WO2012146364A1 - Vaccin à microparticules et/ou nanoparticules pour application prophylactique et/ou thérapeutique - Google Patents

Vaccin à microparticules et/ou nanoparticules pour application prophylactique et/ou thérapeutique Download PDF

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Publication number
WO2012146364A1
WO2012146364A1 PCT/EP2012/001738 EP2012001738W WO2012146364A1 WO 2012146364 A1 WO2012146364 A1 WO 2012146364A1 EP 2012001738 W EP2012001738 W EP 2012001738W WO 2012146364 A1 WO2012146364 A1 WO 2012146364A1
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Prior art keywords
micro
vaccine
cells
nanoparticle
components
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German (de)
English (en)
Inventor
Peter Walden
Jürgen LADEMANN
Barbara Baude
Lars DÄHNE
Karl-Heinz Wiesmüller
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Charite Universitaetsmedizin Berlin
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Charite Universitaetsmedizin Berlin
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Priority to EP12721408.8A priority Critical patent/EP2701740A1/fr
Publication of WO2012146364A1 publication Critical patent/WO2012146364A1/fr
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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K47/646—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 the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a micro- and / or nanoparticle vaccine for prophylactic and / or therapeutic use, in particular for topical application to the skin surface. Moreover, the invention relates to a pharmaceutical preparation containing the micro- and / or nanoparticle vaccine, as well as the use of the micro- or nanoparticle vaccine or such a pharmaceutical preparation for the treatment or prophylaxis of a disease.
  • a topical vaccination in contrast to the simple, flexible and painless handling also the targeted addressing of relevant structures of the immune system in the skin, which allow an effective immunization without burdening the entire organism with the often poorly tolerated substances, and the Avoidance of any risk of infection. Trained medical staff is often expendable.
  • the topical application of medicaments to the skin is based on the direct application of the active ingredient formulation to the surface of the skin, the passage of the optionally carrier-bound active ingredients through the skin and the penetration / diffusion of the active components to the site of action.
  • the topical application of only the adjuvants is described in order to intensify the immune reaction triggered by parenterally administered vaccines.
  • the skin penetration mediation technology used conforms to the above principle involving separation of the upper epidermal layer. Thereafter, the patch is adhered and the exiting wound fluid wets the vaccine, which is then to diffuse into the skin.
  • the transfer through liquid intercellular channels between the keratinocytes are discussed.
  • the hair follicles and sweat glands are expressly granted a "minimal role" in transcutaneous drug delivery, particles are proposed only for better stabilization of suspensions and as depots for the delayed release of the active ingredient components.
  • liposomes have also been used as transport systems for topically applied drugs in and through the barrier of human skin (Barry 2002). Investigations have shown that these vesicular structures do not penetrate the skin barrier as intact units, but release the active ingredients, which then further penetrate on their own.
  • nanoscale carrier systems have been developed, the main purpose of which is to transport the carrier-bound drugs directly to and into the immunocompetent cells.
  • Si0 2 nanoparticles having a size of 25 nm are used as the antigen carrier, the effectiveness of the uptake in the antigen-presenting cells of the lymph nodes and thus of the immunization via the size of the particles being controllable.
  • the addressing of the immunocompetent cells is achieved by size and geometry of the carrier particles, largely independent of their composition. The application is parenteral in both cases by intradermal or subcutaneous injection.
  • the carrier particles, together with the covalently bound active ingredients, are taken up directly by the antigen-presenting cells of the lymph nodes.
  • PAMPs Pathogen-Associated Molecular Patterns
  • bacterial lipopeptides which primarily stimulate the T-cellular immune response, are of great importance (Brown et al., 2005). Because the TLRs for lipopeptides, unlike the TLRs for most other immunomodulators, are expressed on the surfaces of the cells, bacterial lipopeptides are particularly well suited for targeting. In addition, these can be replaced by synthetic analogues, which are characterized by the unusual amino acid S- [2,3-di (hydroxy) -propyl] - [f?] Cysteine at the N-terminus, bound to the two or three fatty acids as a characteristic feature , exhibit.
  • TLR 1 / TLR2 heterodimer also reacts to triacylated synthetic lipopeptides, such as the Pam 3 Cys derivatives (EP 0210412).
  • TLR6 / TLR 2 heterodimers are sensitive to diacylated synthetic lipopeptides, such as the Pam 2 Cys derivatives (EP 0519327).
  • the lipopeptides preferably Pam 3 Cys and Pam 2 Cys derivatives as TLR 2 targeting substance, this mixed with an antigenic component for B lymphocytes or for cytotoxic T cells and other excipients to induce synergistic effects (WO 2007 / 103322, WO 2006/104389, US Pat. No. 7,387,271).
  • WO 2006/040076 also describes a topical application of such an active ingredient formulation.
  • formulations as liposomes or virus like particles which, in addition to antigens and optionally cytostatics, also contain lipopeptides, preferably Pam 3 Cys, are claimed for the treatment of various diseases and allergies (WO 2005/063201, WO 2005/063288).
  • conjugates of Pam 2 Cys and Pam 3 Cys derivatives and antigens as vaccines to specifically bind or infiltrate the antigens to the immunocompetent cells in WO 2007/078879 a combination of a membrane protein of the influenza virus and a Pam 3 Cys derivative, in WO 2009/046498 a DNA A vaccine which is coupled by polar amino acids to a Pam 2 Cys derivative, in WO 2007/079448 a linear tandem molecule consisting of a B cell epitope, a T cell epitope and Pam 3 Cys or in WO 2004/014956 and WO 2004/014957 a molecule of a B-cell or CTL epitope and a helper T-cell epitope, which are bridged by a Pam 3 Cys lipopeptide.
  • WO 2007/079448 a linear tandem molecule consisting of a B cell epitope, a T cell epitope and Pam 3 Cys or in WO 2004/014956 and WO 2004/014957 a
  • a topically applicable micro- and / or nanoparticle vaccine as defined in claim 1.
  • This vaccine can be used for prophylactic and / or therapeutic use in infections, cancer, autoimmune diseases and inflammatory and allergic conditions.
  • a vaccine according to the present invention is typically able to address with the DCs the sensor cells of the immune organ skin directly through the hair follicle as portal of entry and additionally to stimulate by a TLR-based adjuvant. Summary of the invention
  • micro- and / or nanoparticle vaccine also referred to hereinafter as a micro- and / or nanoparticle vaccine, as well as uses and methods based on this vaccine.
  • the present invention relates to novel topical vaccines which, with the aid of a particle-based carrier system, specifically target the dendritic cells (DC) of the skin via the hair follicle as portal of entry and thus induce effective prophylactic and / or therapeutic T-cell-mediated immunity.
  • DC dendritic cells
  • the present invention further relates to the composition of such a vaccine of antigenic determinants for cytotoxic effector T cells and helper T cells, and to the use of synthetic analogs of bacterial lipopeptides as TLR-based adjuvants for enhancing the specific immune response.
  • a combination of the vaccine components is immobilized on micro- and / or nanoscale carrier particles, transported to the skin by these in an active transport process in the hair follicles and temporarily deposited there.
  • the active ingredients released under the physiological conditions in the hair follicle diffuse to the target structures in the skin, stimulate the cell surface receptors of the DCs and induce a targeted vaccine antigen-specific T cell immune response.
  • the new topical microparticles and / or nanoparticle vaccines and their formulations are / are suitable as medicines on the one hand for the prevention and on the other hand for the treatment of diseases such as infections, cancer, autoimmune diseases, chronic inflammations and allergies in humans and animals.
  • the present invention provides a micro and / or nanoparticle vaccine.
  • the vaccine has basic components as individual molecules or conjugates.
  • the vaccine has a first basic component, which is one or more antigenic determinants of cytotoxic effector T cells.
