WO2020233460A1 - Plateforme modulaire de nanosupport à médiation par intéine, son procédé de construction et son application - Google Patents

Plateforme modulaire de nanosupport à médiation par intéine, son procédé de construction et son application Download PDF

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WO2020233460A1
WO2020233460A1 PCT/CN2020/089924 CN2020089924W WO2020233460A1 WO 2020233460 A1 WO2020233460 A1 WO 2020233460A1 CN 2020089924 W CN2020089924 W CN 2020089924W WO 2020233460 A1 WO2020233460 A1 WO 2020233460A1
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intein
protein
nanocarrier
hft
mediated
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Chinese (zh)
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汤书兵
周伟
袁伟明
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广州市妇女儿童医疗中心
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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
    • A61K39/0005—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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins

Definitions

  • the present disclosure relates to a pharmaceutical carrier and a construction method and application thereof, in particular to an intein-mediated nanocarrier module platform and a construction method and application thereof.
  • Vaccines are the most effective and economical means of medical intervention to prevent and control infectious diseases. Attenuated or inactivated pathogens have achieved great success as a traditional classic vaccine, helping humans eliminate smallpox and polio. However, traditional vaccines have potential safety hazards and cannot effectively prevent viruses with high-frequency mutation characteristics, such as HIV and influenza viruses.
  • Subunit vaccines use a certain surface structure component (antigen) of microorganisms to make a vaccine that does not contain nucleic acid and can induce the body to produce antibodies. It has attracted the attention of vaccinologists due to its advantages of easy purification, safety and effectiveness, and stable physical and chemical properties.
  • the subunit vaccine has poor immunogenicity, weak induction of immune response and short duration, which limits its clinical application. With the development of technology, this type of vaccine uses nano-delivery systems and immune adjuvants (referred to as adjuvants) to increase the breadth and strength of the body's immune response.
  • Nano delivery vehicles are usually 5-100nm nanoparticles, which have strong penetrating power and can freely diffuse to reflux lymph nodes where B cells and T cells are located, increasing the probability of antigen being captured by antigen presenting cells (APCs). Nanoparticles have a nanometer size effect, mimic natural pathogens, and are preferentially swallowed by APCs. In addition, nanoparticles promote the maturation of dendritic cells (DCs), promote the secretion of a variety of cytokines, and enhance the immune response of B cells and T cells. In addition, the highly repetitively displayed antigens on the surface of nanoparticles can cross-link B cell receptors, directly activate B cells, and cause high-intensity humoral immune responses.
  • DCs dendritic cells
  • Adjuvants can help immune responses, especially immune enhancers, effectively activate innate immunity and adaptive immunity, and regulate the type, intensity and durability of immune responses. This is because the pathogen-associated molecular pattern (PAMP) is highly conserved and easily recognized by the corresponding receptors on natural immune cells. For example, CpG is recognized by Toll-like receptor 9 (TLR9), and Salmonella flagellin protein is recognized by TLR5, both of which can be activated by myeloid differentiation factor 88 (MyD88) in DCs The NF- ⁇ B signaling pathway stimulates the release of pro-inflammatory factors and triggers a cascade of immune responses.
  • PAMP pathogen-associated molecular pattern
  • CpG CpG is recognized by Toll-like receptor 9 (TLR9)
  • Salmonella flagellin protein is recognized by TLR5, both of which can be activated by myeloid differentiation factor 88 (MyD88) in DCs
  • MyD88 myeloid differentiation factor 88
  • immune enhancers can non-specifically enhance innate immunity and acquired immune response, and therefore may cause systemic inflammation, fever and even hepatosplenomegaly and other diseases.
  • co-localization of immune enhancers and antigens to the same APCs can significantly increase the effective concentration of adjuvants to improve the boosting effect of adjuvants, and reduce the doses of adjuvants and antigens to reduce side effects caused by non-specific immune responses.
  • the modification methods of nanoparticles are gene fusion method and chemical coupling method. Among them, the gene fusion method has high efficiency in loading antigens and adjuvant molecules, convenient operation, and is widely used in vaccine research and development.
  • the loaded antigen may seriously affect the assembly of nanoparticles, leading to the formation of precipitates of recombinant proteins, and a series of renaturation-reassembly is required to re-form nanoparticles.
  • the method of gene fusion is difficult to integrate antigen molecules and adjuvant molecules into the same nanoparticle at the same time.
  • the chemical coupling method can conveniently and polymorphically modify the nanoparticle to make it multifunctional and realize the co-delivery of adjuvant and antigen on the nanoparticle.
  • this method has low efficiency and poor specificity, and side effects are often accompanied by side effects.
  • the purpose of the present disclosure includes, for example, providing a nanocarrier module platform based on intein-mediated nanocarrier module platform.
  • the nanocarrier module platform has good stability, and can maintain the original structure after loading foreign proteins.
  • the co-delivery of immune enhancers and antigens on the nanocarrier module platform significantly improves the boosting effect of adjuvants.
  • a nanocarrier module platform based on intein-mediated which is a recombination formed by the fusion of self-assembled protein X and the C-terminus of the linker gbl-intein
  • the protein gbl-intein c- X is self-assembled into polymer nanoparticles, and the N-terminal of the protein X extends to the surface of the nanoparticles and is exposed to the external environment.
  • the protein X is ferritin or dioxytetrahydropteridine synthase or bacteriophage Q ⁇ nucleocapsid protein or cowpea mosaic virus (Cowpea mosaic virus) nucleocapsid protein or hepatitis E virus (Hepatitis E virus) Nucleocapsid protein.
  • cowpea mosaic virus cowpea mosaic virus
  • Hepatitis E virus Hepatitis E virus
  • the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus (Pyrococcus furiosus) heavy-chain ferritin or ferritin derived from other species.
  • the purpose of the present disclosure includes, for example, providing a method for constructing the aforementioned nanocarrier module platform.
  • the technical solution provided by the present disclosure is as follows: a method for constructing a nanocarrier module platform mediated by intein, consisting of the N-terminus of protein X that can be self-assembled into nanoparticles and the linker gbl- intein C-terminal fusion proteins form a recombinant gbl-intein c -X, the recombinant protein gbl-intein c -X extended to polymer nanoparticles nanoparticle surface is exposed to the external environment from the assembly into the N-terminus of the protein X.
  • the protein X that can be self-assembled into nanoparticles is ferritin or dioxoperidine synthase or bacteriophage Q ⁇ nucleocapsid protein or cowpea mosaic virus nucleocapsid protein or hepatitis E virus nucleocapsid protein protein.
