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WO2009043165A1 - Compositions comprenant un antigène, un composé amphipathique et un support hydrophobe, et leurs utilisations - Google Patents

Compositions comprenant un antigène, un composé amphipathique et un support hydrophobe, et leurs utilisations Download PDF

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Publication number
WO2009043165A1
WO2009043165A1 PCT/CA2008/001747 CA2008001747W WO2009043165A1 WO 2009043165 A1 WO2009043165 A1 WO 2009043165A1 CA 2008001747 W CA2008001747 W CA 2008001747W WO 2009043165 A1 WO2009043165 A1 WO 2009043165A1
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WIPO (PCT)
Prior art keywords
antigen
hydrophobic carrier
composition according
amphipathic compound
water
Prior art date
Application number
PCT/CA2008/001747
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English (en)
Inventor
Marc Mansour
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Immunovaccine Technologies Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunovaccine Technologies Inc. filed Critical Immunovaccine Technologies Inc.
Priority to BRPI0817484-9A priority Critical patent/BRPI0817484B1/pt
Priority to EP08800418A priority patent/EP2195022A4/fr
Priority to JP2010527303A priority patent/JP5591705B2/ja
Priority to CN200880110239.7A priority patent/CN101815529B/zh
Priority to CA2700828A priority patent/CA2700828C/fr
Priority to US12/679,998 priority patent/US20100209452A1/en
Priority to AU2008307042A priority patent/AU2008307042B2/en
Publication of WO2009043165A1 publication Critical patent/WO2009043165A1/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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K39/39—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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K9/0031—Rectum, anus
    • 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
    • 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
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/36—Opioid-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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

  • compositions Comprising an Antigen, an Amphipathic Compound and a Hydrophobic Carrier, and Uses Thereof
  • compositions comprising an antigen, an amphipathic compound, and a hydrophobic carrier.
  • Compositions of the invention have been found to provide enhanced immune responses in vivo.
  • Vaccination generally involves the injection of an antigenic substance or antigen into an animal.
  • the antigenic substance generates an immune response in the animal.
  • the antigen may be e.g. a killed organism such as a bacterium or an inactivated virus, a component of an organism that has antigenic properties, a live organism or virus with low virulence.
  • an adjuvant may function by different mechanisms, including (1) trapping the antigen in the body to cause a slow release, (2) attracting cells of the immune system to the injection site, (3) stimulating cells of the immune system to proliferate and to become activated, and (4) improving antigen dispersion in the recipient's body.
  • Commonly used adjuvants include aluminum salts, water- in-oil and oil-in water emulsions, mineral salts and other compounds that can act as a stimulatory danger signal. Polycations such as diethylaminoethyldextran (DEAE dextran) may also be effective as adjuvants in some cases.
  • the adjuvant may be included in the vaccine as an additive or may be administered separately.
  • compositions are water- in-oil or oil- in-water emulsions.
  • Water- in-oil compositions in particular are effective because of the prolonged presence of such compositions at the injection site, causing the slow release of antigen at the site of immunization.
  • water- in- oil emulsions may become unstable once injected in vivo, causing the separation of the aqueous and oily phases of the composition. This leads to premature or accelerated release of antigens and other components.
  • the invention provides a composition comprising: an antigen; an amphipathic compound; and a hydrophobic carrier; wherein the composition is substantially free of water.
  • the invention provides a process for making the composition as described above, the process comprising: (a) combining an antigen and an amphipathic compound to form a dry mixture; and (b) suspending said mixture in a hydrophobic carrier; wherein the composition is substantially free of water.
  • the invention provides a method comprising administering the composition as described above to a subject.
  • the invention provides a method for inducing an antibody response or cell -mediated immune response in a subject, the method comprising administering to a subject in need thereof the composition as described above .
  • Figure 1 illustrates tumor growth over a 42 -day- period in mice implanted with C3 cells and treated with phosphate buffered saline 8 days post tumor implantation according to the present invention.
  • PTI phosphate buffered saline 8 days post tumor implantation
  • Figure 2 illustrates tumor growth over a 42 -day period in mice implanted with C3 cells and treated on day 8 with a control formulation consisting of FP antigen in a water- in-oil emulsion.
  • Figure 3 illustrates tumor growth over a 42 -day period in mice implanted with C3 cells and treated on day 8 with a water- free composition consisting of FP antigen, DOPC carrier and a hydrophobic carrier (Incomplete Freund's adjuvant) .
  • Figure 4 illustrates tumor growth in mice implanted with C3 cells and treated on day 8 with a control formulation consisting of FP antigen and Pam3Cys adjuvant in a water-in-oil emulsion (incomplete Freund' s adjuvant) .
  • Tumors were monitored weekly for a total of 42 days.
  • Figure 5 illustrates tumor growth over a 42 -day period in mice implanted with C3 cells and treated on day 8 with a water-free composition consisting of FP antigen, Pam3Cys adjuvant, DOPC carrier and a hydrophobic carrier (Incomplete Freund's adjuvant) .
  • Mice (group 5, n 7) implanted with C3 cells and treated on day 8 with a water- free composition consisting of FP antigen, Pair ⁇ Cys adjuvant, DOPC carrier and a hydrophobic carrier (Incomplete Freund's adjuvant) .
  • Tumors were monitored weekly for a total of 42 days.
  • Cellular immune responses were measured by ELISPOT assay and are presented as an average of spot forming units.
  • Figure 7 shows vials (front elevation) containing hydrophobic carrier (vial ISA51) , polyIC formulated according to the invention (vial 21) , and polyIC suspended in the hydrophobic carrier in the absence of the amphipathic compound DOPC (vial 26) .
  • a heterogeneous suspension of insoluble polyIC strands can be easily seen in vial 26.
  • Figure 8 shows vials (bottom plan view) containing hydrophobic carrier (vial ISA51) , peptide antigens formulated according to the invention (vial 30) , and peptide antigens suspended in the hydrophobic carrier in the absence of the amphipathic compound DOPC (vial 35) .
  • Antigen aggregates that could not resuspended in the hydrophobic carrier can be easily seen in vial 35 (circled) .
  • compositions comprising, consisting essentially of, or consisting of: an antigen; an amphipathic compound; and a hydrophobic carrier; wherein the composition is substantially free of water.
  • compositions of the invention comprise one or more antigens.
  • antigen refers to a substance that can bind specifically to an antibody or to a T-cell receptor.
  • Antigens useful in the compositions of the invention include, without limitation, polypeptides, a microorganism or a part thereof, such as a live, attenuated, inactivated or killed bacterium, virus or protozoan, or part thereof .
  • antigen also includes a polynucleotide that encodes the polypeptide that functions as an antigen.
  • Nucleic acid- based vaccination strategies are known, wherein a vaccine composition that contains a polynucleotide is administered to a subject.