  • the vaccine has a second basic component, which is one or more antigenic determinants of helper T cells.
  • the vaccine has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells.
  • the vaccine components are coupled to micro- and / or nanoscale carrier particles.
  • the micro- and / or nanoscale carrier particles are designed in such a way that after topical application to the skin surface they are actively transported into the hair follicles where they are temporarily deposited.
  • the vaccine components induce T cell-mediated immunity via stimulation of the skin's dendritic cells.
  • the micro- and / or nanoparticle vaccine according to the first aspect is a vaccine for medical use.
  • the micro- and / or nanoparticle vaccine is, in some embodiments, for therapeutic and / or prophylactic use.
  • the micro- and / or nanoparticle vaccine is a vaccine for use in a therapeutic and / or prophylactic process.
  • the present invention provides a prophylactic and / or therapeutic method using a micro- and / or nanoparticle vaccine.
  • the method comprises inducing T-cell mediated immunity.
  • Inducing T-cell mediated immunity occurs by stimulating the dendritic cells of the skin by the micro- and / or nanoparticle vaccine.
  • the vaccine has basic components as individual molecules or conjugates.
  • the vaccine has a first basic component, which is one or more antigenic determinants of cytotoxic effector T cells.
  • the vaccine has a second basic component, which is one or more antigenic determinants of helper T cells.
  • the vaccine has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells.
  • the vaccine components are coupled to the micro- and / or nanoscale carrier particles.
  • the vaccine is a vaccine according to the first aspect.
  • the method further typically includes topically applying the micro- and / or nanoparticle vaccine to the skin surface of a subject.
  • the method further comprises allowing the micro- and / or nanoscale particles to be actively transported into the hair follicles.
  • the procedure includes allowing temporary storage of the micro- or nanoscale particles in hair follicles.
  • the vaccine components induce T cell-mediated immunity via stimulation of the skin's dendritic cells.
  • the method according to the second aspect is typically a method for the prophylactic and / or therapeutic treatment of a disease of a subject, ie a disease in humans and / or animals.
  • the method according to the second aspect is a method for the prophylactic and / or therapeutic treatment of a disease in humans and / or animals associated with infections, cancer, an autoimmune disease, or an inflammatory and / or allergic condition.
  • the method according to the second aspect is a method for prophylactically specific immunization against and / or for therapeutic vaccination in a disease.
  • the method according to the second aspect is a method of supporting a conventional vaccine.
  • the method according to the second aspect is one A method of enhancing the immune response following vaccination performed by conventional vaccination techniques.
  • the present invention provides a method of vaccinating a subject.
  • the method comprises administering to a subject a pharmaceutically acceptable quality of a micro and / or nanoparticle composition.
  • the method typically involves topically applying the micro- and / or nanoparticle composition to the skin surface of a subject.
  • administering the micro and / or nanoparticle composition is sufficient to trigger an immune response of the subject.
  • the micro / nanoparticle composition comprises micro- and / or nanoparticles.
  • Basic components of the micro- and / or nanoparticle composition are coupled to the micro- and / or nanoparticles. The basic components are present as single molecules or as conjugates.
  • the micro / nanoparticle composition has a first base component that is one or more antigenic determinants of cytotoxic effector T cells.
  • the micro and / or nanoparticle composition has a second base component which is one or more antigenic determinants of helper T cells.
  • the micro / nanoparticle composition has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells.
  • the micro / nanoparticle composition is a vaccine according to the first aspect.
  • the method further comprises allowing the micro- and / or nanoparticles to be actively transported into the hair follicles.
  • the method involves allowing the micro- and / or nanoparticles to be temporarily deposited in the hair follicles. This allows the basic components of the micro / nanoparticle composition to induce T-cell mediated immunity by stimulating the dendritic cells of the skin.
  • the present invention provides a pharmaceutical preparation.
  • the pharmaceutical preparation contains a micro- and / or nanoparticle vaccine according to the first aspect, alone or in combination.
  • the preparation is in a formulation suitable for topical application.
  • the pharmaceutical preparation contains a therapeutically effective amount of the micro- and / or nanoparticle vaccine.
  • the pharmaceutical preparation according to the fourth aspect is present as a paste, ointment, cream, lotion, emulsion, gel, hydrogel, suspension, solution, spray or patch.
  • a preparation contains further constituents, such as otherwise therapeutically relevant pharmaceutical active ingredients and adjuvants, as well as further pharmaceutically suitable ingredients.
  • other pharmaceutically acceptable ingredients include, but are not limited to, excipients, adjuvants and / or adjuvants. Substances such as detergents, stabilizers and preservatives.
  • Such other ingredients which may be included in the preparation may be mixed, dissolved or associated with a physical or biological carrier.
  • the present invention relates to the use of a micro- and / or nanoparticle vaccine according to the first aspect or a pharmaceutical preparation according to the fourth aspect for a medical indication.
  • Such a medical indication can cause a disease such as an infection, cancer, an autoimmune disease, or an inflammatory and / or allergic condition.
  • a microparticle nanoparticle vaccine or pharmaceutical preparation according to the fifth aspect relates to prophylactic specific immunization against disease and / or therapeutic vaccination in a disease.
  • the use relates to enhancement of the immune response following vaccination performed by conventional vaccination techniques.
  • the use relates to the preparation of a medicament for human and / or animal for immunization.
  • immunization may be a prophylactic specific immunization against a disease.
  • immunization may also be a therapeutic vaccination in a disease.
  • immunization may be to enhance the immune response following vaccination performed by a conventional vaccination procedure.
  • Fig. 1 Principle of pH triggered adsorption and desorption of the polarized by a charge tag vaccine components on a ® in the LBL method with charged polymers coated carrier particles.
  • the release of the vaccine components takes place by recharging the top polymer layer as a function of the pH, as a result of which the electrostatic attraction is abolished.
  • FIG. 2 Dependence of the loading of the carrier particles on the order of addition of the vaccine components and the residence time in the loading solution with sequential addition
  • GILG GILGFVFTL-KKKKK fluorescein, SEQ ID NO: 11 coupled to the fluorescent dye fluoerszein
  • P2C Pam 2 Cys-GDPKHPKSF-KKKKK-Tamra, SEQ ID NO: 9 coupled to the fluorescent dye Tamra
  • Cha aK-L-Cha-VAAWTLKAAa-Aca-C-KKKKK-Cy5, SEQ ID NO: 10 coupled to the fluorescent dye Cy5).
  • Fig. 4 Schematic drawing of a loaded with the individual vaccine components carrier particle.
  • the number of components, the loading of the particles and the real stoichiometric ratios are variably adjustable according to the spectrum of action of the vaccine (the epitopes are shown by way of example for the antigenic determinants).
  • Fig. 5 Schematic drawing of a carrier particle loaded with a conjugate of the vaccine components.
  • the loading of the particles is variably adjustable according to the spectrum of action of the vaccine (the epitopes are shown by way of example for the antigenic determinants).
  • FIG. 6 Penetration of the micro- / nanoparticle vaccine into the hair follicle of the pig ear model (laser scan microscopic image of the cryosections) 60 min after application to the skin.
  • the rhodamine-labeled epitope GILGFVFTL-K4 penetrates further into the hair follicle after detachment from a silica carrier particle (labeled with Cy5).
  • Fig. 7 penetration depth of the silica carrier particles as a function of the particle size compared to the length of the terminal hair / Vellushaar follicles.
  • the target region Infundibulum is reached at about 600 ⁇ depth.
  • FIG. 8 Accumulation and residence time of the silica carrier particles in the hair follicles and in the stratum corneum.
  • FIG. 9 Effect of the oligoamino acid tags on the immunological activity of the CTL epitope GILGFVFTL in the cytotoxicity assay with the human CD8 T cell line 10-21A and the B lymphoblastoid target cell line JY (titration in the radiochrome release assay).
