WO2024186623A1 - Methods of making dried pharmaceutical compositions - Google Patents

Methods of making dried pharmaceutical compositions Download PDF

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Publication number
WO2024186623A1
WO2024186623A1 PCT/US2024/018048 US2024018048W WO2024186623A1 WO 2024186623 A1 WO2024186623 A1 WO 2024186623A1 US 2024018048 W US2024018048 W US 2024018048W WO 2024186623 A1 WO2024186623 A1 WO 2024186623A1
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WIPO (PCT)
Prior art keywords
lipid
lipid vesicle
vesicle particles
mixture
drying
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PCT/US2024/018048
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French (fr)
Inventor
Rajkannan RAJAGOPALAN
Leanne Jennifer LUCAS
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BioVaxys Inc.
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Publication of WO2024186623A1 publication Critical patent/WO2024186623A1/en

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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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid

Definitions

  • vaccines containing antigens are typically not immunogenic enough to generate rapid and prolonged immunity. This can sometimes be overcome with the use of an adjuvant to boost the immune response towards an antigen.
  • adjuvants There are generally two broad categories of adjuvants: delivery systems and immune-stimulants.
  • the delivery system of a vaccine can act as an adjuvant by providing stability and prolonged interaction of the antigen with the immune system.
  • Vaccine compositions may also incorporate molecular compounds with immune- stimulatory activity as adjuvants with the aim of further enhancing immunogenicity of the vaccine by directly activating cells of the immune system.
  • Immune-stimulant adjuvants are defined molecular agonists that are recognized by the immune system via specialized receptors, for example polyI:C polynucleotide and lipid- based adjuvants such as PAM 3 CSK4 stimulate distinct Toll-like receptors. Immune-stimulants can activate the immune system and also direct the type of immune response generated towards a vaccine antigen.
  • the effectiveness of many vaccines is correlated to the generation of antibodies; however, for other types of vaccines a strong cytotoxic immune response primarily mediated by CD8+ T cells is desired.
  • the type of immune response generated towards a vaccine antigen can be manipulated by including immune-stimulants that activate particular receptors found on immune cells that can initiate these responses through generation of cytokines and chemokines.
  • Differences in the properties of immune-stimulants such as polyI:C polynucleotide and lipid-based adjuvants can complicate their incorporation into pharmaceutical compositions.
  • a method for preparing a pharmaceutical composition comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the first dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the second dried preparation to produce a pharmaceutical composition.
  • a method for preparing a pharmaceutical composition comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the second dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the first dried preparation to produce a pharmaceutical composition.
  • a pharmaceutical composition prepared by the method as described herein.
  • a method of inducing an antibody and/or CTL immune response in a subject comprising administering to the subject the pharmaceutical composition as described herein.
  • a use of the pharmaceutical composition as described herein for inducing an antibody and/or CTL immune response in a subject there is provided a method of delivering a therapeutic agent to a subject comprising administering to the subject the pharmaceutical composition as described herein.
  • a use of the pharmaceutical composition as described herein for delivering a therapeutic agent to a subject comprising administering to the subject the pharmaceutical composition as described herein.
  • kits for preparing the pharmaceutical composition as described herein comprising: a container comprising a first dried preparation prepared by the method as described herein; a container comprising second dried preparation prepared by the method as described herein; and a container comprising a hydrophobic carrier.
  • Poly I:C polynucleotide is an oligonucleotide molecule, composed of either DNA or RNA nucleosides, which interacts with Toll-like receptors (TLRs) that can detect nucleic acids, such as TLR3.
  • TLRs Toll-like receptors
  • Lipid-based adjuvants such as PAM 2 Cys and PAM 3 Cys interact with TLR1/2. Stimulating multiple TLR receptors at once has been reported to have an additive or synergistic activating effect on dendritic cells.
  • poly I:C polynucleotide and a lipid-based adjuvant such as PAM2Cys or PAM3Cys in a pharmaceutical composition that can ensure they engage with TLRs on the same cell can provide a potent activating effect to the immune system.
  • Optimal doses of each adjuvant may need to be confirmed for different applications. For example, vaccines for cancer therapy may need stronger immune stimulation than vaccines for prevention of infectious disease.
  • the lipopeptides PAM 2 Cys and PAM 3 Cys comprise a lipid palmitic acid moiety conjugated to a cysteine (Cys) residue that may be extended into a peptide with polar residues such as serine (Ser) and/or lysine (Lys) with a positive surface charge.
  • Cys cysteine
  • polyI:C polynucleotides are made up of single or double stranded molecules containing inosinic acid residues (I) and cytidylic acid residues (C) with a negative surface charge. If needed, these two distinct immune stimulators can be complexed together by electrostatic interactions to a certain extent with concentration restrictions.
  • any one of these immune stimulators may lead to a pH shift and cause unwanted precipitation during formulation and/or in the finished product, constraining the ability to provide desired concentrations of lipid- based adjuvant and polyI:C polynucleotide in a pharmaceutical composition.
  • Using current methods it is only possible to combine polyI:C polynucleotides and PAM 2 Cys and PAM 3 Cys lipid-based adjuvants in a single water-free formulation up to about 0.5 mg/mL concentration.
  • Embodiments of the present invention provide methods of fully incorporating both polyI:C polynucleotide and lipid-based adjuvant (such as PAM 2 Cys and PAM 3 Cys) along with therapeutic agents that overcomes concentration limitations, irrespective of the nature of therapeutic agents and formulation buffer/pH used to prepare the pharmaceutical composition.
  • polyI:C polynucleotide and lipid-based adjuvant such as PAM 2 Cys and PAM 3 Cys
  • PolyI:C polynucleotide are polynucleotide molecules (either RNA or DNA or a combination of DNA and RNA) containing inosinic acid residues (I) and cytidylic acid residues (C), and which induce the production of inflammatory cytokines, such as interferon.
  • the polyI:C polynucleotide is double-stranded. In double-stranded embodiments, they are typically composed of one strand consisting entirely of cytosine-containing nucleotides and one strand consisting entirely of inosine-containing nucleotides, although other configurations are possible.
  • each strand may contain both cytosine-containing and inosine-containing nucleotides.
  • the polyI:C polynucleotide is single- stranded. In single-stranded embodiments, the strand comprises both cytosine-containing and inosine-containing nucleotides. In some instances, either or both strand may additionally contain one or more non-cytosine or non-inosine nucleotides.
  • the polyI:C polynucleotide may be a single-stranded molecule containing inosinic acid residues (I) and cytidylic acid residues (C).
  • the single-stranded polyI:C polynucleotide may be a sequence of repeating dIdC.
  • the sequence of the single-stranded polyI:C may be a 26-mer sequence of (IC) 13 , i.e. ICICICICICICICICICICICICIC.
  • IC ICICICICICICICICICICICICIC
  • a “polyI:C” or “polyI:C polynucleotide” is a double- or single-stranded polynucleotide molecule (RNA or DNA or a combination of DNA and RNA), each strand of which contains at least 6 contiguous inosinic or cytidylic acid residues, or 6 contiguous residues selected from inosinic acid and cytidylic acid in any order (e.g. IICIIC or ICICIC).
  • PolyI:C polynucleotides will typically have a length of about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000 or more nucleotides.
  • Each strand of a double-stranded polyI:C polynucleotide may be a homopolymer of inosinic or cytidylic acid residues, or each strand may be a heteropolymer containing both inosinic and cytidylic acid residues.
  • the polymer may be interrupted by one or more non-inosinic or non-cytidylic acid residues (e.g. uridine), provided there is at least one contiguous region of 6 I, 6 C or 6 I/C residues as described above.
  • each strand of a polyI:C polynucleotide will contain no more than 1 non-I/C residue per 6 I/C residues, more preferably, no more than 1 non-I/C residue per every 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 I/C residues.
  • the inosinic acid or cytidylic acid (or other) residues in the polyI:C polynucleotide may be derivatized or modified as is known in the art, provided the ability of the polyI:C polynucleotide to promote the production of an inflammatory cytokine, such as interferon, is retained.
  • Non-limiting examples of derivatives or modifications include e.g. azido modifications, fluoro modifications, or the use of thioester (or similar) linkages instead of natural phosphodiester linkages to enhance stability in vivo.
  • the polyI:C polynucleotide may also be modified to e.g. enhance its resistance to degradation in vivo by e.g.
  • lipid-based adjuvant is an adjuvant that comprises at least one lipid moiety or lipid component.
  • lipid moiety or “lipid component” refers to any fatty acid (e.g. fatty acyls) or derivative thereof, including for example triglycerides, diglycerides, and monoglycerides.
  • Exemplary fatty acids include, without limitation, palmitoyl, myristoyl, stearoyl and decanoyl groups or any C2 to C30 saturated or unsaturated fatty acyl group, preferably any C14 to C22 saturated or unsaturated fatty acyl group, and more preferably a C16 saturated or unsaturated fatty acyl group.
  • lipid-based adjuvant encompasses adjuvants comprising a fatty acyl group or derivative thereof.
  • Lipid-based adjuvants contain at a minimum at least one lipid moiety, or a synthetic/semi-synthetic lipid moiety analogue, which can be coupled onto an amino acid, an oligopeptide or other molecules (e.g. a carbohydrate, a glycan, a polysaccharide, biotin, Rhodamine, etc.).
  • the lipid-based adjuvant may be, for example, a lipoamino acid, a lipopeptide, a lipoglycan, a lipopolysaccharide or a lipoteichoic acid.
  • a lipid moiety or a structure containing a lipid moiety can be coupled covalently or non-covalently to an antigen to create antigenic compounds with built-in adjuvanting properties.
  • the lipid-based moiety may comprise a cation (e.g. nickel) to provide a positive charge for non-covalent coupling.
  • the lipid moiety or lipid component may be naturally occurring, such as for example a cell-wall component (e.g. lipoprotein) from a Gram-positive or Gram-negative bacteria, Rhodopseudomonas viridis, or mycoplasma.
  • the lipid moiety or lipid component may be synthetic or semi-synthetic
  • the lipid-based adjuvant may comprise palmitic acid (PAM) as at least one of the lipid moieties or components of the adjuvant.
  • PAM palmitic acid
  • Such lipid-based adjuvants are referred to herein as a “palmitic acid adjuvant”. Palmitic acid is a low molecular weight lipid found in the immunologically reactive Braun’s lipoprotein of Escherichia coli. Other common chemical names for palmitic acid include, for example, hexadecanoic acid in IUPAC nomenclature and 1-Pentadecanecarboxylic acid.
  • a palmitic acid adjuvant contains at a minimum at least one palmitic acid moiety, which can be coupled onto an amino acid, an oligopeptide or other molecules.
  • a palmitic acid moiety or a structure containing palmitic acid can be coupled covalently or non-covalently to an antigen to create antigenic compounds with built-in adjuvanting properties.
  • the palmitic acid moiety or a chemical structure containing palmitic acid can be conjugated to a cysteine peptide (Cys) to allow for various structural configurations of the adjuvant, including linear and branched structures.
  • the cysteine residue has been commonly extended by polar residues such as Serine (Ser) and/or lysine (Lys) at the C terminus to create adjuvant compounds with improved solubility.
  • Palmitic acid containing adjuvant compounds could be admixed with an antigen, associated with antigen through non-covalent interactions, or alternatively covalently linked to an antigen, either directly or with the use of a linker/spacer, to generate enhanced immune responses.
  • an antigen associated with antigen through non-covalent interactions
  • an antigen either directly or with the use of a linker/spacer
  • two palmitic acid moieties are attached to a glyceryl backbone and a cysteine residue to create dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys), which can also be used in multiple configurations as described above.
  • PAM2Cys dipalmitoyl-S-glyceryl-cysteine
  • PAM3Cys tripalmitoyl-S-glyceryl-cysteine
  • the lipid-based adjuvant is any type of adjuvant comprising a palmitic acid moiety or component.
  • lipid-based adjuvant is a lipopeptide comprising one or more palmitic acid moieties.
  • the palmitic acid moiety may be modified or manipulated to improve its stability in vitro or in vivo, enhance its binding to receptors (such as for example toll-like receptors as described below) or enhance its biological activity.
  • the palmitic acid adjuvant comprises PAM2Cys.
  • the palmitic acid adjuvant comprises PAM 3 Cys.
  • the palmitic acid adjuvant comprises PAM 2 -Cys-Ser-(Lys)4 or PAM3-Cys-Ser-(Lys)4.
  • the palmitic acid adjuvant is an analog of PAM2-Cys-Ser- (Lys)4 or PAM3-Cys-Ser-(Lys)4 including, without limitation, PAM3Cys-SKKKK ( ⁇ - irradiated), R-PAM3Cys-SKKKK S-PAM3Cys-SKKKK, PAM3Cys-SKKKK(Biotin-Aca-Aca), PAM 3 Cys-SKKKK(Fluorescein-Aca-Aca), PAM 3 Cys-SKKKK, PAM 3 Cys-SKKKK-FLAG-tag, PAM3Cys-SSNAKIDQLSSDVQT, PAM3Cys-SSNKSTT
  • the palmitic acid adjuvant or analog thereof may be used as stereochemically defined compounds or as a mixture of stereoisomers.
  • the lipid-based adjuvant is PAM 3 -Cys-Ser-(Lys)4: that act as TLR agonists may also be used as the lipid-based adjuvant disclosed herein, including without limitation the palmitic acid adjuvants and analogs described above and synthetic diacylated lipoprotein FSL-1 available from InvivoGen (San Diego, California, USA) and EMC Microcollections GmbH (Germany).
  • FSL-1 (Pam2CGDPKHPKSF) is a synthetic lipoprotein that represents the N-terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium.
  • FSL-1 comprises PAM 2 Cys and has a similar framework structure as macrophage activating lipopeptide-2 (MALP-2), a Mycoplasma fermentans derived lipopeptide.
  • MALP-2 macrophage activating lipopeptide-2
  • the lipid-based adjuvant comprises FSL-1 or MALP-2, or the lipid-based adjuvant is FSL-1 or MALP-2.
  • FSL-1 or MALP-2 may be used as stereochemically defined compounds or as a mixture of stereoisomers.
  • the FSL-1 or MALP- 2 may be labelled (e.g.
  • lipid-based adjuvants that comprise palmitic acid may include substructures of TLR2 ligands such as monoacylated lipopeptides. Without limitation, these may include, for example, Pam-Dhc-SKKKK, Pam-CSKKKK, Pam-Dhc-GDPKHPKSF or Pam-CGDPKHPKSF (EMC Microcollections).
  • therapeutic agent is any molecule, substance or compound that is capable of providing a therapeutic activity, response or effect in the treatment or prevention of a disease, disorder or condition, including diagnostic and prophylactic agents.
  • a “first therapeutic agent” is any one or more therapeutic agents which are used in the preparation of the non-sized lipid vesicle particle preparation (i.e. incorporated in the methods before the step of sizing the non-sized lipid vesicle preparation).
  • a “second therapeutic agent” is any one or more therapeutic agents which are used in the methods herein after preparation of the sized lipid vesicle particle preparation (i.e.
  • lipids may first be dissolved and mixed in an organic solvent. In embodiments where different types of lipid are used, this step will allow a homogenous mixture of the lipids to be formed. In an embodiment, these steps may be carried out in chloroform, chloroform:methanol mixtures, tertiary butanol or cyclohexane. In an embodiment, the lipids are prepared at 10-20mg lipid/mL organic solvent; however, higher or lower concentrations may also be used. In some embodiments, after mixing, the organic solvent is removed (e.g. by evaporation) to yield a lipid film.