  • the antigenic polypeptide encoded by the polynucleotide is expressed in the subject, such that the antigenic polypeptide is ultimately present in the subject, just as if the vaccine composition itself had contained the polypeptide.
  • the term "antigen" encompasses such polynucleotides that encode the polypeptide which functions as the antigen.
  • Polypeptides or fragments thereof that may be useful as antigens in the invention include, without limitation, those derived from Cholera toxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, hemagglutinin, neuraminidase, influenza M protein, PfHRP2 , pLDH, aldolase, MSPl, MSP2 , AMAl , Der-p-1, Der-f-1, Adipophilin, AFP, AIM-2, ART-4, BAGE, alpha- fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin DlEp-CAM, EphA2, EphA3, ELF-2, FGF-5, G250, Gonadotropin Releasing Hormone, HER-2, intestinal carboxyl esterase (iCE) , IL13Ralpha2, MAGE-I, MAGE-2,
  • Viruses, or parts thereof, useful as antigens in the invention include, without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Orthoreovirus, Rotavirus,
  • Ebolavirus parainfluenza virus, influenza A virus, influenza B virus, influenza C virus, Measles virus, Mumps virus, Rubella virus, Pneumovirus, Human respiratory syncytial virus, Rabies virus, California encephalitis virus, Japenese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Coronavirus, Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella .
  • Bacteria or parts of thereof useful as antigens in the invention include, without limitation, Anthrax, Brucella, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli 0157: H7, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Legionella, Leptospira, Listeria, Meningococcus, Mycoplasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica.
  • the antigen may alternatively be of protozoan origin, e.g. Plasmodium falciparum, which causes malaria.
  • polypeptide As used herein, the term "polypeptide" or
  • protein means any chain of amino acids, regardless of length (e.g. 4, 6, 8, 10, 20, 50, 100, 200, 500 or more amino acids) or post-translational modification ⁇ e.g. , glycosylation or phosphorylation) . Both terms are used interchangeably.
  • polypeptide and protein are intended to encompass molecules (such as peptidomimetics) mimicking the properties or function of a polypeptide or protein but incorporating modifications to alter the molecule's properties, such as the molecule's stability or biological activity. These modifications include e.g. altered backbones (e.g. inclusion of non-peptidic bonds) and the incorporation of non-naturally occurring amino acids.
  • polynucleotide encompasses a chain of nucleotides of any length (e.g. 9, 12, 18, 24, 30, 60, 150, 300, 600, 1500 or more nucleotides) or number of strands (e.g. single-stranded or double- stranded) .
  • Polynucleotides may be DNA (e.g. genomic DNA or cDNA) or RNA (e.g. mRNA) or combinations thereof. They may be naturally occurring or synthetic (e.g. chemically synthesized) . It is contemplated that the polynucleotide may contain modifications of one or more nitrogenous bases, pentose sugars or phosphate groups in the nucleotide chain. Such modifications are well-known in the art and may be for the purpose of e.g. improving stability of the polynucleotide.
  • the concentration of antigen may be as high as required to effectively stimulate an immune response, with the limitations on the amount of antigen being that the antigen should not precipitate out of the composition, and the antigen must be re-suspendable into the hydrophobic carrier. Further, the concentration of antigen varies depending on the type of antigen and the amount of other components in the composition. One skilled in the art can readily determine the amount of antigen needed in a particular application. For example, for peptide antigens about 0.01 to about 5 mg/ml may be used (based on the total volume of the composition) , with the preferred range being not less than 0.1 and not more than 1.0 mg/ml . For other antigens, such as recombinant proteins, the concentration may be in the range of about 0.01 to about 0.5 mg/ml, with the preferred range being not less than 0.01 and not more than 0.5 mg/ml . Amphipathic compounds
  • compositions of the invention comprise one or more amphipathic compounds.
  • An "amphipathic compound” is a compound having both hydrophilic and hydrophobic parts or characteristics.
  • the hydrophobic portion of an amphipathic compound is typically a large hydrocarbon moiety, such as a long chain of the form CH 3 (CH 2 )H/ with n > 4.
  • the hydrophilic portion of an amphipathic compound is usually either a charged group or a polar uncharged group. Charged groups include anionic and cationic groups.
  • anionic charged groups include the following (wherein the hydrophobic part of the molecule is represented by "R") : carboxylates : RCO 2 " ; sulfates: RSO 4 " ; sulfonates: RSO 3 " ; and phosphates (the charged functionality in phospholipids) .
  • Cationic charged groups include e.g. amines: RNH 3 + ("R” again representing the hydrophobic part of the molecule) .
  • Uncharged polar groups include e.g. alcohols with large R groups, such as diacyl glycerol (DAG) .
  • Amphipathic compounds may have several hydrophobic parts, several hydrophilic parts, or several of both. Proteins and some block copolymers are examples. Steroids, cholesterol, fatty acids, bile acids, and saponins, are also amphipathic compounds useful in the practice of the invention.
  • compositions of the invention may contain a single amphipathic compound or a mixture of amphipathic compounds.
  • the amphipathic compound (s) is a phospholipid or mixture of phospholipids.
  • a "phospholipid” is a member of a group of lipid compounds that yield on hydrolysis phosphoric acid, an alcohol, fatty acid, and nitrogenous base.
  • Phospholipids that may be used in the practice of the invention include phosphoglycerides, which are phospholipids in which two fatty acyl side chains are esterified to two of the three hydroxyl groups of a glycerol molecule.
  • the third hydroxyl group of the glycerol molecule is esterified with phosphate.
  • the phosphate group is usually also esterified to a hydroxyl group on a hydrophilic compound such as ethanolamine, serine, choline, or glycerol.
  • Phospholipids that are phosphoglycerides include, e.g.
  • Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond, to form ceramide . The hydroxyl group of sphingosine is esterified to phosphocholine .
  • sphingomyelin is amphipathic. All of these and other phospholipids may be used in the practice of the invention. In some embodiments, phospholipids having a carbon chain length of between 4 and 24 are used. Lecithin, which also can be used, is a natural mixture of phospholipids typically derived from chicken eggs or sheep's wool. Phospholipids can be purchased from Avanti lipids (Alabastar, AL, USA) , and lipoid LLC (Newark, NJ, USA) .
  • compositions of the invention may comprise one or more emulsifiers.
  • the emulsifier may be a pure emulsifying agent or a mixture of emulsifying agents.
  • the emulsifiers of the present invention are pharmaceutically and/or immunologically acceptable. Emulsifiers generally assist in stabilizing the mixture of amphipathic compound and antigen or the mixture of amphipathic compound, antigen and adjuvant, when the mixtures are resuspended into the hydrophobic carrier.
