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WO2017220099A1 - Adjuvants aux propriétés de drainage modifiées - Google Patents

Adjuvants aux propriétés de drainage modifiées Download PDF

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Publication number
WO2017220099A1
WO2017220099A1 PCT/DK2017/050208 DK2017050208W WO2017220099A1 WO 2017220099 A1 WO2017220099 A1 WO 2017220099A1 DK 2017050208 W DK2017050208 W DK 2017050208W WO 2017220099 A1 WO2017220099 A1 WO 2017220099A1
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poly
liposomes
antigen
trimethylammonium
propane
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PCT/DK2017/050208
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English (en)
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Dennis Engelmann CHRISTENSEN
Peter Lawætz ANDERSEN
Signe Tandrup SCHMIDT
Camilla FOGED
Henrik FRANZYK
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Statens Serum Institut
University Of Copenhagen
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Publication of WO2017220099A1 publication Critical patent/WO2017220099A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • the invention discloses a method of controlling the surface charge of liposomes and thereby the cellular targeting and drainage properties by anchoring hydrophilic coating polymers onto the liposomes by electrostatic interactions using polar tags and a method for coating charged vaccine delivery systems with hydrophilic polymers via attractive electrostatic interactions using oppositely charged residues in the anchoring part of the coating and adjuvants comprising such liposomes to control the net surface charge, drainage properties and cellular targeting.
  • a prerequisite for efficient induction of cellular immunity is the activation of antigen presenting cells (APCs) with ligands for pattern recognition receptors (PRRs).
  • a diverse array of pathogen- associated molecular patterns (PAMPs) binds to the various families of PRRs, e.g. the Toll-like receptors (TLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs).
  • TLRs Toll-like receptors
  • NLRs NOD-like receptors
  • CLRs C-type lectin receptors
  • Different PAMPs induce different types of adaptive immune responses, depending on which signaling pathways are triggered, eventually determiningwhich cytokine milieus and expression of co- stimulatory molecules are induced to direct the T-cell differentiation.
  • DCs Several subtypes of DCs have been identified in mice with different functionalities that can be distinguished via their expression of specific cell surface markers (Figure 2.).
  • conventional DCs comprise of two subsets, namely 1) lymphoid organ-resident DCs which as the name implies stay in the lymphoid organs and collect foreign matters taken there by the lymph and 2) tissue-migratory DCs which sweep the non-lymphoid organs, sample foreigen matters and transport them to the draining lymph nodes where they can activate naive antigen-specific T cells.
  • CD8a+ DCs have thus been shown to be the principal APC responsible for the induction of CD8 T cell immunity against infections with both viruses and intracellular bacteria (Belz, Smith et al. 2004, Heath, Belz et al. 2004, Belz, Shortman et al. 2005). It has furthermore been shown that mice deprived of CD8a+ DCs cannot cross-present virus antigens, resulting in a highly abolished virus-specific CD8 T cell immune response (Hildner, Edelson et al. 2008).
  • liposomes displaying a positive surface charge are more - immunogenic than neutral or negatively charged (anionic) liposomes (Christensen, Korsholm et al. 2007, Korsholm, Agger et al. 2007, Henriksen-Lacey, Bramwell et al. 2010, Christensen, Korsholm et al. 2011).
  • One of their primary merits is that they are taken up more efficiently by APCs because of their ability to adsorb to the anionic heparan sulfate proteoglycan-coated cell surface (Korsholm, Agger et al. 2007).
  • a vaccine containing cationic liposomes will typically form a vaccine depot at the injection site (SOI) and be taken up at the SOI) by migratory DCs resulting in a strong induction of systemic cell-mediated immune (CMI) responses (Christensen, Henriksen-Lacey et al. 2012).
  • Cationic liposomes forms depot e.g. they migrate very slowly from the site of injection. In some cases it is not desirable to have a formation of depots of liposomes at the site of injection (SOI) but it could be advantageous with some drainage to the local lymphnodes.
  • Liposomes are often used as carriers for drugs, e.g. in cancer treatment, carriers of
  • liposomes avoid interaction with the immunesystem.
  • PEGylation is the adsorption or grafting of poly(ethyklene)glycol (PEG) or PEG- containing copolymers to the surface of nanoparticles such as liposomes.
