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WO2003000232A2 - Procede de preparation de vesicules chargees d'oligodeoxynucleotides immunostimulateurs (iss-odn) et utilisations diverses de celles-ci - Google Patents

Procede de preparation de vesicules chargees d'oligodeoxynucleotides immunostimulateurs (iss-odn) et utilisations diverses de celles-ci Download PDF

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WO2003000232A2
WO2003000232A2 PCT/IL2002/000507 IL0200507W WO03000232A2 WO 2003000232 A2 WO2003000232 A2 WO 2003000232A2 IL 0200507 W IL0200507 W IL 0200507W WO 03000232 A2 WO03000232 A2 WO 03000232A2
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odn
iss
lip
combination
liposome
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WO2003000232A3 (fr
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Yechezkel Barenholz
Eliezer Kedar
Yigal Louria-Hayon
Aviva Joseph
Eyal Raz
Kenji Takabayashi
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Yissum Research Development Co of Hebrew University of Jerusalem
University of California Berkeley
University of California San Diego UCSD
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Yissum Research Development Co of Hebrew University of Jerusalem
University of California Berkeley
University of California San Diego UCSD
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Priority to AU2002311617A priority Critical patent/AU2002311617A1/en
Publication of WO2003000232A2 publication Critical patent/WO2003000232A2/fr
Anticipated expiration legal-status Critical
Publication of WO2003000232A3 publication Critical patent/WO2003000232A3/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
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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
    • A61K39/145—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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention generally relates to Hposomal formulations and in particular to a method for the preparation of liposome vesicles loaded with immunostimulatory oligodeoxynucleotides (ISS-ODN) and to the different uses of its products.
  • ISS-ODN immunostimulatory oligodeoxynucleotides
  • lipid vesicles formed by natural or synthetic phospholipids as vehicles for the administration of effective substances.
  • Amphotericin B an effective but toxic antifungal, was the first liposomally formulated agent to be licensed for parenteral use.
  • Antitumor agents like adriamycin have also been incorporated into liposomes, as well as vaccines, adjuvants and biological response modifiers like cytokines and others.
  • Liposomes are also utilized as vehicles in the field of gene transfer [Kastel P.L, and Greenstein R.J., Biotechnol. Annu. Rev. 5:197-220 (2000)]. In another application, liposomes were used for the delivery of therapeutic proteins. N. Sakuragawa et al. [Thrombosis Research 38:681-685, (1985); Clinical Hematology 29(5):655-661 (1988)]report that liposomes containing factor VIII have been prepared for oral administration to patients suffering from von Willebrand's disease.
  • factor VIII The encapsulation of factor VIII was carried out by dissolving the protein factor
  • liposomes loaded with biological structures, biopolymers and/or oligomers are obtained by co-drying a fraction of an amphipathic material in an organic solvent and a fraction of the biological structure(s), biopolymers and/or oligomers, from an aqueous medium.
  • the present invention aims for the providence of a novel method for efficient encapsulation (>60%) of oligomers, particularly those being pharmaceutically active, into lipid membrane vesicles.
  • a group of oligomers of particular interest according to the present invention are oligonucleotides and especially, the immunostimulatory oligodeoxynucleotides and analogs (ISS-ODN or CpG motifs).
  • ISS-ODN are short synthetic oligodeoxynucleotides (6-30 bases) usually containing an active 6-mer sequence that has the general structure of two 5' purines, an unmethylated CpG dinucleotide, and two 3' pyrimidines (Pu-Pu-CpG-Pyr-Pyr).
  • Bacterial DNA and its synthetic ISS-ODN are known to be potent stimulators of both innate immunity and specific adaptive immune responses, including direct activation of monocytes/macrophages, dendritic cells, NK cells and B cells. Further, bacterial DNA and its synthetic ISS-ODN induce the production of pro-inflammatory cytokines (e.g., IL-6, IL-12, IFNs, TNF ⁇ ) and up-regulate the expression of MHC I, MHC II and co-stimulatory molecules [Van Uden J., and Raz, E. in Springer Semin. Immunopathol. 22:1-9 (2000)].
  • pro-inflammatory cytokines e.g., IL-6, IL-12, IFNs, TNF ⁇
  • ISS-ODNs exhibit strong Thl and mucosal adjuvanticity to a wide range of antigens [McCluskie, M.J., et al. Vaccine, 19:2657-2660 (2001)] or allergens [Homer, A.A., et al. Immunol Rev. 179:102-118 (2001)]. Furthermore, pretreatment with ISS-ODN, even without concomitant administration of the relevant antigen, was shown to afford protection (for about 2 weeks) against subsequent infection with intracellular pathogens [Klinman, D.M., et al. in Springer Semin. Immunopathol 22: 173- 183 (2000)], indicating activation of innate immunity.
  • ISS-ODNs The immunostiinulatory activity of ISS-ODNs requires cellular uptake by endocystosis following their binding to a receptor belonging to the Toll-like receptor family, TLR9. Endosomal acidification and digestion of the ODN followed by interaction with specific protein kinases results in rapid generation of reactive oxygen intermediates, leading to activation of MAPK and NF- KB pathways and subsequent cytokine production (Chu, W, et al. Cell 103:909-918 (2000)].
  • mice In mice, doses of 50-100 ⁇ g per mouse of soluble ISS-ODN, and in many cases two or more administrations were required to achieve the desired immunomodulatory effects. This relatively high dose and repeated administration, in theory, may cause adverse reactions resulting from the "cytokine storm" induced [Wagner, H., et al.
  • liposomes can effectively entrap various drugs, which are slowly released over an extended period of time in vivo, and can rapidly and effectively be uptaken by macrophages and dendritic cells, it is suggested that liposomes can serve as an efficient delivery system for ISS-ODN based vaccines [Alving, CR. (1997) in New generation vaccines, 2 nd ed. (Levine, M.M., Woodrow, G.C., Kaper, J.B., and Cobon, G.S., eds.), Marcel Dekker, New York, pp. 207-213; and Kedar, E. and Barenholz, Y (1998) in The biotherapy of cancers: from im unotherapy to gene therapy (Chouaib S, ed.), INSERM, Paris, pp. 333-362].
  • the present invention is based on the surprising finding that step-wise hydration of lipids, a priori freeze dried, with an aqueous solution containing ISS-ODN to be encapsulated in said liposomes, results in a very effective encapsulation of the ISS-ODN as compared to hitherto known encapsulation methods.
  • the present invention provides a method for loading ISS-ODN in liposomes, the method comprising:
  • liposome as used includes all spheres or vesicles of amphipathic substance which may spontaneously or non-spontaneously vesiculate, for example phospholipids, where at least one acyl group is replaced by a complex phosphoric acid ester.
  • loading means any kind of interaction of the oligomeric substances to be loaded with the liposomes, for example, an interaction such as encapsulation, adhesion, entrapment (to the inner or outer wall of the vesicle) or embedding in the liposome membrane, with or without extrusion of the ISS-ODN.
  • liposome-forming lipid ⁇ denotes any physiologically acceptable amphipathic substance that contains groups with characteristically different properties, e.g. both hydrophilic and hydrophobic properties or a mixture of such molecules, and which upon dispersion thereof in an aqueous medium, form vesicles.
