需要的哺乳动物受试者施用预防或治疗有效量的疫苗。
在本发明中，术语“免疫接种”表示主动免疫，即因施用而产生的特异性免疫应答的诱导，该施用为例如经由皮下、皮内、肌肉内、口或经鼻途径的少量的抗原，该抗原被所接种个体识别为外源的，因此在合适的配方中是免疫原性的。因此抗原被用作免疫系统的“触发”，以建立针对该抗原的特异性免疫应答。
依照本发明，免疫接种可以是治疗性或预防性的。举例为证，也许能够通过对不患癌症的个体进行免疫接种而达到针对癌症疾病的出现的预防性保护。可能应用此种预防性接种疫苗的个体的例子是具有罹患癌症的风险增加的个体，尽管这种应用不限于此种个体。处于癌症风险的患者可以已经产生肿瘤，无论是作为原发肿瘤还是转移灶，或者显示出癌症的倾向。
本发明中，"有效量"一词意指当施用给人类或动物时，诱发免疫反应之抗原性/免疫原性组合物的量。本领域技术人员依据常规的程序可以容易地确定有效量。
本发明所提供的疫苗或药物组合物可以制备成无菌粉末形式，例如，所述无菌粉末含有疫苗和甘露醇，可以采取如下方法制备：取微囊，注射用水冲洗，转移至冻干盘中，加入甘露醇和适量注射用水，置冷冻干燥机中冻干；冻干品经过筛混匀，无菌分装，轧盖，即得无菌粉末。在向患者给药前，将无菌粉末混悬于一种可接受的分散溶媒中，所述分散溶媒由助悬剂、pH调节剂、等渗调节剂、表面活性剂中的一种、或几种及注射用水组成，所述助悬剂可以是羧甲基纤维素钠、聚乙烯醇、聚乙烯吡咯烷酮、海藻酸钠、甘油中的一种或多种，所述等渗调节剂可以是氯化钠、葡萄糖、甘露醇、山梨醇中的一种或多种等，所述表面活性剂为非离子型表面活性剂，如聚山梨酯系列(如聚山梨酯80、聚山梨酯60等)。
本发明提供的药物组合物例如疫苗微囊可使用本领域技术人员熟知的方法将本发明的疫苗施用于患者，例如动脉内，静脉内，经皮注射，以及鼻内，经支气管，肌肉内或口服施用。剂量和施用方法根据患者的体重和年龄及施用方法而变化；而本领域技术人员可按需要进行选择。
液体药学组合物通常配制成具有在约3.0-9.0之间的pH，更优选在约4.5-8.5之间并且仍然更优选在约5.0-8.0之间。组合物的pH可以通过使用缓冲液如醋酸盐、柠檬酸盐、磷酸盐、琥珀酸盐、Tris或组氨酸来维持，通常采用的范围从约l mM-50mM。组合物的pH可以另外通过使用生理学可接受的酸或碱来调节。
所述药学组合物以预防有效量或治疗有效量(视情况而定，虽然预防可被视为治疗)给个体，这足以显示出对个体有利。通常，这将引起对个体有利的治疗上有用的活性。所施用的化合物的实际量，以及施用的速率和时间过程将取决于所处理的病症的性质和严重程度。处理的处方例如剂量决定等在全科医生和其它医生的责任范围内，并且通常考虑所处理的病症、个体患者的情况、递送位点、施用方法和医生已知的其他因素。
本文中公开的具体实施方案的范围，这些实施方案仅旨在说明本发明的几个方面。
对比例1、由PLA制备的开孔微球
将100mg分子量为20000道尔顿的外消旋端羧基聚乳酸(PLA，济南岱岗生物工程有限公司)溶于2mL乙酸乙酯中(油相，O)，加入0.5mL0.05％的氯化钠水溶液(内水相W1)，用超声破碎仪制备初乳液(油包水，W1/O)，然后将初乳液倒入15mL含有PVA(购自日本可乐丽)的水溶液(外水相W2)中，用均质仪乳化制备复乳液(水包油包水，W1/O/W2)。之后用垂直混悬仪混悬25min)进行复乳演变。随后将复乳液倒入500mL超纯水中，室温下利用磁力搅拌10min(500转/分钟)使微球固化。最后多次离心(500g，5min)，弃掉上清收集底部微球，再添加1mL超纯水于微球中，置于4℃保存。
吸取PLA微球悬液50uL，滴在锡箔纸上，置于室温晾干。用导电胶将含有样品的锡箔纸粘贴在制样台上，喷金后用SEM观察微球形貌。所制得的PLA微球如图1所示：单纯以PLA制备的微球无明显的多孔结构，不适合用作本发明的多孔微球以进一步开发本发明的封口微球。
实施例1、由PLA和PELA制备开孔微球