  • Cationic liposomes e.g. made from amphiphilic quaternary ammonium compounds are wellknown lipids used in adjuvant compositions. Preferred quaternary ammonium compounds are the bromide- , chloride- , sulfate- , phosphate- or acetate- salt of
  • DDA dimethyldioctadecylammonium
  • DODA dimethyldioctadecenylammonium
  • quaternary ammonium compounds are 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), 1 , 2-dimyristoyl-3-trimethylammonium-propane, 1 ,2- dipalmitoyl-3-trimethylammonium-propane, 1 ,2-distearoyl-3-trimethylammonium-propane and dioleoyl-3-dimethylammonium propane (DOTMA).
  • DOTAP 1,2-dioleoyl-3- trimethylammonium propane
  • DODAP dioleoyl-3-dimethylammonium propane
  • DODAP dioleoyl-3-dimethylammonium propane
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium
  • the cationic liposomes are not colloidally stable in aqueous solution but can be stabilized with a glycolipid in the membrane.
  • a well-known glycolipid for stabilizing the liposomes is alpha, alpha'-trehalose 6, 6' dibehenate (TDB) or alpha, alpha'-trehalose 6,6'-dimycolate (TDM) which both have immunostimulating properties.
  • DDA/TDB DDA/TDB
  • CAF01 DDA/TDB
  • CD8 T cell-inducing liposomal adjuvant which consists of the synthetic TLR3 ligand poly(l:C) and the novel immunostimulator monomycolyl glycerol (MMG) (Andersen, Rosenkrands et al. 2009, Andersen, Agger et al. 2009), associated with liposomes based on the cationic surfactant
  • DDA dimethyldioctadecylammonium
  • the antigen and/or adjuvant should be delivered to APCs not migrating to the site of injection but to lymphnode resident B cells or dendritic cells.
  • lymphnode resident B cells or dendritic cells increasing the free drainage of the vaccine from the site of injection to the lymphnode would be desireable so the vaccine would be delivered directly to resident DCs able to cross present the antigen and induce a CD8 T cell response.
  • a fraction of vaccine has to drain to the local lymph nodes because the vaccine should be delivered to lymph node-resident CD8+ DCs.
  • PEG polyethylene glycol
  • diacylglycerophospholipids highly stable towards hydrolysis under both acidic and basic conditions.
  • WO99/55306 describes the situation in which lipid anchored PEG stabilizes and increases circulation time of a neutral small uni-lamellar vesicles (SUV) carrying blood coagulations factor FVIII or other proteins/peptides associating to neutral lipids and or PEG, after i.v. injection.
  • SUV neutral small uni-lamellar vesicles
  • cationic liposomes PEGylated by lipid anchoring are utilized for gene therapy. Furthermore liposomes serve as delivery system for gene therapy and not for vaccines.
  • Neutral liposomes PEGylated by lipid anchoring are utilized for targeting of neutral liposome particles larger than 300 nm to the spleen carrying e.g. cancer therapeutics and/or adjuvants in order to amplify the immune response.
  • liposomes including PEGylated lipids are applied for the delivery of immunogen-encoding RNA.
  • the mechanism of association between liposomes and PEG is based on apolar interactions.
  • Kaur et al. (Kaur, Bramwell et al. 2012) describes the incorporation of different concentrations of lipid anchored hydrophilic coatings into DDA/TDB liposomes. They report that this leads to an increased drainage of the vaccine to the lymph nodes but also report an altered immune response with decreasing Th1 and increasing Th2 response. This illustrates that PEGylation does increase the potential of PEGylation of Cationic liposomes for targeting cells resident in the lymphoid organs. The reason for the Th2 bias PEGylated adjuvant is suggestively reduced targeting to migratory DCs and thus reduced Th1 induction.
  • Finsinger et al. (Finsinger, Remy et al. 2000) describes the use of PEG with reactive linkers based on anionic peptides, to coat positively charged nonviral gene vectors by electrostatic interactions.
  • the linkers are designed to be cleaved from the PEG at reduced PH resembling cytosolic environment, thus releasing the DNA when the nanoparticle has entered the cytosol of the cells.
  • WO2012/062867; WO2013/033563 is part of the initial formation of the liposomes, which results in a reduced ability to complex vaccine antigens and immunostimulators to the surfaces of the vaccine delivery system, since the antigens ad-mixed to a PEG coated liposome formulation cannot interact with the liposome surface due to sterically hindrance from the PEG chains .