  • the emulsifier may be amphipathic and therefore, the emulsifier may include a broad range of compounds.
  • the emulsifier may be a surfactant, such as for example, a non- ionic surfactant.
  • emulsifiers which may be used include polysorbates, which are oily liquids derived from polyethylene glycolyated sorbital, and sorbitan esters. Polysorbates may include, for example, sorbitan monooleate. Typical emulsifiers include mannide oleate (ArlacelTM A) , lecithin, TweenTM 80, and SpansTM 20, 80, 83 and 85, The emulsifier is generally pre-mixed with the hydrophobic carrier.
  • a hydrophobic carrier which already contains an emulsifier may be used.
  • a hydrophobic carrier such MontanideTM ISA-51 already contains the emulsifier mannide oleate.
  • the hydrophobic carrier may be mixed with emulsifier before combining with the amphipathic compound and antigen.
  • the hydrophobic carrier may be an essentially pure hydrophobic substance or a mixture of hydrophobic substances .
  • Hydrophobic substances that are useful in the compositions as described herein are those that are pharmaceutically and/or immunologically acceptable.
  • the carrier is preferably a liquid but certain hydrophobic substances that are not liquids at atmospheric temperature may be liquefied, for example by warming, and are also useful in this invention.
  • Oils or mixtures of oils are particularly suitable carriers for use in the present invention. Oils should be pharmaceutically and/or immunologically acceptable. Oils may be metabolizable or non-metabolizable, or a mixture of metabolizable and non-metabolizable oils may be used.
  • oils are mineral oil (especially light or low viscosity mineral oil) , vegetable oil (e.g., soybean oil), nut oil (e.g., peanut oil) .
  • a low viscosity mineral oil such as Drakeol ® 6VR may be used in some embodiments.
  • the oil is a mannide oleate in mineral oil solution, commercially available as Montanide ® ISA 51.
  • Other oils may include the Montanide ISA 700 series (Seppic Inc., France) or MAS-I (Mercia
  • the hydrophobic carrier may be mixed with emulsifier before use in the compositions of the present invention.
  • Animal fats and artificial hydrophobic polymeric materials particularly those that are liquid at atmospheric temperature or that can be liquefied relatively easily, may also be used.
  • Liquid fluorocarbons are medically applicable hydrophobic carriers that may also be used in the practice of the invention.
  • composition may further comprise one or more additional components such as, for example, pharmaceutically acceptable adjuvants, excipients, etc., as are known in the art: See, for example, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) and The United States Pharmacopoeia: The National Formulary (USP 24 NF19) published in 1999.
  • additional components such as, for example, pharmaceutically acceptable adjuvants, excipients, etc.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al, Immunology, 2d ed. , Benjamin/Cummings : Menlo Park, CA. , 1984; see Wood and Williams, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 23, pp. 317-323) .
  • Suitable adjuvants include, but are not limited to, alum, other compounds of aluminum, Bacillus of Calmette and Guerin (BCG), TiterMax ® , Ribi ® , incomplete Freund's adjuvant (IFA) , saponin, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, Corynebacteriumparvum, QS-21, Freund's Complete Adjuvant (FCA) , adjuvants of the TLR agonist family such as CpG, polyIC (a double stranded RNA) , falgellin, lipopeptides, peptidoglycans, imidazoquinolines, single stranded RNA, lipopolysaccharides (LPS) , heat shock proteins (HSP) , and ceramides and derivatives such as alpha Gal-cer.
  • BCG Bacillus of Calmette and Guerin
  • IFA incomplete Freund's adjuvant
  • Suitable Adjuvants also include cytokines or chemokines in their polypeptide or DNA coding forms such as, but not limited to, GM-CSF, TNF-alpha, IFN-gamma, IL-2, IL-12, IL-15, IL-21.
  • hydrophobic carrier discussed above, may in some instances function as an adjuvant.
  • the amount of adjuvant used depends on the amount of antigen and on the type of adjuvant. One skilled in the art can readily determine the amount of adjuvant needed in a particular application.
  • composition may also contain one or more additional polypeptides, which may be a short synthetic polypeptide such as a T helper epitope.
  • additional polypeptides which may be a short synthetic polypeptide such as a T helper epitope.
  • the antigen and amphipathic compound are mixed prior to suspension in the hydrophobic carrier.
  • the antigen and amphipathic compound are combined in such a manner that a substantially homogeneous mixture is formed. This may be accomplished by solubilizing the antigen and/or the amphipathic compound in a suitable solvent before the components are combined. Alternatively, the two entities can be mixed together in their dry form (e.g. by milling) .
  • a "substantially homogeneous mixture" of the antigen and amphipathic compound is a mixture in which the amphipathic compound is substantially evenly dispersed within the antigen component.
  • amphipathic compound or mixture of amphipathic compounds are present in the compositions of the invention in a sufficient amount that the antigen may be resuspended in the hydrophobic carrier.
  • the amount of amphipathic compound in the compositions of the present invention may be, for example, from about 0.1 mg to about 250 mg of amphipathic compound per ml of the composition, more preferably about 0.1 to about 120 mg of amphipathic compound per ml of the composition.
  • a polar protic solvent such as an alcohol (e.g. tert-butanol , n-butanol, isopropanol, n-propanol, ethanol or methanol) , water, acetic acid or formic acid, or chloroform may be used.
  • an alcohol e.g. tert-butanol , n-butanol, isopropanol, n-propanol, ethanol or methanol
  • water, acetic acid or formic acid, or chloroform may be used.
  • Antigens such as polypeptides may be solubilized with a polar aprotic solvent such as dimethyl sulfoxide (DMSO) , dimethyl formamide (DMF) , or tetrahydrofuran (THF) .
  • a polar aprotic solvent such as dimethyl sulfoxide (DMSO) , dimethyl formamide (DMF) , or tetrahydrofuran (THF) .
  • DMSO dimethyl sulfoxide
  • DMF dimethyl formamide
  • THF tetrahydrofuran
  • Other solvents, such as non-polar solvents (e.g. hexane) may be used, as well as liquid CO 2 .
  • the same solvent can be used to solubilize both the amphipathic and the antigen of interest.
  • solubilized antigen and solubilized amphipathic compound are then mixed.
  • the antigen and amphipathic compound may be mixed prior to solubilization, and then solubilized together.
  • only one of the amphipathic compound or the antigen is solubilized, and the non-solubilized component added .
  • Solvent is then removed, and this may be accomplished using standard techniques. If a readily evaporated solvent is used, such as ethanol, methanol, or chloroform, a standard evaporation technique, such as rotary evaporation, evaporation under reduced pressure, or freeze drying may be employed. Solvents, such as water, may be removed by e.g. lyophilization, freeze drying or spray drying. Low heat drying that does not compromise the integrity of the components can also be used. Heat can also be used to assist in resuspending the antigen/amphipathic compound mixture prior to its use.