  • the insert shows the values transformed to Clark.
  • the functional avidity values in the table indicate the peptide concentration for half-maximal cytolysis. Factors for activity loss are related to the functional avidity of the untethered peptide.
  • FIG. 10 Comparison of the immunological activity of the particle-bound and the free individual components in the ELISpot assay on IFN- ⁇ with human CD8 T cells. 11 Titration of the immunological activity of mixtures of the particle-bound and the free vaccine components in the ELISpot assay on IFN- ⁇ with human CD8 T cells (ordinate: optical units of the densitometric measurement).
  • TNF- ⁇ , IL-1- ⁇ and IL-6 is an inflammatory cytokine cocktail as a positive control.
  • TNF, IL-4 and IL-6 is an inflammatory cytokine cocktail as a positive control (above: proportion of memory T cells formed, below: proliferation rate).
  • TNF, IL-4 and IL-6 is an inflammatory cytokine cocktail as a positive control (proliferation rate).
  • FIG. 16 Induction of antigen-specific T cell reactions by various combinations of the vaccine components on silica particles using the example of the CMV epitope
  • NLVPMVATV (SEQ ID NO: 6), detected by recombinant tetramerized MHC-peptide complexes.
  • FIG. 17 Induction of antigen-specific T-cell reactions by various combinations of the vaccine components on silica particles using the example of the influenza epitope GILGFVFTL, detected on the basis of the recombinant tetramerized MHC-peptide complexes.
  • the present invention relates to a micro- and / or nanoparticle vaccine and its use.
  • This vaccine comprises a micro- or nanoparticle to which immunologically active components are coupled.
  • the vaccine may also include both a microparticle and a nanoparticle to which immunologically active components are coupled.
  • the vaccine is a nanoparticle to which are coupled three basic components as defined below.
  • the vaccine has a plurality of micro- and / or nanoparticles to which immunologically active components are coupled.
  • microparticle refers to any particle that has an extent of about 5000 nm or less in one or more dimensions, such as an extent, eg, a diameter, in the range of about 500 nm to about 3000 nm, in the range from about 500 nm to about 2000 nm, in the range of about 500 nm to about 1000 nm, in the range of about 700 nm to about 1500 nm, in the range of about 500 nm to about 800 nm, or in the range of about 600 nm to about 900 nm.
  • nanoparticle refers to any particle having an extent in one or more dimensions, eg a diameter of about 500 nm or less, such as about 400 nm or less, about 300 nm or less, for example, in the range of about 1 nm to about 500 nm, about 5 nm to about 450 nm, about 1 nm to about 250 nm or from about 10 nm to about 150 nm.
  • a micro- and such a nanoparticle is suitably dimensioned in order to be transported to hair follicles (see below).
  • a particle contained in a vaccine according to the present invention typically has an extent of about 5000 nm or less in one or more dimensions, such as an extent, eg, a diameter, of about 3000 nm or less, a diameter of about 1000 nm, a diameter of about 700 nm or less, about 500 nm or less, about 250 nm or less, about 100 nm or less, about 80 nm or less, about 60 nm or less, about 50 nm or less , about 35 nm or less, or about 35 nm or less.
  • an extent eg, a diameter, of about 3000 nm or less, a diameter of about 1000 nm, a diameter of about 700 nm or less, about 500 nm or less, about 250 nm or less, about 100 nm or less, about 80 nm or less, about 60 nm or less, about 50 nm or less , about 35 nm or less, or about 35 nm or less.
  • a micro- or nanoparticle may have a maximum extension in the range of about 1 nm to about 1000 nm, such as about 1 nm to about 500 nm, or about 1 nm to about 100 nm.
  • nanoparticles include, but are not limited to, a nanocrystal, a nanosphere, a nanorod, a nanoshell or a nanofilament such as a nanotube or a nanowire.
  • a nanoparticle, as well as a microparticle according to the invention is an inorganic particle.
  • a micro- and / or nanoparticle may contain any suitable material - or consist thereof - such as carbon Semi-metal such as boron, germanium or silicon, or a metal such as gold, silver or copper, including an alloy thereof or a polymer.
  • a corresponding micro- and / or nanoparticle may be of any surface finish.
  • a microparticle and / or nanoparticle may have a polar or apolar surface and thus be hydrophilic or hydrophobic.
  • Hydrophobic material is water repellent or tends to separate from water. Typically, hydrophobic material has a uniform electron density distribution. In contrast, hydrophobic material contains molecules or regions which can enter into dipole-dipole interactions with water molecules and thus have a high wettability for water.
  • a use or method according to the present invention based on the use of the micro- and / or nanoparticles may be applied to any subject, such as e.g. carried out on a mammal.
  • a suitable mammal include, but are not limited to, a mouse, rabbit, rat, guinea pig, hamster, dog, cow, pig, goat, sheep, horse, rhesus monkey Schimpanze, a gorilla or a human.
  • a use or method according to the invention directly addresses the sensor cells of one of the most important organs of the immune system, namely the DCs of the skin, including the Langerhans cells.
  • DCs In their role as mediators of immunity, DCs have two key functions that can be clearly distinguished in time. As immature cells, they are responsible for the uptake and processing of antigens. Only a little later, after activation, they provide as mature cells for the stimulation mainly of T cells and indirectly via the T cells also of B cells, by giving them the processed antigen in the form of MHC-peptide complexes, together with costimulatory molecules, present on their surface while releasing cytokines that support and stabilize immune responses. As guardians and alerters of the immune system, they thus exercise a higher-level control function over the actual actors of the cellular immune response. Only one DC is needed to activate 100 to 3,000 antigen-specific T cells.
  • DCs Because of their ability to induce, enhance, and build immune responses, DCs have become an important cellular tool for immunotherapy. New approaches to fully activate and differentiate these immunocompetent cells can significantly increase the efficacy of prophylactic and therapeutic vaccines, as only mature DCs have a specific T cell-based cytotoxic immune response and the expression of proinflammatory cytokines can trigger. At the same time, mature DCs are able to reverse the effect of tumors on immunosuppressive factors.
  • DCs are the Langerhans cells. These are still inactive antigen-presenting cells that form a continuous network in the epidermis of the skin. Upon contact with the antigen, their activation and differentiation into mature DCs transports the antigens into the regional lymph nodes where they are presented to the T lymphocytes.
  • the DCs of the skin are ideal target structures for topical vaccination.
  • the hair follicle is particularly well suited, since the epidermis is crossed in this area with a particularly dense network of immunocompetent cells, which serves to monitor and defend against by the non-closed hair barrier in the hair follicle penetrating foreign substances.
  • the system of the micro- and / or nanoparticle vaccines according to the invention has succeeded for the first time in rendering carrier-bound active substances into the hair follicles and thus into the deeper layers of the epidermis by targeted utilization of a natural transport mechanism. the stratum spinosum, to convey.
  • the micro- and / or nanoparticles of a vaccine of the present invention are typically configured so that, after being administered by application to the skin surface, it may be allowed for the nanoparticles to be transported into the hair follicles. Components of the vaccine coupled to the micro- and / or nanoparticles thus likewise reach the hair follicle.
  • it may be allowed for the micro- and / or nanoparticles to remain in the hair follicle for a certain period of time (see below).
  • the highest density of immunocompetent cells of the skin is located, so that the vaccine components come in close proximity to the target structures and only have to diffuse a short distance after detachment from the carrier particle.
  • the carrier particles typically have a quadruple function.
  • the dimension of the micro- / nanoparticles is / are significant. It has been shown that one or more particles, in particular one or more spherical particles, in a size range of 100 nm to 3,000 nm, for example 300 nm to 1,500 nm (supra), are / are particularly well suited to the various vaccine components to be transported by the described pumping mechanism to the optimal position in the hair follicle, wherein the penetration depth can be adjusted by the particle size (Fig. 7). After only 1 h, the infundibulum is reached at a depth of 600 ⁇ m, where the highest density of DC of the skin is found in human terminal hair. Further penetration within the next 24 h is insignificant for vaccination success.