  • the lipid film may then be frozen and lyophilized to yield a dry lipid film.
  • the dry lipid film may then be hydrated with an aqueous solution containing therapeutic agents to provide a non-sized lipid vesicle particle preparation.
  • the step of hydration may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour).
  • an aqueous solution of lipids may be combined with a solution containing one or more solubilized therapeutic agents.
  • one or more dry therapeutic agents may be added to, and solubilized in, the aqueous solution of lipids or sized lipid vesicle preparation. These embodiments may be performed with shaking and/or mixing (e.g.
  • therapeutic agents are either solubilized in a solvent (e.g. aqueous or organic) prior to mixing with lipid vesicle particles or therapeutic agents are solubilized upon being mixed with the lipid vesicle particles.
  • a solvent e.g. aqueous or organic
  • therapeutic agents may be added as a dry powder to a solution containing lipid vesicle particles or both the lipid vesicle particles and dry therapeutic agents may be mixed together in a fresh solvent.
  • the therapeutic agent is an antigen, a small molecule drug, an antibody or a functional fragment thereof, an antibody mimetic or a functional fragment thereof, an immunomodulatory agent, a polynucleotide encoding a polypeptide, or an interfering polynucleotide.
  • the therapeutic agent is an antigen.
  • the term “antigen” may be used interchangeably with “immunogen” and may refer to a pathogen, a part of a pathogen, or a molecule that is able to induce an adaptive antibody immune response and/or an adaptive cellular immune response that is specific to said antigen or a portion thereof.
  • the term “antigen” may refer to a pathogen, a part of a pathogen, or a molecule that is specifically recognized by antibodies and/or immunoglobulin receptors of the adaptive antibody immune response and/or the adaptive cellular immune response.
  • the term “peptide antigen” is an antigen as defined above that is a protein or a polypeptide.
  • Adaptive antibody responses are mediated by B cells, which recognize antigen by specific binding of IgM immunoglobulins in their B cell receptors to the antigen. Activated B cells may mature into plasma cells and secrete soluble IgM, IgG, IgA, or IgE antibodies that specifically to bind to the antigen.
  • T cell receptors specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers
  • T cell receptors specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers
  • Antibody and cellular adaptive immune responses are coordinated by CD4+ T helper cells, which recognize polypeptide antigen by specific binding of their T cell receptors (specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers) to peptides from the antigen that are displayed on MHC class I/II molecules on the surface of APC.
  • T cell receptors specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers
  • the immunoglobulin superfamily proteins in B cell receptors and antibodies (IgM, IgD, IgG, IgA, IgE), and in T cell receptors (TCR-alpha/TCR-beta, TCR-gamma/TCR-delta) that specifically bind to antigen are created randomly by processes of genetic recombination during B cell and T cell development.
  • Each B cell and T cell expresses only one species of randomized immunoglobulin superfamily receptor with a specific binding recognition, and B cells and T cells that bind an antigen are clonally selected for expansion in the body during an immune response.
  • the immunoglobulins in B cell receptors and antibodies may further diversify by somatic mutation and clonal selection for variants with high antigen binding.
  • antibodies, B cell receptors, and T cell receptors are immunoglobulin superfamily molecules that enable the immune system to adapt to an antigen, thus forming an adaptive immune response.
  • This adaptive immune response contrasts with innate immune responses that are mediated by conserved immune receptors, such as TLR or other pattern recognition receptors, that are unchanging and have fixed specificity for binding to specific pathogen- associated molecular patterns.
  • the term “antigen” or “peptide antigen” may refer to a molecule that is specifically bound by antibodies, B cell receptors, and/or T cell receptors of the adaptive immune system during an active adaptive immune response.
  • an antigen that is bound by an antibody, a B cell receptor, and/or a T cell receptor is called an “epitope”.
  • Antibodies and B cell receptors may bind to an epitope on various types of molecules including polypeptides, polysaccharides, glycoproteins, and lipoproteins.
  • T cell receptors bind to peptides that are derived from antigen, wherein the peptides are displayed on MHC class I/II molecules.
  • the term “antigen” may refer to a molecule that comprises a B cell epitope and/or a T cell epitope.
  • the antigen may include a polypeptide, a polysaccharide, a glycoprotein, a lipoprotein, a microorganism or a part thereof, such as a live, attenuated, inactivated or killed bacterium, virus or protozoan, or part thereof, an allergen, or an antigen derived from a cancer cell (such as a conserved cancer antigen or a neoantigen).
  • the term “derived from” encompasses, without limitation: an antigen that is isolated or obtained directly from an originating source; a synthetic or recombinantly generated antigen that is identical or substantially related to an antigen from an originating source; or an antigen which is made from an antigen of an originating source or a fragment thereof.
  • an antigen is “from” a source
  • the term “from” may be equated with “derived from”.
  • substantially related means that the antigen may have been modified by chemical, physical or other means (e.g. sequence modification), but that the resultant product remains capable of generating an immune response to the original antigen or to the disease or disorder associated with the original antigen.
  • the term “antigen” also includes a polynucleotide that encodes a 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.
  • Viruses, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, herpes virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, human papillomavirus (HPV), Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, human immunodeficiency virus (HIV), Seneca Valley virus (SVV), Orthoreovirus, Rotavirus, Ebola virus, parainfluenza virus, influenza virus (e.g.
  • influenza virus H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), Measles virus, Mumps virus, Rubella virus, Pneumovirus, respiratory syncytial virus, respiratory syncytial virus (RSV), Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Coronavirus (e.g. Sars-Cov-2), Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella.
  • the peptide antigen is derived from HPV.
  • the HPV peptide antigen is one that is associated with HPV-related cervical cancer or HPV-related head and neck cancer.
  • the peptide antigen is a peptide comprising the sequence RAHYNIVTF (HPV16E7 (H-2Db) peptide 49-57; R9F).
  • the peptide antigen is a peptide comprising the sequence YMLNLGPET (HPV Y9T peptide).
  • the peptide antigen is derived from HIV.
  • the HIV peptide antigen may be derived from the V3 loop of HIV-1 gp120.
  • the HIV peptide antigen may be RGP10 (RGPGRAFVTI).
  • the peptide antigen may be AMQ9 (AMQMLKETI).
  • AMQ9 peptide is the immunodominant MHC class I epitope of gag for mice of the H-2Kd haplotype.
  • the peptide antigen is derived from RSV.
  • the RSV virion a member of the genus Paramyxoviridae, is composed of a single strand of negative-sense RNA with 15,222 nucleotides.
  • the nucleotides encode three transmembrane surface proteins (F, G and small hydrophobic protein or SH), two matrix proteins (M and M2), three nucleocapsid proteins (N, P and L), and two non-structural proteins (NS1 and NS2).
  • the peptide antigen may be derived from any one or more of the RSV proteins.
  • the peptide antigen may be derived from the SH protein of RSV or any other paramyxovirus, or a fragment thereof.
  • the RSV peptide antigen may be any one or more of the RSV peptides described or disclosed in WO 2012/065997, incorporated herein by reference.
  • Bacteria, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli O157: 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.
  • Anthrax Bacteria,
  • the peptide antigen is derived from a Bacillus anthracis.
  • the peptide antigen may for example be derived from anthrax recombinant protective antigen (rPA) (List Biological Laboratories, Inc.; Campbell, CA) or anthrax mutant recombinant protective antigen (mrPA).
  • rPA has an approximate molecular weight of 83,000 daltons (Da) and corresponds a cell binding component of the three-protein exotoxin produced by Bacillus anthracis.
  • the protective antigen mediates the entry of anthrax lethal factor and edema factor into the target cell.
  • the antigen may be derived from the sequence found under GenBank Accession number P13423, or any suitable sequence variant thereof.
  • Protozoa, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi), which causes malaria.
  • the peptide antigen is derived from a Plasmodium species.
  • the peptide antigen may be derived from the circumsporozoite protein (CSP), which is a secreted protein of the sporozoite stage of the malaria parasite (Plasmodium sp.).
  • CSP circumsporozoite protein
  • the amino-acid sequence of CSP consists of an immunodominant central repeat region flanked by conserved motifs at the N- and C-termini that are implicated in protein processing as the parasite travels from the mosquito to the mammalian vector.
  • the structure and function of CSP is highly conserved across the various strains of malaria that infect humans, non-human primates and rodents.
  • the peptide antigen derived from CSP is a malaria virus-like particle (VLP) antigen which comprises circumsporozoite T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen.
  • VLP malaria virus-like particle
  • the peptide antigen may be derived from a cancer or tumor-associated protein, such as for example, a membrane surface-bound cancer antigen.
  • the cancer may be one that is caused by a pathogen, such as a virus.
  • Viruses linked to the development of cancer include, but are not limited to, human papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukaemia virus-1.
  • HPV human papillomaviruses
  • JCV John Cunningham virus
  • EBV Epstein Barr Virus
  • Merkel cell polyomavirus Hepatitis C Virus
  • Hepatitis C Virus Human T cell leukaemia virus-1.
  • the peptide antigen may be derived from a virus that is linked to the development of cancer.
  • the peptide antigen is a cancer-associated antigen.
  • Many cancer or tumor-associated proteins are known in the art such as for example, and without limitation, those described in WO 2016/176761, incorporated by reference herein.
  • the peptide antigen is one or more survivin antigens.
  • Survivin also called baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), is a protein involved in the negative regulation of apoptosis.
  • the peptide antigen is any peptide, polypeptide or variant thereof derived from a survivin protein, or a fragment thereof.
  • the survivin peptide antigen may comprise the full length survivin polypeptide.
  • the survivin peptide antigen may be a survivin peptide comprising a fragment of any length of the survivin protein.
  • Exemplary embodiments include a survivin peptide that comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.
  • the survivin peptide consists of a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide, consisting of 7, 8, 9, 10, 11 consecutive amino acid residues of the survivin protein, respectively.
  • Particular embodiments of the survivin antigen include survivin peptides of about 9 or 10 amino acids.
  • Survivin peptide antigens also encompass variants and functional equivalents of natural survivin peptides.
  • Variants or functional equivalents of a survivin peptide encompass peptides that exhibit amino acid sequences with differences as compared to the specific sequence of the survivin protein, such as one or more amino acid substitutions, deletions or additions, or any combination thereof. The difference may be measured as a reduction in identity as between the survivin protein sequence and the survivin peptide variant or survivin peptide functional equivalent.
  • a pharmaceutical composition of the present invention may include any one or more of the survivin peptides, survivin peptide variants or survivin peptide functional equivalents disclosed in WO 2004/067023; WO 2006/081826 or WO 2016/176761, each of which is incorporated by reference herein.
  • the survivin peptide antigen may be any one or more of FEELTLGEF, FTELTLGEF, LTLGEFLKL, LMLGEFLKL, RISTFKNWPF, RISTFKNWPK, STFKNWPFL, and/or LPPAWQPFL.
  • the peptide antigen is a neoantigen.
  • the term “neoantigen” refers to a class of tumor antigens which arise from tumor-specific mutations in an expressed protein. The neoantigen can be derived from any cancer, tumor or cell thereof.
  • neoantigens the term “derived from” as used herein encompasses, without limitation: a neoantigen that is isolated or obtained directly from an originating source (e.g. a subject); a synthetic or recombinantly generated neoantigen that is identical in sequence to a neoantigen from an originating source; or a neoantigen which is made from a neoantigen of an originating source or a fragment thereof.
  • the mutations in the expressed protein that create the neoantigen may be patient-specific. By “patient-specific”, it is meant that the mutation(s) are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s).
  • neoantigen may comprise one or more neoepitopes.
  • epitope refers to a peptide sequence which can be recognized by the immune system, specifically by antibodies, B cells or T cells.
  • a “neoepitope” is an epitope of a neoantigen which comprises a tumor-specific mutation as compared to the native amino acid sequence.
  • neoepitopes may be identified by screening neoantigens for anchor residues that have the potential to bind patient HLA.
  • the neoepitopes are normally ranked using algorithms, such as NetMHC, that can predict peptide binding to HLA.
  • a "T-cell neoepitope” is to be understood as meaning a mutated peptide sequence which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex.
  • the T-cell neoepitope should typically be one that is amenable to recognition by T cell receptors so that a cell-mediated immune response can occur.
  • a "B-cell neoepitope” is to be understood as meaning a mutated peptide sequence which can be recognized by B cells and/or by antibodies.
  • At least one of the neoepitopes of the neoantigen is a patient-specific neoepitope.
  • patient-specific neoepitope it is meant that the mutation(s) in the neoepitope are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s). Thus, a “patient-specific neoepitope” may be shared by a small or large sub-population of subjects.
  • the neoantigen may be derived from a mutated gene or protein that has previously been associated with cancer phenotypes, such as for example tumor suppressor genes (e.g.
  • the neoantigen may comprise or consist of the neoantigens disclosed in Castle et al. (2012) Exploiting the Mutanome for Tumor Vaccination. Cancer Res, 72(5): 1081-1091, incorporated by reference herein.
  • the neoantigen may be one or more of the Mut1-50 neoantigens disclosed in Table 1 of Castle 2012, or a neoantigen of the same or related protein (e.g. a human homologue).
  • the neoantigen may be one or more of Mut25 (STANYNTSHLNNDVWQIFENPVDWKEK), Mut30 (PSKPSFQEFVDWENVSPELNSTDQPFL) and Mut44 (EFKHIKAFDRTFANNPGPMVVFATPGM), or a neoantigen of the same or related protein (e.g. a human homologue).
  • Mut25 STANYNTSHLNNDVWQIFENPVDWKEK
  • Mut30 PSKPSFQEFVDWENVSPELNSTDQPFL
  • Mut44 EFKHIKAFDRTFANNPGPMVVFATPGM
  • a neoantigen of the same or related protein e.g. a human homologue
  • the antigen may be a peptide derived from the ectodomain of the small hydrophobic protein of respiratory syncytial virus (RSV) as disclosed in WO2012/065997, incorporated herein by reference.
  • RSV respiratory syncytial virus
  • the RSV antigen may be a peptide with the sequence NKLCEYNVFHNKTFELPRARVNT, NKLSEHKTFCNNTLELGQMHQINT, or NKLCDFNDHHTNSLDIRTRLRNDTQLITRAHEGSINQSSN, or a portion or variant thereof
  • the antigen may be a peptide derived from the survivin protein as disclosed in WO2004/067023 and WO2006/081826, incorporated herein by reference.
  • the survivin antigen may be one or more peptides of the sequence FEELTLGEF, FTELTLGEF, LTLGEFLKL, LMLGEFLKL, RISTFKNWPF, RISTFKNWPK, STFKNWPFL, or LPPAWQPFL, or a portion or a variant thereof.
  • the antigen may be a fusion peptide (FP) antigen comprising the sequence RAHYNIVTF (HPV16E7 (H-2Db) peptide 49-57) fused to the universal T-helper epitope is PADRE (pan-DR epitope) comprising the peptide sequence AKXVAAWTLKAAA, wherein X may be phenylalanine or cyclohexylalanine.
  • the therapeutic agent is one or more peptide antigens as described herein.
  • the peptide antigen is a synthetically produced polypeptide.
  • the peptide antigen may be a polypeptide of any length.
  • the peptide antigen may be 5 to 120 amino acids in length, 5 to 100 amino acids in length, 5 to 75 amino acids in length, 5 to 50 amino acids in length, or 5 to 30 amino acids in length. In an embodiment, the peptide antigen may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length. In an embodiment, the peptide antigen is 20 to 30 amino acids in length. In an embodiment, the peptide antigen is 27 amino acids in length. In an embodiment, the peptide antigen is 8 to 40 amino acids in length.