  • This amount of complexing is an important parameter to obtain an effective vaccine, since co-localization of immunostimulator and antigen is a prerequisite for the induction of strong T cell induction (Kamath et al./Christensen et al. 2012).
  • -Most vaccine antigens and some immunostimulators are associated to the cationic adjuvant formulation via electrostatic interactions. If the polymer coating is based on hydrophic interactions as is the case with afore mentioned prior art (i.e. WO96/10391; Kaur et al. 2012; WO99/55306; WO2009/111088;
  • immunostimulator to the vaccine delivery system and then coat the charged particle surface hydrophilic coatings constituting an opposite charge or to add-mix the coating together with the antigen to get partial association between antigen and adjuvant.
  • This invention covers a method of controlling the drainage of a vaccine from the site of injection and the amount of antigen bound to the liposomes by the use of hydrophilic coatings onto vaccine delivery systems by electrostatic interactions using anionic residues in the anchoring part of the coating.
  • This enables the coating to be added to the vaccine formulation as the very last component, thus ensuring that both antigen and adjuvant components which are complexed to the vaccine delivery system will be shielded by the hydrophilic coating.
  • Polymer shielding using apolar association thus reduces the ability to complex the antigen and immunostimulator to the vaccine delivery system to obtain co-delivery to the desired APCs.
  • the invention furthermore covers the design of hydrophilic coatings which can be displaced after vaccination.
  • the kinetics of this displacement can be controlled by the strength of the electrostatic interaction, i.e. by adjusting the amount of anionic residues in the anchoring part of the hydrophilic coating intended for a cationic delivery system.
  • This displacement can furthermore be designed to take place only under specific physiologic conditions, such as acidic environment in the lysosomes, by incorporating anchoring parts that will only be
  • the present invention discloses a method of controlling the surface charge of liposomes or shielding vaccine delivery systems by anchoring hydrophilic coating polymers onto the liposomes by electrostatic interactions using polar tags.
  • the polar tags are oppositely charged residues attached in the anchoring part of the coat.
  • the hydrophilic coating polymers are chosen from poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s.
  • the polar tags are anionic residues such as 1) polyanionic peptides such as peptides containing two or more aspartic or glutamic acid residues. 2) polyphosphates defined as n tetrahedral P04 units linked together by sharing oxygen centres, such that end phosphorus groups share one oxide and the others phosphorus centres share two oxide centres, 3) polyanionic polysaccharide derivatives such as polyanionic cellulose and 4) synthetic sulphated polymers.
  • Negatively charged residues in the anchoring part (tags) of the coat are chosen from
  • polyanionic peptides e.g., peptides containing two or more aspartic or glutamic acid residues, polyphosphates defined as n tetrahedral P04 units linked together by sharing oxygen centres, such that end phosphorus atoms share one oxide and the others phosphorus centres share two oxide centres, polyanionic polysaccharide derivatives such as polyanionic cellulose and synthetic sulphated polymers.
  • the invention also discloses adjuvant formulations comprising liposomes coated with hydrophilic polymer with negatively charged residues in the anchoring part of the coat.
  • the liposomes are preferably cationic liposomes such as dimethyldioctadecylammonium (DDA), dimethyldioctadecenylammonium (DODA), 1 ,2-dioleoyl-3-trimethylammonium propane
  • DOTAP dioleoyl-3- dimethylammonium propane
  • DODAP dioleoyl-3- dimethylammonium propane
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium
  • the liposome can comprise a glycolipid, e.g., TDB or MMG.
  • the liposomes are coated with the hydrophilic polymer after complexing or associating antigens and immunostimulators to the liposomes.
  • Preferred cationic lipids used for preparing cationic liposomes are chosen from
  • DDA dimethyldioctadecylammonium
  • DODA dimethyldioctadecenylammonium
  • DOTAP dimethyldioleoyl- 3-trimethylammonium propane
  • DODAP dioleoyl-3-dimethylammonium propane
  • DODAP dioleoyl-3-dimethylammonium propane
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium
  • Another embodiment of the invention is delivery systems or vaccine adjuvants formulations comprising liposomes coated with hydrophilic polymer anchored polar tags according to said method.