  • this term refers to a single amphipathic substance or to a mixture of such substances.
  • the amphipathic substance includes, inter alia, phospholipids, sphingolipids, glycolipids, such as cerebrosides and gangliosides; PEGylated lipids and sterols, such as cholesterol and others.
  • drying refers to any manner of drying the liposome-forming lipids, which results in the formation of a dry lipid cake. According to one preferred embodiment, drying is achieved by freeze drying, also referred to as lyophilizing. Alternatively, drying may be achieved by spray drying.
  • solubilizing which is used herein interchangeably with the term “dissolving” or “dispersing” may be achieved by a single use of the bulk aqueous medium with which said solubilization is achieved. However, this term preferably refers to step-wise addition of two or more aliquots of the said medium solubilizing the solute.
  • POST post-encapsulation
  • CO co-encapsulation
  • CO is an encapsulation method, which includes co-drying the liposome forming lipid(s) and the ISS-ODN (co- lyophilized) after which they are hydrated together with an aqueous medium.
  • ISS-ODN does not need to be exposed to any organic solvent or detergent that may be destructive to its activity.
  • the method of the present invention enables to obtain vesicles with substantially high loading rate of ISS-ODNs (more than 60%). This feature is unique since it improves efficiency of treatment or prophylaxis with ISS-ODN loaded into the liposomes as well as it enables to reduce the dose and frequency/number of administrations required in order to achieve a desired therapeutic effect. Further, since by the present invention, the liposome-forming lipids and the ISS- ODN are kept separately, it enables combinatorial formulations, i.e. the physician may prescribe any combination of liposome-forming(s) substance and biological agent, and upon need, the pharmacist can easily formulate the selected combination.
  • the freeze-dried lipids are provided with a long shelf-life at 4°C or at room temperature, preserving their entrapment capability for over two years (data not shown), and the hydration of the lipids with the ISS-ODN solution to form the liposomes is very simple and requires only several minutes. Therefore, the Hposomal formulation can be readily prepared before treatment (bedside), ensuring high pharmaceutical stability of the formulation and without leakage of the entrapped material from the liposome.
  • a combination of two compositions including a first composition comprising dry liposome- forming lipids and a second composition comprising ISS-ODN, the combination is for use in the preparation of a pharmaceutical formulation comprising Hposomal ISS-ODN.
  • the present invention also concerns a combination of at least one composition of dry liposome-forming lipid or a dry mixture of liposome-forming lipids; and at least one composition comprising ISS-ODN; further comprising instructions for selection and use of the first and second compositions for the preparation of a therapeutic formulation, said instructions comprising hydrating said dry liposome-forming lipid(s) with an aqueous solution of said ISS-ODN to yield a pharmaceutical formulation comprising liposomes loaded with ISS-ODN; and further comprising instructions prescribing administration of said pharmaceutical formulation to a subject in need of said formulation.
  • the present invention also concerns pharmaceutical formulations comprising as active ingredient a therapeutically effective amount of Hposomal ISS-ODN; the Hposomal ISS-ODN being prepared by the method of the invention as disclosed hereinabove and below.
  • the pharmaceutically "effective amount”, including also a prophylactically effective amount, for purposes herein, is determined by such considerations as are known in the art.
  • the amount refers to that of ISS-ODN must be effective to achieve the desired therapeutic effect.
  • therapeutic methods comprising administration to a subject in need an effective amount of Hposomal ISS-ODN prepared according to the present invention, optionally in combination with other active agents, such as antigens.
  • the therapeutic method comprises the prevention or treatment of a disease.
  • prevention or treatment refers to administering of a therapeutic amount of Hposomal ISS-ODN which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, to prevent the disease form occurring, to prime the immune response against the disease to be treated, or a combination of two or more of the above.
  • treatment in the context used herein refers to prevention of a disease from occurring.
  • the treatment (also preventative treatment) regimen and the specific formulations to be administered will depend on the type of disease to be treated and may be determined by various considerations known to those skilled in the art of medicine, e.g. the physicians.
  • the invention concerns a method for achieving a therapeutic effect, the therapeutic effect comprising stimulating an immune response of an individual, the method comprising administration to said individual an amount of Hposomal ISS-ODN effective to achieve said therapeutic effect, wherein said effect is to an extent greater than that obtained by administration to the individual free ISS-ODN, the Hposomal ISS-IDN being prepared by the method of the invention.
  • the amount administered to the individual is a dosage of up to 2,000 mg of ISS- ODN loaded Hposomal vesicles (measured by phospholipid), per kg body weight (wt).
  • Fig. 1 shows the therapeutic effect of Leishmanial infection in mice by free ISS-ODN (ODN 1018, O), Hposomal M-ODN (ODN 1019, ⁇ ), or Hposomal ISS-ODN,
  • n 10 Balb/c mice per group.
  • Fig. 2 shows the therapeutic effect of Leishmanial infection by a free anti-Leishmania drug, Amphotericin B (Fungizone, O), Hposomal ISS-ODN
  • ODN 1018, ⁇ Hposomal ISS-ODN in combination with Fungizone (•); Saline served as the control ( ⁇ ).
  • Fig. 3 shows the therapeutic effect of Leishmanial infection by free Amphotericin B derivative (AmB-AG, •), Liposomal M-ODN (ODN 1019, ⁇ ), Hposomal ISS-ODN (ODN 1018, O), and liposomal ISS-ODN in combination with AmB-AG ( ⁇ ); Saline served as the control ( ⁇ ).
  • Liposomes can be classified according to various parameters. For example, when size and number of lamellae (structural parameters) are used, four major types of liposomes are identified: multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV) and oligolamellar vesicles (OLV). MLV form spontaneously upon hydration of dried phospholipids above their gel to liquid crystalline phase transition temperature (Tm). Their size and shape are heterogeneous and their exact structure is determined by their method of preparation
  • MLV have an aqueous and lipid component separated by bilayers.
  • SUV are formed from MLV by sonication or by extrusion and are single bilayered ( ⁇ 100 nm). They are the smallest species with a high surveillance and high surface-to- volume ratio and hence have the lowest capture volume of aqueous space to weight of lipid.
  • the third type of liposome according to this classification includes large (> 100 nm) unilamellar vesicles (LUV) having one large aqueous compartment and a single lipid bilayer, while the fourth type of liposome includes oligolamellar vesicles (OLV), which are vesicles containing few lamellae (lipid bilayers).
  • Liposomes are formed from amphipathic compounds, which may spontaneously or non-spontaneously vesiculate.
  • amphipathic compounds typically include triacylglycerols where at least one acyl group is replaced by a polar and/or charged moiety, e.g. phospholipids formed by a complex phosphoric acid ester.
  • Other groups of liposome-forming lipids are the sphingolipids such as sphingomyelin (N-acyl sphingosine-phosphocholine).
  • SPM sphingomyelins
  • examples of sphingomyelins include (but are not limited to) egg-derived SPM, milk-derived SPM, N-palmitoyl-SPM, N-stearoyl-SPM, N-oleoyl-SPM (C18:l), N-nervacyl C (C24:l) SPM, N-lignoceryl SPM (C24:0), and their mixtures.