  • the peptide antigen is 9 or 10 amino acids in length.
  • the therapeutic agent is a small molecule drug.
  • small molecule drug refers an organic or inorganic compound that may be used to treat, cure, prevent or diagnose a disease, disorder or condition.
  • small molecule refers to a low molecular weight compound which may be synthetically produced or obtained from natural sources and has a molecular weight of less than 2000 Daltons (Da), less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da or less than 500 Da.
  • a small molecule drug is typically a chemically manufactured active substance or compound (i.e.
  • small molecule drug does not encompass larger structures, such as polynucleotides, proteins, and polysaccharides, which are made by a biological process.
  • small molecule may refer to compounds or molecules that selectively bind specific biological macromolecules and act as an effector, altering the activity or function of the target.
  • a small molecule drug is a substance or compound that regulates a biological process in the body of a subject, and more particularly within a cell.
  • a small molecule drug may exert its activity in the form in which it is administered, or the small molecule drug may be a prodrug.
  • small molecule drug encompasses both the active form and the prodrug.
  • prodrug refers to a compound or substance that, under physiological conditions, is converted into the therapeutically active agent.
  • a prodrug is a compound or substance that, after administration, is metabolized in the body of a subject into the pharmaceutically active form (e.g. by enzymatic activity in the body of the subject).
  • a common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the pharmaceutically active form.
  • Many small molecule drugs are known in the art and are used as active ingredients in medicaments. The skilled person is aware of numerous small molecule drugs such as those disclosed in the online DrugBank database.
  • a small molecule drug may include a cytotoxic agent, an anti-cancer agent, an anti- tumor agent, a chemotherapeutic agent, an anti-neoplastic agent, an antiviral agent, an antibacterial agent, an anti-inflammatory agent, an immunomodulatory agent (e.g. a cytokine or a chemokine), an immune response checkpoint agent, a biological response modifier, a prodrug, a ligand, an analgesic, a radiopharmaceutical, a radioisotope or a dye for visual detection.
  • a cytotoxic agent may be an agent that kills a target cell by necrosis or apoptosis.
  • a cytotoxic agent may include epacadostat, cyclophosphamide, rapamycin, ifosfamide, afosfamide, melphalan, bendamustine, uramustine, palifosfamide, chlorambucil, busulfan, 4-hydroxycyclophosphamide, bis-chloroethylnitrosourea (BCNU), mitomycin C, yondelis, procarbazine, dacarbazine, temozolomide, cisplatin, carboplatin, oxaliplatin, acyclovir, gemcitabine, 5-fluorouracil, cytosine arabinoside, ganciclovir, camptothecin, topotecan, irinotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, etoposide, teniposide, mitoxantrone, valproic acid, vorin
  • a small molecule drug may include an checkpoint agent.
  • a “checkpoint agent” refers to any compound or molecule that totally or partially modulates (e.g. activates or inhibits) the activity or function of one or more checkpoint molecules (e.g. proteins). Checkpoint molecules regulate various cellular processes.
  • a small molecule drug may include a checkpoint agent that is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B- and T-lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD33, CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), Killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), phosphatidylserine (PS
  • a small molecule drug may include a cell-penetrating peptide, a peptide transduction domain, or a dendritic cell peptide, used as molecular shuttles that can transport other molecules or ions from one location to another.
  • the therapeutic agent is an antibody, a functional equivalent of an antibody or a functional fragment of an antibody.
  • an “antibody” refers to a polypeptide or protein that consists of or comprises antibody domains, which are understood as constant and/or variable domains of the heavy and/or light chains of immunoglobulins, with or without a linker sequence. Antibody domains may be of native structure or modified by mutagenesis or derivatization, e.g.
  • An antibody may comprise a complete (i.e. full-length) immunoglobulin molecule, including e.g. polyclonal, monoclonal, chimeric, humanized and/or human versions having full length heavy and/or light chains.
  • the term “antibody” encompasses any and all isotypes and subclasses, including without limitation the major classes of IgA, IgD, IgE, IgG and IgM, and the subclasses IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • An antibody may be one that is naturally occurring or one that is prepared by any means available to the skilled person, such as for example by using immunoglobulin gene fragment recombinatorial processes.
  • An antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a fully human antibody.
  • a “chimeric antibody” as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, such for example a rodent, while the constant domains of the antibody are derived from a different species, such as a human.
  • CDRs complementarity determining regions
  • the constant domains of the chimeric antibody may be derived from that of an animal, such as for example a cat or dog.
  • a “humanized antibody” as used herein refers to a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains, including human framework region (FR) sequences.
  • the constant domains of the humanized antibody are likewise derived from a human antibody.
  • a “human antibody” as used herein refers to an antibody obtained from transgenic animals (e.g. mice) that have been genetically engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic animal can synthesize human antibodies specific for human antigens, and the animal can be used to produce human antibody-secreting hybridomas.
  • a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art.
  • the term “functional fragment”, with respect to an antibody refers to an antigen-binding portion of an antibody. In this context, by “functional” it is meant that the fragment maintains its ability to bind to the target antigen.
  • Functional fragments of antibodies include a portion of an antibody such as a F(ab') 2 , a F(ab) 2 , a Fab', a Fab, a Fab2, a Fab3, and single domain antibody. Regardless of structure, a functional fragment of an antibody binds with the same antigen that is recognized by the intact antibody.
  • the term “functional fragment”, in relation to antibodies, also includes isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”).
  • Antibody fragments can be incorporated into single domain antibodies (e.g.
  • nanobodies single-chain antibodies
  • maxibodies evibodies
  • minibodies intrabodies
  • diabodies triabodies
  • tetrabodies vNAR, bis-scFv and other like structures.
  • Another form of a functional fragment is a peptide comprising one or more CDRs of an antibody or one or more portions of the CDRs, provided the resultant peptide retains the ability to bind the target antigen.
  • Antibodies for therapeutic use are known in the art such as, for example, anti-CTLA4 antibodies (ipilimumab, tremelimumab, BN- 13, UC10-4F10-11, 9D9 or 9H10), anti-PD1 antibodies (pembrolizumab, nivolumab, pidilizumab, AMP-224, RMP1-4 or J43), and anti-PDL1 antibodies (tezolizumab, avelumab, BMS-936559 or durvalumab).
  • the therapeutic agent is an antibody mimetic, a functional equivalent of an antibody mimetic, or a functional fragment of an antibody mimetic.
  • antibody mimetic refers to compounds which, like antibodies, can specifically and/or selectively bind antigens or other targets, but which are not structurally related to antibodies.
  • Antibody mimetics are usually artificial peptides or proteins and are typically smaller than antibodies, with a molar mass of about 3-20 kDa (whereas antibodies are generally about 150 kDa).
  • Non-limiting examples of antibody mimetics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, nanoCLAMPs, monobodies, affinity reagents and scaffold proteins.
  • the term “functional fragment”, with respect to an antibody mimetic, refers any portion or fragment of an antibody mimetic that maintains the ability to bind to its target molecule.
  • the functional fragment of an antibody mimetic may be, for example, a portion of any of the antibody mimetics as described herein.
  • a “functional equivalent” in the context of an antibody mimetic refers to a polypeptide or other compound or molecule having similar binding characteristics to an antibody mimetic, but not necessarily being a recognizable “fragment” of an antibody mimetic.
  • the therapeutic agent is an immunomodulatory agent.
  • an “immunomodulatory agent” is a molecule or compound that modulates the activity and/or effectiveness of an immune response.
  • “Modulate”, as used herein, means to enhance (upregulate), suppress (downregulate), direct, redirect or reprogram an immune response.
  • the term “modulate” is not intended to mean activate or induce. By this, it is meant that the immunomodulatory agent modulates (enhances, reduces or directs) an immune response that is activated, initiated or induced by a particular antigen, but the immunomodulatory agent is not itself the antigen against which the immune response is directed, nor is the immunomodulatory agent derived from that antigen.
  • An immunomodulatory agent that enhances the immune response may be selected from cytokines (e.g.
  • interleukins and interferons certain interleukins and interferons
  • stem cell growth factors include lymphotoxins, co-stimulatory molecules, hematopoietic factors, colony stimulating factors, erythropoietins, thrombopoietins, and the like, and synthetic analogs of these molecules.
  • An immunomodulatory agent may include: lymphotoxins, such as tumor necrosis factor (TNF); hematopoietic factors, such as interleukin (IL); colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF); interferon, such as interferons-alpha, -beta or –lamda; and stem cell growth factor, such as that designated "SI factor".
  • lymphotoxins such as tumor necrosis factor (TNF); hematopoietic factors, such as interleukin (IL); colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF); interferon, such as interferons-alpha, -beta or –lamda; and stem cell growth factor, such as that designated "SI factor”.
  • cytokines include hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGFP; insulin-like growth factor-I and -II; erythro
  • An immunomodulatory agent may be an immune costimulatory molecule agonist.
  • Immune costimulatory molecules are signaling proteins that play a role in regulating immune response. Some immune costimulatory molecules are receptors located on the surface of a cell that respond to extracellular signaling. When activated, immune costimulatory molecules produce a pro-inflammatory response that can include suppression of regulatory T cells and activation of cytotoxic or killer T cells.
  • Exemplary immune costimulatory molecules include CD27, CD28, CD40, CD122, CD137, CD137/4-1BB, ICOS, IL-10, OX40 TGF-beta, TOR receptor, and glucocorticoid-induced TNFR-related protein GITR.
  • An immune costimulatory molecule agonist may therefore be any compound, molecule or substance that is an agonist of a costimulatory immune molecule as described herein.
  • An immunomodulatory agent may be an immunosuppressive agent.
  • immunosuppressive agent it is meant a molecule or compound that reduces (downregulates) the activity and/or efficacy of the immune response, or directs, redirects or reprograms the immune response in a manner that alleviates an undesired result (e.g. an autoimmune response or allergy).
  • immunosuppressive agent including calcineurin inhibitors, interleukin inhibitors, selective immunosuppressants and THF-alpha inhibitors.
  • An immunomodulatory agent may be an immunosuppressant selected from 5-fluorouracil, 6-thioguanine, adalimumab, anakinra, Atgam, abatacept, alefacept, azathioprine, basiliximab, belatacept, belimumab, benralizumab, brodalumab, canakinumab, certolizumab, chlorambucil, cyclosporine, daclizumab, dimethyl fumerate, dupilumab, eculizumab, efalizumab, ethanercept, everolimus, fingolimod, golimumab, guselkumab, imiquimod, infliximab, ixekizumab, leflunomide, lenlidomide, mechlorethamine, mepolizumab, methotrexate, muromonab-cd3, mycophenolate mofetil,
  • An immunomodulatory agent may be any molecule or compound that is an immunosuppressive cytotoxic drug.
  • An immunosuppressive cytotoxic drug may include a glucocorticoid, a cytostatic (e.g. alkylating agents, antimetabolites), an antibody, a drug acting on immunophilins, an interferon, an opioid, or a TNF binding protein.
  • Immunosuppressive cytotoxic drugs include nitrogen mustards (e.g. cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs (e.g. methotrexate), purine analogs (e.g. azathioprine and mercaptopurine), pyrimidine analogs (e.g.
  • an immunomodulatory agent may be an anti- inflammatory agent.
  • An anti-inflammatory agent may be a non-steroidal anti-inflammatory agent (such as a Cox-1 and/or Cox-2 inhibitor), a non-steroidal anti-inflammatory agent, aspirin, salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, a corticosteroid, or celecoxib.
  • a non-steroidal anti-inflammatory agent such as a Cox-1 and/or Cox-2 inhibitor
  • a non-steroidal anti-inflammatory agent such aspirin, salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone,
  • An immunomodulatory agent may be an anti-rheumatic agent such as prednisone, dexamethasone, chloroquine, hydroxychloroquine, methotrexate, sulfasalazine, cyclosporine, azathioprine, cyclophosphamide, azathioprine, sulfasalazine, penicillamine, aurothioglucose, gold sodium thiomalate, auranofin, methotrexate, mechlorethamine, cyclophosphamide, chlorambucil, or azathioprine.
  • an anti-rheumatic agent such as prednisone, dexamethasone, chloroquine, hydroxychloroquine, methotrexate, sulfasalazine, cyclosporine, azathioprine, cyclophosphamide, azathioprine, sulfas
  • the therapeutic agent is a polynucleotide encoding a polypeptide, or an interfering polynucleotide (such as miRNA, siRNA shRNA, DsiRNA, or a polynucleotide encoding any thereof).
  • a therapeutic agent may be a polynucleotide that contains sequences that correspond largely to the sense or antisense sequence of specific genes or their products, and hence have a direct effect on the expression of these genes and/or their products.
  • the use of polynucleotides that contain gene coding sequences affects the transcription and/or translation of the genes of interest in cells that uptake such polynucleotides.
  • RNA interference polynucleotides affects the expression of specific genes of interest by directly affecting the levels of mRNA in cells that uptake such nucleotides. This differs significantly from other polynucleotide-based molecules such as CpG, and DNA- or RNA- based poly I:C adjuvants, which do not act through the presence of gene specific sequences.
  • an active agent may be a polynucleotide that is not expressed as a protein in a cell, but rather encodes, for example, an antisense RNA, an interfering RNA, a catalytic RNA, or a ribozyme.
  • RNA interference is a sequence specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA such as small (or short) interference RNA (siRNA), short hairpin RNA (shRNA), and single stranded intracellular RNA such as microRNA (miRNA), all of which can cause degradation of homologous mRNAs in a cell.
  • Interfering RNA may be a naturally occurring or synthetic RNA chain of varying length. Interfering RNA can be duplexes, usually but not always limited to, 20 to 25-nt long that have 19 base pair central double stranded domain with terminal 2-base 3’ overhangs.
  • Interfering RNA can be further modified chemically to enhance its in vivo efficacy, induce nuclease-resistance to prevent degradation and enhance stability.
  • the anti-sense strand may have either a free 5’-OH or 5’-phosphate terminus, the latter results in natural Dicer processing and represents the active form of the molecule.
  • Interfering RNA may have phosphorothioate or boranohosphate modification of the internucleoside linkage to improve nuclease stability and prolong life of the duplex when exposed to serum or other nuclease sources.
  • Interfering RNA may have modifications at 2’position, for example, 2’-O-methyl RNA residue incorporation to retain full potency compared with unmodified RNA, retaining stability in serum and significantly reducing the risk of potential IFN responses in the cell. Interfering RNA may also have 2’-fluoro modification, which is usually incorporated selectively at pyrimidine bases, to improve stability and potency.
  • the therapeutic agent is a T-helper peptide.
  • T-helper peptide refers to a peptide that is an epitope strongly recognized by T cells when displayed on MHC class I/II molecules.
  • T-helper peptides are recognized by helper T cells (CD4+ T cells), which play an important role in establishing and maximizing the capabilities of the immune system, and are involved in activating and directing other immune cells, such as for example cytotoxic T cells and B cells. Accordingly, T-helper peptides are capable of enhancing or stimulating an immune response to an antigen.
  • Immunodominant T-helper peptides or universal T-helper peptides are known peptides that are broadly reactive in animal and human populations with widely divergent MHC types. T-helper peptides may be comprised in an antigen, or provided with an antigen to boost the immune response to the antigen.
  • a T helper peptide may include an immunodominant or universal T helper peptide as known in the art such as, for example but not limited to the modified Tetanus toxin peptide A16L (830-844, AQYIKANSKFIGITEL, with an alanine residue added to its amino terminus to enhance stability); PADRE (pan-DR epitope) comprising the peptide sequence AKXVAAWTLKAAA, wherein X may be phenylalanine or cyclohexylalanine; or Tetanus toxin peptide F21E (947-967, FNNFTVSFWLRVPKVSASHLE).