  • the hydrophilic coating polymers are chosen from poly(ethyleneglycol),
  • Preferred adjuvant formulations comprise cationic liposomes made from
  • the cationic liposomes preferably comprises an
  • the adjuvant or delivery system comprises liposomes where the surface charge of liposomes has been altered with a coating of hydrophilic polymers chosen from poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2- hydroxypropyl)methacrylamide] and poly(amino acid)s where the hydrophilic polymer has been electrostatically anchored with polar tags such as polyanionic peptides such as peptides containing two or more aspartic or glutamic acid residues, polyphosphates defined as n tetrahedral P04 units linked together by sharing oxygen centres, such that end phosphorus groups share one oxide and the others phosphorus centres share two oxide centres, polyanionic polysaccharide derivatives such as polyanionic cellulose and synthetic sulphated polymers.
  • hydrophilic polymers chosen from poly(ethyleneglycol), poly(oxazoline), poly(
  • This invention covers a method for coating charged delivery systems with hydrophilic (stealth) polymers via attractive electrostatic interactions using oppositely charged residues in the anchoring part of the coat. This is hypothesised to affect the drainage of the vaccine from the SOI and the degree of adsorption of antigen to the liposomes. This allows for the addition of the shielding after adsorption of antigen/immunostimulator to the liposomes, thus ensuring that both antigen and adjuvant components, which are adsorbed to the vaccine delivery system, may be shielded by the hydrophilic coating.
  • the invention furthermore covers the design of hydrophilic coatings which may be desorbed after vaccination.
  • the kinetics of this desorption process may be controlled by varying the strength of the attractive electrostatic interaction, e.g. by varying the number of charged residues in the anchoring part of the hydrophilic coating intended for an oppositely charged delivery system. This desorption may be designed to take place only under specific
  • Liposomes are defined as closed vesicles structures made up of one or more lipid bilayers surrounding an aqueous core. Each lipid bilayer is composed of two lipid monolayers, each of which has a hydrophobic "tail” region and a hydrophilic "head” region. In the bilayer, the hydrophobic "tails” of the lipid monolayers orient toward the inside of the bilayer, while the hydrophilic "heads” orient toward the outside of the bilayer. Liposomes can have a variety of physicochemical proper-ties such as size, lipid composition, surface charge, fluidity and number of bilayer membranes.
  • liposomes can be categorized as unilamellar vesicles (UV) comprising a single lipid bilayer or multilamellar vesicles (MLV) comprising two or more concentric bilayers each separated from the next by a layer of water.
  • UV unilamellar vesicles
  • MLV multilamellar vesicles
  • Water soluble compounds are entrapped within the aqueous phases/core of the liposomes opposed to lipophilic compounds which are trapped in the core of the lipid bilayer membranes.
  • Liposomes have been used as delivery systems in pharmacology and medicine such as immunoadjuvants, treatment of infectious diseases and inflammations, cancer therapy, radiographic contrast medium and gene therapy. In vaccine technology the delivery system is often an adjuvant.
  • An adjuvant is defined as a substance that non-specifically enhances the immune response to an antigen. Depending on the nature of the adjuvant it can promote a cell- mediated immune response, a humoral immune response or a mixture of the two. Since the enhancement of the immune response is non-specific, it is well understood in the field that the same adjuvant can be used with different antigens to promote responses against different targets e.g. with an antigen from M. tuberculosis to promote immunity against M. tuberculosis or with an antigen derived from a tumor, to promote immunity against tumors of that specific kind.
  • Factors which may have an influence on the adjuvant effect of the liposomes are liposomal size, lipid composition, and surface charge.
  • Dendritic cells can be used as antigen delivery vehicles. Loading of antigen to antigen-presenting cells, such as dendritic cells, have shown to be an effective method for generating active T-cells with a role in antitumor immunity.
  • cationic lipid includes any amphiphilic lipid, including synthetic lipids and lipid analogs, having hydrophobic backbone and polar head group moieties, a net positive charge at physiological pH, and which by itself can form spontaneously into bilayer vesicles or micelles in water.
  • Stealth coating is the coating of adjuvant delivery systems with a "hydrophilic coating polymer” shielding the delivery system from the immune system and interstitial proteins.
  • Hydrophilic coating polymer is defined as hydrophilic head group component selected from polymers based on poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein the polymer may be linear or branched, and wherein the polymer may be optionally substituted.