将95mg分子量为20000道尔顿的外消旋端羧基聚乳酸(PLA，济南岱岗生物工程有限公司)与5mg分子量为38000道尔顿的聚乙二醇(mPEG，Mw：2000)-外消旋端羧基聚乳酸(PLA，Mw：36000)的共聚物(PELA,购自济南岱岗生物工程有限公司)溶于2mL乙酸乙酯中(油相，O)，加入0.5mL0.05％的氯化钠水溶液(内水相W1)，用超声破碎仪制备初乳液(油包水，W1/O)，然后将初乳液倒入15mL含有PVA的水溶液(外水相W2)中，用均质仪乳化制备复乳液(水包油包水，W1/O/W2)。之后用垂直混悬仪混悬25min)进行复乳演变。随后将复乳液倒入500mL超纯水中，室温下利用磁力搅拌10min(500转/分钟)使微球固化。最后多次离心(500g，5min)，弃掉上清收集底部微球，再次添加1mL超纯水于微球中，置于4℃保存。
吸取上述微球悬液50uL，滴在锡箔纸上，置于室温晾干。用导电胶将含有样品的锡箔纸粘贴在制样台上，喷金后用SEM观察微球表面形貌。如图2所示，表面孔径在1-2μm，具有表面开口的结构，其粒径分布如图3所示，微球的平均粒径在60μm。同时为了观察微球内部的结构，用超薄刀片将晾干后的微球切碎。然后将样品粘附在导电胶上喷金，用SEM观察。如图4所示，微球内部同样具有内部多孔且相互贯穿的结构，内部孔径在1-5μm左右。
取30mg冻干后的多孔微球，用压汞仪测定，平行测定三次，计算得出多孔微球的孔隙率和平均孔径分布，多孔微球的孔隙率为82％。如图5所示，微球内部的孔径从800nm-5μm不等，平均孔径在1μm左右。
实施例2、装载抗原的封口微囊的制备
吸取1mL的实施例1制备的干重为30mg开口微球悬液于1.5mL的离心管中，离心去掉上清，加入500μL 50mg/mL 10mg/mL的抗原OVA(购自Sigma公司)或500μL 8mg/mL的MUC1肽段(购自吉尔生化上海有限公司)与微球共混，置于垂直混悬仪上混悬4h(300rpm)，使蛋白质或者肽段利用微球贯穿的孔道充分进入微球空腔内部，之后将垂直混悬仪和微球放在39℃恒温箱中升温封口，过程中混悬速度为100rpm，保证微球在整个升温过程中受热均匀，同时不会发生沉降，反应2h后封口完成，离心(500g，5min)去掉上清，得到装载蛋白质、多肽分子的封口微囊。
吸取含抗原OVA的封口微囊悬液50uL，滴在锡箔纸上，置于室温晾干。用导电胶将含有样品的锡箔纸粘贴在制样台上，喷金后用SEM观察微囊表 面形貌。如图6所示，表面多孔完全封闭，形成封口微囊。同时为了观察封口微囊内部的结构，用超薄刀片将晾干后的微囊切碎。然后将样品粘附在导电胶上喷金，用SEM观察。如图7所示，微囊内部虽然依然具有内部多孔的结构，且孔径在1-5μm左右，但是内部贯穿的多孔结构变成了封闭且独立的多孔结构。
实现微球封口后，进一步评价了封口后微囊中抗原OVA或肽段MUC1的装载率和包埋率，OVA蛋白和MUC1多肽含量的测定方法如下，
提取封口微囊中的蛋白OVA：称取5mg装载OVA的冻干微球(n＝3)，加入1mL的0.1M氢氧化钠水溶液，4℃过夜反应12h，微囊被全部降解后，离心取上清，用0.1M的盐酸滴定使溶液恢复pH中性，从中吸取100μL的上清用BCA试剂盒检测蛋白含量。
提取封口微囊中的多肽MUC1：称取5mg装载MUC1多肽的冻干微囊(n＝3)，首先加入300μL乙腈使材料完全溶解，体系澄清后再加入1.7mL0.015M HCl溶液，最后用0.45μm的滤膜除去体系中的不溶物与杂质，最后用RP-HPLC测定MUC1肽段的浓度。实验条件如下：
检测条件：0.1％三氟乙酸的去离子水(流动相A)；0.1％三氟乙酸的高效液相级乙腈(流动相B)；洗脱梯度：流动B：0％-60％，0-25min；流速：1.0mL/min；检测波长：220nm。
最终微囊载药率(％)的计算方法为封口微囊内的抗原(OVA/MUC1)的质量除以微球的质量(干重)，再乘以100％；微囊包埋率(％)的计算方法为封口微囊内的抗原(OVA/MUC1)质量除以装载前抗原(OVA/MUC1)的质量(浓度乘以体积)，再乘以100％。