  • an immunodominant or universal T helper peptide as known in the art such as, for example but not limited to the modified Tetanus toxin peptide A16L (830-844, AQYIKANSKFIGITEL, with an alanine residue added to its amino terminus to enhance stability); PADRE (pan-DR epitop
  • hydrophobic carrier The pharmaceutical compositions of the present comprise a hydrophobic carrier.
  • the hydrophobic carrier may be an essentially pure hydrophobic substance or a mixture of hydrophobic substances. Hydrophobic substances that are useful in the compositions described herein are those that are pharmaceutically acceptable.
  • the hydrophobic carrier is typically a liquid but certain hydrophobic carriers that are not liquids at standard room temperature (about 18-25 o C) may be liquefied, for example by warming, and may also be useful.
  • the hydrophobic carrier is an oil or a mixture of oils.
  • the hydrophobic carrier is a mineral oil (such as Drakeol TM 6VR or IFA), a vegetable oil (such as soybean oil), a nut oil (such as peanut oil), or a mixture of any thereof.
  • the hydrophobic carrier is Incomplete Freund’s Adjuvant (IFA), a mineral oil-based hydrophobic carrier.
  • the hydrophobic carrier is mannide monooleate in mineral oil, such as commercially available Montanide TM ISA 51 (SEPPIC, France). Montanide TM ISA 51 is a mixture of highly purified mineral oil (Drakeol TM 6VR) and mannide monooleate.
  • the hydrophobic carrier is mannide oleate in non- mineral oil, such as commercially available Montanide TM ISA 720 (SEPPIC, France).
  • the hydrophobic carrier is MS80 oil which is a mixture of mineral oil (Sigma Aldrich) and a sorbitan monooleate (such as Span TM 80), the components of which can be purchased separately and mixed prior to use.
  • the amount of hydrophobic carrier used will depend on the desired volume of the final composition and/or the desired concentration of the components suspended and/or solubilized in the hydrophobic carrier.
  • the compositions of the present invention are water-free or substantially free of water, meaning that the compositions are not emulsions.
  • water-free it is meant that the compositions contain no water at all.
  • the compositions may be substantially free of water.
  • substantially free of water is intended to encompass embodiments where the hydrophobic carrier may still contain small quantities of water, provided that the water is present in the non-continuous phase of the carrier.
  • individual components of the composition may have small quantities of 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.
  • compositions as disclosed herein that are “substantially free of water” contain, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water on a weight/weight basis of the total weight of the carrier component of the composition.
  • the compositions that still contain small quantities of water do not contain a sufficient amount of water such that an emulsion would be formed.
  • an “emulsion” refers to a mixture of two or more liquids that are normally immiscible wherein droplets of one liquid are dispersed in the other.
  • a hydrophobic substance e.g.
  • a dispersion of water droplets in oil is a water-in-oil (W/O) emulsion in which the water (aqueous phase) forms a discontinuous phase and the oil (hydrophobic phase) forms a continuous phase.
  • W/O water-in-oil emulsion
  • Water-in-oil emulsion or “W/O”, as used herein, refers to an emulsion of a hydrophobic phase in an aqueous phase.
  • a dispersion of oil droplets in water is an oil-in-water (O/W) emulsion in which the oil (hydrophobic phase) forms a discontinuous phase and the water (aqueous phase) forms a continuous phase.
  • Oil-in-water emulsion or “O/W”, as used herein, refers to an emulsion of a hydrophobic phase in an aqueous phase.
  • Conventional emulsion refers to emulsions are composed of numerous emulsifier-coated fluid droplets dispersed within another immiscible fluid medium, wherein the emulsifier forms an interfacial thin layer surrounding individual droplets that create interactions with hydrophilic and hydrophobic phases.
  • the pharmaceutical compositions and dried preparations of the present invention comprise at least lipid.
  • the lipid is an amphipathic lipid or a mixture of amphipathic lipids.
  • any amphipathic lipid may be used, particularly suitable lipids may include those with at least one fatty acid chain containing at least 4 carbons, and typically about 4 to 28 carbons in length.
  • the fatty acid chain may contain any number of saturated and/or unsaturated bonds.
  • amphipathic lipids which may be used in the pharmaceutical compositions of the present invention, and the pharmaceutical compositions of the present invention may contain a single type of amphipathic lipid or a mixture of different types of amphipathic lipids.
  • the amphipathic lipid may be a natural lipid or a synthetic lipid.
  • amphipathic lipids for use in the present invention include phospholipids, sphingolipids, sphingomyelin, cerobrocides, gangliosides, ether lipids, sterols, cholesterol, glycerophospholipid, cardiolipin, cationic lipids, anionic lipids, and lipids modified with poly (ethylene glycol) and other polymers.
  • Synthetic lipids may include, without limitation, the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids.
  • An “amphipathic lipid” is a lipid having both hydrophilic and hydrophobic parts or characteristics.
  • the term “amphipathic” may be used interchangeably with “amphiphile” or “amphiphilic”.
  • the hydrophobic portion of an amphipathic lipid may be a large hydrocarbon moiety, either linear or cyclic, such as a long chain of the form CH3(CH2)n, with n > 4.
  • the hydrophilic portion of an amphipathic lipid may be either a charged group or a polar uncharged group.
  • Charged groups include anionic and cationic groups. Examples of anionic charged groups include the following (wherein the hydrophobic part of the molecule is represented by "R"): carboxylates: RCO 2 ⁇ ; sulfates: RSO 4 ⁇ ; sulfonates: RSO3 ⁇ ; and phosphates (the charged functionality in phospholipids).
  • Cationic charged groups include, for example, amines: RNH3 + ("R" again representing the hydrophobic part of the molecule).
  • Uncharged polar groups include, for example, alcohols with large R groups, such as diacyl glycerol (DAG).
  • Amphipathic lipids may have several hydrophobic parts, several hydrophilic parts, or several of both. Cholesterol is also an amphiphilic lipid.
  • the lipid is a phospholipid or a mixture of phospholipids. Broadly defined, 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 include, for example and without limitation, those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine (for example DOPC; 1,2-Dioleoyl-sn-glycero-3-phosphocholine) and phosphoinositol.
  • the phospholipid may be phosphatidylcholine or a mixture of lipids comprising phosphatidylcholine.
  • the phospholipid may be DOPC or a mixture of phospholipids such as a lecithin (for example Lipoid S100 lecitihin).
  • Lecithin is a mixture of phospholipids typically derived from biological sources such as eggs, soybean, and other vegetable sources.
  • the phospholipid is sphingomyelin.
  • 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.
  • the at least one lipid is present in the pharmaceutical composition at a concentration of 60-160 mg/mL such as, for example 132 mg/mL.
  • the at least one lipid is present in the pharmaceutical composition at a concentration of at least 60 mg/mL, at least 70 mg/mL, at least 80 mg/mL, at least 90 mg/mL, at least 100 mg/mL, at least 110 mg/mL, at least 120 mg/mL, at least 132 mg/mL, at least 140 mg/mL, at least 150 mg/mL, or at least 160 mg/mL.
  • the at least one lipid is a mixture of a phospholipid and a sterol such as cholesterol.
  • the at least one lipid is a mixture of DOPC and cholesterol.
  • the at least one lipid is a mixture of lecithin and cholesterol.
  • the cholesterol or other sterol is used in an amount equivalent to about 2%, 5%, 10%, 12%, 15%, or 20% of the weight of phospholipid or mixture of phospholipids. In some embodiments, the amount of cholesterol or other sterol used is determined as the amount to sufficiently stabilize the formation of lipid-based structures formed by the phospholipid when suspended in the hydrophobic carrier.
  • the term “lipid vesicle particle” may be used interchangeably with “lipid vesicle”.
  • a lipid vesicle particle refers to a complex or structure having an internal environment separated from the external environment by a continuous layer of enveloping lipids.
  • the expression “layer of enveloping lipids” can mean a single layer lipid membrane (e.g. as found on a micelle or reverse micelle), a bilayer lipid membrane (e.g. as found on a liposome) or any multilayer membrane formed from single and/or bilayer lipid membranes.
  • the layer of enveloping lipids is typically a single layer, bilayer or multilayer throughout its circumference, but it is contemplated that other conformations may be possible such that the layer has different configurations over its circumference.
  • the lipid vesicle particle may contain, within its internal environment, other vesicle structures (i.e. it may be multivesicular).
  • lipid vesicle particle encompasses many different types of structures, including without limitation micelles, reverse micelles, unilamellar liposomes, multilamellar liposomes and multivesicular liposomes.
  • the lipid vesicle particles may take on various different shapes, and the shape may change at any given time (e.g. upon drying, sizing, or mixing with therapeutic agents).
  • lipid vesicle particles are spherical or substantially spherical structures.
  • substantially spherical it is meant that the lipid vesicles are close to spherical, but may not be a perfect sphere.
  • lipid vesicle particles include, without limitation, oval, oblong, square, rectangular, triangular, cuboid, crescent, diamond, cylinder or hemisphere shapes. Any regular or irregular shape may be formed.
  • a single lipid vesicle particle may comprise different shapes if it is multivesicular.
  • the outer vesicle shape may be oblong or rectangular while an inner vesicle may be spherical.
  • Exemplary embodiments of lipid vesicle particles include, without limitation, single layer vesicular structures (e.g. micelles or reverse micelles) and bilayer vesicular structures (e.g.
  • single layer it is meant that the lipids do not form a bilayer, but rather remain in a layer with the hydrophobic part oriented on one side and the hydrophilic part oriented on the opposite side.
  • bilayer it is meant that the lipids form a two-layered sheet, such as with the hydrophobic part of each layer internally oriented toward the center of the bilayer with the hydrophilic part externally oriented.
  • the opposite configuration is also possible, i.e. with the hydrophilic part of each layer internally oriented toward the center of the bilayer with the hydrophobic part externally oriented.
  • the term “multilayer” is meant to encompass any combination of single and bilayer structures.
  • the form adopted may depend upon the specific lipid that is used.
  • the lipid vesicle particles may be formed from single layer lipid membranes, bilayer lipid membranes and/or multilayer lipid membranes.
  • the lipid membranes are predominantly comprised of and formed by lipids, but may also comprise additional components.
  • the lipid membrane may include stabilizing molecules to aid in maintaining the integrity of the structure. Any available stabilizing molecule may be used such as, for example, a sterol such as cholesterol.
  • the lipid vesicle particles may be formed by lipids, such as amphipathic lipids, as disclosed herein.
  • Lipid vesicle particles may be comprised of a single layer lipid assembly.
  • the compositions disclosed herein may comprise a single type of lipid vesicle particle having a single layer lipid assembly or comprise a mixture of different such lipid vesicle particles.
  • Lipid vesicle particles having a single layer lipid assembly may comprise aggregates of lipids with the hydrophobic part of the lipids oriented outwards toward the hydrophobic carrier and the hydrophilic part of the lipids aggregating as a core. These structures do not necessarily form a continuous lipid layer membrane.
  • Lipid vesicle particles having a single layer lipid assembly comprise reverse micelles.
  • Lipid vesicle particles may be bilayer vesicular structures, such as for example, a liposome. Liposomes are completely closed lipid bilayer membranes.
  • Liposomes may be unilamellar vesicles (possessing a single bilayer membrane), multilamellar vesicles (characterized by multimembrane bilayers whereby each bilayer may or may not be separated from the next by an aqueous layer) or multivesicular vesicles (possessing one or more vesicles within a vesicle).
  • Liposomes form in aqueous or partially aqueous environments, and may form when the compositions herein are not water-free or may form in the aqueous solvents and solutions used in the preparation of pharmaceutical compositions.
  • the lipid vesicle particles are liposomes.
  • the liposomes are unilamellar, multilamellar, multivesicular or a mixture thereof.
  • the mean particle size of the liposomes is ⁇ 80 nm.
  • the mean particle size of the liposomes used in the methods disclosed herein is in the range of 80 nm to 120 nm, with a PDI of ⁇ 0.1.
  • mean refers to the arithmetic mean of the particle size of the lipid vesicle particles in a given population. It is a synonym for average.
  • lipid vesicle particles may not be perfectly spherical, and therefore the “particle size” of a given vesicle particle may not be an exact measure of its diameter.
  • the particle size may be defined by other means known in the art, including for example: the diameter of the sphere of equal area or the largest perpendicular distance between parallel tangents touching opposite sides of the particle (Feret’s statistical diameter).
  • the sized lipid vesicle particles have a mean particle size of less than or equal to 120 nanometers (i.e. ⁇ 120 nm) and a polydispersity index (PDI) of ⁇ 0.1.
  • the sized lipid vesicle particles have a mean particle size of ⁇ 115 nm, more particularly still ⁇ 110 nm and more particularly still ⁇ 100 nm.
  • the mean particle size of the sized lipid vesicle particles is between 50 nm and 120 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is between 80 nm and 120 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is between about 80 nm and about 115 nm, about 85 nm and about 115 nm, about 90 nm and about 115 nm, about 95 nm and about 115 nm, about 100 nm and about 115 nm or about 105 nm and about 115 nm.
  • the mean particle size of the sized lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114
  • the mean particle size is 120 nm.
  • polydispersity index is a measure of the size distribution of the lipid vesicle particles. It is known in the art that the term “polydispersity” may be used interchangeably with “dispersity”.
  • the PDI can be calculated by determining the mean particle size of the lipid vesicle particles and the standard deviation from that size. There are techniques and instruments available for measuring the PDI of lipid vesicle particles. For example, DLS is a well-established technique for measuring the particle size and size distribution of particles in the submicron size range, with available technology to measure particle sizes of less than 1 nm (LS Instruments, CH; Malvern Instruments, UK).
  • Lipid vesicle particles having a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1 may be prepared and provided by any suitable means.
  • the lipid vesicle particles are prepared in a manner in which their size is controlled in order to achieve the mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • lipid vesicle particles are subjected to one or more sizing steps or protocols to achieve the mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • the lipid vesicle particles may be prepared and provided by any combination of controlling their size during manufacture, performing sizing steps and/or any other means available in the art.
  • the lipid vesicle particles must be subjected to one or more active steps of sizing in order to achieve the mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • the sizing is performed by filter-extrusion whereby lipid vesicle particles are passed through a filter membrane or a series of filter membranes (e.g. polycarbonate membranes) of appropriate pore size.
  • sized lipid vesicle particles refers to lipid vesicle particles that have been prepared by a means in which their size is controlled to attain a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1 and/or they are sized to meet the criteria of having a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • the skilled person will be well aware of techniques available for providing lipid vesicle particles having a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • non- sized lipid vesicle particles or a “non-sized lipid vesicle particle preparation” means that the lipid vesicle particles have not be subject to procedures that limit their size to meet the defined size criteria, and/or they do not have a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • the sized lipid vesicle particles may be prepared from a lipid precursor that naturally forms lipid vesicle particles of the required size.
  • the sized lipid vesicle particles may be prepared using Presome® (Nippon Fine Chemical, Japan).
  • Presome® is a dry powder precursor made up of different lipid combinations.
  • Presome® is supplied ready to be wetted in a suitable buffer to prepare liposomes.
  • the liposomes formed from Presome® have an average particle size of about 93 nm, and sizing procedures (e.g. membrane extrusion, high pressure homogenization, etc.) can be used to achieve the required mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1.