  • reversible adsorption may be based on electrostatic interactions, van der Waals bonds, hydrophobic interactions, and / or hydrogen bonds. For example, an interaction can occur between a polarized surface of carrier particles and charged regions of the vaccine components, which leads to adsorption. If necessary, negative or positive net charge regions may be incorporated into vaccine components in the form of one or more additional molecular tags, called tags.
  • the pH shift within the hair follicle can advantageously be used.
  • Particles were designed whose surface potential ( ⁇ -potential) changes in dependence on the pH in such a way that the vaccine components stably bind to the carrier particles during loading and transport and desorb again under the physiological conditions in the hair follicles.
  • particles were coated in the layer-by-layer (LBL) process.
  • LBL ® technology uses the electrostatic interaction between charged polymers (polyelectrolytes) and surfaces.
  • a reloading of the particles takes place, whereby the deposited amount of polymer by itself to a thickness of about 2-7 nm regulated.
  • the process can be repeated with an oppositely charged polymer. In this way, any number of layers can be built up on substrates.
  • any biocompatible polymer can be used.
  • the cationic polymers used are poly (allylamine hydrochloride) (PAH), poly (diallyldimethylammonium chloride) (PDA) and poly (acryloyloxyethyltrimethylammonium chloride) (PAOET) and as anionic polymers, the sodium salts of poly (vinyl sulfate) (PVS), sulfoethyl cellulose and polystyrene sulfonate (PSS) have been found to be particularly suitable.
  • PAH poly (allylamine hydrochloride)
  • PDA diallyldimethylammonium chloride
  • PAOET poly (acryloyloxyethyltrimethylammonium chloride)
  • anionic polymers the sodium salts of poly (vinyl sulfate) (PVS), sulfoethyl cellulose and polystyrene sulfonate (PSS) have been found to be particularly suitable.
  • LBL® surfaces having 2 to 20 layers can be used, using a weak polyanion or polycation for at least the outermost layer which alters its charge as a function of pH, such as chitosan.
  • a weak polyanion or polycation for at least the outermost layer which alters its charge as a function of pH, such as chitosan.
  • Natural proteins are also suitable as pH-switchable materials, depending on their isoelectric point.
  • an appendage having charged molecular regions such as amino acids.
  • Amino acids are known as organic molecules having a carboxylic acid group (carboxyl group) and an amino group, typically ⁇ - or ⁇ -amino acids.
  • an appendage may have about two to about 20, about three to about 15, or about 4-10 charged ⁇ -amino acids, or be composed of such amino acids. Any number of amino acids can be used, including naturally occurring amino acids and isomers thereof.
  • An appendage that has a positive or negative charge under physiological conditions is also referred to below as a charge tag.
  • such an appendage is over a peptide segment of up to about 8 amino acids in length, e.g. 1,2,3,4,5,6 or 7 amino acids coupled to a vaccine component.
  • a corresponding peptide segment can also be referred to as a spacer.
  • Such a charge tag contains one or more amino acids which have a side chain which have a positive or negative total charge under physiological conditions or consists of such amino acids.
  • a chemical group or an amino acid having a total positive charge under physiological conditions is also referred to as a basic group or amino acid.
  • a chemical group or an amino acid with a total negative charge is also referred to as an acidic group or amino acid.
  • the basic amino acids lysine, 5-hydroxy-lysine, pyrrolysine, hypusine (N6- (4-amino-2-hydroxybutyl) -lysine), histidine, ornithine, and / or arginine may be used as positively charged amino acids under physiological conditions become.
  • the presence of such amino acid units can serve for positive polarization.
  • Glutamic acid-aspartic acid or sulfotyrosine units are illustrative examples of amino acids that can be used for negative polarization. They can ensure that the vaccine components accumulate in analogy to the polymers on the oppositely charged particles and are desorbed when the particle surface is recharged by pH.
  • the length and composition of the charge tags as well as an optionally insertable spacer must be individually tailored to the respective vaccine component, since these molecules may be sterically hindered or further interactions, such as hydrogen bonds or hydrophobic interactions, may occur with the polymer surfaces.
  • the capacity and efficiency of the loading process as well as the separation kinetics can be individually adjusted by modifying the charge tags.
  • PAH / PSS PAH / PMAA coated silica particles
  • PAH / PSS PAH / PMAA coated silica particles
  • the loading of the LBL® particles in some embodiments takes place under basic conditions, the desorption of the vaccine components in such embodiments typically occurs after the transport process in the acidic environment of the hair follicle and vice versa. Due to the increased hydrogen ion concentration occurs below about pH 7 to protonate the PMAA ⁇ - groups, whereby the outer LBL discharges ® layer or recharges cationic by said underlying PAH layer. This leads to repulsion of the positively polarized vaccine components from the particle surface ( Figure 1). An additional positive effect on the release of the vaccine is the increased salt concentration in the hair follicle.
  • the individual vaccine components modified with corresponding oligoamino acid tags per 1 mg of silica carrier particles were adsorbed by this method.
  • the loading for each vaccine component can be adjusted individually.
  • the stoichiometric composition of the microparticle and / or nanoparticle vaccine according to the invention can be adjusted in accordance with the individual medical indication, the form of administration and the efficiency of the vaccine components (Example B).
  • the simultaneous loading of a carrier particle with different, differently interacting vaccine components in a defined molar ratio, as well as their simultaneous and uniform release at the site of action could be realized for the micro- and / or nanoparticle vaccine according to the invention, using the example of some active ingredient combinations.
  • a system be found particles which enables stable, effective and uniform adsorption of the vaccine components and their uniform release in the hair follicle - could therefore with the example described in the embodiments LBL ®.
  • the yields of the released in the hair follicles active ingredients in the order of 30 - 90% of the adsorbed amount are in a sufficient for vaccination order of magnitude.
  • Unwanted aggregation effects could be low loading methods, the choice of coating materials and the order of the polymer layers can be prevented.
  • composition of the subject typically topical, micro- and / or nanoparticle vaccine of the present invention may be minimized Components and lowest levels of drugs are limited.
  • a synthetic minimal vaccine which consists of the following essential basic components for addressing various synergistic targets:
  • a vaccine according to the present invention these components are interchangeable according to the medical indication and combined in any ratio.
  • any antigenic determinants for T cells e.g. multiple peptides, polyepitopes, biopolymers, or other antigenic information carriers, such as a nucleic acid, e.g. DNA or RNA, a lipid and / or an oligo- or polysaccharide.
  • antigenic determinant refers to one or more regions of an antigen or hapten that can be bound by an immunoglobulin, a T cell receptor, or a binding reagent such as a lipocalin mutein
  • An antigenic determinant has at least one epitope (see below)
  • the antigenic determinants for CTL epitopes are selected according to the desired immunization
  • the helper T-cell epitope Pan-DR-E is universal for all HLA-DR allotypes, but must be adapted to the specific species and may be advantageous when a vaccine contains a plurality of antigen determinants / epitopes to achieve adequate vaccine protection in an immunogenetically heterogeneous population.
  • nucleic acid refers to any nucleic acid in any configuration, such as single-stranded, double-stranded, or a combination thereof, to nucleic acids include DNA molecules, eg, genomic DNA or cDNA, RNA molecules, such as mRNA, analogues of DNA or RNA prepared by nucleotide analogues or by chemical nucleic acid synthesis, such as protein nucleic acid (PNA) .
  • a nucleic acid may, of course, contain non-native nucleotide analogs or be coupled to an affinity tag or label. Numerous nucleotide analogs that may be included are known to those skilled in the art.