  • a readily evaporated solvent such as ethanol, methanol, or chloroform
  • a standard evaporation technique such as rotary evaporation, evaporation under reduced pressure, or freeze drying
  • Solvents such as water, may be removed by e.g. lyophilization, freeze drying or spray drying. Low heat drying that does not compromise the integrity of the components can also be used. Heat can also be used to assist in resus
  • solubilized antigen and solubilized amphipathic compound are mixed thoroughly and then dried as described above.
  • the dried mixture is a substantially homogeneous mixture of antigen and amphipathic compound wherein the amphipathic compound is substantially evenly dispersed within the antigen component. If instead the substantially homogeneous mixture of antigen and amphipathic compound is formed by a solvent -free process such as dry milling, there is of course no need for a drying step .
  • the hydrophobic carrier may contain an emulsifier, as described in detail above, which is provided in an amount sufficient to re-suspend the dry mixture of antigen and amphipathic compound in the hydrophobic carrier and maintaining the antigen and amphipathic compound in suspension in the hydrophobic carrier.
  • the emulsifier may be present at about 5% to about 15% weight/weight or weight/volume of the hydrophobic carrier.
  • Additional components as described above may be added at any stage in the formulation process.
  • one or more such additional components may be combined with the antigen or amphipathic compound either before or after solubilization, or added to the solubilized mixture.
  • Additional components, such as an adjuvant may instead be added to or combined with the dried mixture of antigen and amphipathic compound, or combined with the hydrophobic carrier either before or after suspension of the dry mixture of antigen and amphipathic compound in the hydrophobic carrier.
  • any additional components may be added either before or after milling.
  • a strong immune response is obtainable when the antigen is suspended in the hydrophobic carrier as described, in the absence of substantial quantities of water. It is not expected that antigens could be placed in a hydrophobic carrier unless formulated in the composition described herein. In practice, it may be difficult to obtain completely water-free compositions. That is, although all or substantially all water is removed, such as by evaporation, lyophilization or any other suitable drying technique, at the appropriate stage in the formulation process, there may be small amounts of water remaining. For example, individual components of the composition may have bound water that may not be completely removed by processes such as lyophilization or evaporation and certain hydrophobic carriers may contain small amounts of water dissolved therein.
  • water When water is present, for example, in the form of an emulsion in the composition, it is expected that some amount of antigen may become partitioned into the water. Accordingly, the presence of water in the composition decreases the amount of antigen suspended in the hydrophobic carrier and thus, water is undesirable in the final composition.
  • the finished composition is substantially free of water.
  • substantially free of water is meant that the proportion of antigen suspended in (e.g. dissolved in) water relative to the total amount of antigen in the composition (weight/weight) is low enough that the quantity of antigen suspended in water by itself is incapable of mounting an equivalent immune response to that provided by the composition as a whole.
  • the quantity of antigen that is suspended in the hydrophobic carrier is sufficiently high that an equivalent immune response can be generated with that quantity (i.e. the total quantity minus the antigen quantity present in a residual water component) .
  • the efficacy of the composition i.e. its ability to produce the desired biological response
  • the efficacy of the composition is at least 80%, 85%, 90% or 95% as great as induced by the same composition in which none of the antigen is present in a residual water component .
  • compositions of the invention are sufficiently free of water that no water-in-oil emulsion that is visible to the naked eye is formed. For instance, the presence of an undesirable water-in-oil emulsion might be detected by a non-transparent , cloudy or opaque appearance to the composition.
  • compositions of the invention typically have a clear or transparent appearance and are free of visible particulate matter, such as precipitated or aggregated antigen that is not suspended in the hydrophobic carrier.
  • compositions as described herein may be formulated in any form suitable for delivery of an antigen to a subject, including, by way of non-limiting examples, a form that is suitable for oral, nasal, rectal or parenteral administration.
  • Parenteral administration includes, without limitation, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial , intrapulmonary, intrathecal, and topical modes of administration.
  • kits of the invention contains one or more of the compositions of the invention.
  • the kit can further comprise one or more additional reagents, packaging material, containers for holding the components of the kit, and an instruction set or user manual detailing preferred methods of using the kit components for a desired purpose .
  • compositions of the invention may be provided in which the dry mixture of the antigen and amphipathic compound is packaged in a first container and the hydrophobic carrier is packaged in a second container.
  • the dry mixture of antigen and amphipathic compound may then be suspended in the hydrophobic carrier shortly before administration to a subject.
  • the invention finds application in any instance in which it is desired to administer an antigen to a subject.
  • the subject may be a vertebrate, such as a fish, bird or mammal, preferably a human.
  • compositions of the invention may be administered to a subject in order to raise an antibody response to the antigen.
  • an “antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lamdba.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a protein containing four polypeptides.
  • Each antibody structural unit is composed of two identical pairs of polypeptide chains, each having one "light” and one "heavy” chain.
  • the N-terminus of each chain defines a variable region primarily responsible for antigen recognition.
  • Antibody structural units e.g. of the IgA and IgM classes
  • Antibodies are the antigen-specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells) . Engagement of antigen with antibody expressed on the surface of B cells can induce an antibody response comprising stimulation of B cells to become activated, to undergo mitosis and to terminally differentiate into plasma cells, which are specialized for synthesis and secretion of antigen-specific antibody.
  • antibody response refers to an increase in the amount of antigen-specific antibodies in the body of a subject in response to introduction of the antigen into the body of the subject.
  • One method of evaluating an antibody response is to measure the titers of antibodies reactive with a particular antigen. This may be performed using a variety of methods known in the art such as enzyme-linked immunosorbent assay (ELISA) of antibody-containing substances obtained from animals. For example, the titers of serum antibodies which bind to a particular antigen may be determined in a subject both before and after exposure to the antigen. A statistically significant increase in the titer of antigen-specific antibodies following exposure to the antigen would indicate the subject had mounted an antibody response to the antigen.
  • ELISA enzyme-linked immunosorbent assay
  • compositions of the invention may be administered to a subject in order to raise a cell -mediated immune response to the antigen.
  • a cell -mediated immune response refers to an increase in the amount of antigen- specific cytotoxic T- lymphocytes, macrophages, natural killer cells, or cytokines in the body of a subject in response to introduction of the antigen into the body of the subject.
  • humoral immunity for which the protective function of immunization could be found in the humor (cell- free bodily fluid or serum that contain antibodies)
  • cellular immunity for which the protective function of immunization was associated with cells.
  • Cell-mediated immunity is an immune response that involves the activation of macrophages, natural killer cells (NK) , antigen-specific cytotoxic T- lymphocytes, and the release of various cytokines in response to a x non-self antigen.