  • Presome® may for example be wetted in sodium acetate, pH 9.0 ⁇ 0.5 to form liposomes.
  • the Presome® bulk dry powder may be made from DOPC/cholesterol (10:1 (w/w)) or DOPC alone.
  • lipid vesicle particles of any size may be employed.
  • conventional liposome forming processes may be used, such as the hydration of solvent-solubilized lipids. Exemplary methods of preparing liposomes are discussed, for example, in Gregoriadis 1990; and Frezard 1999.
  • the non-sized lipid vesicle particle preparation is subjected to a sizing procedure to obtain lipid vesicle particles having a mean particle size of ⁇ 120 nm and a PDI of ⁇ 0.1.
  • a sizing procedure there are various techniques available for sizing lipid vesicle particles (see e.g. Akbarzadeh 2013).
  • the non-sized lipid vesicle particle preparation may be sized by high pressure homogenization (microfluidizers), sonication or membrane based extrusion.
  • the sizing of the non-sized lipid vesicle particle preparation is performed using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1, and the lipid vesicle particles obtained via high pressure homogenization can then be further sized down using membrane based extrusion.
  • membrane based extrusion is performed by passing the sized lipid vesicle particle preparation 5-20 times through a 0.1 um polycarbonate membrane or, alternatively, 5-20 times through a 0.08 um polycarbonate membrane, thereby attaining a mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1.
  • the sized lipid vesicle particles may be prepared by adding the lipids to a suitable solvent (e.g. sodium phosphate, 50 mM, pH 7.0), shaking and/or stirring the lipid mixture (e.g. at 300 RPM for about 1 hour) and using membrane based extrusion to obtain the sized lipid vesicle particles.
  • a suitable solvent e.g. sodium phosphate, 50 mM, pH 7.0
  • shaking and/or stirring the lipid mixture e.g. at 300 RPM for about 1 hour
  • Exemplary, non-limiting embodiments of membrane based extrusion include: (i) passing a non-sized lipid vesicle particle preparation 20-40 times through a 0.2 ⁇ m polycarbonate membrane, and then 10-20 times through a 0.1 ⁇ m polycarbonate membrane; or (ii) passing a non-sized lipid vesicle particle preparation 20-40 times through a 0.2 ⁇ m polycarbonate membrane, then 10-20 times through a 0.1 ⁇ m polycarbonate membrane, and then 10-20 times through a 0.08 ⁇ m polycarbonate membrane.
  • the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 ⁇ m polycarbonate membrane, and then 10 times through a 0.1 ⁇ m polycarbonate membrane.
  • the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 ⁇ m polycarbonate membrane, then 10 times through a 0.1 ⁇ m polycarbonate membrane, and then 15 times through a 0.08 ⁇ m polycarbonate membrane.
  • the term “dried preparation” refers to a mixture of components as described herein that is dried by a technique known in the art in order to remove all or substantially all of a solvent.
  • the term “dried preparation” does not necessarily mean that the preparation is completely dry. For example, depending on the solvent or solvents used in the methods disclosed herein, it is possible that a small component of volatile and/or non-volatile material will remain in the dried preparation. In an embodiment, the non-volatile material will remain.
  • dried preparation it is meant that the preparation no longer contains substantial quantities of water and/or organic solvent. The process used to dry the preparation should be capable of removing substantially all water and/or organic solvent.
  • the dried preparation is completely free of water and/or organic solvent.
  • the dried preparation may contain a residual moisture content based on the limitations of the drying process (e.g. lyophilization). This residual moisture content will typically be less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.05% or less by weight of the dried preparation. This residual moisture content will not be more than 5% by weight of the dried preparation as this would result in a product that is not clear.
  • Various methods may be used to dry compositions are known in the art. In an embodiment, the drying is performed by lyophilization, spray freeze-drying, or spray drying.
  • the drying is performed by lyophilization.
  • lyophilization works by freezing the material and then reducing the surrounding pressure to allow the volatile solvent (e.g. water) in the material to sublime directly from the solid phase to the gas phase. Any conventional freeze-drying procedure may be used to carry out the drying step of the methods disclosed herein.
  • the lyophilization is performed by sequential steps of loading, freezing, evacuation and drying (e.g. primary drying and secondary drying).
  • lyophilization of the sized lipid vesicle particle/therapeutic agent mixture can be performed within a sealed bag in a benchtop freeze dryer.
  • This may be particularly advantageous because it reduces the number of steps that must be performed in a sterile laboratory environment and allows for the rapid manufacture of smaller batch sizes.
  • aseptically filled vials containing the mixture can be loaded and sealed within a sterile bag under sterile conditions.
  • These sterile, sealed units can then undergo lyophilization in an open laboratory (i.e. non-sterile environment) using a benchtop freeze dryer.
  • the freeze dryer is a benchtop freeze dryer.
  • the freeze dryer is a Virtis benchtop lyophilizer.
  • the freeze dryer is in an open laboratory (i.e. non-sterile environment).
  • Sterilization may be performed by any method known in the art.
  • the sterilization is performed by sterile filtration, steam heat sterilization, irradiation (e.g. gamma irradiation) or chemical sterilization.
  • the sterilization is performed by sterile filtration.
  • the sterile filtration is be performed before drying. Any conventional procedure for sterile filtration may be employed so long as it does not affect the solubility and stability of the therapeutic agents, lipid-based adjuvant, and/or polyI:C polynucleotide in the sized lipid vesicle particle/therapeutic agent mixture.
  • it may be desirable to perform the sterile filtration under low pressure conditions e.g.
  • the serial filtration may be performed using commercially available sterile filtration membranes (e.g. MilliporeSigma). In an embodiment, the sterile filtration is performed using a 0.22 ⁇ m-rated membrane, a 0.2 ⁇ m-rated membrane and/or a 0.1 ⁇ m-rated membrane. In an embodiment, the sterile filtration is performed by a single passage of the mixture through a single filtration membrane. In another embodiment, the sterile filtration is performed by serially passing the mixture sequentially through a combination of the same or different sized filtration membranes.
  • sterile filtration membranes e.g. MilliporeSigma
  • the methods disclosed herein may further comprise a step of confirming that the sized lipid vesicle particles have retained a mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1.
  • a step of confirming that the sized lipid vesicle particles have retained a mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1 there are several techniques, instruments and services that are available to measure the mean particle size and PDI of lipid vesicle particles, such as for example and without limitation TEM, SEM, AFM, FTIR, XPS, XRD, MALDI-TOF-MS, NMR and DLS.
  • the step of confirming the size and PDI of the lipid vesicle particles is performed using a DLS ZETASIZER NANO-S particle size analyzer.
  • the methods disclosed herein may further comprise a step of evaluating the stability of the lipids, therapeutic agent(s), lipid-based adjuvant, and/or polyI:C polynucleotide before and/or after the drying step.
  • the stability of the components may be measured by any known means or method.
  • stability of the dried preparation may be determined by the appearance of the dried preparation (lyophilisate) or measurement of the content of the components over time (e.g. by HPLC, RP-HPLC, IEX-HPLC, etc.).
  • HPLC is a technique which can be used to separate, identify and quantify each component in a mixture.
  • stability may evaluated upon solubilization in a hydrophobic carrier by various methods, such as for example: appearance of the solubilized product; identification and quantification of lipids, therapeutic agent(s), lipid-based adjuvant, and/or polyI:C polynucleotide, impurities or degradants (e.g. by RP-HPLC, IEX-HPLC, etc.); particle size of the lipid vesicle particles (e.g.
  • lipids may first be dissolved and mixed in an organic solvent. In embodiments where different types of lipid are used, this step will allow a homogenous mixture of the lipids to be formed. In an embodiment, these steps may be carried out in chloroform, chloroform:methanol mixtures, tertiary butanol or cyclohexane.
  • the lipids are prepared at 10-20mg lipid/mL organic solvent; however, higher or lower concentrations may also be used.
  • the organic solvent is removed (e.g. by evaporation) to yield a lipid film.
  • the lipid film may then be frozen and lyophilized to yield a dry lipid film.
  • the dry lipid film may then be hydrated with an aqueous solution containing therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant to provide a non-sized lipid vesicle particle preparation.
  • the step of hydration may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour).
  • an aqueous solution of lipids may be combined with at least one solution containing therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant.
  • one or more dry therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be added to, and solubilized in, the aqueous solution of lipids or sized lipid vesicle preparation. These embodiments may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour).
  • therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant are either solubilized in a solvent (e.g. aqueous or organic) prior to mixing with lipid vesicle particles or therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant are solubilized upon being mixed with the lipid vesicle particles.
  • a solvent e.g. aqueous or organic
  • therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be added as a dry powder to a solution containing lipid vesicle particles or both the lipid vesicle particles and therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be mixed together in a fresh solvent.
  • the pharmaceutical compositions disclosed herein may find application in any instance in which it is desired to administer therapeutic agents to a subject.
  • the subject may be a vertebrate, such as a fish, bird or mammal.
  • the subject is a mammal.
  • the subject is a human.
  • the pharmaceutical compositions may be used in methods for treating, preventing or diagnosing a disease, disorder or condition to which the therapeutic agent is targeted.
  • the pharmaceutical compositions may be used in methods for delivering a therapeutic agent to a subject.
  • the methods comprise administering to a subject the pharmaceutical composition as described herein.
  • the pharmaceutical compositions may be used in methods for modulating an immune response in a subject.
  • modulating is intended to refer to both immunostimulation (e.g. inducing or enhancing an immune response) and immunosuppression (e.g. preventing or decreasing an immune response).
  • the method would involve one or the other of immunostimulation or immunosuppression, but it is possible that the method could be directed to both.
  • the “immune response” may either be a cell-mediated (CTL) immune response or an antibody (humoral) immune response.
  • the pharmaceutical compositions disclosed herein may be used for inducing a cell-mediated immune response to the therapeutic agents (e.g. antigens).
  • to “induce” an immune response is to elicit and/or potentiate an immune response.
  • the pharmaceutical compositions disclosed herein may be used for inducing an antibody immune response to the therapeutic agents (e.g. antigens).
  • an “antibody immune response” or “humoral immune response” (used interchangeably herein), as opposed to cell-mediated immunity, is mediated by secreted antibodies which are produced in the cells of the B lymphocyte lineage (B cells).
  • B cells B lymphocyte lineage
  • Such secreted antibodies bind to antigens, such as for example those on the surfaces of foreign substances, pathogens (e.g.
  • the pharmaceutical compositions disclosed herein may be useful for treating or preventing diseases and/or disorders ameliorated by a cell-mediated immune response or a humoral immune response.
  • the pharmaceutical compositions disclosed herein may find application in any instance in which it is desired to administer therapeutic agents (e.g. antigens) to a subject to induce a cell-mediated immune response or a humoral immune response.
  • the pharmaceutical compositions is for inducing an antibody immune response and/or cell-mediated immune response to the therapeutic agents (e.g. antigens) in said subject.
  • pharmaceutical compositions is for the treatment and/or prevention of an infectious disease or cancer.
  • Treating” or “treatment of”, or “preventing” or “prevention of”, as used herein, refers to an approach for obtaining beneficial or desired results.
  • Beneficial or desired 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 (e.g. suppression), delay or slowing of disease onset, conferring protective immunity against a disease-causing agent and amelioration or palliation of the disease state.
  • Treating can be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, or is known to have already been exposed to an infectious agent, whereas “preventing” typically occurs in a subject who does not have a disease or disorder, or is not known to have been exposed to an infectious agent. As will be appreciated, there may be overlap in treatment and prevention.
  • the pharmaceutical compositions disclosed herein may be used for treating and/or preventing an infectious disease, such as caused by a viral infection, in a subject in need thereof.
  • the subject may be infected with a virus or may be at risk of developing a viral infection.
  • Viral infections that may be treated and/or prevented by the use or administration of a pharmaceutical composition as disclosed herein, without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus 1 , Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human papillomavirus (HPV), Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Orthoreovirus, Rotavirus, Ebola virus, parainfluenza virus, influenza A virus, influenza B virus, influenza C virus, Measles virus, Mumps virus, Rubella virus, Pneumovirus, respiratory syncytial virus (RSV), Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic chor
  • exemplary bacterial pathogens may include Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli O157: 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.
  • Anthrax Bacillus anthracis
  • Brucella Bacillus anthracis
  • the bacterial infection is Anthrax.
  • exemplary protozoan pathogens may include those of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi), which cause malaria.
  • the pharmaceutical compositions disclosed herein may be for use in treating and/or preventing cancer in a subject in need thereof. The subject may have cancer or may be at risk of developing cancer.
  • cancer refers to cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized.
  • cancer or “tumor” includes metastatic as well as non-metastatic cancer or tumors.
  • a cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • cancers that may be capable of being treated and/or prevented by the use or administration of a pharmaceutical composition as disclosed herein include carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors.
  • particularly suitable embodiments may include glioblastoma, multiple myeloma, ovarian cancer, breast cancer, fallopian tube cancer, prostate cancer or peritoneal cancer.
  • the cancer may be caused by a pathogen, such as a virus.
  • Viruses linked to the development of cancer are known to the skilled person and include, but are not limited to, human papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukaemia virus-1.
  • the cancer is one that expresses one or more tumor- specific neoantigens.
  • the cancer is breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, peritoneal cancer, glioblastoma or diffuse large B cell lymphoma.
  • the pharmaceutical compositions disclosed herein may be useful for either the treatment or prophylaxis of cancer; for example, a reduction of the severity of cancer (e.g. size of the tumor, aggressiveness and/or invasiveness, malignancy, etc.) or the prevention of cancer recurrences.
  • the pharmaceutical composition as disclosed herein may be administered by any suitable route.
  • the route of administration is injection, such as subcutaneous injection.
  • kits The pharmaceutical compositions disclosed herein are optionally provided to a user as a kit comprising the individual components which may be assembled to produce the pharmaceutical composition.
  • the kit is for preparing a composition for the treatment, prevention and/or diagnosis of a disease, disorder or condition or for the delivery of a therapeutic agent.
  • the kits can further comprise one or more additional reagents, packaging materials, and an instruction set or user manual detailing preferred methods of using the kit components.
  • the containers are vials.
  • the invention is further illustrated by the following non-limiting examples.
  • Example 1 [0118] First, a vial containing DPX-FP (IMV-Inc, Dartmouth, Canada) was reconstituted in 0.45 mL of Montanide ISA 51 oil diluent (SEPPIC, France) by soaking for 5 minutes and shaking for 2 minuntes, then vortexing for one minute.
  • SEPPIC Montanide ISA 51 oil diluent
  • DPX-PAM3CSK4 IMV-Inc, Dartmouth, Canada
  • DPX-PAM3CSK4 IMV-Inc, Dartmouth, Canada
  • reconstitution was completed by soaking for 5 minutes, shaking for 2 minutes, and vortexing for one minute to obtain the final concentrations of: FP peptide 9 ug/50 uL dose; DNA based polyI:C polynucleotide (dIdC) 18 ug/50 uL dose; PAM3CSK42 ug/50 uL dose; and Lipid 12.5 mg/50 uL dose.
  • dIdC DNA based polyI:C polynucleotide
  • the DPX-FP product vial was previously prepared by adding FP (NeoMPS) and DNA based polyI:C polynucleotide (dIdC) stock (Biospring) to a lipid-mixture solution, mixing well and freeze-drying.
  • FP NeoMPS
  • dIdC DNA based polyI:C polynucleotide
  • Biospring DNA based polyI:C polynucleotide
  • a lipid-mixture 132 mg/mL) containing DOPC and cholesterol in a 10:1 ratio (w:w) (Lipoid GmBH, Germany) was dissolved in 40% tertiary-butanol by shaking well at 300 RPM at room temperature for 1 hour or until dissolved.