如图8和图9所示，随着初始抗原OVA蛋白和MUC1肽段浓度的增加，微囊装载率呈线性增加的趋势。当抗原浓度为100mg/mL时，微囊的装载率高达20％。同时随着抗原浓度的增加，封口微囊的包埋率始终均维持在相对稳定的水平，说明微囊的包埋率只取决于微囊的空腔体积。这种装载率线性增加和包埋率相对稳定的特点将有利于调控抗原与微囊之间的比例，从而满足不同的装载体系的需求。
实施例3、装载抗原与MPLA的封口微囊的制备
首先，将1mg MPLA(购自Sigma)溶于500μL氯仿(CHCl 3)中，然 后将100μL氯仿加入1.9mL油相乙酸乙酯，再将95mg分子量为20000道尔顿的外消旋端羧基聚乳酸(PLA,购自济南岱岗生物工程有限公司))与5mg分子量为38000道尔顿的聚乙二醇(mPEG，Mw：2000)-外消旋端羧基聚乳酸(PLA，Mw：36000)的共聚物(PELA,购自济南岱岗生物工程有限公司)溶于2mL上述含有氯仿和MPLA的油相中，加入0.5mL 0.05％的氯化钠水溶液(内水相W1)，用超声破碎仪制备初乳液(油包水，W1/O)，然后将初乳液倒入15mL含有PVA的水溶液(外水相W2)中，用均质仪乳化制备复乳液(水包油包水，W1/O/W2)。之后用垂直混悬仪混悬25min进行复乳演变。随后将复乳液倒入500mL超纯水中，室温下利用磁力搅拌(500rpm)使微球固化。最后多次离心(500g，5min)，弃掉上清收集底部微球，再次添加1mL超纯水于微球中，置于4℃保存。
装载抗原和MPLA的封口微囊的制备过程如下：吸取1mL装载MPLA的微球悬液于1.5mL的离心管中，离心去掉上清，剩余微球体积500μL。然后加入500μL的10mg/mL的抗原OVA(购自Sigma公司)或500μL 8mg/mL的MUC1肽段(购自吉尔生化上海有限公司)与微球共混，置于垂直混悬仪上混悬4h(300rpm)，使肽段利用微球贯穿的孔道充分进入微球空腔内部，之后放在39℃恒温箱中升温封口，反应2h后封口完成。离心(500g，5min)去掉上清，得到装载抗原和MPLA的封口微囊。经过计算，微囊中OVA的实际装载率为2％，肽段MUC1的实际装载率为1.7％，MPLA的实际装载率为1‰。
实施例4、装载趋化因子的封口微囊的制备
吸取干重为30mg的实施例2中制备的开口微球悬液于1.5mL的离心管中，离心去掉上清，剩余微球体积500μL，加入500uL 1mg/mL的GM-CSF趋化因子(购自Peprotech)溶液，使其与微球共混，置于垂直混悬仪上混悬4h(300转/分钟)，使趋化因子充分进入微球空腔内部，之后放在39℃恒温箱中升温愈合，反应两小时后封口完成。离心去掉上清，得到趋化因子的封口微囊。经过计算，微囊中GM-CSF的实际装载率为2.4‰。
实施例5、装载抗原的封口微囊的抗原释放与细胞募集的协同作用
首先在雌性6-8周的C57BL/6小鼠(n＝4，购自北京维通利华实验动物有限公司)背部皮下注射不同疫苗制剂，分别设置了单纯荧光抗原((染料Cy5标记的抗原OVA，OVA-Cy5)组、荧光抗原与多孔微球共混的共混组、荧光抗原封装在封口微囊内的包埋组。所有组别微球与抗原的注射剂量均分别为3mg与60μg。在代谢的不同阶段(1h、2h、4h、8h、0.5d、1d、2d、4d、7d、10d、14d、17d、21d、25d和30d)，然后利用小动物活体成像系统来观测抗原在多孔微球和封口微球中不同的代谢行为。
如图10所示，单纯的OVA抗原在第3d的时候局部荧光强度几乎全部消失，说明抗原代谢速率很快，基本代谢完全。共混组在24h内的突释为50％，因为在代谢的早期，游离在微球外部的抗原会快速地被组织代谢，所以代谢速度比较快；之后多孔微球内的抗原会进一步释放，然而第5d时依然有较强的荧光强度。从图中可以观察到，依然存在10％的抗原没有被代谢，原因是微球的局部滞留和孔道的延阻效应，抗原代谢得到显著的延缓，总的代谢时间延长到了7d左右。相比之下，包埋组在检测的两周内始终保持较强的荧光强度，在定量图中可以观察到，早期代谢没有明显的突释效应，24h内的释放为10％，原因是封口微囊表面孔的封闭，抗原只可能从暂时从材料表面的纳米孔道中缓慢的释放出来，所以3d仅仅代谢了20％的抗原；随着代谢时间的延长，载体表面及内部结构发生变化，微囊内的抗原大量释放，7d释放了50％左右的抗原；随后代谢速率进一步变缓，代谢了14d后依然具备很强的荧光强度，如图10所示，依然存在35％的抗原没有被代谢。