  • the molecular weight for the hydrophilic coating ranges from 350 g/mole to 5000 g/mole.
  • Anchoring of the polymer is defined as the linking of the hydrophilic coating polymer to the liposomes surface. This classically happens through lipidation of the polymer and insertion of the polymer linked lipids into the liposomal membrane. This anchoring is referred to as apolar association.
  • Anchoring applied in this invention is electrostatic interaction in which the polymer is covalently linked to polar tags of opposite charge than the liposome surface rendering electrostatic association between polymer and liposome membrane possible.
  • the polar tag can be based on peptides, charged polymers, small molecules etc.
  • the core of the delivery system is comprised of liposomes which are preferably cationic liposomes, which are made from cationic lipids.
  • a preferred cationic lipid comprises a quaternary amine with a halogen counter ion of the formula NR1 R2R3R4-hal, wherein R1 and R2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R3 and R4 independently each is an alken containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms, and hal is a halogen atom, not comprising any nucleic acid as adjuvant.
  • the R1 and R2 groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R3 and R4 may be dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl nonadecenyl and eicocenyl groups.
  • C-"12-C20 alkenes are possible because even though the R3 and R4 groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g.
  • halogen atom "hal" is preferably bromine or chlorine because the other halogens, fluorine and iodine, may have undesirable biochemical, physiological and injurious effects, but for some experimental purposes, where such effects can be accepted, they may also be selected.
  • cationic lipid compounds suitable as adjuvant formulations are1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), 1 , 2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1 ,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1 ,2-distearoyl-3- trimethylammonium-propane (DSTAP) and dioleoyl-3-dimethylammonium propane (DODAP), Cholesteryl 3b-N-(dimethylaminoethyl)carbamate hydrochloride (DC-Choi) and N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA).
  • DOTAP trehalose 6'6'-dibehenate
  • TDB trehalose 6'6'-dibehenate
  • the adjuvant or delivery system can additionally comprise an immunostimulator.
  • the immunostimulator is preferably selected from the group of so-called pathogen-associated molecular patterns (PAMPs) which comprises e.g. TLR-ligands e.g. MPL (mono-phosphoryl lipid A) or derivatives thereof, polyinosinic polycytidylic acid (poly-IC) or derivatives thereof, flagellin, CpG oligodeoxynucleotides, Resiquimod, Imiquimod, Gardiquimod, nucleotide- binding oligomerization domain NOD-like receptors e.g. muramyldipeptide, C-type lectins e.g.
  • PAMPs pathogen-associated molecular patterns
  • TLR-ligands e.g. MPL (mono-phosphoryl lipid A) or derivatives thereof, polyinosinic polycytidylic acid (poly-IC) or derivatives thereof
  • the immunomodulator can also be selected from the group of pathogen-associated molecular patterns for which no receptor has been identified yet e.g MMG or derivatives thereof (PCT/DK2008/000239 which is hereby incorporated as reference), zymosan, tamoxifen, or ligands for other pathogen-pattern recognition receptors such as muramyl dipeptide (MDP) or analogs thereof.
  • MDP muramyl dipeptide
  • the liposomes are made by the thin film method or by high shear mixing as described in WO2013004234.
  • an antigenic component When used as a vaccine adjuvant an antigenic component is added to the adjuvant dispersion.
  • An antigenic component or substance is a molecule, which reacts with preformed antibody and/or the specific receptors on T and B cells.
  • a molecule that can stimulate the development of specific T or B cells leading to the formation of a memory population of immune cells that will promote a faster "memory" response if the antigen is encountered a second time by immune cells. Since memory populations are rarely clonal, in practice this means that an antigen is any molecule or collection of molecules, which can stimulate an increase in immune responses when it is re-encountered by immune cells from an individual who has previously been exposed to it.
  • the antigenic component or substance can be a polypeptide or a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays de-scribed herein.
  • the immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope.
  • the hydrophilic coating comprises polymers based on poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2- hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein the polymer may be linear or branched, and wherein the polymer may be optionally substituted.
  • the molecular weight for the hydrophilic coating ranges from 350 g/mole to 5000 g/mole.