  • any commonly known liposome-forming lipid may be suitable for use by the method of the present invention.
  • the liposome-forming lipids can also be produced synthetically.
  • the source of the lipid or its method of synthesis is not critical: any naturally occurring lipid, with and without modification, or a synthetic phosphatide can be used.
  • the lipids are phospholipids.
  • specific phosphatides are L- ⁇ -(distearoyl) lecithin, L- - (diapalmitoyl) lecithin, L- ⁇ -phosphatide acid, L- ⁇ -(dilauroy ⁇ )-phosphatidic acid, L- ⁇ (dimyristoyl) phosphatidic acid, L- ⁇ (dioleoyl)phosphatidic acid, DL- ⁇ (dipalmitoyl) phosphatidic acid, L- ⁇ (distearoyl) phosphatidic acid, and the various types of L- ⁇ - phosphatidylcholines prepared from brain, liver, egg yolk, heart, soybean and the like, or synthetically, and salts thereof.
  • PC phosphatidylcholines
  • zwitterionic amphiphates which form micelles by themselves or when mixed with the PCs such as alkyl analogues of PC.
  • lecithines also known as phosphatidylcholines
  • PC soy phosphatidylcholine
  • the phospholipids can vary in purity and can also be hydrogenated either fully or partially. Hydrogenation reduces the level of unwanted peroxidation, and modifies and controls the gel to liquid/crystalline phase main transition temperature (T m ) which effects packing and leakage.
  • T m liquid/crystalline phase main transition temperature
  • the liposomes can be "tailored" to the requirements of any specific reservoir including various biological fluids, which maintain their stability without aggregation or chromatographic separation, and thereby remain well dispersed and suspended in the injected fluid.
  • the fluidity in situ changes due to the composition, temperature, salinity, bivalent ions and presence of proteins.
  • the liposomes can be used with or without any other solvent or surfactant.
  • the liposomes may contain a combination of lipid components, as long as this combination does not induce instability, aggregation and/or chromatographic separation (demixing). This can be determined by routine experimentation, known to those in the art.
  • a variety of methods for producing the different types of liposomes are known and available of such methods include, for example:
  • the present invention provides a novel, fast and simple method for preparing liposomes efficiently loaded (i.e. at least 60% loading) with ISS-ODN.
  • effective encapsulation of the molecule may reduce the number of doses and frequency of administrations required in order to achieve a desired therapeutic effect.
  • Liposomal ISS-ODN as obtained by the present invention may be utilized, for example, as a vaccine adjuvant against pathogens and cancer; in therapeutic treatment or prevention of diseases caused by certain infectious microorganisms; in the treatment or prevention of allergic diseases; or to boost innate immunity.
  • the ISS-ODN is an endotoxin-free ISS-ODN with a phosphorothioate (PS) or phosphodiester (PO) backbone.
  • PS phosphorothioate
  • PO phosphodiester
  • liposomal ISS-ODN are prepared by: (a) solubilizing at least one liposome-forming lipid in a solvent and drying same to effect a dry liposome-forming lipid or a dry mixture of liposome-forming lipids;
  • the method of the invention provides a highly effective entrapment of the biologically active material in the liposomes, typically equal or greater than 60% (from the initial amount of ISS-ODN employed by the method).
  • the lipids are preferably freeze dried, i.e. by lyophilization thereof, resulting in a powder with a unique arrangement of the lipids enabling the effective loading of the ISS-ODN upon hydration into liposomes formed by the hydration.
  • the lipids may be any substance that forms liposomes upon dispersion thereof in an aqueous medium.
  • Preferred liposome-fo ⁇ ning amphipathic substances are of natural sources, semi-synthetic or fully synthetic molecules; negatively or positively charged phospholipids, sphingolipids, or other lipids optionally combined with a sterol, such as cholesterol, and/or with lipopolymers, such as PEGylated lipids.
  • the liposome-forming amphipathic substances may include saturated or unsaturated amphiphiles.
  • amphiphiles are phospholipids including, without being limited thereto, fully hydrogenated, partially hydrogenated or non-hydrogenated soybean derived phospholipids, egg yolk phospholipids, dimyristoyl 5 phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomeylins, and mixtures of the above.
  • DMPC dimyristoyl 5 phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • phosphatidylglycerol phosphatidylinositol
  • phosphatidylserine sphingomeylins, and mixtures of the above.
  • a preferred phospholipid combination according to the invention includes a mixture of DMPC and DMPG at a molar ratio of DMPC DMPG between about 1 :20 and 10 20:1. Such mixtures may be combined with cholesterol, and/or PEGylated lipids.
  • PEGylated lipids are commercially available.
  • Preferred PEGylated lipids include, without being limited thereto, DSPE-PEG [Haran, G., et al. Biochim. Biophys. Acta 1151:201-215 (1993)] or dihexadecyl phosphatidyl PEG 2000 (DHP-PEG 2000 ) [Tirosh, O., et al. Biophys. J. 74:1371-1379 (1998); US Patent No. 6,165,501]
  • the solvent according to the invention is any solvent with which lipids may solubilize or be dissolved.
  • solvents include, inter alia, the water miscible polar solvent, tertiary-butanol and the water immiscible apolar solvent, cyclohexane.
  • the ISS-ODN in a medium isoosmotic to plasma such as 0.9% sodium chloride, or cryprotectant, which is a
  • the aqueous solution according to the present invention is a physiologically acceptable aqueous medium in which ISS-ODN can solubilize, dissolve, or disperse and may be selected from 0.9% NaCl by weight (Saline), buffered Saline such as phosphate-buffered Saline (PBS), 5% dextrose, buffered
  • Saline 0.9% NaCl by weight
  • PBS phosphate-buffered Saline
  • dextrose buffered
  • the ISS-ODN can solubilize in pyrogen-free sterile water (at times referred to as 'water for injection') and after hydration of the dry amphipathic substance, the resulting dispersion is adapted to the physiological conditions suitable for administration.
  • a combination of two compositions including a first composition comprising dry liposome-forming lipids and a second composition comprising ISS-ODN, the combination is for use in the preparation of a pharmaceutical formulation comprising liposomal ISS-ODN, the preparation being in accordance with the method of the present invention.
  • the combination of the invention may be obtained in the form of a package.
  • the present invention also concerns a combination at least one first composition of dry liposome-forming lipids; and at least one second composition of ISS- ODN (either dry or in an aqueous solution); in the form of a package further comprising instructions for use of the first and second compositions for the preparation of a pharmaceutical formulation, said instructions comprising hydrating the dry lipid(s) of said first composition with an aqueous solution of ISS-ODN to obtain liposomes loaded with said ISS-ODN (preferably more than 60%); and the package further comprising instructions prescribing administration of said pharmaceutical formulation to a subject in need thereof.
  • the dry liposome-forming lipids and the ISS-ODN are each contained in a separate vial.
  • the package may contain more than one of said first composition of dry lipid(s) in separate vials and more than one of second composition comprising ISS-ODN, the instructions for selection and use of the compositions will depend on the specific Hposome/ISS-ODN formulation of interest. These instructions may be addressed to the physician, to the pharmacists or even to a subject of the treated individual.
  • the package may further comprise an aqueous medium, e.g. a physiologically acceptable aqueous medium, in which the ISS-ODN is dissolved or diluted.