伴随着抗原的释放，微囊注射后因原位滞留效应会导致炎症应答，进而募集炎症相关的细胞吞噬载体及释放出来的抗原。为了进一步观察细胞募集的现象，在雌性6-8周的C57BL/6小鼠(n＝4，购自北京维通利华实验动物有限公司)背部注入实施例2中的空白封口微囊，在体内不同的代谢时间处死小鼠。剖开小鼠背部注入微囊的部位，用镊子取出夹取皮下包裹载体的局部皮下组织，然后用剪刀剪去组织，将其置于4％的福尔马林溶液中浸泡24h。随后石蜡包埋、切片、苏木精&伊红(H&E)染色和封片，制备出H&E染色样品，最后用病理切片全景扫描和分析系统(Vectra 3.0，购自PerkinElmer)观察注射部位炎症反应。不同的代谢时间处死小鼠。取出皮下包裹载体的局部组织，并制备H&E染色样品，最后用病理切片全景扫描和分析系统观察注射部位炎症反应。
如图11a所示，第3d时，细胞(深色点状物)逐渐被募集到了微囊(浅色圈状物)周围；随着降解时间的延长，微球周围的细胞不断增加，炎症反应增强，甚至在某些微囊内部也会观察到炎症细胞的浸润。利用Inform软件统计点的数量和微囊的数量，通过定量计算局部每个微囊募集的细胞数。通过分析，如图11b所示，14d时平均每个微球可以募集20个左右的细胞。因此在整个代谢的过程中，虽然由于降解产物的累积导致局部炎症应答逐渐的增强。同时小鼠解剖中也没有发现局部组织出现红肿，肉芽以及流脓的现象，说明封口微囊产生的炎症反应是安全的。
分别设置了Cy5染料标记OVA(Cy5-OVA)的单纯抗原组、Cy5-OVA与多孔微球共混的共混组、Cy5-OVA装载在封口微囊内的包埋组。分别注入6-8周雌性的C57BL/6小鼠大腿肌肉内(n＝4，购自北京维通利华实验动物有限公司)。5d后处死小鼠，取出局部包含微球的肌肉组织，用剪刀剪成小块，研磨成悬液，在4℃的PBS中重悬，离心去除上清。再加入1mL的混合酶溶液(1mg/mL胶原酶D与100U/mL的重组DNase I溶于RPMI 1640培养基中)，37℃反应30min。之后再用PBS清洗，20μm的细胞筛过滤杂质，制得单细胞悬液。按照流式细胞样品染色的操作步骤，分别在样品中加入抗鼠的FITC-CD11c和eFlour450-F4/80的荧光抗体标记DC和巨噬细胞。进一步用BD LSRFortessa流式细胞仪检测OVA +细胞，CD11c +OVA +细胞和F4/80 +OVA +细胞，并用Flowjo软件分析处理。
如图12所示，单纯抗原组(对照组)，OVA +的细胞数目和胞内OVA的荧光强度均很弱，说明局部的抗原在第5d几乎被代谢完全。共混组中多孔微球的滞留效应使抗原利用率显著提高，OVA +的细胞数目提高了10倍左右，细胞对OVA的内吞量提高了两倍，对应的抗原利用率提高了20倍左右；包埋组由于其出色的抗原滞留效应和抗原有效释放能力，OVA +的细胞数目提升了50倍左右，OVA内吞量提高了4倍以上，抗原利用率最终提高了200倍以上。以上结果表明封口微囊可以更有效的调控微囊体系中抗原释放与细胞募集行为，使两者协同作用，进一步提升抗原的利用率，抗原利用率计算公式：OVA +细胞数目×荧光强度，用单独OVA组的抗原利用率进行归一化处理。该协同作用不仅可以促使更多的细胞吞噬抗原，而且可以提高细胞对抗原的内吞量，最大程度地提高抗原的利用率。
实施例6、装载抗原的封口微囊的酸性微环境对细胞募集的影响。
聚乳酸微囊降解产物乳酸会导致局部微环境的酸化，酸化的程度、变化以及持续的时间都可能会对后期的免疫效应产生影响，因此需要精确地监控局部微环境的酸化过程及变化。SNARF R○-1是一种pH(监测范围6-9)敏感型的探针，在488nm处激发，在640nm和580nm两处波长下接收，荧光强度比值I640/I580对应体系中的pH值。该比值越小，说明体系的pH越低。因此通过在微球中装载SNARF ○R-1，利用激光共聚焦显微镜可以实时监测聚乳酸微囊降解产物乳酸对局部微环境pH的改变。