  • ferritin is human-derived heavy-chain ferritin or heavy-chain ferritin of Pyrococcus furiosus or ferritin derived from other species.
  • the purpose of the present disclosure includes, for example, providing applications of the aforementioned nanocarrier module platform.
  • the technical solution provided by the present disclosure is as follows: the application of the intein-mediated nanocarrier module platform as a drug and/or vaccine delivery system as described above.
  • the present disclosure has at least the following advantages:
  • the nanocarrier module platform provided by the present disclosure is a recombinant protein gbl-intein c- X formed by introducing a linker gbl-intein C into the N end of protein X to self-assemble into polymer nanoparticles, and the N end of protein X extends to the nanometer
  • the surface of the particles is exposed to the external environment, and foreign proteins such as antigen proteins, immune enhancers, polypeptide epitopes and antibodies are efficiently and specifically covalently cross-linked to the N-terminus of protein X through intein-mediated protein editing technology to form a drug And/or vaccine delivery systems, including vaccine delivery systems, adjuvant delivery systems, and vaccine-adjuvant co-delivery systems.
  • the nanocarrier module platform provided by the present disclosure has good stability and remains stable at pH 2-8. After loading antigen proteins, immune enhancers, polypeptide epitopes and antibodies and other foreign proteins, the assembly of the nanoparticles is not destroyed, that is, the structure is not destroyed, and no refolding-reassembly is required. It not only improves the preparation efficiency of vaccine delivery systems, etc., but also solves the problem of the effect of loaded protein on the stability of protein self-assembled nanoparticles and the difficulty of co-delivering adjuvant and antigen molecules with nanoparticles. It realizes that the nano-formulation can efficiently deliver antigen and immune enhancer at the same time, and has good specificity at the same time.
  • the nanocarrier module platform provided by the present disclosure has high loading efficiency of foreign proteins such as antigen proteins, immune enhancers, polypeptide epitopes and antibodies, and has low side effects.
  • the recombinant protein gbl-intein c- X provided by the present disclosure is highly expressed in Escherichia coli, is easy to prepare, has low manufacturing cost, and greatly reduces manufacturing cost.
  • the nanocarrier module platform provided by the present disclosure can be used in the fields of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody-conjugated drugs.
  • Figure 1 is a schematic diagram of the construction principle of the nanocarrier module.
  • Figure 2 is an analysis diagram showing that the introduction of the linker gbl-intein C does not affect the stability of the nanoparticles;
  • Figure 2a shows the electrophoresis of the three recombinant proteins
  • Figure 2b shows the electrophoresis of gb1-intein C -HFT and gb1-intein C -PFT after precipitation with ammonium sulfate
  • Figure 2c shows the electrophoresis of the purified gb1-intein C -HFT
  • Figure 2d is purified by gel filtration gb1-intein C -HFT UV gel
  • Figure 2e electron micrograph of purified gb1-intein C -HFT detecting a TEM
  • Fig. 2f is a TEM examination of purified gb1-intein C -PFT SEM picture.
  • Figure 3 is an analysis diagram of GFP protein and strep2 after being efficiently covalently coupled to HFT;
  • Figure 3a is the electrophoresis diagram of the GFP-HFT content of the target product detected by the Coomassie brilliant blue method (CB assay);
  • Figure 3b is the electrophoresis diagram of the coupling efficiency of GFP and HFT using Western-blotting assay (WB assay);
  • Figure 3c is the Coomassie brilliant blue The electrophoresis diagram of the content of strep2-HFT of the target product by the method;
  • Figure 3d is the electrophoresis diagram of the coupling efficiency of strep2 polypeptide and HFT using Western-blotting.
  • Figure 4 is an analysis diagram showing that loading GFP protein and strep2 polypeptide by self-shearing does not affect the stability of the nanoparticle structure
  • Figure 4a is a Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Figure 4b is a UV curve diagram of gel filtration purified GFP-HFT
  • Figure 4c is an electron micrograph of TEM detection of purified GFP-HFT
  • 4d is an electrophoretic analysis chart of gel filtration purified strep2-HFT
  • FIG. 4e is a UV curve chart of gel filtration purification of strep2-HFT
  • FIG. 4f is an electron micrograph of TEM detection and purification of strep2-HFT.
  • Fig. 5 is a schematic diagram of preparing different nanodevices by splicing nanoparticles and exogenous proteins provided by the present disclosure through intein-mediated protein editing technology.
  • Figure 6 is an analysis diagram showing that nanoparticles can efficiently load polypeptide epitopes and flagellin;
  • Figure 6a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT
  • Figure 6b is the UV curve diagram of the gel filtration purification nano vaccine SP70-HFT
  • Figure 6c is the transmission electron micrograph of the nano vaccine SP70-HFT
  • Figure 6d is SP70 and CBLB co-delivery nano-preparation SP70-CBLB-HFT gel filtration purification electrophoresis analysis diagram
  • Figure 6e is the gel filtration purification co-delivery nano-preparation SP70-CBLB-HFT UV curve diagram
  • Figure 6f is the co-delivery nano-preparation SP70 -CBLB-HFT transmission electron microscope image
  • Figure 6g is the gel filtration purification electrophoresis analysis image of nano adjuvant CBLB-HFT
  • Figure 6h is the UV curve diagram of gel filtration purification nano adjuvant CBLB-HFT
  • Figure 6i is nano adjuvant Transmission electron micrograph of agent CBLB-HFT.
  • Figure 7 is an analysis diagram of whether modular nanocarriers can efficiently load CpG
  • Figure 7a is the electrophoresis analysis diagram of rhizavidin-HFT gel filtration purification
  • Figure 7b is the UV curve diagram of rhizavidin-HFT gel filtration purification
  • Figure 7c is the transmission electron microscope image of rhizavidin-HFT
  • Figure 7d is the SP70-rhizavidin-HFT Gel filtration purification electrophoresis analysis diagram
  • Figure 7e is the UV curve of SP70-rhizavidin-HFT gel filtration purification
  • Figure 7f is the transmission electron microscope image of SP70-rhizavidin-HFT
  • Figure 7g is the two-dimensional type of SP70-rhizavidin-HFT Average graph
  • Figure 7h is the electrophoresis analysis graph of target protein rhizavidin-HFT combined with biotinylated CpG.
  • Figure 8 is an analysis diagram of the impact of different vaccine formulations on the immune effect using the SP70 epitope as a model
  • Figures 8a-c are ELISA analysis of CpG adjuvants delivered in different ways and CBLB induced IgG titers;
  • Figure 8d in vitro cell titration analysis of antibody neutralization titers induced by different vaccine formulations;
  • Figure 8e is the immune response induced by co-delivery of nano formulations.