  • the aqueous medium may be obtained separately, as it is typically a commercially available medium. Selection of the medium suitable for use will depend on considerations known to those versed in the art and, therefore, do not need to be further discussed herein.
  • the package comprises two or more compositions of dry lipids and optionally two or more compositions of ISS-ODN, thereby enabling to construct different pharmaceutical combinations according to the desired effect to be achieved and instructions prescribed by the medical practitioner.
  • the package may be for use by the physician, by the pharmacist or, at times, by the subject in need of the liposomal formulation.
  • the invention also concerns pharmaceutical formulations comprising as active ingredient a therapeutically effective amount of liposomal ISS-ODN and optionally a pharmaceutically acceptable additive, the liposomal ISS-ODN being prepared by the method of the invention.
  • the package may contain at least: (a) one first composition of a dry liposome-forming lipid and (b) liposomal ISS-ODN prepared by the CO method.
  • Mixture (b) may also be in a form of aqueous dispersion.
  • the pharmaceutical formulation of the invention is basically the liposomal formulation obtainable by the method of the invention but adapted for administration to the individual in need of a treatment or prevention of an identified disease.
  • the liposomal ISS-ODN is administered and dosed in accordance with good medical practice, taking into account the nature of the active ingredient, the clinical condition of the individual patient, the site, route and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the liposomal ISS-ODN of the invention may be administered in various ways. It can be administered in combination with pharmaceutically acceptable diluents, excipients, additives and adjuvants, as known in the art, e.g. for the purposes of adding flavors, colors, lubrication or the like to the liposomal formulation.
  • the pharmaceutically acceptable diluent/s, excipient/s, additive/s employed according to the invention generally refer to inert, non-toxic substances which preferably do not react with the liposomal formulation of the present invention.
  • the formulation of the invention may comprise other biologically active agents, such as antigens.
  • the additional agents may be in a free form or also encapsulated in liposomes (in the same or different liposomes of the immunoadjuvant) and may be administered simultaneously, concomitant or within a predefined time interval from administration of the immunoadjuvant.
  • the antigen may be, inter alia, derived from a killed or modified (e.g. genetically) organism or virus.
  • the liposomal ISS-ODN can be administered orally, intranasally, or parenterally including intravenously, intraarterially, intramuscularly, intraperitoneally, subcutaneously, intradermally, and intrathecally, and by infusion techniques.
  • the liposomal ISS-ODN of the invention may be made into aerosol formulations for administration by inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
  • the manner of administration will depend on different considerations known to the man of the art, e.g. on the type of agent to be loaded into the liposome.
  • a method for the prevention or treatment of a disease is also provided by the present invention, the method including administration to a subject in need of liposomal ISS-ODN of the invention.
  • the dosage for the liposomal ISS-ODN include up to 2,000 mg of loaded liposomal vesicles, measured by lipid, per kg body weight of the treatment subject. It should be noted, however, that the accurate dosage can vary dramatically, the variation depends on e.g. the type and efficacy of the ISS- ODN entrapped by the liposome, the efficiency of encapsulation (albeit being high with the method of the invention), the route of administration, the disease to be treated and the like. The respective parameters may be easily optimized by those skilled in the art and can thus be regarded as being routine experiments. The invention will now be further explained by the following non-limiting examples.
  • DMPC and DMPG with or without cholesterol.
  • Two types of liposomes containing CpG were formed, for the purpose of comparison of the method of preparation of the present invention with another hitherto known method.
  • the two encapsulation methods employed are designated herein as post encapsulation (the method of the present invention) and co-encapsulation.
  • Post encapsulation A lyophilized mixture of the lipids was hydrated with the ISS-ODN, a priori dissolved in pure water, and the liposomal preparation was diluted in 0.9% NaCl (Saline) or phosphate-buffered Saline (PBS). 2. Co-encapsulation: The solubilized lipids in tertiary butanol and ISS-ODN in pure water were co-lyophilized overnight and then hydrated with 0.9% Saline. Materials and Reagents
  • Influenza subunit vaccines HN - Subunit preparations containing mainly the influenza viral surface proteins hemagglutinin (H) and neuraminidase (N), 80-90% and
  • H1N1 A/Beijing/262/95
  • H1N1 A/New Caledonia/20/99
  • H3N2 A/Panama2007/99
  • B/Yamanashi/166/98 were provided by Dr's. R. Gl ⁇ ck and R. Zurbriggen, Berna Biotech, Bern, Switzerland.
  • Hepatitis B vaccines Hb
  • yeast Hansenula polymorpha derived hepatitis B's antigen (HBsAg, Rhein Biotech, Dusseldorf, Germany, Diminsky et al. 1997).
  • Tuberculosis vaccine -Combined vaccines composed of M. tuberculosis derived recombinant proteins (ESAT-6, L7/L12 and 85 B, or Sod, 85B and CFP21) mixed with Ribi adjuvant (Sigma, USA), or without Ribi adjuvant.
  • ESAT-6, L7/L12 and 85 B, or Sod, 85B and CFP21 M. tuberculosis derived recombinant proteins
  • Ribi adjuvant Sigma, USA
  • DMPC Dimyristoyl phosphatidylcholine
  • DMPG Dimyristoyl phosphatidylglycerol
  • the subunit preparations were used each separately or combined then diluted in sterile phosphate-buffered saline (PBS pH 7.4) for injection or intranasal administration (0.15-1 ⁇ g protein/dose of each viral strain).
  • PBS pH 7.4 sterile phosphate-buffered saline
  • ISS-ODN Liposomal ISS-ODN
  • M-ODN M-ODN
  • soluble ODN was added in a minimal volume (e.g. for 10 mg-30mg lipid, 25-50 ⁇ l of ODN solution was added). This was then vortexed vigorously for about 1 min. until a paste was obtained. The paste was then gradually diluted by further vortexing with sterile PBS or Saline to obtain the required concentration. This method corresponds to the post encapsulation method of the present invention.
  • the liposomal preparation was centrifuged at 4°C, for lhr. at 45,000 rpm.
  • the liposome pellet and the supernatant were subjected to a 2-phase lipid extraction procedure [Bligh, E.J. and Dyer, W.J. (1959) Canadian J. Biochem. Physiol. 37:911-917].
  • the amounts of free and encapsulated ODN and liposomal phospholipids were assessed by organic phosphorus determination [Barenholz, Y and Amselem, S. (1993) in Liposome technology, 2 n ed., Vol I. (Gregoriadis G, ed.), CRC Press, Boca Raton, FL, pp. 501-525 (1993)].
  • the free/liposomal ISS-ODN and M-ODN were used at 5- 25 ⁇ g/mouse/dose.
  • the POST technique described above was compared to another procedure for encapsulating ISS-ODN in liposomes that were prepared by co-lyophilization of a mixture consisting of the solubihzed lipids (in tertiary butanol) and ISS-ODN in aqueous solution, followed by hydration with 0.9% NaCl(referred to herein as the co-encapsulation method).