  • Anionic anchor can consist of any anionic chemical/biological compound which will interact with the cationic surface. Examples hereof are: 1) polyanionic peptides such as peptides containing two or more aspartic or glutamic acid residues. 2) polyphosphates defined as n tetrahedral P04 units linked together by sharing oxygen centres, such that end phosphorus groups share one oxide and the others phosphorus centres share two oxide centres, 3) polyanionic
  • polysaccharide derivatives such as polyanionic cellulose and 4) synthetic sulphated polymers. Furthermore this invention relates to the binding of this coating by the aid of electrostatic interactions, thus permitting the complexing of other vaccine components, such as antigens and immunostimulators, to the cationic liposomes before the formation of a hydrophilic coating.
  • the hydrophilic coating of cationic liposome based vaccine adjuvants shield the liposomes from the instant interaction with interstitial proteins after injection.
  • the invention provides enhanced passive diffusion of the vaccine into the lymphatics to reduce/prevent aggregation at the injection site caused by interaction between matrix proteins and the cationic lipid surface by associating hydrophilic polymer coatings by electrostatic interactions obtained by covalently binding an anionic anchor residue to the end of the polymer.
  • the invention also covers the use of hydrophilic polymer coatings, which upon exposure to extracellular fluid will be shredded from the liposome surface due to i.e. competition with other anionic compounds or pH changes modifying the charge of the anionic anchor and thus the adsorption strength.
  • Another embodiment of the invention encompass the use of anionic anchors on the hydrophilic coating polymers, which dependent on their size and overall charge are more or less prone to be shredded from the surface of the cationic liposomes upon injection.
  • the vaccine delivery systems comprises cationic liposomes, such as the DDA based liposomes used in CAF01 and CAF09 which are coated with hydrophilic coatings, such as poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2- hydroxypropyl)methacrylamide] and poly(amino acid)s.
  • hydrophilic coatings such as poly(ethyleneglycol), poly(oxazoline), poly(ethylene oxide), polyvinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2- hydroxypropyl)methacrylamide] and poly(amino acid)s.
  • the delivery system will carry antigen and immunostimulators such as TDB, MMG, MPL, pIC, CpG, Resiquimod, Imiquimod, MDP etc complexed to the delivery system, which will only be exposed to the in vivo environment when the hydrophilic coating is displaced from the vaccine. This can e.g. happen when the vaccine is exposed to interstitial proteins or when the vaccine ends up in the lysosome of APCs.
  • antigen and immunostimulators such as TDB, MMG, MPL, pIC, CpG, Resiquimod, Imiquimod, MDP etc complexed to the delivery system, which will only be exposed to the in vivo environment when the hydrophilic coating is displaced from the vaccine. This can e.g. happen when the vaccine is exposed to interstitial proteins or when the vaccine ends up in the lysosome of APCs.
  • Figure 1 side-by-side immunization creates increased Ab responses
  • Figure 2 PEGylation of CAF09 increase CD8 T cell responses
  • Figure 3 Illustration of the invention rationale
  • FIG. 7 Mice were immunized with the model-antigen OVA, OVA adjuvanted with CAF09 and OVA adjuvanted with CAF09-PEG-peptide (CAF09-PP) Top: Association of OVA with lymphocytes in the draining lymph nodes upon intramuscular immunization. Bottom:
  • FIG. 8 Mice were immunized with the model-antigen OVA, OVA adjuvanted with CAF09 and OVA adjuvanted with CAF09-PEG-peptide (CAF09-PP). Inflammatory responses at the site of injection 24 h after immunization.
  • Figure 9 Illustration and examples of how the order of add-mixing of hydrophilic anionic tag and antigen can affect the antigen/adjuvant interaction.
  • Figure 10 Evaluation of how the order of add-mixing of hydrophilic anionic tag and antigen can affect the immune profile of a vaccine.