  • Figure 9 is an analysis diagram of protein editing and cutting technology that can efficiently load HA antigen on HFT/PFT carriers
  • Figure 9a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis
  • Figure 9b is the electrophoretic analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing shearing technology
  • Figure 9c is the UV curve of gel filtration purification of HA-HFT Figure
  • Figure 9d is a transmission electron microscope image of HA-HFT
  • Figure 9e is an electrophoresis analysis image of HA-PFT prepared based on protein editing and shearing technology purified by gel filtration
  • Figure 9f is a UV curve of HA-PFT purified by gel filtration Figure
  • Figure 9g is a transmission electron microscope image of HA-PFT.
  • Figure 10 is an analysis diagram of the nano adjuvant CpG-HFT enhancing HA specific humoral immune response and regulating IgG type.
  • Figure 10a shows that ELISA analysis reveals that both nanocarriers and nanoadjuvants can enhance HA specific humoral immune response
  • Figure 10b-d shows that ELISA analysis reveals that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and nano
  • the antibody recognition ability induced by the adjuvant and nano vaccine mixed preparation is higher than that of the nano vaccine itself.
  • the present disclosure provides a nanocarrier module platform mediated by intein, wherein the nanocarrier module platform mediated by intein is the fusion of protein X and the C-terminus of the linker gbl-intein to form a recombinant protein gbl-intein c- X is a polymer nanoparticle formed by molecular self-assembly, and the N end of the protein X extends to the surface of the nanoparticle.
  • the protein X that can extend from the N-terminus to the surface of the nanoparticle self-assembled nanoparticle is ferritin or dioxoperidine synthase or bacteriophage Q ⁇ nucleocapsid protein or cowpea Mosaic virus nucleocapsid protein or hepatitis E virus nucleocapsid protein.
  • the protein X is a protein whose N-terminal stretches to the surface of the nanoparticle after self-assembly into a nanoparticle.
  • the protein X is selected from ferritin, dioxoperidine synthase, bacteriophage Q ⁇ nucleocapsid protein, cowpea mosaic virus nucleocapsid protein, and hepatitis E virus nucleus At least one of the group consisting of capsid proteins.
  • the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or other species-derived ferritin.
  • the present disclosure provides a method for constructing a nanocarrier module platform mediated by intein, wherein the protein X of the self-assembled nanoparticle can be stretched from the N end to the surface of the nanoparticle and the C end of the linker gbl-intein is fused to form a recombinant protein gbl -intein c -X self-assembles into a 24-mer structure.
  • the protein X that can extend from the N-terminal to the surface of the nanoparticle self-assembled nanoparticle in step 1 is ferritin or dioxoperidine synthase or bacteriophage Q ⁇ nucleocoat Shell protein or cowpea mosaic virus nucleocapsid protein or hepatitis E virus nucleocapsid protein.
  • the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or other species-derived ferritin.
  • the present disclosure provides the application of the intein-mediated nanocarrier module platform as a drug and/or vaccine delivery vehicle as described herein.
  • the present disclosure provides the application of the intein-mediated nanocarrier module platform as a delivery vehicle in the preparation of drugs and/or vaccines as described herein.
  • the present disclosure provides applications of the intein-mediated nanocarrier module platform as described herein in the delivery of drugs and/or vaccines.
  • the present disclosure provides the intein-mediated nanocarrier module platform as described herein for use as a drug and/or vaccine delivery vehicle.
  • the present disclosure provides a method for delivering drugs and/or vaccines, including administering the intein-mediated nanocarrier module platform described herein loaded with drugs and/or vaccines to subjects in need.
  • the subject is a human.
  • the drug is a protein or peptide.
  • the drug is an antibody.
  • the vaccine includes an antigen.
  • the vaccine further includes an immune enhancer.
  • the antigen is an antigen protein or an epitope.
  • the present disclosure provides a protein drug, including:
  • the protein active ingredient is connected to the N-terminal of the protein X in the intein-based nanocarrier module platform.
  • the protein active ingredient is a vaccine.
  • the vaccine includes an antigen and/or an immune enhancer.
  • the antigen is an antigen protein or an epitope.
  • the gbl-intein connected to the N-terminus of protein X extends to the surface of the nanoparticle, And/or exposure to the external environment.
  • the N-terminus of gbl-intein connected to the N-terminus of protein X extends to the nanometer Particle surface, and/or exposure to the external environment.
  • the protein X is a protein having the following characteristics: in a nanoparticle formed by self-assembly of the protein, the N-terminal of the protein extends to the surface of the nanoparticle and/or is exposed to the external environment.
  • Protein X extending from the N-terminal to the surface of the nanoparticle self-assembled nanoparticle refers to a protein having the following characteristics: in the nanoparticle formed by self-assembly of the protein, the The N-terminus of the protein extends to the surface of the nanoparticle and/or is exposed to the external environment.
  • immunostimulatory structure can be found in or composed of the following molecules: including but not limited to lipopolysaccharide; phosphatidylcholine; cytokine adjuvant; glycans, including peptidoglycans; teichoic acid, including lipoteichoic acid; Proteins, including lipoproteins and lipopeptides; outer membrane protein (OMP), outer surface protein (OSP) and other protein components of bacterial cell walls; bacterial DNA; single-stranded and double-stranded viral RNA; unmethylated CpG-DNA; Mannans; mycobacterial membranes; porins; and a variety of other bacterial and fungal cell wall components, including those found in yeast.
  • OMP outer membrane protein
  • OSP outer surface protein
  • vaccine may refer to a composition comprising an antigen and optionally other auxiliary molecules, the purpose of which is to administer the composition to a subject to stimulate an immune response specific to the antigen and preferably to cause immune memory, resulting in At some point in the future, when subjects encounter this antigen, they will have an immune response.
  • auxiliary molecules are other molecules that act as adjuvants for non-specific immunostimulatory molecules and improve the pharmacokinetic and/or pharmacodynamic properties of the antigen.
  • vaccines usually contain certain parts of disease-causing organisms (appropriately attenuated or killed) or pathogenic organisms as antigens.
  • An attenuated organism such as an attenuated virus or an attenuated bacteria, is manipulated to lose some or all of its ability to grow in its natural host.
  • the experiments and examples of the present disclosure involve the gene synthesis of the following proteins and polypeptides in Shanghai Tolo Harbor Biotechnology Co., Ltd., and primer synthesis in Nanjing GenScript Biotechnology Co., Ltd.