具体操作如下：在雌性C57/BL6小鼠(n＝6)背部皮下注射包埋了SNARF ○R-1的封口微囊，分别在第3d、5d、7d和14d处死小鼠。取出皮下包裹微球的组织，置于激光扫描共聚焦显微镜下观察局部组织pH变化情况。观察其荧光图像，并计算荧光强度比值I640/I580，并根据标曲计算出实际的pH值。
实验发现，代谢3d后局部微环境从中性变为了酸性的微环境。如图13所示，局部pH由中性的7.2变为了弱酸性的6.5。伴随着材料的持续代谢，并没有发现局部酸性环境的进一步酸化，而是稳定维持在pH 6.5左右，即没有发生因为普通非多孔类聚乳酸类微球逐渐降解导致的乳酸堆积问题。普通非多孔类聚乳酸类微球的材料内部与外部同时发生降解时，内部代谢产生的乳酸会由于传质受阻的原因发生累积，从而内部的pH会低于外部，导致局部乳酸堆积，容易产生可能的pH突然降低或者不稳定的现象。本发明封口微囊因为其独特的内部多孔贯穿结构，有效的避免了上述乳酸堆积和传质受阻的问题，使内部材料降解产生的乳酸高效地与外部置换；当机体对乳酸的代谢与聚乳酸材料降解产生的乳酸在体内实现动态平衡，局部的pH就会维持在相对稳定的水平，从而为局部细胞创造一个相对稳定的微环境来发挥功能，同时有助于分析稳态对局部细胞的影响。
在很多生理炎症情况下，炎症部位都会募集大量的细胞，同时炎症部位伴随着酸化，因此酸性应该与细胞募集行为存在一定的关联，因此重点考察中性微环境(Neutral Microenvironment,NM)和酸性微环境(Acidic Microenvironment,AM)对细胞募集的影响。通过在聚乳酸微囊中装载强碱弱酸盐NaHCO 3，利用其与微囊降解产物乳酸的酸碱中合作用消除局部的酸性，在局部构建中性的微环境，以此对比聚乳酸微囊的酸性微环境。
酸性环境通过增加细胞募集与刺激细胞对抗原的内吞提高了抗原利用率，同时也说明仅有抗原释放行为与细胞募集行为的协同配合对抗原利用率的提升是可以改进的，例如需要一个刺激免疫细胞大量吞噬抗原的有益微环境才能进一步提高抗原的利用率。因此需要抗原释放行为、细胞募集行为、DC数目与类型、刺激内吞的酸性微环境多因素的共同作用，才能使抗原利用率最大化。共混组和装载NaHCO 3的包埋组均说明了上述任何因素都是不可或缺的，只有本发明封口微囊可以同时调动所有这些因素，协同的发挥各自的功能，使抗原被最高效的利用。
当DC吞噬抗原以后，抗原经过加工和处理变成抗原肽，以抗原肽-主要组织相容性复合物(MHC)的形式表达于APC表面，供T细胞识别。然而，当APC只吞噬了抗原，缺乏外在刺激物刺激时，APC不会有效的表达共刺激分子(CD80、CD86、CD40)来激活T细胞。同时在大多数情况下，DC吞噬外来抗原以后，其对抗原的加工和递呈主要通过溶酶体途径，该过程受MHC-II类分子限制，主要介导T细胞体液免疫相关的反应；在某些特殊的情况下，外源性抗原可通过胞质溶胶递呈途径，即MHC I类分子途径递呈，从而直接激活细胞毒性T细胞(CTL)，进而最直接有效的杀死靶细胞。因此对于治疗性肿瘤疫苗而言，需要使抗原更多的以MHC-I类分子进行递呈，才能更有效地杀伤肿瘤。
酸性环境可以显著地提高APC，特别是DC对抗原的内吞，然而APC的激活、抗原被递呈的数量与途径还需要进一步考察。具体操作如下：首先分别用20mM NaHCO 3和PBS溶液溶解OVA，制备封口微囊，混合方法与封口方法参见抗原的装载。分别注入6-8周雌性C57BL/6小鼠大腿肌肉中(n＝6，购自北京维通利华实验动物有限公司)，第5d解剖后取出含有愈合微囊的组织。用剪刀剪成碎块，再加入1mL的混合酶溶液(1mg/mL胶原酶D与100U/mL的重组DNase I溶于RPMI 1640培养基中)，37℃反应30min。再置于细胞匀浆机中匀浆，制成单细胞悬液，离心去掉上清，收集细胞用20μm的细胞筛过滤去除杂质，用手持细胞计数器计数。然后用流式抗体进行染色标记，分别用PE-CD11c、APC-CD80、APC-Cy7-CD86、eFlour 450-MHC-I、BV655-MHC-II标记DC表面共刺激分子与主要组织相容性复合物的标志，以检测局部DC被激活的比例。