  • ODN 1018 and the influenza antigens (HN) were encapsulated, each separately or combined, in liposomes composed of DMPC alone, DMPC/DMPG (9:1 molar ratio), or DMPC/cholesterol (6:4 molar ratio), using the post- or the co-encapsulation techniques at a Hpid/ODN w/w ratio of 100/1, and at a lipid/protein(HN) w/w ratio of 300/1. Both techniques resulted in 60-80% encapsulation of ODN 1018 in all lipid compositions tested, when separate vesicles were used for ISS-ODN and the influenza antigens. For co-encapsulation of ODN 1018 and HN within the same vesicles, the CO technique was superior over the POST technique (70% vs 30% ODN 1018 encapsulation).
  • HN influenza antigens
  • MLV liposomal formulations
  • Table 1 The data presented in Table 1 is a compiled summary of 3 separate experiments, using subunit vaccines derived from influenza A/Panama/2007/99 (H3N2) and B/Yamanashi/166/98 strains.
  • the lipids dissolved in tertiary butanol and lyophilized overnight.
  • the dried lipid powder was then hydrated by stepwise addition (in 25-50 ⁇ l aliquotes) and vortexing of HN and/or ODN solution.
  • the solubihzed lipids solubihzed in tertiary butanol as described above
  • the ODN solution co-lyophilized overnight
  • HN stepwise (as above) with the HN solution or saline. Due to HN instability upon lyophilization, HN was encapsulated using the POST technique only.
  • the Hpid/ODN (w/w) ratio was 100/1
  • the lipid/HN (w/w) ratio was 300/1.
  • liposomal ISS-ODN was prepared at 100:1 and 300:1 lipid: ODN w/w ratios using saline or citrate buffer (containing 150 mM NaCl in 5 mM sodium citrate) pH 6.5-6.7 as final media for storage. The preparations were stored for up to 5 months at 4°C and at room temperature. ISS-ODN leakage and liposome lipid stability were tested 1, 3 and 5 months after preparation. The lipids in the dried lipid "cake" used to prepare the liposomal vaccine by the "post" encapsulation method were found to be chemically stable when stored at 4°C for at least 2 years.
  • Liposome lipid stability was determined by thin layer chromatography (TLC) and by the release of non-esterified fatty acids (NEFA) as a result of aryl ester hydrolysis. No significant lipid degradation ( ⁇ 5% hydrolysis) was noted up to 3 months at both temperatures for both saline and citrate buffer. A marked degradation (>10%) was found at 5 months, but only in unbuffered saline at room temperature. Much lower level of degradation was found in saline/citrate buffer than in unbuffered saline. Formulations prepared at a Hpid:ODN w/w ratio of 300:1 were slightly less stable and leakier than those prepared at a 100:1 ratio.
  • Liposomal HN Liposomal HN
  • HN-loaded large multilamellar vesicle (MLV) liposomes (mean diameter, 1.5 ⁇ m) were prepared by using the POST-encapsulation method as described above in connection with preparation of Lip ISS-ODN by hydrating the dry lipid cake with the HN solution.
  • HN was encapsulated with the ISS-ODN in the same vesicles using the POST technique for HN and the POST or CO technique for ISS-ODN, as summarized above (Table 1).
  • Liposomes co-loaded with HN and ISS-ODN 1018 were prepared by (a) hydration of the dry lipids, performed as above, with an aqueous solution mixture containing HN + ISS-ODN 1018 (the POST technique), (b) co-lyophilization of the lipid + ISS-ODN solution (CO), followed by hydration with the HN solution (POST). The liposomes were then further diluted in sterile saline or PBS and stored at 4°C.
  • Encapsulation efficiency was assessed by spinning the loaded liposomes at 4°C for 30 min. at 14,000 ⁇ m (under these conditions free HN does not precipitate while the majority of the MLV liposomes do), and by determining the protein concentration of the supernatant and of the liposomal fraction using a modified Lowry determination assay [Peterson G.L., Methods Enzymol. 91:95-119 (1983)].
  • ODN encapsulation was determined as described above and it was 60-80% when encapsulated alone by both procedures or when encapsulated together with HN in the same liposomes using procedure (b) described above.
  • ISS-ODN encapsulation was 20-30%) when encapsulated together with HN using procedure (a) described above (Table 1).
  • the lipid integrity was found to be very high
  • Example 1 Enhancement of the systemic anti-viral humoral response by liposomal ISS-ODN (Lip ISS-ODN) co-administered intramuscularly (i.m.) with influenza subunit vaccines
  • mice Female Balb/c mice, 6-8-weeks-old (5-6 per group) were vaccinated once (0.1 ml) i.m, with a divalent influenza subunit vaccine composed of the viral surface proteins hemagglutinin and neuraminidase (HN) derived from the A/Beijing/262/95-like (H1N1) and B/Yamanashi/166/98-like strains (0.15 ⁇ g protein of each strain).
  • HN antigens were given either in soluble form or entrapped in liposomes (Lip HN), alone or in combination with free ISS-ODN (ODN 1018) or Lip ISS-ODN (5, 12.5, 25 ⁇ g, Table 2).
  • the liposomes comprised DMPC:DMPG (9:1 mole ratio), and HN and ISS-ODN were encapsulated by the POST technique. Preparation of the Lip HN and Lip ISS-ODN is described herein before. Two additional groups (group 9 and group 10, Table 2) were immunized with liposomes co-entrapping the HN antigens and the ISS-ODN. Mutant ODN (M-ODN) lacking the immunostimulatory sequence served as control. Sera were tested 3 weeks (21 days) and 3 months (90 days) postvaccination for hemagglutination-inhibiting (HI) Abs [Sever, J. (1962). J Immunol.
  • Vaccine and dose A/Beijing (Day 21) a B/Yamanashi (Da r 21) a A/Beijing (Day 90) a B/Yamanashi (Day 90)
  • HN+Lip ISS-ODN 12.5 ⁇ g b 96+36 (100%) 72+18 (100%) 288+72 (100%) 96+61 (100%)
  • HN+Lip M-ODN 25 ⁇ g 30+10 (40%) 22+11 (20%) 176+88 (100%) 20+14 (20%) a In parentheses, % seroconversion : % of mice achieving a HI titer >40.
  • HN+ISS-ODN 25 ⁇ g ⁇ 10 64 ⁇ 10 70 ⁇ 10 350 ⁇ 10 450
  • HN+Lip ISS-ODN 12.5 ⁇ g ⁇ 10 560 ⁇ 10 400 ⁇ 10 2500 ⁇ 10 4200
  • HN+M-ODN 25 ⁇ g ⁇ 10 ⁇ 10 ⁇ 10 ⁇ 10 190 50 200 20
  • HN+Lip M-ODN 25 ⁇ g ⁇ 10 ⁇ 10 ⁇ 10 ⁇ 10 300 20 300 10
  • Table 2 shows the results of hemagglutination inhibition (HI), while Table 3 shows the results of the ELISA of antigen-specific IgGl and IgG2a Abs.
  • mice co-immunized with Lip ISS-ODN were significantly greater than those of mice vaccinated with antigen alone (groups 1, 2) or with antigen combined with soluble ISS-ODN (groups 3-5).
  • the HI titers obtained with Lip ISS-ODN were 2-8-fold higher than with soluble ISS-ODN.
  • the seroconversion (HI titer >40) rate was 0-40% using soluble ISS-ODN or M-ODN and 100% with Lip ISS-ODN.