  • GFP SEQ ID NO.1 strep2 SEQ ID NO.2 gp41-1-intein C (referred to as the intein C) SEQ ID NO.3 gp41-1-intein N , (abbreviated as intein N ) SEQ ID NO.4 SP70 SEQ ID NO.5 gb1 SEQ ID NO.6 Rhizavidin SEQ ID NO.7 CBLB SEQ ID NO.8 H1HA10 SEQ ID NO.9 HFT SEQ ID NO.10 PFT SEQ ID NO.11 GFP-intein N -gb1 SEQ ID NO.12 strep2-gp41-1-intein N -gb1 SEQ ID NO.13 SP70-gp41-1-intein N -gb1 SEQ ID NO.14 rhizavidin-gp41-1-intein N -gb1 SEQ ID NO.15 CBLB-gp41-1-intein N SEQ ID NO.16 SP70-CBLB-gp41-1
  • HFT is human ferritin heavy chain (human ferritin heavy chain or human heavy chain of ferrtin), also known as human ferritin heavy chain
  • PFT is Pyrococcus furiosus ferrtin heavy chain or Pyrococcus furiosus heavy chain of ferrtin), also known as Pyrococcus furiosus ferritin heavy chain.
  • GFP-intein N -gb1, strep2-gp41-1-intein N -gb1, SP70-gp41-1-intein N -gb1, rhizavidin-gp41-1-int N -gb1, CBLB-gp41-1-intein N , SP70-CBLB-gp41-1-intein N , H1HA10-gp41-1-intein N- gb1 are intein N- related "cargo" fusion proteins.
  • the gene of the "cargo" fusion protein is amplified by overlapping PCR method, and the 5'and 3'primers used have NcoI and XhoI restriction sites respectively.
  • the fusion gene was digested with the same enzymes and inserted into the linearized pET28a vector to obtain a recombinant vector, and the resulting recombinant vector was transformed into Escherichia coli JM109 for recombinant cloning construction.
  • gb1-gp41-1-intein C- HFT and gb1-gp41-1-intein C- PFT are intein C- related "carrier" fusion proteins.
  • the gene of the "carrier" fusion protein is also amplified by overlapping PCR method, and the 5'primer and 3'primer used have EcoRI and XhoI restriction sites respectively.
  • the digested fragment was inserted into the pET28a linearization vector, it was transformed into E. coli JM109.
  • the plasmid was extracted and transformed into E. coli BL21(DE3) plysS for protein expression. Pick a single monoclonal strain, inoculate it into 20ml of kanachloramphenicol bi-anti-LB medium, and cultivate overnight at 37°C on a shaker.
  • the primers used in this disclosure are used to prepare nanodevices with different functions.
  • the antigen or adjuvant protein purification involved in the present disclosure all proteins are purified by Ni 2+ affinity chromatography. Different recombinant proteins have slightly different purification methods because of their different properties and solubility. GFP-intein N- gb1, strep2-intein N- gb1, SP70-intein N -gb1, H1HA10-inteinN-gb1 and rhizavidin-inteinN-gb1 are soluble proteins, the supernatant of bacterial lysate can be directly added to Ni 2+ resin Carry out affinity chromatography purification.
  • the bacterial supernatant was lysed and mixed with 1ml resin on a shaker at 4°C for 30 minutes. Then, the non-specific binding protein flows out with the liquid gravity. Next, add 20ml NT buffer containing 10mM, 20mM, 40mM imidazole to wash the resin to remove non-specific binding proteins. Then, 500 mM imidazole was added to the NT buffer for elution. Finally, the eluted product was dialyzed overnight at 4°C in 1L NT buffer. Most of the CBLB-intein N and SP70-CBLB-intein N formed a precipitate.
  • CBLB-intein N and SP70-CBLB-intein N form inclusion body precipitates, which can be resuspended in NT buffer containing 2M urea, and the supernatant after centrifugation of the resuspension is purified by soluble protein purification.
  • intein was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 1).
  • the N-terminal of the heavy chain ferritin (ferrti, shown as C in Figure 1) is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- ferrtin.
  • the recombinant proteins gb1-intein C- HFT (marked as 2 in Figure 2a-2b) and gb1-intein C -PFT (marked as 3 in Figure 2a-2b) were constructed with HFT and PFT, respectively.
  • the abscissa of all UV profile of gel filtration in this disclosure represents the elution volume, and the ordinate represents the change in absorbance at A280 nm of ultraviolet light.
  • the electrophoresis band at 35kDa-40kDa is very obvious, and the expression of recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT are both significantly increased, and the solubility is high. It can be seen from the results that the introduction of gb1 can greatly increase the expression of heavy chain ferritin in cells and improve its solubility, which is conducive to the formation of large-scale production. Therefore, the recombinant protein gb1-intein C- ferrtin nanoparticles can be used to prepare nanocarrier platforms. feasibility.
  • Figure 2b-2d The purification results are shown in Figure 2b-2d.
  • Figure 2b is the electrophoresis diagram of gb1-intein C -HFT and gb1-intein C -PFT after precipitation with ammonium sulfate. Up: supernatant , Pellet: ammonium sulfate precipitated product;
  • Figure 2c shows the electrophoresis after purification of gb1-intein C- HFT, among which pellet: ammonium sulfate enriched precipitate resuspended product, Purification of gb1-intein C- HFT by gel filtration: gel Purification of gb1-intein C -HFT by filtration method, gel filtration of gb1-intein C -HFT: Gel filtration of purification of gb1-intein C -HFT, 9-17: Separate collection of the number of samples for purification of gb1-intein C -HFT
  • Figure 2d
  • the purified recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT were observed by transmission electron microscope (TEM), the magnification of the transmission electron microscope was 110k, and the length of the ruler was 20nm.
  • TEM transmission electron microscope
  • Figure 2e is the electron micrograph of the purified gb1-intein C- HFT by TEM (negative staining preparation)
  • Figure 2f is the electron micrograph of the purified gb1-intein C- PFT by TEM
  • Fig. 2e and Fig. 2f show that both gb1-intein C- HFT and gb1-intein C- PFT exist in the form of nanoparticles.
  • the scale used to observe negatively stained samples by transmission electron microscopy represents 20nm, and the arrow indicates the nanoparticle.
  • Both recombinant proteins exist in the form of 10-20nm spherical nanoparticles. It can be seen that the recombinant protein gb1-intein C- ferrtin forms nanoparticles through molecular self-assembly. It is determined that the recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT can self-assemble into nanoparticles with a diameter of 18nm with 24 monomers.
  • Example 1 Taking the recombinant protein gb1-intein C- HFT nanoparticles constructed in Example 1 as an example, they were respectively contained with the exogenous protein GFP protein and strep2 polypeptide tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) Peptide-mediated protein splicing modification was performed to observe the stability of the nanoparticle.