如图14所示，酸性环境中，DC被显著地被激活。CD86 +DC中MHC-I分子的表达量是MHC-II的40倍之多，说明DC更多地以MHC-I的方式来递呈抗原，从而说明微囊具备高效促进抗原交叉递呈的能力。然而在中性环境中，CD86 +DC中MHC-I的比例下降了超过一倍多，因此说明环境在微囊促进抗原的交叉递呈中发挥着非常重要的作用。
实施例7、封口微囊基于E.G7淋巴瘤实体瘤的动物实验效果
首先构建E.G7荷瘤小鼠(购自北京维通利华实验动物有限公司)，将1*10 6的E.G7肿瘤细胞(购自ATCC)，注入6-8周雌性C57BL/6小鼠腋下，然后将荷瘤小鼠按每组6只随机分组，第4d时肿瘤体积在50-60mm 3时进行疫苗接种。疫苗设置组别如下：PBS、OVA低剂量多次(20μg,7d、14d各加强免疫一次)、OVA高剂量单次(60μg)、多孔微球与OVA共混(3mg微球、60μg OVA)、装载OVA的封口微囊组(制备方法参见实施例2)(3mg微球、60μg OVA)、包埋OVA与装载MPLA的封口微球组(制备方法参见实施例3)(3mg微球、60μg OVA、3μg MPLA)。自肿瘤模型构建之日起，隔天观察小鼠毛发、体重和生存情况，并用游标卡尺测量肿瘤的长度和宽度，根据如下公式计算肿瘤体积：V＝1/2×L×W 2(L：长度；W：宽度)。
如图15的肿瘤生长曲线所示，单纯抗原组中，低剂量多次组和高剂量单次组均难以抑制肿瘤生长，25d的生存率(SR)只有17％；共混组在微球的佐剂效应下提升生存率至50％，但是肿瘤在早期的生长较快；包埋组的肿瘤在早期就能够被明显地抑制，生长速率缓慢，小鼠25d的生存率高达100％。说明微囊体系在只用抗原，没有添加额外的分子佐剂的前提下，通过肿瘤抗原的充分利用就能够取得较好的治疗效果。当在愈合微囊的材料中装载微量的佐剂MPLA(3μg)时，不仅生存率高达100％，而且荷瘤小鼠的生存率得到更加明显地抑制，在26d时50％的小鼠的肿瘤体积基本与治疗初期相差不大，且超过60％的小鼠生存超过40d，进一步证实了微球体系与MPLA体系的兼容性可以进一步提升肿瘤抑制的效果。
实施例8、封口微囊基于B16黑色素瘤实体瘤的动物实验效果
首先构建B16荷瘤小鼠(购自北京维通利华实验动物有限公司)，将5*10 5B16黑色素瘤细胞(由吉林大学提供)，注入6-8周雌性C57BL/6小鼠腋下，然 后将荷瘤小鼠按每组6只随机分组，第4d时肿瘤面积＞10mm 2时疫苗接种。设置组别如下：PBS组、MUC1肽段组(50μg)、封口微囊装载MUC1肽段的包埋组(微球3mg、肽段50μg)、包埋MUC1与装载MPLA的封口微囊组(微球3mg、肽段60μg、MPLA 3μg)。自肿瘤模型构建之日起，隔天观察小鼠毛发、体重和生存情况，并用游标卡尺测量肿瘤的长度和宽度，根据如下公式计算肿瘤体积：V＝1/2×L×W 2(L：长度；W：宽度)。
如图16的肿瘤生长曲线所示，PBS和高剂量单次组均不能有效抑制黑色素瘤的生长，而封口微球可以显著地抑制肿瘤的生长，33％的小鼠肿瘤体积小于500mm 3，且在监测的22d内生存率为100％。装载MPLA的封口微球表现出最优的抗肿瘤效果，22d时50％的小鼠中肿瘤几乎没有生长，个别小鼠黑色素瘤甚至消失。
实施例9、封口微囊基于B16黑色素瘤转移瘤的动物实验效果
首先构建B16转移模型小鼠(购自北京维通利华实验动物有限公司)，将2*10 5B16黑色素瘤细胞(由吉林大学提供))，注入6-8周雌性C57BL/6小鼠尾静脉中，然后将小鼠按每组6只随机分组，第4d时进行疫苗接种。具体免疫策略与实施例8中一致。在接种后的18d，每组分别处死2只小鼠，观察脏器中肺、肾中黑色素瘤的转移情况，并计数和统计脏器中的转移灶。
如图17所示，PBS组中肺部出现了大量的黑色素瘤转移，计数发现每只小鼠的肺部分布了100个左右的转移灶，，同时肾脏组织长出了总体积100mm 3的转移灶，证实黑色素瘤转移模型构建成功。疫苗免疫的18d后，解剖封口微球组的小鼠发现，相比PBS组，肺部转移灶减少从100个到15-20个，肾脏肿瘤体积从100mm 3减少到了10mm 3。封口微球加入MPLA后抗肿瘤转移效果得到了进一步的提升，肺部几乎没有明显的转移灶，肾脏中也没有肿瘤生成，所以微囊体系与TLR受体激动剂的搭配产生了最佳的抗肿瘤转移效果。