  • NK natural killer cells
  • cytokines in response to a x non-self antigen.
  • Cellular immunity is an important component of adaptive immune response and following recognition of antigen by cells through their interaction with antigen-presenting cells such as dendritic cells, B lymphocytes and to a lesser extent, macrophages, protects the body by various mechanisms such as :
  • cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of foreign antigen on their surface, such as virus- infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens;
  • Cell -mediated immunity is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also plays a major role in transplant rejection.
  • i) Antigen presenting cells Dendritic cells and B-cells (and to a lesser extent macrophages) are equipped with special immuno- stimulatory receptors that allow for enhanced activation of T cells, and are termed professional antigen presenting cells (APC) .
  • APC professional antigen presenting cells
  • immuno- stimulatory molecules are up-regulated on these cells following infection or vaccination, during the process of antigen presentation to effector cells such as CD4 and CD8 cytotoxic T cells.
  • co-stimulatory molecules such as CD80, CD86, MHC class I or MHC class II
  • APC such as CDlIc for dendritic cells
  • Cytotoxic T cells (also known as Tc, killer T cell, or cytotoxic T-lymphocyte (CTL) ) are a sub-group of T cells which induce the death of cells that are infected with viruses (and other pathogens), or expressing tumor antigens. These CTLs directly attack other cells carrying certain foreign or abnormal molecules on their surface. The ability of such cellular cytotoxicity can be detected using in vitro cytolytic assays (chromium release assay) . Thus, induction of adaptive cellular immunity can be demonstrated by the presence of such cytotoxic T cells, wherein, when antigen loaded target cells are lysed by specific CTLs that are generated in vivo following vaccination or infection.
  • CTL cytotoxic T-lymphocyte
  • Naive cytotoxic T cells are activated when their T-cell receptor (TCR) strongly interacts with a peptide- bound MHC class I molecule. This affinity depends on the type and orientation of the antigen/MHC complex, and is what keeps the CTL and infected cell bound together. Once activated the CTL undergoes a process called clonal expansion in which it gains functionality, and divides rapidly, to produce an army of "armed" -effector cells. Activated CTL will then travel throughout the body in search of cells bearing that unique MHC Class I + peptide. This could be used to identify such CTLs in vitro by using peptide-MHC Class I tetramers in flow cytometric assays.
  • TCR T-cell receptor
  • effector CTL release perforin and granulysin cytotoxins which form pores in the target cell ' s plasma membrane, allowing ions and water to flow into the infected cell, and causing it to burst or lyse.
  • Release of these molecules from CTL can be used as a measure of successful induction of cellular immue response following vaccination. This can be done by enzyme linked immunosorbant assay (ELISA) or enzyme linked immunospot assay (ELISPOT) where CTLs can be quantitatively measured. Since CTLs are also capable of producing important cytokines such as IFN-g, quantitative measurement of IFN-g-producing CD8 cells can be achieved by ELISPOT and by flowcytometric measurement of intracellular IFN-g in these cells.
  • ELISA enzyme linked immunosorbant assay
  • ELISPOT enzyme linked immunospot assay
  • CD4+ "helper" T-cells CD4+ lymphocytes, or helper T cells, are immune response mediators, and play an important role in establishing and maximizing the capabilities of the adaptive immune response. These cells have no cytotoxic or phagocytic activity; and cannot kill infected cells or clear pathogens, but, in essence "manage" the immune response, by directing other cells to perform these tasks.
  • Two types of effector CD4+ T helper cell responses can be induced by a professional APC, designated ThI and Th2 , each designed to eliminate different types of pathogens.
  • Helper T cells express T-cell receptors (TCR) that recognize antigen bound to Class II MHC molecules.
  • TCR T-cell receptors
  • helper T-cell causes it to release cytokines, which influences the activity of many cell types, including the APC that activated it.
  • Helper T-cells require a much milder activation stimulus than cytotoxic T-cells.
  • Helper T-cells can provide extra signals that "help" activate cytotoxic cells.
  • Two types of effector CD4+ T helper cell responses can be induced by a professional APC, designated ThI and Th2 , each designed to eliminate different types of pathogens. The measure of cytokines associated with ThI or Th2 responses will give a measure of successful vaccination.
  • Thl-cytokines such as IFN-g, IL-2, IL-12, TNF-a and others, or Th2- cytokines such as IL-4, IL- 5, ILlO among others .
  • cytokines released from regional lymph nodes gives a good indication of successful immunization.
  • APC immune effector cells
  • CD4 and CD8 T cells several cytokines are released by lymph node cells.
  • antigen-specific immune response can be detected by measuring release if certain important cytokines such as IFN-g, IL-2, IL-12, TNF-a and GM-CSF. This could be done by ELISA using culture supernatants and recombinant cytokines as standards .
  • the invention finds broad application in the prevention and treatment of any disease susceptible to prevention and/or treatment by way of administration of an antigen.
  • Representative applications of the invention include cancer treatment and prevention, gene therapy, adjuvant therapy, infectious disease treatment and prevention, allergy treatment and prevention, autoimmune disease treatment and prevention, neuron-degenerative disease treatment, and artheriosclerosis treatment, drug dependence treatment and prevention, hormone control for disease treatment and prevention, control of a biological process for the purpose of contraception.
  • Prevention or treatment of disease includes obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, conferring protective immunity against a disease-causing agent and amelioration or palliation of the disease state.
  • Prevention or treatment can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily, although more preferably, it involves preventing the occurrence of disease such as by preventing infection in a subject.
  • HPV 16 E7 (H-2Db) peptide RAHYNIVTF49-57 (SEQ ID NO: 1) containing a CTL epitope was fused to PADRE containing a CD4+ helper epitope by Dalton Chemical Laboratories Inc. (Toronto, Ontario, Canada) .
  • This peptide is hereafter designated FP and was used as antigen in vaccine at 50 micrograms/ 100 microliters dose.
  • FP has been used in vaccine studies to prevent or eliminate C3 tumors in mice .
  • dioleoyl phosphatidylcholine (DOPC) was solubilized in tert-butanol .
  • FP was first solubilized in dimethyl sulfoxide, although a water suspension of FP can also be used. FP was then added to the DOPC/tert-butanol mixture. Where indicated, a synthetic lipopeptide-based immune stimulating compound (the adjuvant) was resuspended in water and added to the DOPC/FP/ tert-butanol mixture. A dry homogenous mixture of antigen (with or without adjuvant) was prepared by removing the solvent and water present in the formulation by lyophilization.
  • the dry mixture was then suspended in Incomplete Freund's adjuvant, a mineral oil-based model hydrophobic carrier.