  • EXAMPLE 2 In order to verify that PEGylation of the CAF09 adjuvant increase the CD8 responses mice (4- 8/group) were immunized s.c. with a dose of 10 ⁇ g unadjuvanted OVA (Ag) or OVA adjuvanted with CAF09 (+CAF09) at a dose of 250/50/50 ⁇ g DDA/MMG/poly(l:C), or OVA adjuvanted with CAF09 incorporating 10% DSPE-PEG, all in a dose-volume of 200 ⁇ in TRIS-buffer. Mice received three immunizations with two-week intervals, and the immune responses were evaluated eight days after the final immunization. Blood lymphocytes were separated and stained for antigen specific CD8+ T cells using siinfekl tetramer. As illustrated in figure 2, the PEGylation significantly increased the Ag specific CD8 T cell responses. EXAMPLE 3:
  • Coating of cationic liposomes with hydrophilic coatings using lipid linkers has do be done during the preparation of the liposomes, which reduces, not only the depot effect after immunization, but also the ability to associate with antigens and immunostimulatory compounds through electrostatic interaction (figure 3 A)
  • the present invention utilizing hydrophilic coating through electrostatic interaction by linking PEG to anionic tags, such as polyanionic peptides, polyphosphates etc. will make it possible to either associate antigens and immunostimulators before coating to enable full association (figure 3B), simultaneously with the hydrophilic coating to obtain partial association of the antigens and immunostimulators in a competitional manner or after the hydrophilic coating to reduce association of the antigens and immunostimulators.
  • Examples of compounds to be used for hydrophilic coatings of cationic surfaces through electrostatic interactions could be one or more polyethylene glycol (PEG) chains linked to peptides of varying charge.
  • Figure 4 illustrates examples hereof where one (monoPEG) or two (bisPEG) chains were associated to peptides with 2-4 repeat sequences of GDGDY thus carrying 4-8 anions.
  • EXAMPLE 5
  • mice were immunized s.c. with a dose of 10 ⁇ g unadjuvanted CTH522 (Ag) or Ag adjuvanted with
  • lymphocytes and B cells in the draining lymph nodes was evaluated by using flow cytometry. At 24 hours after immunization, significantly more antigen was associated with lymphocytes in the mice immunized with OVA/CAF09-PEG-peptide, as compared to mice immunized with
  • OVA/CAF09 The increased pro-inflammatory response may arise from dissociation of poly(l:C), PEG-peptides and/or OVA from the liposomes due to competition with interstitial proteins.
  • EXAMPLE 9 the increased pro-inflammatory response may arise from dissociation of poly(l:C), PEG-peptides and/or OVA from the liposomes due to competition with interstitial proteins.
  • Order of add-mixing vaccine Ag and hydrophilic anionic tag can influence the vaccine association to the cationic liposomes: Admixing the Ag before hydrophilic anionic tag will make full association possible. Admixing Ag after, on the other side can block the Ag association whereas admixing Ag and hydrophilic anionic tag simultaneously will facilitate partial Ag association. None of these situations are absolute, and the outcome will be influenced by the nature of the Ag, the pi of the AT, the molecular size of hydrophilic anionic tag and the concentrations of each individual component. One example is shown in the graphs Figure 9, where ⁇ OVA was added to CAF01 , either before or after addition of different concentrations of PEG peptide (aa10-bisPEG; PP).
  • mice (4/group) were immunized s.c. with a dose of 1 ⁇ g unadjuvanted CTH522 (Ag) or CTH522 adjuvanted with CAF01 at a dose of 250/50 ⁇ g DDA/TDB or CTH522 adjuvanted with CAF01 with 10 mole% aa10-bisPEG (PP) added before or after antigen adsorption to the adjuvant, all in a dose-volume of 200 ⁇ in TRIS-buffer.
  • Mice received two immunizations with three-week intervals, and the immune responses were evaluated 14 days after the final immunization.

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Abstract

L'invention porte également sur un procédé de contrôle de la charge de surface de liposomes par ancrage de revêtement de polymères hydrophiles sur les liposomes par des interactions électrostatiques utilisant des marqueurs polaires et des adjuvants ayant des propriétés de drainage modifiées comprenant de tels liposomes.
PCT/DK2017/050208 2016-06-24 2017-06-23 Adjuvants aux propriétés de drainage modifiées WO2017220099A1 (fr)

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WO2021209562A1 (fr) * 2020-04-15 2021-10-21 Statens Serum Institut Composition liposomale pour la prévention ou le traitement précoce d'une infection pathogène

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021209562A1 (fr) * 2020-04-15 2021-10-21 Statens Serum Institut Composition liposomale pour la prévention ou le traitement précoce d'une infection pathogène
CN115515561A (zh) * 2020-04-15 2022-12-23 史坦恩斯血清研究所 预防或早期治疗病原性感染的脂质体组合物

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