  • a single vaccine dose containing even 5 ⁇ g of Lip ISS-ODN results in very high HI titers and specific IgG2a levels, with no IgGl response, namely a Thl type. This enhanced response lasts for at least 3 months.
  • the results of this experiment were reproduced in two additional experiments, using divalent and trivalent (adding the A/Sydney/5-97-like, H3N2, to the divalent preparation) vaccines.
  • the HI titers on days 21-90 were 4-10-fold higher with Lip ISS-ODN (10 ⁇ g) than with non-liposomal ISS- ODN.
  • Lip ISS-ODN 1018 prepared by the POST technique and Lip ISS-ODN prepared by the CO technique had an equal adjuvant activity following intramuscular co-administration with the influenza HN antigens (data not shown).
  • Vaccine HI titer (mean ⁇ SD) against: Mean IgG titer against: A/Beijing B/Yamanashi A/Beijing B/Yamanashi
  • HN+ISS-ODN 25 ⁇ g 896+350 (100%) 368+260 (100%) 400 1,000 600 3,000
  • HN+Lip ISS-ODN 25 ⁇ g 3000+1500 (100%) 1900+800 (100%) 600 8,000 1,000 10,000 HN+M-ODN 25 ⁇ g 640+0 (100%) 170+200 (50%) 500 ⁇ 10 800 100 ⁇ »-*-.
  • Vaccine Increase in footpad Incidence of mice with thickness (mm) a DTH b
  • Lip ISS-ODN A single dose of 5 ⁇ g Lip ISS-ODN was by far more potent (4-11 times, Tables 2,3) for the two viral strains than 25 ⁇ g of free ISS-ODN.
  • the enhanced potency was reflected in both humoral and cellular (DTH) responses.
  • Lip ISS- ODN mainly enhanced Thl-biased immunity, based on the relative IgGl and IgG2a levels upon i.m. administration.
  • results disclosed herein and additional results show that the same increment in the humoral response was obtained when free or liposomal antigen (HN) was administered together i.m. with Lip ISS-ODN (in separate liposomes), and when the vaccine consisted of antigen and ISS-ODN entrapped together in the same liposomes using the post technique.
  • HN liposomal antigen
  • results differ from those reported recently by two groups on liposomal plasmid DNA and liposomal ISS-ODN. Gursel et al. [G ⁇ rsel, M., et al. (1999) Vaccine.
  • a liposomal divalent vaccine (Lip HN, 0.5 ⁇ g of each strain) was administered twice i.n., spaced 1 week, alone and in combination with free or Lip ISS-ODN (10 ⁇ g), or with free cholera toxin (CT, 1 ⁇ g), considered a powerful (yet toxic) mucosal adjuvant.
  • HN and ISS-ODN were inco ⁇ orated into the same DMPC/DMPG liposomes by the POST technique.
  • Serum, nasal wash and lung homogenate were tested for antigen-specific IgGl, IgG2a and IgA Abs four weeks after the second vaccine dose, and protective immunity to viral challenge was assessed six weeks post-vaccination by determining lung virus titer.
  • the mean Ig titer was tested by ELISA on pooled samples.
  • Lip HN+CT 30 75 ⁇ 20 300 100 50 -
  • soluble ISS-ODN had no adjuvant effect (except for a low increase in serum IgG2a against one viral strain).
  • Lip ISS-ODN induced significant levels of antigen-specific IgGl, IgG2a and IgA in serum, nasal wash and lung.
  • the response to the two viral strains included in the vaccine varied, however.
  • Lip ISS-ODN and CT elicited similar levels of semm and mucosal IgG2a and IgA; the latter inducing higher levels of IgGl as well.
  • Lip ISS-ODN, but not soluble ISS-ODN is capable of boosting both systemic and mucosal antigen-specific IgG2a and IgA Abs.
  • mice of this experiment were challenged i.n. with a recombinant ABeijing virus.
  • mice were lightly anesthetized with Halotane and 25 ⁇ l live virus suspension per nostril was administered.
  • the test virus was the reassortant vims X-127 (A-Beijing/262/95 (H1N1) x X-31 (A/Hong Kong/1/68 x A/PR/8/34), which is infectious to mice, 10 7 EID 50 (egg- infectious dose 50%).
  • mice were sacrificed on day 4 post-infection, the lungs were removed, washed x 3 in cold PBS, and homogenates in PBS of each group were pooled and centrifuged at 2000 ⁇ m for 30 min. at 4°C and the supematants collected. Serial tenfold dilutions were performed and 0.2 ml of each dilution was injected in duplicate into the allantoic sac of 10-11 days-old fertilized chicken eggs. After 48 hrs., at 37°C the eggs were stored overnight at 4°C. Then 0.1 ml of allantoic fluid was removed and checked for viral presence by hemagglutination with chicken erythrocytes (0.1 ml of 0.5%) suspension) for 30 min at room temperature.
  • the lung vims titer is determined as the highest dilution of lung homogenate producing vims in the allantoic fluid (positive hemagglutination). A titer of 0.5 indicates 1 egg was positive and 1 egg was negative at the highest dilution. Table 8-Protection against influenza vims infection 6 weeks following intranasal vaccination with a divalent influenza vaccine co-administered with free/liposomal ISS-ODN
  • Vaccine fn 5 Lung virus vacc i ne ⁇ n a titer floglO
  • Table 8 shows that, as compared with unimmunized mice, the lung vims titer of mice vaccinated with Lip HN alone, Lip HN+free ISS-ODN and Lip (HN+ISS-ODN) was reduced by 1, 2 and 3.5 logs, respectively (and 3 logs with CT).
  • the 30-fold difference in lung vims titer between free and liposomal ISS-ODN is in good agreement with the differences seen in HI, IgG2a and IgA titers between these groups.
  • Table 9 shows that, as compared with unimmunized mice, the lung vims titer of mice vaccinated with Lip HN alone, Lip HN+free ISS-ODN and Lip
  • ISS-ODN 1018 encapsulated by the POST technique and Lip ISS-ODN 1018 encapsulated by the CO technique.
  • H1N influenza A/New Caledonia/20/99
  • groups 6,7 separate vesicles were formed for HN and ODN 1018; in groups 8, 9 HN and ODN 1018 were encapsulated together within the same vesicles.
  • HN was encapsulated by the POST technique.
  • Liposomes (MLV) consisted of DMPC/DMPG (9:1 mole ratio). Response was measured 4 weeks after the second vaccine dose. CT-cholera toxin.
  • Lip ISS-ODN was superior to free ISS-ODN with regard to HI and IgG2a titers.
  • Vaccine Encapsulation Mean HI titer a Mean IgGl Mean IgG2a method of titer titer 0 ODN
  • mice were immunized once intramuscularily (i.m.) with a monovalent subunit vaccine (0.5 ⁇ g) derived from A/New Caledonia/20/99-like (HlNl), alone and combined with 10 ⁇ g of free or lip ISS-ODN (MLV liposomes (1.5 ⁇ m) consisted of DMPC:DMPG at 9:1 mole ratio, Hpid:ODN w/w ratio of 100:1 and prepared as described above). Mice injected with lip ISS-ODN alone served as control.