实施例10、封口微囊装载肿瘤新生抗原基于4T1乳腺癌的动物实验效果
所选肿瘤新生抗原是8种多肽按等比例的组合(所述8种多肽的氨基酸序列分别是：SPNRSWVSL、HPMYLFLSM、VAVKVNFYVI、KAPHNFQFV、YHYVLNSMV、EYSAMTTRGTI、GSPPRFFYM和CPQTHAVVL)，该多肽 的制备过程如下：通过对4T1肿瘤细胞系及正常Balb/c小鼠组织(由国家生化工程重点实验室提供)进行比对测序筛选出肿瘤突变基因，然后用计算机算法预测出突变多肽(交由深圳裕策生物科技公司)，随后进行多肽合成(交由金斯瑞生物科技公司)。
AS04佐剂组制备方法如下：首先将MPLA溶解于0.5％的三乙醇胺溶液中，随后加热到65℃并保持5min以促进溶解，随后用超声探头以60W功率1min超声三次，溶解完全后通过HCl调整pH至7.4左右。随后将MPLA水溶液与铝佐剂进行混合，超声30s，加入本发明肿瘤新生抗原混匀，确保每只小鼠疫苗各组分用量为MPLA(3μg)，铝佐剂(100μg)及肿瘤新生抗原200μg于100μL生理盐水溶液。
其中，包埋肿瘤新生抗原与装载MPLA的封口微囊组的制备方法参见本发明实施例3的描述。
构建4T1原位瘤模型小鼠(购自北京维通利华实验动物有限公司)，将5*10 5 4T1乳腺癌细胞(购自ATCC)，注入6-8周雌性Balb/c小鼠右下乳腺脂肪垫中，然后将小鼠按每组6只随机分组，第4d时进行疫苗免疫。
设置组别如下：PBS组、单纯肿瘤新生抗原组(200μg)、装载肿瘤新生抗原的AS04佐剂组(铝佐剂100μg、肿瘤新生抗原200μg、MPLA 3μg)、包埋肿瘤新生抗原与装载MPLA的封口微囊组(微球3mg、肿瘤新生抗原200μg、MPLA 3μg)。
自肿瘤模型构建之日起，隔天观察小鼠毛发、体重和生存情况，并用游标卡尺测量肿瘤的长度和宽度，根据如下公式计算肿瘤体积：V＝1/2×L×W 2(L：长度；W：宽度)。
[根据细则91更正 05.11.2019]　
如图18的肿瘤生长曲线所示，PBS和单纯肿瘤新生抗原组均不能有效抑制乳腺癌的生长，AS04佐剂组对肿瘤生长有一定的抑制作用，展现出比较明显的肿瘤延迟生长。相比而言，封口微囊组表现出最优的抗肿瘤效果，28d时83％的小鼠中肿瘤体积小于500mm3。
实施例11、封口微囊装载肿瘤新生抗原基于4T1乳腺癌术后复发的动物实验效果
构建4T1-luc乳腺癌术后复发模型小鼠(购自北京维通利华实验动物有限公司)，将Luciferase(荧光素酶)转染的5*10 5 4T1-luc乳腺癌细胞(购自ATCC)，注入6-8周雌性Balb/c小鼠右下乳腺脂肪垫中，待肿瘤生长至200mm 3后，对小鼠进行肿瘤手术切除，通过生物发光成像控制残留肿瘤组织大小相近，然后将小鼠按每组6只随机分组，第2d时进行疫苗免疫。
具体免疫策略与实施例10中的免疫策略一致。自肿瘤模型构建之日起，隔天观察小鼠毛发、体重和生存情况，用生物发光成像监测肿瘤复发情况，并用游标卡尺测量肿瘤的长度和宽度，根据如下公式计算肿瘤体积：V＝1/2×L×W 2(L：长度；W：宽度)。
[根据细则91更正 05.11.2019]　
如图19的肿瘤生长曲线所示，PBS和单纯肿瘤新生抗原组均不能有效抑制乳腺癌的术后复发，所有小鼠均发生了肿瘤复发，肿瘤进展较快且迅速死亡。AS04佐剂组防止了部分小鼠的肿瘤复发，13％的小鼠在第22d仍无可见肿瘤生长，但仍在长期监测中死亡。封口微球组则表现出最优的术后抗复发效果，22d时仅一只小鼠发生肿瘤复发，83％的小鼠均无肿瘤复发。如图22生物发光成像图片及统计图所示，手术切除后，肿瘤病灶残留体积相近，而经后续不同治疗策略后，肿瘤复发展现出明显的差异。封口微球组在第14d通过生物发光成像观察不到任何肿瘤病灶，其他组均全部或部分出现肿瘤复发。
实施例12、装载趋化因子的封口微囊募集细胞的效果比较