  • strep2 polypeptide tag Trp-Ser-His-Pro-Gln-Phe-Glu-Lys
  • the C ends of the GFP protein and strep2 polypeptide tags were introduced into the linker intein N- gb1 through gene fusion, respectively, to form GFP-intein N- gb1 and strep2-intein N- gb1.
  • the GFP-labeled antibody, strep2-labeled antibody, and HFT antibody are used to detect changes in the reaction substrate and target product in the reaction system.
  • Western-blotting detection data show that the trans-shearing efficiency is very high and the reaction is complete ( Figure 3b and 3d). It can be seen that both GFP and strep2 can be efficiently and specifically covalently cross-linked to the HFT protein.
  • Figure 4a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Figure 4b is the UV curve diagram of gel filtration purified GFP-HFT
  • Figure 4c is the TEM detection and purification
  • Figure 4d is the electrophoresis analysis image of gel filtration purified strep2-HFT (Coomassie brilliant blue staining)
  • Figure 4e is the UV curve of gel filtration purified strep2-HFT
  • 4f is the electron microscope image of the purified strep2-HFT detected by TEM (negative staining preparation); before: before the intein self-cleavage, after: after the intein self-cleavage, Figure 4a 8-19 and 4d 9- 20: Separately collect
  • the proteins GFP-HFT and GFP-intein N- gb1 with very similar molecular weights can be separated well, and the elution position of the protein GFP-HFT is earlier than GFP-intein N- gb1, the arrow in Figure 4b
  • the peak of GFP-HFT indicates that the protein GFP-HFT exists as a macromolecular polymer.
  • the proteins strep2-HFT and strep2-intein N -gb1 with very similar molecular weights can also be separated well, and the protein strep2-HFT eluted earlier than strep2-intein N -gb1, the arrow in Figure 4e
  • the peak of GFP-HFT indicates that the protein strrep2-HFT exists in the form of a macromolecular polymer.
  • the separated and purified target products GFP-HFT and strep2-HFT were observed by transmission electron microscope, and both GFP-HFT and strep2-HFT existed in the form of nanoparticles ( Figure 4c, 4f).
  • proteins or peptides can be efficiently sheared into HFT nanoparticles without affecting the stability of the nanoparticles, and the nanocarrier module platform can be efficiently covalently coupled with exogenous proteins.
  • enterovirus 71 enterovirus 71
  • enterovirus 71 enterovirus 71
  • nanocarrier module technology to allow polymorphic modification of the characteristics of nanoparticles, nanoparticles with different functions were prepared, in order to study the influence of different vaccine formulations on antigen immunogenicity, the establishment of nano adjuvants and vaccine-adjuvant co-delivery nano formulations And nano vaccines, see Figure 5 for details.
  • CpG or flagellin were used as adjuvants to prepare corresponding preparations.
  • Flagellin specifically uses CBLB.
  • CBLB is a truncated form of flagellin and has its adjuvant function.
  • FIG. 6a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT
  • Figure 6b is the gel filtration purification nano vaccine SP70-HFT
  • Figure 6c is the transmission electron micrograph of the nano vaccine SP70-HFT
  • Figure 6d is the gel filtration purification electrophoresis analysis of the SP70 and CBLB co-delivered nano preparation SP70-CBLB-HFT
  • Figure 6e is the gel filtration purification
  • Figure 6f is the transmission electron microscope image of the co-delivery nano-preparation SP70-CBLB-HFT (negative staining)
  • Figure 6g is the gel filtration purification of the nano adjuvant CBLB-HFT Electrophoresis analysis diagram
  • Figure 6h is the UV curve of the purified nano-adjuvant CBLB-HFT by
  • Nano vaccine 1 Intein is introduced into Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 1). The N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT. 24 recombinant protein gb1-intein C- HFT molecules self-assembled into 24-mer nanoparticles. 2Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1. In the 2mM DTT solution, the molar ratio is 1:1 by adding nanoparticles and protein SP70-intein N- gb1, and SP70-HFT nanoparticles are obtained by protein splicing, which is the nano vaccine delivery system.
  • Intein N is introduced into the C-end of CBLB to form the protein CBLB-intein N.
  • CBLB-intein N is added at a molar ratio of HFT to CBLB 1:1, and CBLB-HFT is obtained by protein splicing and self-assembly Form CBLB-HFT nanoparticles.
  • CpG cannot be directly covalently linked to HFT, but biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin. Biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin.
  • Intein N- gb1 is introduced into the C-terminus of avidin rhizavidin to form the protein rhizavidin-intein N- gb1.
  • Nanoparticles and rhizavidin-intein N- gb1 are added to the 2mM DTT solution to obtain rhizavidin-HFT through protein splicing and self-assembly Form rhizavidin-HFT nanoparticles.
  • Figure 7 shows the analysis diagram of whether the modular nanocarrier can efficiently load CpG; among them, Figure 7a is the gel filtration purification electrophoresis analysis diagram of rhizavidin-HFT; Figure 7b is the UV curve diagram of rhizavidin-HFT gel filtration purification; 7c is the transmission electron microscope image of rhizavidin-HFT (negative staining); Figure 7d is the gel filtration purification electrophoresis analysis image of SP70-rhizavidin-HFT; Figure 7e is the UV curve of SP70-rhizavidin-HFT gel filtration purification; Figure 7f is a transmission electron microscope image of SP70-rhizavidin-HFT (negative staining); Figure 7g is a two-dimensional class average image of SP70-rhizavidin-HFT; Figure 7h is an electrophoretic analysis image of target protein rhizavidin-HFT combined with biotinylated CpG ; Before: before the intein self-cleavage
  • rhizavidin can be efficiently coupled to HFT, and the target product can be purified by gel filtration.
  • the rhizavidin-HFT purified by gel filtration was observed by transmission electron microscope in the form of spherical nanoparticles ( Figure 7c).
  • rhizavidin and SP70 are coupled at a molar ratio of 1:5, and it does not affect the nanoparticle structure, as shown in Figure 7d-f.
  • the white circle indicates that the target protein self-assembles into spherical nano-sized particles, and the discretely distributed white spots in the circle are rhizavidin protein (the electron density is greater than the peptide) , And the average number of bright spots is consistent with the concentration of rhizavidin-intein N -gb1 and SP70-intein N -gb1 in the initial reaction.
  • the target product rhizavidin-HFT was combined with sufficient biotinylated CpG, the protein band migrated, revealing that CpG was successfully loaded.
  • the biotinylated CpG can be covalently attached to the nanoparticle by the following method.