  • a monovalent subunit vaccine 0.5 ⁇ g
  • HlNl A/New Caledonia/20/99-like
  • MMV liposomes (1.5 ⁇ m) consisted of DMPC:DMPG at 9:1 mole ratio, Hpid:ODN w/w ratio of 100:1 and prepared as described above).
  • Humoral (hemagglutination inhibition [HI] titer, semm and lung IgGl, IgG2a, IgA and IgE titer, Table 10) and cellular (proliferation, cytokine production, cytotoxicity, Table 11) responses were assessed 4 and 6 weeks post-vaccination using spleen cells.
  • cells 0.5 x 10 /well were incubated in U- shaped 96-well plates, in triplicate, with or without the HN antigen (0.5 ⁇ g/well), in a final volume of 0.2 ml of RPMI 1640 medium supplemented with 5%> FCS. After 72 h, cultures were pulsed with 1 ⁇ C H-thymidine for 16 h.
  • IFN ⁇ production cells For determination of IFN ⁇ production cells, (2.5 x 10 /well) were incubated in 24- well plates, in duplicate, with or without the HN antigen (10 ⁇ g/well), in a final volume of 1 ml of RPMI 1640 + FCS. Supematants were collected after 72 h and tested by ELISA for murine IFN ⁇ and IL-4.
  • responding splenocytes For determining cytotoxic activity, responding splenocytes, (2.5 x 10 6 /well) were incubated in 24-well plates, in duplicate, with or without the HN antigen (10 ⁇ g/well) in a final volume of 1 ml of RPMI 1640 + 10% FCS + 5 x 10 5 M 2-mercaptoethanol.
  • cytotoxicity was tested in a 4 h Cr release assay (at an effector/target cell ratio of 15/1, in triplicate) against labeled non-infected P815 cells and P815 cells infected with the reassortant vims X-127 (HlNl, which is infectious to mouse cells and cross-reactive with A/New Caledonia), using 0.2 ml of vims-containing allantoic fluid for 5x10 cells, for 2 h at 37°C. Specific cytotoxicity was calculated after subtracting the cytotoxicity against non-infected P815. Table 10-The humoral response of BALB/c mice immunized i.m. with a monovalent influenza subunit vaccine, alone and combined wit free/liposomal ISS-ODN
  • Stimulation index mean cpm of cells cultured with antigen/mean cpm of cells cultured without antigen; c The values presented were obtained in antigen-containing cultures; no significant cytotoxicity ( ⁇ 5%) was seen in cells culture without antigen. Cytotoxicity against non-infected p8l5 target cells was subtracted.
  • lip ISS-ODN Using lip ISS-ODN as an adjuvant, the semm and lung IgG2a levels were 10 and 6 times higher, respectively, than those achieved with free ISS-ODN.
  • lip ISS-ODN (10 ⁇ g) is 4-10 times more efficient than free ISS-ODN.
  • free ISS-ODN had no adjuvant activity in any of the three tests performed.
  • IFN ⁇ (>20 pg/ml) was detected only in cultures containing the antigen. No IL-4 ( ⁇ 20 pg/ml) was detected in any of the groups.
  • IL-4 ⁇ 20 pg/ml was detected in any of the groups.
  • in vitro stimulated splenocytes of mice co- immunized with antigen and lip ISS-ODN exhibited a 4.2-, 4.4- and 5.3-fold greater proliferative response, interferon ⁇ production and specific cytotoxic activity, respectively.
  • stimulation of naive splenocytes with concanavalin A (2.5 ⁇ g/well) produced 2000 pg/ml of IFN ⁇ and 70 pg/ml of IL-4 (data not shown).
  • lip ISS-ODN triggers a much stronger Thl-dominant response than free ISS-ODN.
  • Table 12 a comparison was made between free/lip ISS-ODN (1018), free/lip M-ODN (1019) and free/lip recombinant human IL-2 (previously used by us as an adjuvant in this model) as adjuvants for the A/New Caledonia subunit vaccine.
  • mice were immunized i.m. as detailed above and pooled sera were tested by ELISA 3 and 8 weeks post-vaccination.
  • Antigen-specific IgG titers were calculated as detailed in connection with Table 3. Empty liposomes alone were not tested.
  • mice immunized with Lip ISS-ODN once or twice i.n. and i.m. No swelling or ulceration was noticed at the i.m. injection site.
  • Example 2 Enhancement of the systemic anti-viral humoral response and cellular response by liposomal ISS-ODN 1018 (Lip ISS-ODN) co-administered with Hepatitis B vaccine
  • HBsAg recombinant HBsAg
  • naked antigen the HBsAg particles alone
  • alum aluminum hydroxide gel adjuvant, Superfros Biosectior, Frederikssund, Denmark, used as the human vaccine, with an alum/antigen w/w ratio of 25:1), each alone and combined with free/lip ISS-ODN (10 ⁇ g, DMPC/DMPG 9:1 mole ratio, lipid:ODN w/w ratio of 100:1).
  • free/lip ISS-ODN 10 ⁇ g, DMPC/DMPG 9:1 mole ratio, lipid:ODN w/w ratio of 100:1).
  • the antigen was co- administered with lip M-ODN (lacking the CpG motif) or empty liposomes.
  • IgG2a antibodies, and HBsAg neutralizing antibodies Animals were bled on days 21 (for primary response) and 35 (for secondary response).
  • Specific, neutralizing anti-HBs antibodies were measured using a commercial microparticle enzyme immunoassay (IMx AUSAB, Abbott Laboratories, USA) on semm samples diluted 1/2-1/10.
  • the cellular response was tested on day 56 using pooled spenocytes of each group.
  • the results are presented in the following Table 14.
  • IFN ⁇ and IL-4 production cells (5 x 10 6 /well) were incubated in duplicate in 24-well plats in a final volume of 1 ml of complete DMEM medium + 5%> FCS, alone and together 5 ⁇ g of HBsAg. Supematants were collected after 48 h and IFN ⁇ and IL-4 were tested by ELISA, using the Opt EIA Set (Pharmingen, USA). In parentheses, cytokine levels in non-stimulated cultures (incubated without antigen).
  • Cells were incubated alone and together with HBsAg 4 ⁇ g/well (c) or 1 ⁇ g/well (d) for 96 h and then pulsed with 1 ⁇ Ci 3 H-tymidine overnight.
  • Alum- HBsAg + lip ISS-ODN 1400 (0) 16 (0) 46 14 3.3/3.0 a 0 ⁇ 10 pg/ml of either cytokine.
  • Stimulation index (SI) mean cpm cells + antigen/mean cpm cells w/o antigen . ( c 4 ⁇ g/well, 1 ⁇ g/well).
  • Vaccination with alum-adsorbed antigen (group 8) induced, as expected, high levels of both IgGl and anti-HBs antibodies and a strong Th2 response (IgG2a/IgGl ratio ⁇ 0.03).
  • alum-adsorbed antigen (group 8) induced, as expected, high levels of both IgGl and anti-HBs antibodies and a strong Th2 response (IgG2a/IgGl ratio ⁇ 0.03).