  • mice groups of mice (7 to 10 mice per group) were injected with half a million C3 cells subcutaneously in the left flank above the base of the tail. Eight days post-implantation, all mice were injected with vaccine formulations (100 microliters per dose) subcutaneously in the right flank.
  • mice Five groups of mice were vaccinated as follows: Group 1 mice served as control mice and were injected with phosphate buffered saline to allow the normal progression of tumors; group 2 mice were vaccinated with a standard water-in-oil mineral oil -based emulsion (incomplete Freund's adjuvant) containing FP antigen (50 micrograms per dose) in the aqueous component of the emulsion; group 3 mice were vaccinated with a homogeneous water- free formulation prepared as described above and containing FP antigen (50 micrograms per dose) , DOPC (12 micrograms per dose) and 100 microliters of a hydrophobic carrier (Incomplete freund's adjuvant); group 4 mice were vaccinated with a standard water-in-oil mineral oil-based emulsion (incomplete Freund's adjuvant) containing FP antigen (50 micrograms per dose) and Pam3Cys adjuvant (50 micrograms per dose) in the aque
  • mice in group 1 implanted with C3 cells developed tumors with tumors reaching 1881 cubic millimeters in size on day 42 post tumor implantation.
  • Control vaccinations consisting of FP antigen in a water-in-oil emulsion controlled tumor growth in mice (group 2) , with tumors progressing to an average size of 548 cubic millimeters on day 42.
  • Average tumor size in group 3 mice was 73 cubic millimeter on day 42 post tumor implantation, with 6 out of 8 mice being rendered tumor free.
  • the addition of an adjuvant, Pam3Cys in this example improved efficacy of the water-in-oil vaccine formulation used for group 2 slightly (group 4 average tumor size on day 42 was 320 cubic millimeters versus group 2 average tumor size of 548 cubic millimeters) .
  • the addition of Pam3Cys adjuvant improved the efficacy of the water-free vaccine formulation further (group 5 vaccine) , with an average tumor size of 14 cubic millimeters on day 42, with 6 out of 7 mice being rendered tumor- free.
  • HPV 16 E7 (H-2Db) peptide RAHYNIVTF49-57 SEQ ID NO: 1
  • Vaccine efficacy was assessed by Enzyme-linked Immunospot assay (ELISPOT) , a method that allows the ex vivo detection of antigen-specific cellular immune responses in splenocytes harvested from immunized C57BL/6 mice.
  • the ELISPOT assay is useful for assessing the presence/absence of an antigen-specific immune response but has its limitations when used as a correlate of vaccine efficacy against a target in vivo. Briefly, on day 8 post- immunization, a 96-well nitrocellulose plate was coated with capture antibody, a purified anti -mouse IFN-gamma antibody, by incubation overnight at 4 "C 7 then blocked with complete media.
  • Splenocytes were added to wells at an initial concentration of 5x105 cells/well in a volume of 100 ⁇ l and a row of serial dilutions prepared. Cells in a dilution series were stimulated with the specific peptide RAHYNIVTF49-57 (10 ⁇ g/ml) . The plate was incubated overnight at 37 * C/5% CO 2 . Next day, the plate was incubated with detection antibody (a biotinylated anti-mouse IFN- ⁇ antibody), for 2 hours at room temperature. Unbound detection antibody was removed by washing and the enzyme conjugate (Streptavidin-HRP) was added.
  • detection antibody a biotinylated anti-mouse IFN- ⁇ antibody
  • dioleoyl phosphatidylcholine (DOPC) was solubilized in tert-butanol .
  • FP was first solubilized in dimethyl sulfoxide, although a water suspension of FP can also be used.
  • FP was then added to the DOPC/tert-butanol mixture.
  • a dry homogenous mixture of antigen and DOPC was prepared by removing the solvent present in the formulation by lyophilization.
  • the dry mixture was then suspended in Incomplete Freund's adjuvant, a mineral oil-based model hydrophobic carrier.
  • the efficacy of this formulation was compared to one consisting of antigen in typical water-in-oil emulsion such as an incomplete Freund's adjuvant -based emulsion consisting primarily of water (containing the antigen) in a continuous mineral -oil based oil carrier.
  • the efficacy of the formulation was also compared to one consisting the antigen diluted directly from a antigen/dimethyl sulfoxide stock solution into typical water-in-oil emulsion such as an incomplete Freund's adjuvant -based emulsion.
  • mice mice (4 mice per group) were injected with vaccine formulations (100 microliters per dose) subcutaneously in the right flank.
  • Three groups of mice were vaccinated as follows: Group 1 mice served as control mice and were vaccinated with a standard water-in-oil mineral oil-based emulsion (incomplete Freund's adjuvant) containing FP antigen (20 micrograms per dose) in the aqueous component of the emulsion; group 2 mice were vaccinated with a homogeneous water- free formulation prepared as described above and containing FP antigen (20 micrograms per dose) , DOPC (12 micrograms per dose) and 100 microliters of a hydrophobic carrier (Incomplete freund's adjuvant); group 3 mice served as control mice and were vaccinated with a homogeneous water- free formulation lacking the amphipathic carrier (DOPC) but containing FP antigen (20 micrograms per dose) , and 100 microliters of a
  • DOPC amphipathic carrier
  • poly IC double stranded RNA (Pierce, Milwaukee, USA) was used as a representative molecule that has physical and chemical characteristics that are similar to those of a genetic antigen construct (a nucleotide based plasmid or RNA molecule) .
  • PoIyIC also serves as a representative of nucleotide based adjuvants that may be co- formulated with antigen in the invention.
  • DOPC dioleoyl phosphatidylcholine
  • polyI:C was first solubilized in water at a concentration of 5mg/ml . Then 80 ul of polylrC (0.4 mg) were further diluted in 320 ul of water. The polyl : C dilution was then added and mixed to the DOPC/tert-butanol mixture in vial 21. A dry homogenous mixture of DOPC/adjuvant was prepared by removing the solvent and water present in the formulation by lyophilization. A control formulation containing polyIC alone (vial 26) was made in the same manner as described above, with the exception that DOPC was not added to tert butanol .
  • vials 21 and 26 were then suspended by adding 0.88 ml of a hydrophobic carrier to vial 21, and 1 ml of hydrophobic carrier to vial 26.
  • the hydrophobic carrier used was a mineral oil containing mannide oleate and known as Montanide ® ISA 51 (Seppic, France) .
  • the dry mixture was resuspended in the hydrophobic carrier by vortexing for approximately 3 minutes (vial 21) , or a minimum of 30 minutes (vial 26) .
  • Vials 21 and 26 were compared by visual inspection along side a vial containing 1 ml of hydrophobic carrier alone (vial labelled ISA51) .
  • vial 21 appeared similar to that of ISA51, with no visible particulate material present. This suggested that the nucleotide molecules were effectively suspended in the hydrophobic carrier in the presence of DOPC.