  • the intein intein
  • the Streptococcus G protein B1 domain tag gb1
  • the N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT.
  • 24 recombinant protein gb1-intein C- HFT molecules self-assembled into 24-mer nanoparticles.
  • 2Intein N- gb1 is introduced into the C-terminal of rhizavidin to form the protein rhizavidin-intein N- gb1.
  • the molar ratio is 1:1:1 by adding nanoparticles, biotinylated CpG and protein rhizavidin-intein N- gb1, obtained by protein splicing and the high affinity interaction between antibiotic protein and biotinylated CpG CpG-HFT nanoparticles are nano adjuvant drug delivery systems.
  • Figure 6a-b shows the results of Tricine-SDS-PAGE and UV curve analysis gel filtration separation and purification of the target product SP70-HFT.
  • the results show that this method can purify a relatively pure target product; before and after are respectively denoted as: samples before and after shearing, and numbers 8-19 represent the sample number (1ml/tube) of the collected sample for gel filtration purification.
  • Figure 6c TEM analysis of the target product SP70-HFT exists in the form of nanoparticles, and the arrow indicates the target product.
  • Figure 6d-f and Figure 6g-i are the purification and quality detection of the target product SP70-CBLB-HFT and CBLB-HFT by gel filtration, respectively.
  • Figures 6a, 6d and 6g show that the unsheared gb1-intein C- HFT bands are almost invisible, indicating that the self-shearing efficiency of the nanoparticles is high.
  • Figures 6c, 6f and 6i show that the three target products all exist in the form of nanoparticles, indicating that the nanoparticles provided in the present disclosure can be covalently cross-linked with antigens and adjuvants through self-shearing, thereby imparting different functions to the nanoparticles.
  • intein was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 1).
  • the N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT.
  • 24 recombinant protein gb1-intein C- HFT molecules self-assembled into 24-mer nanoparticles.
  • 2Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1.
  • the molar ratio of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 1:5, and the molar ratio of nanoparticles to the mixture of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 6. :1:5, the molar ratio of biotinylated CpG and protein rhizavidin-intein N- gb1 is 1:1.
  • SP70-CpG-HFT group used 0.4 ⁇ g/bottle of biotinylated CpG and 10 ⁇ g/bottle SP70-rhizavidin-HFT at a molar ratio of 1:1 to mix for 30 minutes in an ice bath to immunize mice.
  • SP70-HFT+CpG-HFT group used nano vaccine SP70-HFT and nano adjuvant CpG-HFT to immunize mice together, using doses of 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Nano-vaccine SP70-HFT group SP70-HFT immunized mice with a dose of 10 ⁇ g/mouse.
  • SP70-HFT+CpG group SP70-HFT and 0.4 ⁇ g/mouse of biotinylated CpG co-immunized mice.
  • Five mice in each group were vaccinated 3 times with an interval of 2 weeks between each inoculation. Two weeks after the third immunization, blood was collected from the orbit for immune analysis.
  • SP70-HFT Nano vaccine
  • SP70-HFT+CpG Nano vaccine and free CpG mixture
  • SP70-CpG-HFT SP70 and CpG co-delivery nano formulation
  • SP70-HFT+CpG-HFT Nano vaccine and nano adjuvant Agent combination preparation.
  • mice After inoculating mice with different dosage forms, the mice were vaccinated 3 times with an interval of 2 weeks. Blood was taken from the orbit for ELISA analysis after 2 weeks of the third immunization.
  • ELISA analysis of different ways to deliver CpG adjuvant induces IgG titers.
  • the low-dose free 0.4 ⁇ g/CpG has no obvious adjuvant effect
  • the nano adjuvant CpG-HFT has obvious boosting effect
  • the adjuvant effect of SP70-CpG-HFT co-delivery nano formulation is the best. It can be seen that the low-dose 0.4 ⁇ g/free CpG adjuvant has no obvious effect.
  • nano-adjuvant CpG-HFT delivered with nanoparticles and the antigen-adjuvant co-delivered nano-particle SP70-CpG-HFT adjuvant effect is significant, and the co-delivery of nano-formulations induces the most efficient B cell immune response. This is because the co-delivery of nanoformulations ensures that the antigen and the adjuvant are localized to the same antigen presenting cell (APC) to the fullest extent possible to fully exert the boosting effect of the adjuvant.
  • ELISA was used to detect IgG antibody titers of different subtypes (1:1000).
  • the IgG subtype analysis results showed that the co-delivery of the nanoformulation SP70-CpG-HFT also significantly enhanced the IgG titers of Th1 and Th2 types, as shown in Table 1. Th2 type IgG1 accounted for the highest proportion.
  • CpG As an adjuvant, CpG has a better boosting effect on B cell immune response than flagellin; comparing co-delivered CpG and flagellin, it is found that CpG induces higher antibody titers; see Figure 8c for details.
  • mice in each test group Take the sera of mice in each test group from points 1 to 2 of this experimental example, serially dilute the serum (50 ⁇ l) in a 2-fold gradient, and place it in a CO 2 incubator at 37°C with 100TCID50 EV71G082 (50 ⁇ l) After standing for 1 hour, 15,000 human embryo rhabdomyosarcoma cells (RD cells) were added. CPE was observed 3 days after infection, and the neutralizing antibody titer was counted.
  • RD cells human embryo rhabdomyosarcoma cells
  • the antibody neutralization titer analysis found that the antibody titer of SP70-CpG-HFT, SP70-HFT+CpG-HFT, SP70-CBLB-HFT and SP70-HFT+CBLB-HFT were significantly higher than SP70-HFT, among them, the antibody titer induced by SP70-CpG-HFT is the highest.
  • the SP70 epitope is used as a model to analyze the impact of different vaccine formulations on the immune effect.
  • the analysis diagram is shown in Figure 8.
  • Figure 8a-c shows the ELISA analysis of different ways of delivering CpG adjuvant and CBLB to induce IgG titers;
  • Figure 8a is The effect of different ways of delivering CpG on SP70IgG antibody titer.
  • the nano-vaccine mixed with free CpG SP70-HFT+CpG
  • nano-vaccine and nano-adjuvant CpG-HFT mixed preparation and SP70 and CpG co-delivery nano-preparation SP70-CpG-HFT can significantly increase the specific antibody titer.
  • the effect of co-delivery of nano preparations is higher than the antibody titer induced by the mixed preparation of nano vaccine and nano adjuvant.
  • Figure 8b reveals that when CBLB is used as an adjuvant, the co-delivery of the nano-formulation induces a higher specific antibody titre than the nano-vaccine SP70-HFT and nano-adjuvant CBLB-HFT mixed formulation.