  • ISS-ODN groups 9, 10
  • a mixed Thl-Th2 response ratio, 0.56-1.4
  • Liposomal ISS-ODN was also 2-7 more effective than free ISS-ODN in combination with alum-HBsAg for the various antibodies tested.
  • the cellular responses were tested at 8 weeks, 5 weeks after the second vaccine dose (Table 14). Splenocytes were incubated alone and together with the antigen, and IFN ⁇ , IL-4, cytotoxicity and proliferative response were measured. For the cytotoxic response, cultures were also carried out with 30 IU/ml of recombinant human IL-2 (Chiron, USA). IFN ⁇ was produced only in antigen-stimulated cultures. Vaccination with HBsAg administered together with free ISS-ODN increased IFN ⁇ production 4.7- fold compared with antigen alone; lip ISS-ODN induced 5 and 22 times more IFN ⁇ than free ISS-ODN (groups 3, 4) and antigen alone (group 2), respectively.
  • Example 3 Enhancement of the systemic humoral response by liposomal ISS- ODN (Lip ISS-ODN) administered together with Tuberculosis vaccines
  • lip ISS-ODN 1018 was tested in mice as an adjuvant for a new recombinant M. tuberculosis vaccine.
  • a combined vaccine composed of M. tuberculosis recombinant proteins (ESAT-6, L7/L12 and 85 B) mixed with Ribi adjuvant (Sigma, USA) was administered s.c. twice to female BALB/c mice (6-weeks old), alone and together with lip ISS-ODN (10 ⁇ g/dose, prepared as described above). Free ISS-ODN was not tested in this experiment.
  • Antigen-specific IgGl and IgG2a were quantified by ELISA 3 weeks after the second vaccine dose. The results are shown in the following Table 15.
  • mice were vaccinated subcutaneously on days 0 and 21 using a mixture of 3 different antigens (5 ⁇ g each) derived from M. tuberculosis. ODN 1018-10 ⁇ g/dose.
  • the results presented in Table 16 show that lip ISS-ODN was 2-2.5-fold more potent for two of the three antigens tested.
  • lip ISS-ODN may prove beneficial as a parenteral Thl adjuvant for future tuberculosis vaccines.
  • a weakly immunogenic murine mammary carcinoma (4T1) was used in 2 experiments.
  • Tumor challenge was injected subcutaneously 10 days after the last vaccination (injection of 5x10 live tumor cells) and mice were inspected for tumor incidence and size 4 weeks later.
  • Saline, free ISS-ODN, lip ISS-ODN or lip M-ODN (lacking the CpG motif) were administered intramuscularily (i.m., 0.1 ml) at 20 ⁇ g/dose on days 1, 7, 14 (cycle 1) and 72 and 82 (cycle 2) post infection (lip ISS-ODN or lip M-ODN comprising DMPC/DMPG at mole ratio of 9:1 and lipid:ODN ratio (w/w) of 100:1, prepared as described above).
  • Subcutaneous lesions were measured weekly by caliper.
  • mice were infected as above, then treated with a subcurative dose of anti-leishmanial dmgs (Amphotericin B (Fig. 2) and Amphotericin B derivative (Fig. 3)), alone and combined with lip ISS-ODN.
  • Other groups were treated with either lip ISS-ODN or lip M-ODN (lacking the CpG motif) only. As shown in Figs. 2 and 3, a synergistic therapeutic effect was achieved in the group co-treated with the dmg and the lip ISS-ODN.
  • Liposomal M-ODN 20 ⁇ g 1.35 ⁇ 1.67 (2/8) 13.2 ⁇ 10.1 (1/8)

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Abstract

La présente invention concerne un procédé simple et rapide permettant le piégeage efficace d'un ISS-ODN actif dans des liposomes, avec plus de 60 % de charge). Le procédé décrit dans cette invention consiste à sécher une suspension d'un matériau amphipathique, puis, à hydrater cette suspension avec une solution aqueuse contenant l'ISS-ODN, en piégeant ce dernier dans des liposomes formés à partir du lipide. Cette invention concerne un procédé efficace permettant de charger et de retenir l'ISS-ODN dans des vésicules liposomales. Des animaux traités avec l'ISS-ODN liposomale présentent un rythme de développement de la maladie plus lent par rapport à un ISS-ODN libre ou à des liposomes chargés avec un ISS-ODN mutant (lip M-ODN).
PCT/IL2002/000507 2001-06-25 2002-06-25 Procede de preparation de vesicules chargees d'oligodeoxynucleotides immunostimulateurs (iss-odn) et utilisations diverses de celles-ci Ceased WO2003000232A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087097A3 (fr) * 2003-03-31 2005-01-13 Yissum Res Dev Co Ensembles de lipides contenant des amphiphiles de formation non liposomique
WO2007049279A3 (fr) * 2005-10-26 2007-08-16 Yissum Res Dev Co Combinaison de liposomes et utilisations
US7524828B2 (en) 1994-07-15 2009-04-28 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7674777B2 (en) 1994-07-15 2010-03-09 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7956043B2 (en) 2002-12-11 2011-06-07 Coley Pharmaceutical Group, Inc. 5′ CpG nucleic acids and methods of use
US8148340B2 (en) 1994-07-15 2012-04-03 The United States Of America As Represented By The Department Of Health And Human Services Immunomodulatory oligonucleotides
US9326953B2 (en) 2003-04-25 2016-05-03 The Penn State Research Foundation Method and system for systemic delivery of growth arresting, lipid-derived bioactive compounds

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0172007B1 (fr) * 1984-08-10 1991-05-22 Syntex (U.S.A.) Inc. Liposomes stables à médicaments hydrosolubles
JPS6176414A (ja) * 1984-09-21 1986-04-18 Shionogi & Co Ltd リポソーム製剤の製法
US6083923A (en) * 1997-10-31 2000-07-04 Isis Pharmaceuticals Inc. Liposomal oligonucleotide compositions for modulating RAS gene expression
AU2870300A (en) * 1999-02-05 2000-08-25 Genzyme Corporation Use of cationic lipids to generate anti-tumor immunity
PL354879A1 (en) * 1999-08-27 2004-03-08 Inex Pharmaceuticals Corp. Compositions for stimulating cytokine secretion and inducing an immune response

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7524828B2 (en) 1994-07-15 2009-04-28 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7674777B2 (en) 1994-07-15 2010-03-09 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7888327B2 (en) 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions
US8129351B2 (en) 1994-07-15 2012-03-06 The University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US8148340B2 (en) 1994-07-15 2012-04-03 The United States Of America As Represented By The Department Of Health And Human Services Immunomodulatory oligonucleotides
US8258106B2 (en) 1994-07-15 2012-09-04 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7956043B2 (en) 2002-12-11 2011-06-07 Coley Pharmaceutical Group, Inc. 5′ CpG nucleic acids and methods of use
WO2004087097A3 (fr) * 2003-03-31 2005-01-13 Yissum Res Dev Co Ensembles de lipides contenant des amphiphiles de formation non liposomique
US9326953B2 (en) 2003-04-25 2016-05-03 The Penn State Research Foundation Method and system for systemic delivery of growth arresting, lipid-derived bioactive compounds
WO2007049279A3 (fr) * 2005-10-26 2007-08-16 Yissum Res Dev Co Combinaison de liposomes et utilisations

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