  • FP stock (10 mg/mL) was prepared in DMSO and DNA based polyI:C polynucleotide adjuvant stock (10 mg/mL) was prepared in sterile water.
  • DNA based polyI:C polynucleotide adjuvant stock 10 mg/mL was prepared in sterile water.
  • 10 ⁇ L of FP stock was added to obtain 0.1 mg/mL final fill concentration, shaken well at 300 RPM for 5 minutes.
  • 20 ⁇ L of DNA based polyI:C polynucleotide (dIdC) stock was added to obtain 0.2 mg/mL final fill concentration, shaken well at 300 RPM for 5 minutes.
  • the volume was adjusted to 1.0 mL with 40% tertiary-butanol, freeze-dried, and stored at -20 C.
  • the dried preparation is reconstituted in 0.45 mL oil diluent when needed.
  • the DPX-PAM3CSK4 vial was prepared as follows: Briefly, a homogenous lipid- mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture and was added to sodium phosphate, 100 mM, pH 6.0 with shaking at 300 RPM for about 1 hour.
  • the mixture was then sized by passing the material 25 times through a 0.2 ⁇ m polycarbonate membrane, then 10 times through a 0.1 ⁇ m polycarbonate membrane, and then 15 times through a 0.08 ⁇ m polycarbonate membrane to attain a particle size of ⁇ 100 nm with a polydispersity index (PDI) of ⁇ 0.1.
  • the lipopeptide adjuvant PAM 3 CSK4 Polypeptide group, San Diego, USA
  • the mixture was then sterile filtered, aseptically filled in 1.0 mL aliquots into sterile glass vials, freeze-dried, and stored at -20°C.
  • Example 2 [0122] First, a vial containing DPX-MVP-S (IMV-Inc, Dartmouth, Canada) was reconstituted in 0.7 mL of Montanide ISA 51 oil (SEPPIC, France) by soaking for 5 minutes, shaking for 2 mins, and vortexing for one minute.
  • DPX-MVP-S IMV-Inc, Dartmouth, Canada
  • DPX-PAM3CSK4 IMV-Inc, Dartmouth, Canada
  • Restitution was completed by soaking for 5 minutes, shaking for 2 minutes, and vortexing for one minute to obtain the final concentrations of: MVP-S peptide each at 45 ug/50 uL dose; T-helper peptide A16L at 22.5 ug/50 uL dose; DNA based polyI:C polynucleotide (dIdC) 18 ug/50 uL dose; PAM 3 CSK42 ug/50 uL dose; and Lipid 12.5 mg/50 uL dose.
  • dIdC DNA based polyI:C polynucleotide
  • the DPX-MVP-S vial was prepared as follows: Briefly, DNA based polyI:C polynucleotide (dIdC) (BioSpring GmbH (Frankfurt, Germany) and peptide antigens (PolyPeptide Laboratories (San Diego, CA, USA) stock solutions were prepared as listed below: Stock# Component Solvent 5) in the following order: (4), (2), (3), (5) and then (1). The pH was adjusted to 10.0 ⁇ 0.5.
  • a 10:1 (w:w), homogenous lipid-mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture and added to the peptide/polynucleotide solution to form an intermediate bulk (non-sized) and mixed using a Silverson high speed mixer.
  • the pH was adjusted to 10.0 ⁇ 0.5, if required.
  • the intermediate bulk was then sized using an Emulsiflex C5 or C55 extruder by passing the material 35 times through a 0.2 ⁇ m polycarbonate membrane and then 10 times through a 0.1 ⁇ m polycarbonate membrane to attain a particle size of ⁇ 120 nm with a PDI of ⁇ 0.1.
  • the peptide stock solutions (6) and (7) were then added to the sized lipid vesicle particle bulk immediately after preparation.
  • the final pH of the solution was adjusted to 7.0 ⁇ 0.5.
  • the final preparation was then sterile filtered, aseptically filled (1.6 mL aliquots) into sterile glass vials, freeze-dried, and stored at -20°C.
  • the dried preparation is reconstituted in 0.7 mL oil diluent when needed.
  • the DPX-PAM3CSK4 vial was prepared as follows: Briefly, homogenous lipid- mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture was added to sodium phosphate, 100 mM, pH 6.0 with shaking at 300 RPM for about 1 hour. The mixture was then sized by passing the material 25 times through a 0.2 ⁇ m polycarbonate membrane, then 10 times through a 0.1 ⁇ m polycarbonate membrane, and then 15 times through a 0.08 ⁇ m polycarbonate membrane to attain a particle size of ⁇ 100 nm with a PDI of ⁇ 0.1.
  • the lipopeptide adjuvant PAM3CSK4 (Polypeptide group, San Diego, USA) was then added at 0.02 mg/mL to the sized lipid vesicles with shaking at 300 RPM for about 15 minutes. The mixture was then sterile filtered, aseptically filled to 1.0 mL aliquots into sterile glass vials, freeze-dried, and stored at -20°C. The dried preparation is reconstituted in 0.45 mL oil diluent when needed.

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Abstract

The present application relates to methods for preparing pharmaceutical compositions comprising therapeutic agents, lipids, a lipid-based adjuvant, and a polyI:C polynucleotide.

Description

METHODS OF MAKING DRIED PHARMACEUTICAL COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 63/449,779 filed March 3, 2023, which is incorporated by reference herein. FIELD [0002] The present application relates to methods for preparing pharmaceutical compositions comprising therapeutic agents, lipids, a lipid-based adjuvant, and a polyI:C polynucleotide. BACKGROUND [0003] In the pharmaceutical field, the effective delivery of therapeutic agents often poses difficulties and challenges, particularly in respect of the complexities of emerging delivery platforms designed to enhance the efficacy of therapeutic agents. For these specialized delivery platforms that employ unique components, new hurdles arise that do not exist for conventional pharmaceutical compositions. This is certainly the case for delivery platforms using water-free, hydrophobic carriers. These challenges are compounded when it is desirable to formulate pharmaceutical compositions comprising multiple components with diverse properties. [0004] In the case of pharmaceutical compositions comprising antigens (i.e. vaccine compositions), vaccines containing antigens are typically not immunogenic enough to generate rapid and prolonged immunity. This can sometimes be overcome with the use of an adjuvant to boost the immune response towards an antigen. There are generally two broad categories of adjuvants: delivery systems and immune-stimulants. The delivery system of a vaccine can act as an adjuvant by providing stability and prolonged interaction of the antigen with the immune system. Vaccine compositions may also incorporate molecular compounds with immune- stimulatory activity as adjuvants with the aim of further enhancing immunogenicity of the vaccine by directly activating cells of the immune system. [0005] Immune-stimulant adjuvants are defined molecular agonists that are recognized by the immune system via specialized receptors, for example polyI:C polynucleotide and lipid- based adjuvants such as PAM3CSK4 stimulate distinct Toll-like receptors. Immune-stimulants can activate the immune system and also direct the type of immune response generated towards a vaccine antigen. For example, the effectiveness of many vaccines is correlated to the generation of antibodies; however, for other types of vaccines a strong cytotoxic immune response primarily mediated by CD8+ T cells is desired. The type of immune response generated towards a vaccine antigen can be manipulated by including immune-stimulants that activate particular receptors found on immune cells that can initiate these responses through generation of cytokines and chemokines. [0006] Differences in the properties of immune-stimulants such as polyI:C polynucleotide and lipid-based adjuvants can complicate their incorporation into pharmaceutical compositions. SUMMARY [0007] In an embodiment, there is provided a method for preparing a pharmaceutical composition, comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the first dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the second dried preparation to produce a pharmaceutical composition. [0008] In an embodiment, there is provided a method for preparing a pharmaceutical composition, comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the second dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the first dried preparation to produce a pharmaceutical composition. [0009] In an embodiment, there is provided a pharmaceutical composition prepared by the method as described herein. [0010] In an embodiment, there is provided a method of inducing an antibody and/or CTL immune response in a subject comprising administering to the subject the pharmaceutical composition as described herein. [0011] In an embodiment, there is provided a use of the pharmaceutical composition as described herein for inducing an antibody and/or CTL immune response in a subject. [0012] In an embodiment, there is provided a method of delivering a therapeutic agent to a subject comprising administering to the subject the pharmaceutical composition as described herein. [0013] In an embodiment, there is provided a use of the pharmaceutical composition as described herein for delivering a therapeutic agent to a subject. [0014] In an embodiment, there is provided a kit for preparing the pharmaceutical composition as described herein, the kit comprising: a container comprising a first dried preparation prepared by the method as described herein; a container comprising second dried preparation prepared by the method as described herein; and a container comprising a hydrophobic carrier. [0015] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description in conjunction with the accompanying figures. DETAILED DESCRIPTION [0016] Poly I:C polynucleotide is an oligonucleotide molecule, composed of either DNA or RNA nucleosides, which interacts with Toll-like receptors (TLRs) that can detect nucleic acids, such as TLR3. Lipid-based adjuvants such as PAM2Cys and PAM3Cys interact with TLR1/2. Stimulating multiple TLR receptors at once has been reported to have an additive or synergistic activating effect on dendritic cells. Therefore, combining poly I:C polynucleotide and a lipid-based adjuvant such as PAM2Cys or PAM3Cys in a pharmaceutical composition that can ensure they engage with TLRs on the same cell can provide a potent activating effect to the immune system. Optimal doses of each adjuvant may need to be confirmed for different applications. For example, vaccines for cancer therapy may need stronger immune stimulation than vaccines for prevention of infectious disease. [0017] The lipopeptides PAM2Cys and PAM3Cys comprise a lipid palmitic acid moiety conjugated to a cysteine (Cys) residue that may be extended into a peptide with polar residues such as serine (Ser) and/or lysine (Lys) with a positive surface charge. By contrast, polyI:C polynucleotides are made up of single or double stranded molecules containing inosinic acid residues (I) and cytidylic acid residues (C) with a negative surface charge. If needed, these two distinct immune stimulators can be complexed together by electrostatic interactions to a certain extent with concentration restrictions. However, increasing the concentration of any one of these immune stimulators may lead to a pH shift and cause unwanted precipitation during formulation and/or in the finished product, constraining the ability to provide desired concentrations of lipid- based adjuvant and polyI:C polynucleotide in a pharmaceutical composition. Using current methods, it is only possible to combine polyI:C polynucleotides and PAM2Cys and PAM3Cys lipid-based adjuvants in a single water-free formulation up to about 0.5 mg/mL concentration. [0018] Embodiments of the present invention provide methods of fully incorporating both polyI:C polynucleotide and lipid-based adjuvant (such as PAM2Cys and PAM3Cys) along with therapeutic agents that overcomes concentration limitations, irrespective of the nature of therapeutic agents and formulation buffer/pH used to prepare the pharmaceutical composition. Using the methods of the present invention, higher concentrations of polyI:C polynucleotide and/or lipid-based adjuvant can successfully be incorporated into pharmaceutical compositions. [0019] PolyI:C polynucleotide [0020] PolyI:C polynucleotides are polynucleotide molecules (either RNA or DNA or a combination of DNA and RNA) containing inosinic acid residues (I) and cytidylic acid residues (C), and which induce the production of inflammatory cytokines, such as interferon. In some embodiments, the polyI:C polynucleotide is double-stranded. In double-stranded embodiments, they are typically composed of one strand consisting entirely of cytosine-containing nucleotides and one strand consisting entirely of inosine-containing nucleotides, although other configurations are possible. For instance, each strand may contain both cytosine-containing and inosine-containing nucleotides. In some embodiments, the polyI:C polynucleotide is single- stranded. In single-stranded embodiments, the strand comprises both cytosine-containing and inosine-containing nucleotides. In some instances, either or both strand may additionally contain one or more non-cytosine or non-inosine nucleotides. [0021] In some embodiments, the polyI:C polynucleotide may be a single-stranded molecule containing inosinic acid residues (I) and cytidylic acid residues (C). As an example, and without limitation, the single-stranded polyI:C polynucleotide may be a sequence of repeating dIdC. In a particular embodiment, the sequence of the single-stranded polyI:C may be a 26-mer sequence of (IC)13, i.e. ICICICICICICICICICICICICIC. As the skilled person will appreciate, due to their nature (e.g. complementarity), it is anticipated that these single-stranded molecules of repeating IC or dIdC may form homodimers. [0022] Accordingly, as used herein, a “polyI:C” or “polyI:C polynucleotide” is a double- or single-stranded polynucleotide molecule (RNA or DNA or a combination of DNA and RNA), each strand of which contains at least 6 contiguous inosinic or cytidylic acid residues, or 6 contiguous residues selected from inosinic acid and cytidylic acid in any order (e.g. IICIIC or ICICIC). PolyI:C polynucleotides will typically have a length of about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000 or more nucleotides. [0023] Each strand of a double-stranded polyI:C polynucleotide may be a homopolymer of inosinic or cytidylic acid residues, or each strand may be a heteropolymer containing both inosinic and cytidylic acid residues. In either case, the polymer may be interrupted by one or more non-inosinic or non-cytidylic acid residues (e.g. uridine), provided there is at least one contiguous region of 6 I, 6 C or 6 I/C residues as described above. Typically, each strand of a polyI:C polynucleotide will contain no more than 1 non-I/C residue per 6 I/C residues, more preferably, no more than 1 non-I/C residue per every 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 I/C residues. [0024] The inosinic acid or cytidylic acid (or other) residues in the polyI:C polynucleotide may be derivatized or modified as is known in the art, provided the ability of the polyI:C polynucleotide to promote the production of an inflammatory cytokine, such as interferon, is retained. Non-limiting examples of derivatives or modifications include e.g. azido modifications, fluoro modifications, or the use of thioester (or similar) linkages instead of natural phosphodiester linkages to enhance stability in vivo. The polyI:C polynucleotide may also be modified to e.g. enhance its resistance to degradation in vivo by e.g. complexing the molecule with positively charged poly-lysine and carboxymethylcellulose, or with a positively charged synthetic peptide. [0025] Lipid-based adjuvant [0026] As used herein, a lipid-based adjuvant is an adjuvant that comprises at least one lipid moiety or lipid component. [0027] The expression “lipid moiety” or “lipid component” refers to any fatty acid (e.g. fatty acyls) or derivative thereof, including for example triglycerides, diglycerides, and monoglycerides. Exemplary fatty acids include, without limitation, palmitoyl, myristoyl, stearoyl and decanoyl groups or any C2 to C30 saturated or unsaturated fatty acyl group, preferably any C14 to C22 saturated or unsaturated fatty acyl group, and more preferably a C16 saturated or unsaturated fatty acyl group. Thus, as referred to herein, the expression “lipid-based adjuvant” encompasses adjuvants comprising a fatty acyl group or derivative thereof. [0028] Lipid-based adjuvants contain at a minimum at least one lipid moiety, or a synthetic/semi-synthetic lipid moiety analogue, which can be coupled onto an amino acid, an oligopeptide or other molecules (e.g. a carbohydrate, a glycan, a polysaccharide, biotin, Rhodamine, etc.). Thus, without limitation, the lipid-based adjuvant may be, for example, a lipoamino acid, a lipopeptide, a lipoglycan, a lipopolysaccharide or a lipoteichoic acid. Moreover, a lipid moiety or a structure containing a lipid moiety can be coupled covalently or non-covalently to an antigen to create antigenic compounds with built-in adjuvanting properties. For example, and without limitation, the lipid-based moiety may comprise a cation (e.g. nickel) to provide a positive charge for non-covalent coupling. [0029] In some embodiments, the lipid moiety or lipid component may be naturally occurring, such as for example a cell-wall component (e.g. lipoprotein) from a Gram-positive or Gram-negative bacteria, Rhodopseudomonas viridis, or mycoplasma. In other embodiments, the lipid moiety or lipid component may be synthetic or semi-synthetic [0030] The lipid-based adjuvant may comprise palmitic acid (PAM) as at least one of the lipid moieties or components of the adjuvant. Such lipid-based adjuvants are referred to herein as a “palmitic acid adjuvant”. Palmitic