  • Figure 8c shows that the adjuvant effect of CpG is higher than that of CBLB.
  • Figure 8d In vitro cell titration analysis of antibody neutralization titers induced by different vaccine preparations. The results of antibody neutralization titers showed that the co-delivery of nanoformulations induced the highest antibody neutralization ability, and the neutralization ability of the antibodies stimulated by the nanovaccine and nanoadjuvant mixed formulation was the second, which was significantly higher than the nanovaccine itself. And CpG adjuvant induced antibody neutralization titer is better than CBLB adjuvant.
  • Figure 8e In vivo lethal protection analysis reveals that co-delivery of nanoformulations can provide the most effective immune protection (75%), followed by nanovaccine and nanoadjuvant (50%), both of which are higher than nanovaccine (25 %). The results of immunological analysis showed that the immune response induced by co-delivery of nano-formulations was the most effective, and the mixture of nano-adjuvant and nano-vaccine also had significant adjuvant effects.
  • Influenza virus hemagglutinin stem region is an important target of universal influenza vaccine, in which antibodies induced by H1HA10 trimer expressed by E. coli can neutralize different subtypes of strains.
  • H1HA10 abbreviated as HA
  • ferrtin were fused using the existing gene fusion method to obtain the recombinant protein HA-HFT.
  • Figure 9 the protein editing and shearing technology can efficiently load HA antigen on HFT/PFT vector; among them, Figure 9a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis; in the figure, control: pET28a vector blank control bacteria lysis All: bacterial lysate; up: bacterial lysate supernatant; HA-HFT: direct gene fusion of HA (H1HA10, abbreviated as HA) with HFT to form a recombinant protein.
  • Figure 9b is the electrophoresis analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing and shearing technology.
  • Figure 9c is a UV curve diagram of HA-HFT purified by gel filtration.
  • Figure 9d is a transmission electron micrograph of HA-HFT (negative staining).
  • Figure 9e is an electrophoretic analysis diagram of gel filtration purification of HA-PFT prepared based on protein editing and shearing technology.
  • Figure 9f shows the UV curve of HA-PFT purified by gel filtration.
  • Figure 9g is a transmission electron micrograph of HA-PFT (negative dye preparation). Before: before the intein self-cleavage, after: after the intein is cut, the numbers 8-19 in Figure 9b and 9e: the numbers (1ml/tube) of the target product samples collected by the gel filtration purification.
  • the supernatant of the bacterial lysate does not contain the recombinant protein HA-HFT, and it can be seen that the recombinant protein forms an inclusion body precipitate.
  • HA is covalently coupled to HFT to form HA-HFT.
  • HFT HA-HFT prepared based on protein editing and shearing exists in the form of nanoparticles.
  • HA-PFT prepared based on protein editing and shearing also exists in the form of nanoparticles, which means that PFT can also carry HA .
  • mice in each group were immunized three times, with an interval of 2 weeks between each inoculation, and blood was collected from the orbit for immunoassay after 3 weeks of immunization.
  • the HA group used H1HA10-intein N to immunize mice with a dose of 10 ⁇ g/mouse.
  • the HA-HFT+CpG-HFT group used the nano-vaccine HA-HFT and the nano-adjuvant CpG-HFT to immunize mice together, and the dosages were 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Nano-vaccine HA-HFT group HA-HFT immunized mice with a dose of 10 ⁇ g/mouse.
  • Nano-vaccine HA-PFT group HA-PFT immunized mice with a dose of 10 ⁇ g/mouse.
  • mice were immunized with the nano-vaccine HA-PFT and the nano-adjuvant CpG-HFT at the doses of 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Fig. 10 The analysis diagram of nano adjuvant CpG-HFT enhancing HA specific humoral immune response and regulating IgG type is shown in Fig. 10; among them, Fig. 10a is an ELISA analysis revealing that both nanocarrier and nano adjuvant can enhance HA specific humoral immune response.
  • nano-vaccine HA-HFT can increase HA-specific IgG antibody titer by about 10 times, and the mixed preparation of nano-adjuvant CpG-HFT and nano-vaccine can further increase specific antibody titer (about 10 times).
  • the effect of PFT as a carrier is higher than that of HFT, which may be caused by the low homology of PFT and mouse ferritin, which has a certain adjuvant effect.
  • Human HFT is highly homologous to mouse ferritin (99% identical in amino acid sequence), and HFT protein itself has no adjuvant effect.
  • Figure 10b-d ELISA analysis revealed that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and the antibody recognition ability induced by the nano-adjuvant and nano-vaccine mixture is higher than the nano-vaccine itself.
  • the primary antibody is the serum of mice vaccinated with 3 doses of vaccine, and the secondary antibody is a mouse secondary antibody labeled with horseradish peroxidase HRP).
  • Figure 10a shows that the delivery of HA by nanoparticles significantly improves the body's B cell immune response in response to HA, which is 10 times higher than HA-induced antibody titer, and when used in combination with the nano adjuvant CpG-HFT, the specific antibody gradient is again increased by 10 times, further increasing Response level.
  • the antibody titer induced by PFT carrier is higher than that of HFT carrier, suggesting that PFT has low similarity with mouse ferrtin, and PFT carrier may have certain adjuvant function.
  • Figures 10b-d show that HA-specific related antibodies can recognize influenza strains of different subtypes, including: H1NI (abbreviated P), H3N2 and H7N9 (Anhui strain, abbreviated Ahpri).
  • HA H1HA10-intein N ; HA-HFT: HFT nanoparticles deliver H1HA10; HA-HFT+CpG-HFT: HA-HFT and CpG-HFT combined preparation; HA-PFT: PFT nanoparticles deliver H1HA10; HA-PFT +CpG-HFT: HA-PFT and CpG-HFT combined preparation (Note: Since gb1 specifically binds to all types of IgG, H1HA10-intein N protein is used when detecting HA-specific antibody titer. Use protein editing scissors When cutting, gb1-based can significantly improve the solubility of the protein, so use H1HA10-intein N -gb1).
  • the nanocarrier module platform of the present disclosure can efficiently display antigen proteins, immune enhancers, and polypeptide epitopes on the surface of nanoparticles, and is used for the development of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines. field.
  • the problem of the influence of loaded protein on the stability of protein self-assembled nanoparticles and the difficulty of co-delivering adjuvant and antigen molecules with nanoparticles is solved.
  • the N-terminal of the human heavy chain ferritin (HFT) gene was fused with the gbl-intein C gene, and the target gene was amplified by overlap PCR.
  • the 5'and 3'primers contained EcoRI and XhoI restriction sites, respectively.