  • a nucleotide analog is a nucleotide having a modification to, for example, the base, the sugar and / or the phosphate group.
  • a substitution of 2'-OH residues of siRNA with 2'F, 2'O-Me or 2'H residues increases the wVO stability of the RNA in question.
  • Modifications to the base group include natural and synthetic modifications of A, C, G and T / U, various purine or pyrimidine bases such as uracil-5-yl, hypoxanthine-9-yl, and 2-aminoadenine-9-yl, as well as non Purine or non-purine bases.
  • Other nucleotide analogues serve as universal bases.
  • universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases are able to form a base pair with each base. Often, base modifications can be combined with, for example, a sugar modification, eg, 2-O-methoxyethyl, for example, to achieve unique properties such as improved duplex stability.
  • a sugar modification eg, 2-O-methoxyethyl
  • the third basic component of a vaccine of the present invention is an immunomodulator, i. a compound that activates or slows down or inhibits a subject's immune response.
  • An example of an immunomodulator is immunostimulator-lipopolysaccharide (LPS).
  • Another example of an immunomodulator is cationic lipid and lipid protamine DNA, which are described in U.S. Pat. 7,303,881.
  • Another example of an immunomodulator is a synthetic bacterial lipopeptide. Lipopeptides are part of the bacterial cell wall and are recognized by the Toll-like receptors TLR 1, 2, 6 and 10 of the dendritic and other immunocompetent cells.
  • a corresponding intracellular mechanism usually the expression of specific, the antigen-specific defense of pathogens serving genes is triggered in the TLR carrier cells, which leads to the differentiation of pathogen-specific T cells.
  • the nonspecific immune response is initiated, which subsequently follows the response of the acquired immune system to the formation of specific antibodies.
  • Examples of a synthetic analog of a bacterial lipopeptide having immunostimulatory effect are e.g. disclosed in the German patent application DE 102009034779 A1.
  • the lipopeptide immunomodulator can be particularly advantageous, since with a micro- and / or nanoparticle vaccine according to the invention specifically the DCs of the skin are addressed, which have a high density of all TLR and in their guard function for Entrittspforte skin via particularly effective mechanisms as the first line of defense of the innate immune system.
  • the TLRs for the lipopeptides are expressed on the surfaces of the cells so that the lipopeptides can act directly on the cells, if appropriate also particle-bound, without being recorded in advance.
  • an immunomodulator is murabutide (N-acetylmuramyl-L-alanyl-D-glutamine-alpha-n-butyl-ester), a derivative of the bacterial muramyl dipeptide.
  • immunomodulators that are immunosuppressants are cyclosporin and methotrexate.
  • Two other immunomodulating modulators are 4- (3-chlorophenyl) -N- (4- (trifluoromethyl) phenyl) pyrimidin-2-amine (VAF347) and fingoli- mod (720, 2-amino-2- (2- (4- octylphenyl) ethyl) propane-1,3-diol).
  • the direct coupling of the antigen-specific vaccine components with the immunomodulatory effective adjuvant allows a highly focused abolition of the conditioned immunosuppressive state of the skin and thus a marked increase in the T cell induced by the micro- and / or nanoparticle vaccine according to the invention -based, specific immune response. This further minimizes the possibility of immunological side effects.
  • a lipopeptide immunomodulator according to the present invention has the following general structure (I):
  • R1 and R2 which may be the same or different, represent a saturated or unsaturated acyl radical containing from about 6 to 28 carbon atoms or from about 7 to about 25, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 C atoms, for example for a saturated acyl radical having 16 C atoms,
  • R3 is a saturated or unsaturated acyl radical containing from about 6 to 28 carbon atoms or from about 7 to about 25, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 C atoms, for example for a saturated acyl radical having 16 C atoms, or H and R4 is a from 1 to 25 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues, physiologically acceptable and in the treated species not per se immunogenic peptide, for example with the amino acid sequence GDPKHPKSF (SEQ ID NO: 1).
  • SEQ ID NO: 1 amino acid sequence GDPKHPKSF
  • Treating species refers to the species that belongs to the subject being treated or in this context, the use of other known adjuvants, such as squalene or alumina, is not excluded.
  • the induction of the desired immunological reaction is fundamentally dependent on the simultaneous administration of all three components, which however should be coupled to the same or different carrier particles depending on the situation and antigen (embodiment D).
  • the vaccine of the invention consists of a plurality of micro / nanoparticles.
  • each component can be bound to the polarized particle surface via its own charge tag (Figure 4).
  • conjugates are the following general structures (II), (III) or (IV).
  • n is in each case a natural number from 0 to 8, for example 1, 2, 3, 4, 5, 6 or 7.
  • R 1, R 2, R 3, R 4, R 5 and R 6 are molecular units which may be identical or different can.
  • R1, R2, R3, R4, R5 and R6 are independently selected peptide regions having amino acid sequences that are from 0 to about 8 amino acids in length, such as 1, 2, 3, 4, 5, 6 or 7 amino acids. Any such peptide region can serve as a molecular spacer and accordingly can be referred to as an amino acid spacer.
  • T cell epitope and T cell epitope 2 represent an amino acid sequence of an epitope for cytotoxic effector T cells or an epitope for helper T cells. T cell epitope and T cell epitope 2 may be the same or different.
  • An “epitope” is antigenic and thus may be understood to be defining as an "antigenic structure” or an “antigenic determinant.”
  • Immunoglobulins and T cell receptors can recognize and bind an epitope, and an epitope is also a region of an antigen to which An epitope is a region of a molecule that may be linear, ie, an amino acid sequence, for example, in a linearly contiguous form, that is derived from, for example, an immunoglobulin, a T cell receptor, or a binding reagent such as a lipocalin mutein Imunglobulin or T cell receptor recognized epitope.
  • a linear epitope generally contains at least three, and typically at least about 5, eg, 8-10 amino acids.
  • An epitope may also be a conformationally-dependent epitope in which, unlike a linear epitope, the primary amino acid sequence is not the only component of the recognized epitope, for example because it can not be recognized as such by an immunoglobulin or T cell receptor.
  • An immunoglobulin, a T-cell receptor, or a binding reagent such as a lipocalin mutein recognize a three-dimensional structure of the antigen in a conformationally-dependent epitope.
  • a conformationally-dependent epitope typically contains a greater number of amino acids than a linear epitope.
  • a conformation-dependent epitope can be determined, for example, by X-ray crystallography, two-dimensional NMR spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy.
  • lipopeptide stands for an immunomodulator according to formula (I)
  • day stands for a sequence of 4 to 12, e.g. 4-10, including about 5, about 6, about 7, about 8, or about 9 basic (positive charge) or acid (negative charge) amino acids.
  • Such a conjugate is then immobilized on the polarized carrier particle over the charge tag, as shown in FIG.
  • vaccines such or similar conjugates are advantageous, depends on the medical indication as well as the contained vaccine components and antigen determinants. In the investigations it could be shown that the spatial proximity of certain epitopes in some cases has an advantageous effect on the immunization success, in other cases has no influence.
  • the form of binding to the carrier particle charge tags, single / mixed loading
  • the type of conjugation must also be specifically adjusted.
  • the present invention therefore relates to a micro- and / or nanoparticle vaccine which is suitable for prophylactic and / or therapeutic use.
  • the vaccine consists of at least the basic components
  • CTL epitopes polyepitopes, biopolymers, proteins, DNA, RNA, polysaccharides,
  • Glycolipids glycoproteins or similar carriers of antigenic information
  • DCs preferably synthetic analogs of bacterial lipopeptides
  • the vaccine components are reversibly coupled by a charge tag to micro / nano-scale carrier particles
  • the immunological potential of the micro- and / or nanoparticle vaccines according to the invention was determined by means of in vitro experiments with model vaccines containing the CTL epitopes GILGFVFTL (SEQ ID NO: 5) from the influenza matrix protein M1 or NLVPMVATV (SEQ ID NO: 6 ) out the cytomegalovirus CMV early antigen pp65 containing helper T-cell epitope Pan-DR-E (aK-L-Cha-VAAWTLKAAa-Aca-C) and Pam 2 Cys-GDPKHPKSF as immunomodulator (Examples C and D) ).