  • vial 26 lacking DOPC and containing only the nucleotide molecules and hydrophobic carrier, contained a heterogenous suspension of nucleotide molecules in the hydrophobic carrier that can be easily detected by visual inspection.
  • the hydrophilic nucleotide molecules could not be suspended in the hydrophobic carrier. This clearly demonstrates that a molecule such as DOPC that is amphipathic in nature, facilitated the formulation of hydrophilic nucleotide-based molecules in a hydrophobic carrier in the absence of a significant quantity of water.
  • vials 30 and 35 were then suspended by adding 0.88 ml of a hydrophobic carrier to vial 30, and 1 ml of hydrophobic carrier to vial 35.
  • the hydrophobic carrier used was a mineral oil containing mannide oleate and known as Montanide ® ISA 51 (Seppic,
  • the dry mixture was resuspended in the hydrophobic carrier by vortexing for approximately 30 seconds (vial 30), or a minimum of 30 minutes (vial 35) .
  • Vials 30 and 35 were compared by visual inspection along side a vial containing 1 ml of hydrophobic carrier alone (vial labelled ISA51) .
  • vial 30 Upon visual inspection ( Figure 8) , the contents of vial 30 appeared similar to that of ISA51, with no visible particulate material present. This suggested that the peptides were effectively suspended in the hydrophobic carrier in the presence of DOPC. In contrast, vial 35 lacking DOPC and containing only the peptides and hydrophobic carrier, contained a heterogeneous suspension of peptide aggregates in the hydrophobic carrier that can be easily detected by visual inspection. In the absence of DOPC, peptide-based antigens could not be suspended in the hydrophobic carrier. This clearly demonstrates that a molecule such as DOPC that is amphipathic in nature, facilitated the formulation of peptide based antigens in a hydrophobic carrier in the absence of a significant quantity of water.

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Abstract

La présente invention porte sur des compositions comprenant un antigène, un composé amphipathique et un support hydrophobe, l'antigène étant mis en suspension dans ledit support hydrophobe en l'absence substantielle d'eau. L'invention porte également sur des procédés d'utilisation de ces compositions pour induire un anticorps ou une réponse à médiation par une cellule dans un sujet.
PCT/CA2008/001747 2007-10-03 2008-10-02 Compositions comprenant un antigène, un composé amphipathique et un support hydrophobe, et leurs utilisations WO2009043165A1 (fr)

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BRPI0817484-9A BRPI0817484B1 (pt) 2007-10-03 2008-10-02 Composição de vacina sem água, processo de produção e o uso dos mesmos
EP08800418A EP2195022A4 (fr) 2007-10-03 2008-10-02 Compositions comprenant un antigène, un composé amphipathique et un support hydrophobe, et leurs utilisations
JP2010527303A JP5591705B2 (ja) 2007-10-03 2008-10-02 抗原、両親媒性化合物及び疎水性担体を含有する組成物、並びにその使用
CN200880110239.7A CN101815529B (zh) 2007-10-03 2008-10-02 包含抗原、两性化合物和疏水载体的组合物及其应用
CA2700828A CA2700828C (fr) 2007-10-03 2008-10-02 Compositions comprenant un antigene, un compose amphipathique et un support hydrophobe, et leurs utilisations
US12/679,998 US20100209452A1 (en) 2007-10-03 2008-10-02 Compositions comprising an antigen, an amphipathic compound and a hydrophobic carrier, and uses thereof
AU2008307042A AU2008307042B2 (en) 2007-10-03 2008-10-02 Compositions comprising an antigen, an amphipathic compound and a hydrophobic carrier, and uses thereof

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105044328A (zh) * 2015-05-28 2015-11-11 首都医科大学附属北京佑安医院 Elispot预包被培养板及其制备方法
WO2017083963A1 (fr) * 2015-11-18 2017-05-26 Immunovaccine Technologies Inc. Systèmes adjuvant et compositions vaccinales sans eau comprenant un adjuvant de type polyi:c et un adjuvant lipidique
WO2017190242A1 (fr) * 2016-05-04 2017-11-09 Immunovaccine Technologies Inc. Compositions de vaccin comprenant un composé amphipathique, un néoantigène et un support hydrophobe, et leurs procédés d'utilisation
WO2018058230A1 (fr) * 2016-09-27 2018-04-05 Immunovaccine Technologies Inc. Procédés d'utilisation de compositions d'épitope de lymphocytes b à faible volume de dose pour induire une réponse immunitaire à anticorps chez des sujets humains
US10022441B2 (en) 2013-03-27 2018-07-17 Immunovaccine Technologies, Inc. Method for improving the efficacy of a survivin vaccine in the treatment of cancer
US10105435B2 (en) 2011-10-06 2018-10-23 Immunovaccine Technologies Inc. Liposome compositions comprising an adjuvant that activates or increases the activity of TLR2 and uses thereof
WO2019178677A1 (fr) * 2018-03-20 2019-09-26 Immunovaccine Technologies Inc. Méthodes et compositions pour l'administration ciblée d'agents actifs et d'agents immunomodulateurs à des ganglions lymphatiques
US10533033B2 (en) 2015-01-06 2020-01-14 Immunovaccine Technologies Inc. Lipid A mimics, methods of preparation, and uses thereof
US11235069B2 (en) 2007-09-27 2022-02-01 Immunovaccine Technologies Inc. Use of liposomes in a carrier comprising a continuous hydrophobic phase for delivery of polynucleotides in vivo
WO2022177993A1 (fr) * 2021-02-16 2022-08-25 Vaccitech North America, Inc. Nanoparticules auto-assemblées à base de peptides amphiphiles
US11717563B2 (en) 2008-06-05 2023-08-08 Immunovaccine Technologies Inc. Compositions comprising liposomes, an antigen, a polynucleotide and a carrier comprising a continuous phase of a hydrophobic substance

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038175A1 (fr) * 2000-11-07 2002-05-16 Immunovaccine Technologies Inc. Vaccins a reponse immunitaire accrue et leurs procedes de preparation
US6956021B1 (en) * 1998-08-25 2005-10-18 Advanced Inhalation Research, Inc. Stable spray-dried protein formulations
US7306787B2 (en) * 1997-09-29 2007-12-11 Nektar Therapeutics Engineered particles and methods of use

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU94040856A (ru) * 1992-03-03 1996-08-20 Дайити Фармасьютикал Ко. Вакцина для перорального введения