  • the fragments digested with EcoRI and XhoI were inserted into the pET28a linearized vector and transformed into E. coli JM109.
  • the plasmid was extracted and transformed into E. coli BL21(DE3)plysS for protein expression. Pick a single monoclonal strain, inoculate it into 20ml of kanachloramphenicol bi-anti-LB medium, and cultivate overnight at 37°C on a shaker.
  • the system uses Superpose 6Increase molecular exclusion separation and purification to obtain the recombinant protein gbl-intein c- HFT, the recombinant protein gbl-intein c- HFT self-assembles into 24-mer nanoparticles, the diameter is 18nm, the N-terminal of HFT and the connected linker gbl-intein c stretches to the surface of the nanoparticle.
  • the nanoparticle is a nanocarrier module platform.
  • the C-terminus of the polypeptide epitope SP70 is fused to the N-terminus of the intein to form the recombinant protein SP70-intein N- gb1, which serves as an exogenous protein.
  • Recombinant protein SP70-intein N- gb1 and gbl-intein c- HFT nanoparticles are mixed to form a reaction system, in which the recombinant protein SP70-intein N- gb1 and human heavy chain ferritin have an equimolar ratio, and the concentrations in the reaction system are respectively It is 30 ⁇ M.
  • intein N specifically recognizes and removes the linker gbl-intein c exposed on the surface of the nanoparticle, and the peptide epitope SP70 is covalently cross-linked with the N-terminus of human heavy chain ferritin to form the protein SP70-PFT, thus forming a monovalent Antigen delivery system.
  • Example 2 The difference from Example 1 is that the N-terminus of the heavy chain ferritin PFT gene of Pyrococcus furiosus is fused with the C-terminus of the linker gbl-intein gene, and then the target gene is amplified by the overlap PCR method, 5'primer and 3'primer respectively With EcoRI and XhoI restriction sites. Then after constructing the plasmid, transforming and sequencing, the plasmid was extracted and transformed into E. coli BL21(DE3)plysS for protein expression. After culture, induction, extraction and purification, the recombinant protein gbl-intein C- PFT was obtained.
  • the recombinant protein gbl-intein c- PFT self-assembled into 24-mer nanoparticles with a diameter of 18nm.
  • the N-terminus of the PFT and the connected connector gbl- The intein C stretches to the surface of the nanoparticles.
  • the nanoparticle is a nanocarrier module platform.
  • the C-terminus of flagellin is fused to the N-terminus of the intein to form the recombinant protein CBLB-intein N , which acts as an exogenous protein.
  • Recombinant protein CBLB-intein N and gbl-intein C- PFT nanoparticles are mixed to form a reaction system, in which the recombinant protein CBLB-intein N and PFT have an equal molar ratio.
  • the concentration of recombinant protein CBLB-intein N is 20 ⁇ M.
  • the concentration of PFT is 10 ⁇ M.
  • intein N specifically recognizes and excises the linker gbl-intein C exposed on the surface of the nanoparticle, and CBLB is covalently cross-linked with the N-terminus of PFT to form a protein CBLB-PFT, thus constituting a monovalent adjuvant delivery system.
  • the CpG-loaded rhizavidin protein and the C-terminus of the influenza virus hemagglutinin stem region H1HA10 of the same amount are fused to the N-terminus of the intein to form a recombinant protein.
  • the recombinant protein is mixed with the nanoparticles of Example 2 to form a reaction system, wherein the recombinant protein and PFT have an equimolar ratio.
  • the concentration of the recombinant protein is 10 ⁇ M and the concentration of PFT is 10 ⁇ M.
  • intein N specifically recognizes and excises the linker gbl-intein C exposed on the surface of the nanoparticle, and the recombinant protein is covalently cross-linked with the N-terminus of the PFT to form a recombinant protein-PFT, thereby forming a monovalent antigen delivery system.
  • the nanocarrier module platform provided by the present disclosure is a recombinant protein gbl-intein c- X formed by introducing a linker gbl-intein C into the N-terminus of protein X to self-assemble into polymer nanoparticles, and the N-terminus of protein X is stretched to expose the surface of the nanoparticle.
  • foreign proteins such as antigen proteins, immune enhancers, polypeptide epitopes and antibodies are efficiently and specifically covalently cross-linked to the N-terminus of protein X through intein-mediated protein editing technology to form drugs and/or Vaccine delivery systems include vaccine delivery systems, adjuvant delivery systems, and vaccine-adjuvant co-delivery systems.
  • the nanocarrier module platform provided by the present disclosure has good stability and remains stable at pH 2-8. After loading antigen proteins, immune enhancers, polypeptide epitopes and antibodies and other foreign proteins, the assembly of the nanoparticles is not destroyed, that is, the structure is not destroyed, and no refolding-reassembly is required. It not only improves the preparation efficiency of vaccine delivery systems, etc., but also solves the problem of the effect of loaded protein on the stability of protein self-assembled nanoparticles and the difficulty of co-delivering adjuvant and antigen molecules with nanoparticles. It realizes that the nano-formulation can efficiently deliver antigen and immune enhancer at the same time, and has good specificity at the same time.
  • the nano-carrier module platform provided by the present disclosure has high loading efficiency of foreign proteins such as antigen proteins, immune enhancers, polypeptide epitopes and antibodies, and has low side reactions.
  • the recombinant protein gbl-intein c- X provided by the present disclosure is highly expressed in Escherichia coli, is easy to prepare, has low manufacturing cost, and greatly reduces manufacturing cost.
  • the nanocarrier module platform provided by the present disclosure can be used in the fields of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody conjugate drugs.

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Abstract

Plateforme modulaire de nanosupport à médiation par intéine, son procédé de construction et son application. La plateforme modulaire de nanosupport permet de co-administrer un immunopotentialisateur et un antigène sur le même nanosupport, ce qui améliore significativement l'effet d'assistance d'un adjuvant. La plateforme modulaire de nanosupport à médiation par intéine est constituée de nanoparticules polymères auto-assemblées à partir d'une protéine recombinée gbl-intéinec-X par l'intermédiaire de molécules formées par fusion d'une protéine X avec l'extrémité C-terminale d'un adaptateur gbl-intéine. L'extrémité N-terminale de la protéine X se prolonge jusqu'à la surface des nanoparticules.
PCT/CN2020/089924 2019-05-21 2020-05-13 Plateforme modulaire de nanosupport à médiation par intéine, son procédé de construction et son application WO2020233460A1 (fr)

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WO2020233685A1 (fr) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 Nanovecteur à médiation intéine et son application, et nano-préparation capable de délivrer simultanément un antigène et un immunopotentialisateur

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