  • the carrier particles are deposited in the hair follicles for about 5 to 10 days and by a
  • the particle-bound vaccine components of the model vaccines in human effector T cells elicit a specific T cell mediated immune response that is qualitatively and quantitatively comparable to the response to the unbound vaccine components and also the charge tags have no deleterious effect on the have immunological activity of the vaccine components;
  • helper T cell epitope Pan-DR-E additionally stimulates the T cells and, via the T cells, also the DCs;
  • helper T-cell epitope Pan-DR-E and lipopeptide immunomodulator enhances or even makes possible the proliferation and differentiation of naive T cells.
  • Another object of the invention is the preparation of the vaccine components according to the invention and their modification with charge tags (see above), as illustrated in Example A.
  • the synthesis of the epitopes, as well as the charge tags, in some embodiments consisting of a series of 4-10 basic (positive charge) or acid (negative charge) amino acids, can be carried out in solution or solid phase synthesis according to methods of protecting group chemistry known to those skilled in the art ,
  • the charge tags attached N-terminal or C-terminal, in some embodiments C-terminal, are already considered in the construction of the amino acid sequence.
  • a payday may consist of a sequence of identical amino acid units or of a sequence of different amino acid units.
  • a charge tag contains or consists of negatively charged but not positively charged amino acid units or positively charged but not negatively charged amino acid units.
  • a charge tag may also have uncharged amino acid units. Usually, however, one charge day contains more charged than uncharged amino acid units.
  • a vaccine or a nanoparticle or microparticle of the invention contains a charge tag that is a series of four, five, six, or seven lysine units, a series of four, five, six, or seven pyrrolysine units, a series of four , has five, six or seven histidine units and / or a sequence of four, five, six or seven arginine units or consists thereof.
  • a vaccine or a nanoparticle or microparticle of the invention contains a payday that is a series of four, five, six or seven sulfotyrosine units, a series of four, five, six or seven aspartate units and / or a sequence of four, five, six or seven glutamate units or consists thereof.
  • the target compound is obtained as a stereochemically defined derivative consisting of L-amino acids, of the modified amino acid S- [2,3-di (hydroxy) - (2f-J-propyl] - [/!] - cysteine and of two or three Acyl residues is constructed and has a higher biological activity.
  • a vaccine component according to the invention can also be modified as a so-called "prodrug compound", for example as prodrug ester, prodrug peptides or the like
  • a prodrug compound is understood as meaning a molecule which in some cases is itself pharmacologically inactive, but in In other cases, a prodrug compound is less active than a pharmacologically (fully) active agent formed therefrom in the body of the subject, thus allowing a prodrug compound to function as a precursor molecule
  • a prodrug compound may contain residues that are cleaved from the molecule in vivo, for example, by an enzymatically catalyzed reaction such as hydrolysis or oxidation, Thus, a hydroxy group present in a pharmacologically active compound may be present in a prodrug.
  • Verbindun g may be in the form of an ester, an amine may be in the form of an amide, or a carboxyl group may be in the form of an ester or an amide.
  • a carboxyl group present in a pharmacologically active compound can also be present as a hydroxy group which can be oxidized in vivo.
  • coupling of cell-penetration promoting molecules, e.g. Biotin or maleimidopropionic acid, optionally via suitable spacer molecules, to the primary amino group or the acylation of the amino group, the bioavailability and thus the effectiveness of the compounds of the invention can be improved.
  • Another object of the invention is the use of a micro- and / or nanoparticle vaccine according to the invention for the preparation of a pharmaceutical preparation for humans and / or animals for a medical application.
  • One such application may be the prophylactic specific immunization against and / or for therapeutic vaccination in a disease such as an infection, cancer, autoimmune disease, or an inflammatory and / or allergic condition, as well as for the support of a conventional vaccine.
  • Such an immunostimulatory active composition containing a therapeutically effective amount of at least one microparticle and / or nanoparticle vaccine according to the invention may further comprise one or more immunological-action-enhancing adjuvants, pharmaceutically suitable excipients, auxiliaries and / or additives, such as, for example, detergents, Solubilizers, stabilizers or preservatives, as well as otherwise otherwise treatment-relevant agents or skin care products.
  • immunological-action-enhancing adjuvants such as, for example, detergents, Solubilizers, stabilizers or preservatives, as well as otherwise otherwise treatment-relevant agents or skin care products.
  • Pharmaceutically acceptable materials are the substances known to be useful in the pharmaceutical and food technology industries and in adjacent fields, in particular those listed in the relevant pharmacopoeias whose properties do not preclude physiological application.
  • micro- / nanoparticle vaccine The effects produced by a micro- / nanoparticle vaccine according to the invention are also dependent on its formulation.
  • a corresponding pharmaceutical preparation should allow the direct application of the drug to the untreated skin.
  • suitable formulations for the micro- and / or nanoparticle vaccines according to the invention for example, pastes, ointments, creams, lotions, emulsions, gels, hydrogels, suspensions, solutions, sprays or patches, or for example aqueous or ethanolic gels and suspensions or oil Water emulsions.
  • a sustained-release formulation is also suitable which can be prepared by methods of pharmaceutical galenics known to the person skilled in the art.
  • composition of a pharmaceutical preparation according to the invention is carried out in the usual way by methods known to those skilled in the art, as described in the relevant pharmacopoeias, wherein the components can be mixed together, dissolved or associated with a physical or biological carrier.
  • a pharmaceutical preparation according to the invention can additionally be sterilized.
  • the invention also relates to the use of a micro- and / or nanoparticle vaccine according to the invention or a pharmaceutical preparation containing it for the treatment and prevention of one or more human and animal diseases associated with infections, cancer, autoimmune diseases or inflammatory and allergic conditions stand.
  • a subject that is to say a person or an animal requiring such a treatment, has a therapeutically effective amount of a microparticle and / or nanoparticle vaccine according to the invention or a pharmaceutical preparation according to the invention applied directly to the untreated skin.
  • the formulation can be massaged in with a finger or optionally with a massage applicator.
  • additional templates can be used for the size of the treated area of the skin.
  • the order quantity can be specified by the packaging unit.
  • the efficiency of uptake of the micro- and / or nanoparticle vaccine may optionally be increased. Occlusal volumes or patches are other ways to make the drug application.
  • micro- / nanoparticle vaccines and pharmaceutical preparations according to the invention can be used alone, in combination with other micro / nanoparticle vaccines or pharmaceutical preparations contained in the present invention or in combination with one or more active substances which are suitable for the respective medical indication are relevant.
  • the temporal sequence of the application is not restricted. It may be administered either at the same time, before or after the other active substances, as a separate medicinal product or combination preparation and on the same or different routes of administration.
  • the application of different vaccines or parts thereof at different locations and at different times corresponds to the subject matter of the invention.
  • the exact therapeutically effective amount for a subject to be treated, as well as the molar composition of the vaccine or vaccine combination, will depend on various factors, including the nature and course of the disease, size, stature, age and health of the patient, the route of administration, and the specifics used Antigen determinants and, if appropriate, of the other medicaments used. Thus it does not make sense to specify the exact quantity at this time. However, one can in principle start from a 1 to 3-fold application of the pharmaceutical preparations according to the invention over a period of a few weeks to a half year.
  • the amount of active ingredient used should usually be in the range of 0.5 pg to 2.0 mg per application and vaccine component.