在雌性6-8周的C57BL/6小鼠(n＝4，购自北京维通利华实验动物有限公司)背部分别注入3mg微球的空白封口微囊与装载了7μg趋化因子GM-CSF的封口微囊，体内代谢5天后处死小鼠。剖开小鼠背部注入微囊的部位，用镊子夹取包裹载体的局部皮下组织，然后用剪刀剪去组织，将其置于4％的福尔马林溶液中浸泡24h。随后石蜡包埋、切片、苏木精&伊红(H&E)染色和封片，制备出H&E染色样品，最后用病理切片全景扫描和分析系统(Vectra 3.0,购自PerkinElmer)观察注射部位炎症反应。
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如图21所示，体内代谢5天后，只有少量的炎症细胞(黑色点状物)募集到空白微球(浅色圆状物)周围。而装载趋化因子GM-CSF的微囊可以募集到明显更多的细胞到材料周围，意味着所述装载趋化因子GM-CSF的微囊 中释放出来的抗原可以更多的被细胞所吞噬，从而增强抗原递呈的效率和最终的免疫效果。

Claims (14)

  1. 一种疫苗，其包含抗原以及可生物降解的聚合物共混物基质，所述聚合物共混物含有疏水聚合物和两亲嵌段共聚物，所述疫苗以微囊的形式存在，微囊内部含有多腔室结构，所述微囊的平均粒径优选是是10-100μm，更优选是30-60μm，所述微囊由以下方法制备：先由聚合物共混物制备开孔微球，然后使其与含抗原的溶液混合，接着使装载有抗原溶液的开孔微球封口，形成装载有抗原的封口微囊。
  2. 权利要求1所述的疫苗，其中所述疫苗用于对哺乳动物受试者中的肿瘤或肝炎的预防性或治疗性处理，该疫苗例如是治疗性疫苗，优选是抗肿瘤疫苗或治疗性乙肝疫苗，优选的抗原是肿瘤抗原或乙肝表面抗原。
  3. 权利要求1所述的疫苗，其含有趋化因子，所述趋化因子例如是粒细胞集落因子(GM-CSF)、巨噬细胞炎症蛋白3α(MIP-3α)和单核细胞趋化蛋白1(MCP-1)，优选，在所述含抗原的溶液中含有趋化因子，该趋化因子经开孔微球封口被装载在封口微囊中。
  4. 权利要求1所述的疫苗，其中所述疏水聚合物是丙交酯聚合物、乙交酯聚合物、丙交酯-乙交酯共聚物、聚己内酯、聚原酸酯和/或聚酸酐，优选是其分子量重均分子量为5,000-100,000道尔顿，更优选是10,000-50,000道尔顿。
  5. 权利要求1所述的疫苗，其中所述两亲嵌段共聚物是聚乙二醇或单甲氧基乙二醇与丙交酯和/或乙交酯的共聚物，优选是其分子量重均分子量为5,000-10,000道尔顿，更优选是10,000-50,000道尔顿。
  6. 权利要求1所述的疫苗，其中所述两亲嵌段共聚物占微囊中聚合物共混物基质的重量含量比至少是5％，优选至少是10％，至少是15％或至少是20％。
  7. 权利要求1所述的疫苗，其含有免疫刺激增强剂，例如单磷酸脂A、胞嘧啶-鸟嘌呤寡聚脱氧核苷酸和/或聚肌胞苷酸。
  8. 权利要求1所述的疫苗，其中通过将装载有抗原的开孔微球升温至接近微球的玻璃化温度，优选例如低于微球的玻璃化温度的1～2℃，从而使其封口。
  9. 权利要求1所述的疫苗，其中所述开孔微球由复乳溶剂去除法制备。
  10. 权利要求1所述的疫苗，其开所述开孔微球的孔隙率至少在40％以上，例如在至少50％以上，优选在至少60％，更优选70％以上，优选具有例如孔径800nm-5μm的孔道。
  11. 一种药物组合物，其含上述任一项疫苗或上述任一项的封口微囊，以及药学上可以接受的载体。
  12. 上述任一项疫苗、上述任一项开孔微球或上述任一项的封口微囊在制备抗肿瘤疫苗或治疗性疫苗中的应用。
  13. 上述任一项疫苗或上述任一项的封口微囊在制备抗肿瘤药物、治疗肝炎例如乙型肝炎、或诱导针对肿瘤细胞或肿瘤的细胞毒性细胞应答的药物中的应用。
  14. 一种在患者体内诱导针对肿瘤细胞或肿瘤的细胞毒性细胞应答的方法，包括向该患者施用有效量的疫苗优选抗肿瘤疫苗。
PCT/CN2019/101078 2018-08-20 2019-08-16 一种基于微囊的疫苗 WO2020038298A1 (zh)

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