US5736141A (en) * 1992-06-05 1998-04-07 Dalhousie University Method to prevent fertilization in mammals by administering a single dose of zona pellucida derived antigens, liposome and Freund's adjuvant
GB9422990D0 (en) * 1994-11-15 1995-01-04 Cortecs Ltd Immunogenic compositions
US6110492A (en) * 1997-05-28 2000-08-29 Jenner Biotherapies, Inc. Immunogenic compositions
CA2523032A1 (fr) * 2005-10-07 2007-04-07 Immunovaccine Technologies Inc. Vaccins pour la therapie du cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7306787B2 (en) * 1997-09-29 2007-12-11 Nektar Therapeutics Engineered particles and methods of use
US6956021B1 (en) * 1998-08-25 2005-10-18 Advanced Inhalation Research, Inc. Stable spray-dried protein formulations
WO2002038175A1 (fr) * 2000-11-07 2002-05-16 Immunovaccine Technologies Inc. Vaccins a reponse immunitaire accrue et leurs procedes de preparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2195022A4 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11235069B2 (en) 2007-09-27 2022-02-01 Immunovaccine Technologies Inc. Use of liposomes in a carrier comprising a continuous hydrophobic phase for delivery of polynucleotides in vivo
US11717563B2 (en) 2008-06-05 2023-08-08 Immunovaccine Technologies Inc. Compositions comprising liposomes, an antigen, a polynucleotide and a carrier comprising a continuous phase of a hydrophobic substance
US10105435B2 (en) 2011-10-06 2018-10-23 Immunovaccine Technologies Inc. Liposome compositions comprising an adjuvant that activates or increases the activity of TLR2 and uses thereof
US11077184B2 (en) 2011-10-06 2021-08-03 Immunovaccine Technologies Inc. Liposome compositions comprising PAM2Cys or PAM3Cys adjuvant and methods for inducing a humoral immune response
US10729766B2 (en) 2013-03-27 2020-08-04 Immunovaccine Technologies Inc. Method for improving the efficacy of a survivin vaccine in the treatment of cancer
EP3421047A1 (fr) 2013-03-27 2019-01-02 ImmunoVaccine Technologies Inc. Procédé d'amélioration de l'efficacité d'un vaccin à base de survivine pour le traitement du cancer
US10022441B2 (en) 2013-03-27 2018-07-17 Immunovaccine Technologies, Inc. Method for improving the efficacy of a survivin vaccine in the treatment of cancer
US10988500B2 (en) 2015-01-06 2021-04-27 Immunovaccine Technologies Inc. Lipid A mimics, methods of preparation, and uses thereof
US10533033B2 (en) 2015-01-06 2020-01-14 Immunovaccine Technologies Inc. Lipid A mimics, methods of preparation, and uses thereof
CN105044328A (zh) * 2015-05-28 2015-11-11 首都医科大学附属北京佑安医院 Elispot预包被培养板及其制备方法
EP3377099A4 (fr) * 2015-11-18 2019-07-24 ImmunoVaccine Technologies Inc. Systèmes adjuvant et compositions vaccinales sans eau comprenant un adjuvant de type polyi:c et un adjuvant lipidique
WO2017083963A1 (fr) * 2015-11-18 2017-05-26 Immunovaccine Technologies Inc. Systèmes adjuvant et compositions vaccinales sans eau comprenant un adjuvant de type polyi:c et un adjuvant lipidique
US11160861B2 (en) 2015-11-18 2021-11-02 Immunovaccine Technologies Inc. Adjuvanting systems and water-free vaccine compositions comprising a polyI:C polynucleotide adjuvant and a lipid-based adjuvant
US11260116B2 (en) 2016-05-04 2022-03-01 Immunovaccine Technologies Inc. Vaccine compositions comprising an amphipathic compound, a neoantigen and a hydrophobic carrier, and methods of use thereof
WO2017190242A1 (fr) * 2016-05-04 2017-11-09 Immunovaccine Technologies Inc. Compositions de vaccin comprenant un composé amphipathique, un néoantigène et un support hydrophobe, et leurs procédés d'utilisation
EP3452083A4 (fr) * 2016-05-04 2020-01-01 ImmunoVaccine Technologies Inc. Compositions de vaccin comprenant un composé amphipathique, un néoantigène et un support hydrophobe, et leurs procédés d'utilisation
AU2017260610B2 (en) * 2016-05-04 2022-10-27 Immunovaccine Technologies Inc. Vaccine compositions comprising an amphipathic compound, a neoantigen and a hydrophobic carrier, and methods of use thereof
WO2018058230A1 (fr) * 2016-09-27 2018-04-05 Immunovaccine Technologies Inc. Procédés d'utilisation de compositions d'épitope de lymphocytes b à faible volume de dose pour induire une réponse immunitaire à anticorps chez des sujets humains
EP3518963A4 (fr) * 2016-09-27 2020-06-10 ImmunoVaccine Technologies Inc. Procédés d'utilisation de compositions d'épitope de lymphocytes b à faible volume de dose pour induire une réponse immunitaire à anticorps chez des sujets humains
US11406705B2 (en) 2016-09-27 2022-08-09 Immunovaccine Technologies Inc. Methods of using low dose volume B-cell epitope compositions for inducing an antibody immune response in human subjects
US12042537B2 (en) 2016-09-27 2024-07-23 BioVaxys Inc. Methods of using low dose volume B-cell epitope compositions for inducing an antibody immune response in human subjects
CN112188887A (zh) * 2018-03-20 2021-01-05 免疫疫苗技术公司 用于将活性剂和免疫调节剂靶向递送至淋巴结的方法和组合物
US20210147544A1 (en) * 2018-03-20 2021-05-20 Immunovaccine Technologies Inc. Methods and compositions for targeted delivery of active agents and immunomodulatory agents to lymph nodes
EP3768242A4 (fr) * 2018-03-20 2021-12-08 ImmunoVaccine Technologies Inc. Méthodes et compositions pour l'administration ciblée d'agents actifs et d'agents immunomodulateurs à des ganglions lymphatiques
WO2019178677A1 (fr) * 2018-03-20 2019-09-26 Immunovaccine Technologies Inc. Méthodes et compositions pour l'administration ciblée d'agents actifs et d'agents immunomodulateurs à des ganglions lymphatiques
WO2022177993A1 (fr) * 2021-02-16 2022-08-25 Vaccitech North America, Inc. Nanoparticules auto-assemblées à base de peptides amphiphiles

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BRPI0817484A2 (pt) 2015-03-24
CN101815529B (zh) 2016-03-09
CA2700828C (fr) 2017-01-24
AU2008307042B2 (en) 2014-01-30
AU2008307042A1 (en) 2009-04-09
CN101815529A (zh) 2010-08-25
EP2195022A4 (fr) 2012-08-01
CA2700828A1 (fr) 2009-04-09
BRPI0817484B1 (pt) 2021-08-17
JP5591705B2 (ja) 2014-09-17
JP2010540570A (ja) 2010-12-24
EP2195022A1 (fr) 2010-06-16

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