  • the exact dosage specification depends on the specific application and composition of the micro- and / or nanoparticle vaccine and requires further investigation in the clinical trials.
  • the main advantages of a vaccine and a method or an application of the present invention in which this is used consist in the non-invasive application, the direct transport of the vaccine components through the hair follicle to the target structures and the amplification of the immune reaction by the lipopeptide Immunomodulators.
  • the topical application form of a microparticle and / or nanoparticle vaccine according to the invention is simple and flexible to handle and, as such, independent of medical personnel.
  • topical vaccination with a micro- and / or
  • Nanoparticle vaccine superior to the previously dominant systemic and invasive route of administration.
  • a micro- and / or nanoparticle vaccine according to the invention is on the one hand suitable for the prevention of infectious diseases. Due to the unspecific approach of the vaccination strategy according to the invention, a wide range of known infectious diseases can be covered by the selection and combination of T cell epitopes and antigens for B cells.
  • a micro- and / or nanoparticle vaccine according to the invention has decisive advantages in the self-medication of chronically ill patients and in the immunization of vaccine intolerances.
  • topical vaccination is extremely beneficial for the vaccination of children as it saves the children the painful experience associated with the injection.
  • the non-invasive principle of the micro- and / or nanoparticle vaccine significantly reduces the risk of infection in vaccines and avoids significant amounts of clinical waste.
  • a micro- and / or nanoparticle vaccine according to the invention a new dimension of vaccine development is opened, which allows a rational vaccine design based on the known immunological knowledge and the specific selection of the epitopes.
  • the principle underlying the invention for overcoming the skin barrier is also open to further development as a general principle for topically applicable drug delivery systems.
  • the general method for preparing the vaccine components according to the invention in particular the peptidic epitopes and the synthetic analogs of bacterial lipopeptides, as well as their modification with the peptidic charge tags with the methods of solid phase synthesis will be described.
  • Structural modifications can be made by skipping individual synthetic steps or adding steps known to the skilled synthetic chemist.
  • a) Synthesis of the Amino Acid Sequences with Automated Solid-Phase Synthesis The synthesis of the peptides proceeds according to classical techniques known from the literature in solution or in an automated process on the solid phase. The solid phase synthesis procedures are the same for almost all amino acid sequences. For this reason, in the example, only the basic synthesis procedure is described, without going into the peculiarities of individual peptides.
  • the peptides are synthesized using a synthesis robot starting from a Fmoc-L-Lys (Boc) -loaded synthesis resin according to the Fmoc peptide synthesis method. If other amino acids are used for the C-terminal charge tag, one starts in an analogous manner with the corresponding Fmoc-protected derivatives.
  • the loaded trityl chloride-polystyrene resin (8 mg, 7 pmol) is weighed into a reactor.
  • the amino acids are dissolved in a 1-hydroxybenzotriazole solution (0.5 M in N, N-dimethylformamide) to 0.5 molar solutions.
  • the filled reactors and vials are placed on the intended positions of the synthesis robot.
  • the cleavage of the Fmoc protective group takes place according to the program sequence shown in Tab. Tab. 1: Cycle for cleavage of the Fmoc protective group.
  • the program sequence of the synthesizer robot is shown in the following table.
  • reaction solution is stirred for 3 h at room temperature and then evaporated to dryness using the rotary evaporator.
  • the solution is then evaporated to half the volume on a rotary evaporator, mixed with potassium hydrogen sulfate solution (5%, 5.2 mL) and stored at -2 ° C. for 16 h.
  • the product is extracted with dichloromethane (3x50 mL). After drying with sodium sulfate, the organic phase is evaporated on a rotary evaporator to dryness.
  • a side chain protected peptide GDPKHPKSF-KKKK (SEQ ID NO: 2) loaded synthesis resin (5.22 pmol) is added with N, N-dimethylformamide (100 pL) and allowed to swell in a reactor for 5 minutes.
  • the resin (5.22 pmol) loaded with Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2f-S) -propyl] - [ ⁇ ] cysteinyl-GDPKHPKSF-KKKK is treated with N, N-dimethylformamide ( 100 pL) and allowed to swell for 5 min in a reactor.
  • N-dimethylformamide supernatant After filtering off the N, N-dimethylformamide supernatant, ⁇ , ⁇ -diisopropylethylamine (3 M in N, N-dimethylformamide, 24.4 pL, 73.2 pmol, 14 equivalents), palmitic acid (1 M in N, N-dimethylformamide / dichloromethane (1: 1); 104 ⁇ _; 104 ⁇ ; 20 equivalents) and N, N-diisopropylcarbodiimide (3 M in N, N-dimethylformamide; 24.4 L; 73.2 ⁇ mol; 14 equivalents) are pipetted to the resin.
  • the cleavage of the Fmoc and the side-chain protective groups is carried out by the amino acid sequences of the epitope peptides.
  • the cleavage solution trifluoroacetic acid / triisopropylsilane / water (92.5: 5: 2.5, 300 L) is pipetted to the resin (2.6 pmol) and the suspension left at room temperature for 2 h. Subsequently, the cleavage solution is filtered with compressed air into a test tube. Subsequently, another 200 ⁇ _ cleavage solution is pipetted to the resin and the suspension is left for another 30 minutes at room temperature. After re-filtration of the cleavage solution with compressed air, the resin is washed with dichloromethane (200 ⁇ L). The filtrates and the washings are combined and evaporated on a rotary evaporator. The residue is digested with diethyl ether (3 ⁇ 300 ⁇ _) and then centrifuged. The sediment is taken up in tert-butyl alcohol / water (4: 1, 2 mL) and freeze-dried (18 h).
  • the resin (2.6 pmol) loaded with S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [/ -] cysteinyl-GDPKHPKSF-KKKK (SEQ ID NO: 3) is labeled with N, N-dimethylformamide (100 ⁇ _) and allowed to swell for 5 min in a reactor.
  • the cleavage of the product from the resin is carried out according to the method described under h).
  • Example B Reversible immobilization of the vaccine components on the carrier particles
  • the vaccine components were labeled with different fluorescent dyes and the decrease in color in the supernatant of the loading solutions was monitored by UV / vis or fluorescence spectroscopy. The results are shown in the following table and Fig. 2.
  • the unlabeled vaccine components were adsorbed on the surface of the carrier particles in analogy to the respectively optimized loading strategy.
  • the particles were incorporated at pH 8 (10 mM TRIS buffer) in aqueous agarose gel 0.5%. After adding a preservative (ethyl paraben, benzalkonium chloride, ethyl hydrobenzoate), the finished and storage-stable formulations were stored until use.
  • a preservative ethyl paraben, benzalkonium chloride, ethyl hydrobenzoate
  • Example C Transport of the micro / nanoparticle vaccine into the hair follicles and to the
  • the penetration depth of the particles is controllable by the particle size, wherein the nature of the particles plays almost no role. While particles with a diameter of approx. 500 nm to 600 nm reach penetration depths of> 1000 pm, smaller and larger particles penetrate much less deeply (FIG. 7). Explanation for this phenomenon is a mainly mechanical effect. Since the thickness of the hair shells is also about 500 nm, a gear pump effect can be assumed, which is initiated by the movement of the hair in the hair follicle. This is the more pronounced, the more similar particle size and Haarschuppendicke are. In vitro, this effect can be simulated by massage.
  • the target structures of the topical vaccine, the DCs, are particularly abundant in the infundibulum area of the hair follicles. This is located at a depth of up to 600 pm and is reached by the vaccine components already after 1 h. Within In the next 24 h a minimal further diffusion takes place, which is of little importance for the vaccination.
  • spherical silica particles with a diameter of 300 nm to 1,500 nm have proven to be particularly suitable carrier particles. b) Long-term storage of the carrier particles in the hair follicle
  • GILGFVFTL (SEQ ID NO: 5) from the influenza matrix protein M1 (GILG) or
  • NLVPMVATV (SEQ ID NO: 6) from the cytomegalovirus CMV early antigen pp65
  • PBMC peripheral white blood cells
  • iDC immature dendritic cells
  • mDC mature dendritic cells
  • the model epitope GILG was titrated with different variants of C-terminal extension by histidine (H), lysine (K) or glutamate (E) over 12 orders of magnitude and the induction of cytolytic activity in the human cytotoxic T-cell clone 10-21A tested against the B-lymphoblastoid cell line JY.
  • H histidine
  • K lysine
  • E glutamate
  • the modification of the C-terminus of the epitope is expected to reduce the stimulatory capacity.
  • the epitope with the H 4 -tag is 45-fold, that with K t -tag 131-fold and that with E 4 -tag 708-fold less potent than the unmodified epitope.
  • the vaccination effect i. the stimulation of the adaptive immune system, and especially the effector T cells, depends on the successful induction of the differentiation of iDCs into mDCs and their activation into potent immunostimulatory cells. This process can be monitored by expression of costimulatory surface molecules on the DCs.
  • DCs which were differentiated and activated as described under c) with differently loaded carrier particles or which were not or only partially matured because of the lacking immunomodulator, were labeled with T cells of the same donor from whose PBMC the DCs were generated Incubated for 7 days. The T cells were then examined by flow cytometry for proliferation and differentiation. Separate detection of the CD45RA-expressing naive T cells and the CD45RO-expressing memory T cells were separated from PBMC on the basis of specific antibodies using magnetic separation and incubated separately with the DCs.
  • CFSE Proliferation was detected by CFSE assay.
  • the T cells are labeled with the cell-stable fluorescent dye CFSE before stimulation.
  • the dye is diluted 1: 1 so that the rate of proliferation can be determined from the decrease in cell-associated fluorescence.
  • the differentiation of naive to memory T cells could be detected with differentiation markers.
  • naive T cells Figure 14
  • DCs preincubated with particles loaded with the P2C immunomodulator induce proliferation but hardly any differentiation, similar to an inflammatory cytokine cocktail (TNF + IL-4 + IL-6).
  • the helper T cell epitope PADRE has a similar but much weaker effect.
  • DCs pretreated with P2C and PADRE-bearing particles induce both high proliferation and differentiation of a high proportion of naive T cells, and thus exactly the immunological effect expected of a prophylactic vaccine.
  • the DCs induce proliferation after incubation with the immunomodulator P2C or the helper T cell epitope PADRE, as well as with the cytokine cocktail.
  • the immunomodulator P2C or the helper T cell epitope PADRE as well as with the cytokine cocktail.
  • no qualitative differentiation can be observed here, but a clear enhancement of the effect when PADRE and P2C-bearing particles are combined. This effect reflects the situation of therapeutic vaccination or revaccination of immune individuals.
  • human peripheral blood iDCs were cultured for two days with carrier particles coated with the T-cell epitopes NLVPMVATV [SEQ ID NO: 6] ( Figure 16) or GILGFVFTL [SEQ ID NO: 5] ( Figure 17), the immunomodulator P2C, or combinations thereof, and then combined with CD8 T cells and incubated for 7 days. Thereafter, the proliferation of effector T cells with specificity for influenza or CMV was investigated by recirculating tetramerized HC-peptide complexes (HLA-A2.1 molecules carrying the respective peptides GILG and NLVP, respectively) by flow cytometry in the cell cultures. The iDCs and effector T cells were each generated from peripheral blood of the same healthy donor.
  • the strongest immune stimulation was achieved with a mixture of particles carrying the epitope and the immunomodulator separately. So here is no coupling request. A mixture of the particles is more advantageous.
  • induction of antigen-specific T cells depends on the concomitant administration of the specific epitope and the immunomodulator, which, depending on the situation and antigen, should be coupled to the same particles or uncoupled.
  • the few tetramer and CD8-positive cells that have already been detected in the T-cell cultures with or without iDCs show that the donors in these examples have long been in contact with the viruses from which the epitopes originate have had. The experiments carried out thus correspond to the situation of a therapeutic or Revakzinleiter.
  • the investigated concentration range from 1 f M to 500 ⁇ for the model epitopes Pan-DR-E, GILGFVFTL (SEQ ID NO: 5) and NLVPMVATV (SEQ ID NO: 6 ) and for the lipopeptide adjuvants no evidence of a toxic effect of the micro- / nanoparticle vaccine according to the invention.

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Abstract

L'invention concerne de nouveaux vaccins topiques qui, à l'aide d'un système support à base de particules, visent spécifiquement les cellules dendritiques de la peau et induisent ainsi une immunité prophylactique et/ou thérapeutique efficace induite par les lymphocytes T. L'invention concerne en outre la composition d'un tel vaccin à partir de déterminants antigéniques pour des lymphocytes T cytotoxiques effecteurs et auxiliaires, ainsi que l'utilisation d'analogues synthétiques de liptopeptides bactériens comme adjuvants à base de TLR afin de renforcer la réponse immunitaire spécifique. Une combinaison de composantes vaccinales est immobilisée sur des particules supports à l'échelle micrométrique et/ou nanométrique ; après application sur la peau, elle est transportée par celles-ci dans les follicules pileux, sous la forme d'un processus actif de transport, et elle y est déposée temporairement. Les substances actives libérées dans le follicule pileux en fonction des conditions physiologiques diffusent en direction des structures ciblées de la peau, stimulent les récepteurs superficiels des cellules dendritiques et induisent une réponse immunitaire voulue des lymphocytes T. Les nouveaux vaccins topiques à microparticules et/ou nanoparticules et leurs formulations conviennent d'une part pour la prévention et d'autre part pour le traitement de maladies telles que des infections, le cancer, les maladies autoimmunes, les inflammations chroniques et les allergies, chez l'homme et l'animal.
PCT/EP2012/001738 2011-04-23 2012-04-23 Vaccin à microparticules et/ou nanoparticules pour application prophylactique et/ou thérapeutique Ceased WO2012146364A1 (fr)

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DE102016005550A1 (de) 2016-05-09 2017-11-09 Emc Microcollections Gmbh Adjuvans zur lnduzierung einer zellulären lmmunantwort
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WO2023040121A1 (fr) * 2021-09-18 2023-03-23 苏州尔生生物医药有限公司 Système de vaccin pour la prévention ou le traitement du cancer sur la base de cellules cancéreuses multiples et/ou de composants cellulaires entiers de tissu tumoral, et sa préparation et son application
WO2025179633A1 (fr) * 2024-02-28 2025-09-04 无锡博斯顿生物医药有限公司 Vaccin pour induire une tolérance immunologique, son procédé de préparation et son utilisation
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DE102016005550A1 (de) 2016-05-09 2017-11-09 Emc Microcollections Gmbh Adjuvans zur lnduzierung einer zellulären lmmunantwort
DE102016005550B4 (de) 2016-05-09 2024-09-26 Hans-Georg Rammensee Adjuvans zur lnduzierung einer zellulären lmmunantwort
RU2633928C1 (ru) * 2016-08-24 2017-10-19 Федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский национальный исследовательский государственный университет имени Н.Г. Чернышевского" Способ трансдермальной доставки биологически активных веществ
WO2023040121A1 (fr) * 2021-09-18 2023-03-23 苏州尔生生物医药有限公司 Système de vaccin pour la prévention ou le traitement du cancer sur la base de cellules cancéreuses multiples et/ou de composants cellulaires entiers de tissu tumoral, et sa préparation et son application
WO2025179633A1 (fr) * 2024-02-28 2025-09-04 无锡博斯顿生物医药有限公司 Vaccin pour induire une tolérance immunologique, son procédé de préparation et son utilisation

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