acid is a low molecular weight lipid found in the immunologically reactive Braun’s lipoprotein of Escherichia coli. Other common chemical names for palmitic acid include, for example, hexadecanoic acid in IUPAC nomenclature and 1-Pentadecanecarboxylic acid. The molecular formula of palmitic acid is CH3(CH2)14CO2H. As will be understood to those skilled in the art, it is possible that the lipid chain of palmitic acid may be altered. Exemplary compounds which may be used herein as palmitic acid adjuvants, and methods for their synthesis, are described for example in United States Patent Publications US 2008/0233143; US 2010/0129385; and US 2011/0200632, the disclosures of which are incorporated herein. [0031] As described above for lipid moieties generally, a palmitic acid adjuvant contains at a minimum at least one palmitic acid moiety, which can be coupled onto an amino acid, an oligopeptide or other molecules. A palmitic acid moiety or a structure containing palmitic acid can be coupled covalently or non-covalently to an antigen to create antigenic compounds with built-in adjuvanting properties. The palmitic acid moiety or a chemical structure containing palmitic acid can be conjugated to a cysteine peptide (Cys) to allow for various structural configurations of the adjuvant, including linear and branched structures. The cysteine residue has been commonly extended by polar residues such as Serine (Ser) and/or lysine (Lys) at the C terminus to create adjuvant compounds with improved solubility. Palmitic acid containing adjuvant compounds could be admixed with an antigen, associated with antigen through non-covalent interactions, or alternatively covalently linked to an antigen, either directly or with the use of a linker/spacer, to generate enhanced immune responses. Most commonly, two palmitic acid moieties are attached to a glyceryl backbone and a cysteine residue to create dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys), which can also be used in multiple configurations as described above. [0032] In an embodiment, the lipid-based adjuvant is any type of adjuvant comprising a palmitic acid moiety or component. In an embodiment, lipid-based adjuvant is a lipopeptide comprising one or more palmitic acid moieties. The palmitic acid moiety may be modified or manipulated to improve its stability in vitro or in vivo, enhance its binding to receptors (such as for example toll-like receptors as described below) or enhance its biological activity. [0033] In an embodiment, the palmitic acid adjuvant comprises PAM2Cys. In another embodiment, the palmitic acid adjuvant comprises PAM3Cys. [0034] In an embodiment, the palmitic acid adjuvant comprises PAM2-Cys-Ser-(Lys)4 or PAM3-Cys-Ser-(Lys)4. [0035] In some embodiments, the palmitic acid adjuvant is an analog of PAM2-Cys-Ser- (Lys)4 or PAM3-Cys-Ser-(Lys)4 including, without limitation, PAM3Cys-SKKKK (β- irradiated), R-PAM3Cys-SKKKK S-PAM3Cys-SKKKK, PAM3Cys-SKKKK(Biotin-Aca-Aca), PAM3Cys-SKKKK(Fluorescein-Aca-Aca), PAM3Cys-SKKKK, PAM3Cys-SKKKK-FLAG-tag, PAM3Cys-SSNAKIDQLSSDVQT, PAM3Cys-SSNKSTTGSGETTTA, PAM3Cys- SSTKPVSQDTSPKPA, PAM3Cys-SSGSKPSGGPLPDAK, PAM3Cys-SSGNKSAPSSSASSS, PAM3Cys-GSHQMKSEGHANMQL, PAM3Cys-SSSNNDAAGNGAAQT, PAM3Cys-KQNVSSLDEKNSVSV, PAM3Cys-NNSGKDGNTSANSAD, PAM3Cys- NNGGPELKSDEVAKS, PAM3Cys-SQEPAAPAAEATPAG, PAM3Cys- SSSKSSDSSAPKAYG, PAM3Cys-AQEKEAKSELDYDQT, Pam2Cys-SKKKK (mixture of RR and RS stereoisomers), R-Pam2Cys-SKKKK (RR stereoisomer), S-Pam2Cys-SKKKK (RS stereoisomer), PamCys(Pam)-SKKKK, Pam2Cys-SKKKK(Biotin-Aca-Aca)-NH2, Pam2Cys- SKKKK(Fluorescein-Aca-Aca)-NH2, PAM2Cys-SKKKK(Rhodamine-Aca-Aca)-NH2, and PAM2Cys-SKKKK-FLAG-tag. Where appropriate, the palmitic acid adjuvant or analog thereof may used as stereochemically defined compounds or as a mixture of stereoisomers. [0036] In an embodiment, the lipid-based adjuvant is PAM3-Cys-Ser-(Lys)4:
Figure imgf000009_0001
that act as TLR agonists may also be used as the lipid-based adjuvant disclosed herein, including without limitation the palmitic acid adjuvants and analogs described above and synthetic diacylated lipoprotein FSL-1 available from InvivoGen (San Diego, California, USA) and EMC Microcollections GmbH (Germany). FSL-1 (Pam2CGDPKHPKSF) is a synthetic lipoprotein that represents the N-terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium. FSL-1 comprises PAM2Cys and has a similar framework structure as macrophage activating lipopeptide-2 (MALP-2), a Mycoplasma fermentans derived lipopeptide. [0038] In an embodiment, the lipid-based adjuvant comprises FSL-1 or MALP-2, or the lipid-based adjuvant is FSL-1 or MALP-2. Where appropriate, FSL-1 or MALP-2 may be used as stereochemically defined compounds or as a mixture of stereoisomers. The FSL-1 or MALP- 2 may be labelled (e.g. biotin, Fluorescein, Rhodamine, etc.). FSL-1 is also available as a FSL-1 Ala-scan collection (EMC Microcollections) comprising nine different FSL-1-Ala compounds. [0039] Further embodiments of lipid-based adjuvants that comprise palmitic acid may include substructures of TLR2 ligands such as monoacylated lipopeptides. Without limitation, these may include, for example, Pam-Dhc-SKKKK, Pam-CSKKKK, Pam-Dhc-GDPKHPKSF or Pam-CGDPKHPKSF (EMC Microcollections). [0040] Therapeutic agent [0041] As used herein, the term “therapeutic agent” is any molecule, substance or compound that is capable of providing a therapeutic activity, response or effect in the treatment or prevention of a disease, disorder or condition, including diagnostic and prophylactic agents. [0042] In relation to the methods disclosed herein providing a dried preparation, a “first therapeutic agent” is any one or more therapeutic agents which are used in the preparation of the non-sized lipid vesicle particle preparation (i.e. incorporated in the methods before the step of sizing the non-sized lipid vesicle preparation). In contrast, a “second therapeutic agent” is any one or more therapeutic agents which are used in the methods herein after preparation of the sized lipid vesicle particle preparation (i.e. incorporated in the methods after the step of sizing the non-sized lipid vesicle preparation). In embodiments of the methods disclosed herein, lipids may first be dissolved and mixed in an organic solvent. In embodiments where different types of lipid are used, this step will allow a homogenous mixture of the lipids to be formed. In an embodiment, these steps may be carried out in chloroform, chloroform:methanol mixtures, tertiary butanol or cyclohexane. In an embodiment, the lipids are prepared at 10-20mg lipid/mL organic solvent; however, higher or lower concentrations may also be used. In some embodiments, after mixing, the organic solvent is removed (e.g. by evaporation) to yield a lipid film. The lipid film may then be frozen and lyophilized to yield a dry lipid film. The dry lipid film may then be hydrated with an aqueous solution containing therapeutic agents to provide a non-sized lipid vesicle particle preparation. The step of hydration may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour). In yet another embodiment of the methods disclosed herein, an aqueous solution of lipids may be combined with a solution containing one or more solubilized therapeutic agents. In another embodiment, one or more dry therapeutic agents may be added to, and solubilized in, the aqueous solution of lipids or sized lipid vesicle preparation. These embodiments may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour). The above procedures are exemplary methods for providing a non-sized lipid vesicle particle preparation comprising non-sized lipid vesicles and one or more first therapeutic agents. [0043] In some embodiments, therapeutic agents are either solubilized in a solvent (e.g. aqueous or organic) prior to mixing with lipid vesicle particles or therapeutic agents are solubilized upon being mixed with the lipid vesicle particles. In this latter embodiment, therapeutic agents may be added as a dry powder to a solution containing lipid vesicle particles or both the lipid vesicle particles and dry therapeutic agents may be mixed together in a fresh solvent. [0044] In an embodiment, the therapeutic agent is an antigen, a small molecule drug, an antibody or a functional fragment thereof, an antibody mimetic or a functional fragment thereof, an immunomodulatory agent, a polynucleotide encoding a polypeptide, or an interfering polynucleotide. [0045] In some embodiments, the therapeutic agent is an antigen. As used herein, the term “antigen” may be used interchangeably with “immunogen” and may refer to a pathogen, a part of a pathogen, or a molecule that is able to induce an adaptive antibody immune response and/or an adaptive cellular immune response that is specific to said antigen or a portion thereof. The term “antigen” may refer to a pathogen, a part of a pathogen, or a molecule that is specifically recognized by antibodies and/or immunoglobulin receptors of the adaptive antibody immune response and/or the adaptive cellular immune response. As used herein, the term “peptide antigen” is an antigen as defined above that is a protein or a polypeptide. Adaptive antibody responses are mediated by B cells, which recognize antigen by specific binding of IgM immunoglobulins in their B cell receptors to the antigen. Activated B cells may mature into plasma cells and secrete soluble IgM, IgG, IgA, or IgE antibodies that specifically to bind to the antigen. Cellular immune responses are mediated by CD8+ cytotoxic T cells, which recognize polypeptide antigen by specific binding of T cell receptors (specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers) to peptides from the antigen that are displayed on MHC class I molecules on the surface of target cells. Antibody and cellular adaptive immune responses are coordinated by CD4+ T helper cells, which recognize polypeptide antigen by specific binding of their T cell receptors (specifically the immunoglobulin superfamily TCR-alpha/TCR-beta or TCR-gamma/TCR-delta heterodimers) to peptides from the antigen that are displayed on MHC class I/II molecules on the surface of APC. The immunoglobulin superfamily proteins in B cell receptors and antibodies (IgM, IgD, IgG, IgA, IgE), and in T cell receptors (TCR-alpha/TCR-beta, TCR-gamma/TCR-delta) that specifically bind to antigen are created randomly by processes of genetic recombination during B cell and T cell development. Each B cell and T cell expresses only one species of randomized immunoglobulin superfamily receptor with a specific binding recognition, and B cells and T cells that bind an antigen are clonally selected for expansion in the body during an immune response. Further, the immunoglobulins in B cell receptors and antibodies may further diversify by somatic mutation and clonal selection for variants with high antigen binding. By these processes, antibodies, B cell receptors, and T cell receptors are immunoglobulin superfamily molecules that enable the immune system to adapt to an antigen, thus forming an adaptive immune response. This adaptive immune response contrasts with innate immune responses that are mediated by conserved immune receptors, such as TLR or other pattern recognition receptors, that are unchanging and have fixed specificity for binding to specific pathogen- associated molecular patterns. Thus, as used herein, the term “antigen” or “peptide antigen” may refer to a molecule that is specifically bound by antibodies, B cell receptors, and/or T cell receptors of the adaptive immune system during an active adaptive immune response. The specific portion of an antigen that is bound by an antibody, a B cell receptor, and/or a T cell receptor is called an “epitope”. Antibodies and B cell receptors may bind to an epitope on various types of molecules including polypeptides, polysaccharides, glycoproteins, and lipoproteins. T cell receptors bind to peptides that are derived from antigen, wherein the peptides are displayed on MHC class I/II molecules. Thus, the term “antigen” may refer to a molecule that comprises a B cell epitope and/or a T cell epitope. [0046] In some embodiments, the antigen may include a polypeptide, a polysaccharide, a glycoprotein, a lipoprotein, a microorganism or a part thereof, such as a live, attenuated, inactivated or killed bacterium, virus or protozoan, or part thereof, an allergen, or an antigen derived from a cancer cell (such as a conserved cancer antigen or a neoantigen). As used herein, the term “derived from” encompasses, without limitation: an antigen that is isolated or obtained directly from an originating source; a synthetic or recombinantly generated antigen that is identical or substantially related to an antigen from an originating source; or an antigen which is made from an antigen of an originating source or a fragment thereof. When it is stated that an antigen is “from” a source, the term “from” may be equated with “derived from”. The term “substantially related”, as this context, means that the antigen may have been modified by chemical, physical or other means (e.g. sequence modification), but that the resultant product remains capable of generating an immune response to the original antigen or to the disease or disorder associated with the original antigen. As used herein, the term “antigen” also includes a polynucleotide that encodes a 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. [0047] Viruses, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, herpes virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, human papillomavirus (HPV), Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, human immunodeficiency virus (HIV), Seneca Valley virus (SVV), Orthoreovirus, Rotavirus, Ebola virus, parainfluenza virus, influenza virus (e.g. H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), Measles virus, Mumps virus, Rubella virus, Pneumovirus, respiratory syncytial virus, respiratory syncytial virus (RSV), Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Coronavirus (e.g. Sars-Cov-2), Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella. [0048] In an embodiment, the peptide antigen is derived from HPV. In an embodiment, the HPV peptide antigen is one that is associated with HPV-related cervical cancer or HPV-related head and neck cancer. In an embodiment, the peptide antigen is a peptide comprising the sequence RAHYNIVTF (HPV16E7 (H-2Db) peptide 49-57; R9F). In an embodiment, the peptide antigen is a peptide comprising the sequence YMLNLGPET (HPV Y9T peptide). [0049] In an embodiment, the peptide antigen is derived from HIV. In an embodiment, the HIV peptide antigen may be derived from the V3 loop of HIV-1 gp120. In an embodiment, the HIV peptide antigen may be RGP10 (RGPGRAFVTI). In another embodiment, the peptide antigen may be AMQ9 (AMQMLKETI). AMQ9 peptide is the immunodominant MHC class I epitope of gag for mice of the H-2Kd haplotype. [0050] In an embodiment, the peptide antigen is derived from RSV. The RSV virion, a member of the genus Paramyxoviridae, is composed of a single strand of negative-sense RNA with 15,222 nucleotides. The nucleotides encode three transmembrane surface proteins (F, G and small hydrophobic protein or SH), two matrix proteins (M and M2), three nucleocapsid proteins (N, P and L), and two non-structural proteins (NS1 and NS2). In an embodiment, the peptide antigen may be derived from any one or more of the RSV proteins. In a particular embodiment, the peptide antigen may be derived from the SH protein of RSV or any other paramyxovirus, or a fragment thereof. The RSV peptide antigen may be any one or more of the RSV peptides described or disclosed in WO 2012/065997, incorporated herein by reference. [0051] Bacteria, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli O157: 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. [0052] In an embodiment, the peptide antigen is derived from a Bacillus anthracis. Without limitation, the peptide antigen may for example be derived from anthrax recombinant protective antigen (rPA) (List Biological Laboratories, Inc.; Campbell, CA) or anthrax mutant recombinant protective antigen (mrPA). rPA has an approximate molecular weight of 83,000 daltons (Da) and corresponds a cell binding component of the three-protein exotoxin produced by Bacillus anthracis. The protective antigen mediates the entry of anthrax lethal factor and edema factor into the target cell. In some embodiments the antigen may be derived from the sequence found under GenBank Accession number P13423, or any suitable sequence variant thereof. [0053] Protozoa, or parts thereof, from which a peptide antigen may be derived include for example, and without limitation, the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi), which causes malaria. [0054] In an embodiment, the peptide antigen is derived from a Plasmodium species. For example, and without limitation, the peptide antigen may be derived from the circumsporozoite protein (CSP), which is a secreted protein of the sporozoite stage of the malaria parasite (Plasmodium sp.). The amino-acid sequence of CSP consists of an immunodominant central repeat region flanked by conserved motifs at the N- and C-termini that are implicated in protein processing as the parasite travels from the mosquito to the mammalian vector. The structure and function of CSP is highly conserved across the various strains of malaria that infect humans, non-human primates and rodents. In an embodiment, the peptide antigen derived from CSP is a malaria virus-like particle (VLP) antigen which comprises circumsporozoite T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen. [0055] In another embodiment, the peptide antigen may be derived from a cancer or tumor-associated protein, such as for example, a membrane surface-bound cancer antigen. In an embodiment, the cancer may be one that is caused by a pathogen, such as a virus. Viruses linked to the development of cancer are known to the skilled person and include, but are not limited to, human papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukaemia virus-1. Thus, in an embodiment, the peptide antigen may be derived from a virus that is linked to the development of cancer. In an embodiment, the peptide antigen is a cancer-associated antigen. Many cancer or tumor-associated proteins are known in the art such as for example, and without limitation, those described in WO 2016/176761, incorporated by reference herein. [0056] In an embodiment, the peptide antigen is one or more survivin antigens. Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), is a protein involved in the negative regulation of apoptosis. In an embodiment, the peptide antigen is any peptide, polypeptide or variant thereof derived from a survivin protein, or a fragment thereof. In an embodiment, the survivin peptide antigen may comprise the full length survivin polypeptide. Alternatively, the survivin peptide antigen may be a survivin peptide comprising a fragment of any length of the survivin protein. Exemplary embodiments include a survivin peptide that comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In specific embodiments, the survivin peptide consists of a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide, consisting of 7, 8, 9, 10, 11 consecutive amino acid residues of the survivin protein, respectively. Particular embodiments of the survivin antigen include survivin peptides of about 9 or 10 amino acids. Survivin peptide antigens also encompass variants and functional equivalents of natural survivin peptides. Variants or functional equivalents of a survivin peptide encompass peptides that exhibit amino acid sequences with differences as compared to the specific sequence of the survivin protein, such as one or more amino acid substitutions, deletions or additions, or any combination thereof. The difference may be measured as a reduction in identity as between the survivin protein sequence and the survivin peptide variant or survivin peptide functional equivalent. In an embodiment, a pharmaceutical composition of the present invention may include any one or more of the survivin peptides, survivin peptide variants or survivin peptide functional equivalents disclosed in WO 2004/067023; WO 2006/081826 or WO 2016/176761, each of which is incorporated by reference herein. In an embodiment, the survivin peptide antigen may be any one or more of FEELTLGEF, FTELTLGEF, LTLGEFLKL, LMLGEFLKL, RISTFKNWPF, RISTFKNWPK, STFKNWPFL, and/or LPPAWQPFL. [0057] In an embodiment, the peptide antigen is a neoantigen. As used herein, the term “neoantigen” refers to a class of tumor antigens which arise from tumor-specific mutations in an expressed protein. The neoantigen can be derived from any cancer, tumor or cell thereof. In the context of neoantigens, the term “derived from” as used herein encompasses, without limitation: a neoantigen that is isolated or obtained directly from an originating source (e.g. a subject); a synthetic or recombinantly generated neoantigen that is identical in sequence to a neoantigen from an originating source; or a neoantigen which is made from a neoantigen of an originating source or a fragment thereof. The mutations in the expressed protein that create the neoantigen may be patient-specific. By “patient-specific”, it is meant that the mutation(s) are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s). Thus, a “patient-specific” mutation may be shared by a small or large sub-population of subjects. The neoantigen may comprise one or more neoepitopes. As used herein, the term “epitope” refers to a peptide sequence which can be recognized by the immune system, specifically by antibodies, B cells or T cells. A “neoepitope” is an epitope of a neoantigen which comprises a tumor-specific mutation as compared to the native amino acid sequence. Generally, neoepitopes may be identified by screening neoantigens for anchor residues that have the potential to bind patient HLA. The neoepitopes are normally ranked using algorithms, such as NetMHC, that can predict peptide binding to HLA. A "T-cell neoepitope" is to be understood as meaning a mutated peptide sequence which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex. The T-cell neoepitope should typically be one that is amenable to recognition by T cell receptors so that a cell-mediated immune response can occur. A "B-cell neoepitope" is to be understood as meaning a mutated peptide sequence which can be recognized by B cells and/or by antibodies. In some embodiments, at least one of the neoepitopes of the neoantigen is a patient-specific neoepitope. As used herein, by “patient-specific neoepitope”, it is meant that the mutation(s) in the neoepitope are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s). Thus, a “patient-specific neoepitope” may be shared by a small or large sub-population of subjects. In an embodiment, the neoantigen may be derived from a mutated gene or protein that has previously been associated with cancer phenotypes, such as for example tumor suppressor genes (e.g. p53); DNA repair pathway proteins (e.g. BRCA2) and oncogenes. In some embodiments, the neoantigen may comprise or consist of the neoantigens disclosed in Castle et al. (2012) Exploiting the Mutanome for Tumor Vaccination. Cancer Res, 72(5): 1081-1091, incorporated by reference herein. In an embodiment, the neoantigen may be one or more of the Mut1-50 neoantigens disclosed in Table 1 of Castle 2012, or a neoantigen of the same or related protein (e.g. a human homologue). In an embodiment, the neoantigen may be one or more of Mut25 (STANYNTSHLNNDVWQIFENPVDWKEK), Mut30 (PSKPSFQEFVDWENVSPELNSTDQPFL) and Mut44 (EFKHIKAFDRTFANNPGPMVVFATPGM), or a neoantigen of the same or related protein (e.g. a human homologue). [0058] Many peptide antigens are known in the art and may be suitable for use as therapeutic agents of the present invention. In an embodiment, the antigen may be a peptide derived from the ectodomain of the small hydrophobic protein of respiratory syncytial virus (RSV) as disclosed in WO2012/065997, incorporated herein by reference. For example, the RSV antigen may be a peptide with the sequence NKLCEYNVFHNKTFELPRARVNT, NKLSEHKTFCNNTLELGQMHQINT, or NKLCDFNDHHTNSLDIRTRLRNDTQLITRAHEGSINQSSN, or a portion or variant thereof, In an embodiment, the antigen may be a peptide derived from the survivin protein as disclosed in WO2004/067023 and WO2006/081826, incorporated herein by reference. For example, the survivin antigen may be one or more peptides of the sequence FEELTLGEF, FTELTLGEF, LTLGEFLKL, LMLGEFLKL, RISTFKNWPF, RISTFKNWPK, STFKNWPFL, or LPPAWQPFL, or a portion or a variant thereof. In an embodiment, the antigen may be a fusion peptide (FP) antigen comprising the sequence RAHYNIVTF (HPV16E7 (H-2Db) peptide 49-57) fused to the universal T-helper epitope is PADRE (pan-DR epitope) comprising the peptide sequence AKXVAAWTLKAAA, wherein X may be phenylalanine or cyclohexylalanine. [0059] In a particular embodiment, the therapeutic agent is one or more peptide antigens as described herein. In an embodiment, the peptide antigen is a synthetically produced polypeptide. The peptide antigen may be a polypeptide of any length. In an embodiment, the peptide antigen may be 5 to 120 amino acids in length, 5 to 100 amino acids in length, 5 to 75 amino acids in length, 5 to 50 amino acids in length, or 5 to 30 amino acids in length. In an embodiment, the peptide antigen may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length. In an embodiment, the peptide antigen is 20 to 30 amino acids in length. In an embodiment, the peptide antigen is 27 amino acids in length. In an embodiment, the peptide antigen is 8 to 40 amino acids in length. In an embodiment, the peptide antigen is 9 or 10 amino acids in length. [0060] In some embodiments, the therapeutic agent is a small molecule drug. The term “small molecule drug” refers an organic or inorganic compound that may be used to treat, cure, prevent or diagnose a disease, disorder or condition. As used herein, the term "small molecule" refers to a low molecular weight compound which may be synthetically produced or obtained from natural sources and has a molecular weight of less than 2000 Daltons (Da), less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da or less than 500 Da. A small molecule drug is typically a chemically manufactured active substance or compound (i.e. it is not produced by a biological process). Generally, these compounds are synthesized by chemical reactions between different organic and/or inorganic compounds. As used herein, the term “small molecule drug” does not encompass larger structures, such as polynucleotides, proteins, and polysaccharides, which are made by a biological process. The term “small molecule” may refer to compounds or molecules that selectively bind specific biological macromolecules and act as an effector, altering the activity or function of the target. Thus, a small molecule drug is a substance or compound that regulates a biological process in the body of a subject, and more particularly within a cell. A small molecule drug may exert its activity in the form in which it is administered, or the small molecule drug may be a prodrug. In this regard, the term “small molecule drug”, as used herein, encompasses both the active form and the prodrug. The term “prodrug” refers to a compound or substance that, under physiological conditions, is converted into the therapeutically active agent. A prodrug is a compound or substance that, after administration, is metabolized in the body of a subject into the pharmaceutically active form (e.g. by enzymatic activity in the body of the subject). A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the pharmaceutically active form. Many small molecule drugs are known in the art and are used as active ingredients in medicaments. The skilled person is aware of numerous small molecule drugs such as those disclosed in the online DrugBank database. A small molecule drug may include a cytotoxic agent, an anti-cancer agent, an anti- tumor agent, a chemotherapeutic agent, an anti-neoplastic agent, an antiviral agent, an antibacterial agent, an anti-inflammatory agent, an immunomodulatory agent (e.g. a cytokine or a chemokine), an immune response checkpoint agent, a biological response modifier, a prodrug, a ligand, an analgesic, a radiopharmaceutical, a radioisotope or a dye for visual detection. A cytotoxic agent may be an agent that kills a target cell by necrosis or apoptosis. A cytotoxic agent may include epacadostat, cyclophosphamide, rapamycin, ifosfamide, afosfamide, melphalan, bendamustine, uramustine, palifosfamide, chlorambucil, busulfan, 4-hydroxycyclophosphamide, bis-chloroethylnitrosourea (BCNU), mitomycin C, yondelis, procarbazine, dacarbazine, temozolomide, cisplatin, carboplatin, oxaliplatin, acyclovir, gemcitabine, 5-fluorouracil, cytosine arabinoside, ganciclovir, camptothecin, topotecan, irinotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, etoposide, teniposide, mitoxantrone, valproic acid, vorinostat, methotrexate, tacrolimus, or pixantrone. A small molecule drug may include an checkpoint agent. As used herein, a “checkpoint agent” refers to any compound or molecule that totally or partially modulates (e.g. activates or inhibits) the activity or function of one or more checkpoint molecules (e.g. proteins). Checkpoint molecules regulate various cellular processes. A small molecule drug may include a checkpoint agent that is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B- and T-lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD33, CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), Killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), phosphatidylserine (PS), OX-40, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SLAM, TIGIT, TIM3, TNF-α, VISTA, VTCN1, or any combination thereof. A small molecule drug may include a cell-penetrating peptide, a peptide transduction domain, or a dendritic cell peptide, used as molecular shuttles that can transport other molecules or ions from one location to another. [0061] In some embodiments, the therapeutic agent is an antibody, a functional equivalent of an antibody or a functional fragment of an antibody. Broadly, an “antibody” refers to a polypeptide or protein that consists of or comprises antibody domains, which are understood as constant and/or variable domains of the heavy and/or light chains of immunoglobulins, with or without a linker sequence. Antibody domains may be of native structure or modified by mutagenesis or derivatization, e.g. to modify binding specificity or any other property. An antibody may comprise a complete (i.e. full-length) immunoglobulin molecule, including e.g. polyclonal, monoclonal, chimeric, humanized and/or human versions having full length heavy and/or light chains. The term “antibody” encompasses any and all isotypes and subclasses, including without limitation the major classes of IgA, IgD, IgE, IgG and IgM, and the subclasses IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. An antibody may be one that is naturally occurring or one that is prepared by any means available to the skilled person, such as for example by using immunoglobulin gene fragment recombinatorial processes. An antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a fully human antibody. A “chimeric antibody” as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, such for example a rodent, while the constant domains of the antibody are derived from a different species, such as a human. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of an animal, such as for example a cat or dog. A “humanized antibody” as used herein refers to a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains, including human framework region (FR) sequences. The constant domains of the humanized antibody are likewise derived from a human antibody. Without limitation, a “human antibody” as used herein refers to an antibody obtained from transgenic animals (e.g. mice) that have been genetically engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic animal can synthesize human antibodies specific for human antigens, and the animal can be used to produce human antibody-secreting hybridomas. A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. As used herein, the term “functional fragment”, with respect to an antibody, refers to an antigen-binding portion of an antibody. In this context, by “functional” it is meant that the fragment maintains its ability to bind to the target antigen. Functional fragments of antibodies include a portion of an antibody such as a F(ab')2, a F(ab)2, a Fab', a Fab, a Fab2, a Fab3, and single domain antibody. Regardless of structure, a functional fragment of an antibody binds with the same antigen that is recognized by the intact antibody. The term “functional fragment”, in relation to antibodies, also includes isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). Antibody fragments can be incorporated into single domain antibodies (e.g. nanobodies), single-chain antibodies, maxibodies, evibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNAR, bis-scFv and other like structures. Another form of a functional fragment is a peptide comprising one or more CDRs of an antibody or one or more portions of the CDRs, provided the resultant peptide retains the ability to bind the target antigen. Antibodies for therapeutic use are known in the art such as, for example, anti-CTLA4 antibodies (ipilimumab, tremelimumab, BN- 13, UC10-4F10-11, 9D9 or 9H10), anti-PD1 antibodies (pembrolizumab, nivolumab, pidilizumab, AMP-224, RMP1-4 or J43), and anti-PDL1 antibodies (tezolizumab, avelumab, BMS-936559 or durvalumab). [0062] In some embodiments, the therapeutic agent is an antibody mimetic, a functional equivalent of an antibody mimetic, or a functional fragment of an antibody mimetic. As used herein, the term “antibody mimetic” refers to compounds which, like antibodies, can specifically and/or selectively bind antigens or other targets, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins and are typically smaller than antibodies, with a molar mass of about 3-20 kDa (whereas antibodies are generally about 150 kDa). Non-limiting examples of antibody mimetics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, nanoCLAMPs, monobodies, affinity reagents and scaffold proteins. As used herein, the term “functional fragment”, with respect to an antibody mimetic, refers any portion or fragment of an antibody mimetic that maintains the ability to bind to its target molecule. The functional fragment of an antibody mimetic may be, for example, a portion of any of the antibody mimetics as described herein. As used herein, a “functional equivalent” in the context of an antibody mimetic refers to a polypeptide or other compound or molecule having similar binding characteristics to an antibody mimetic, but not necessarily being a recognizable “fragment” of an antibody mimetic. [0063] In some embodiments, the therapeutic agent is an immunomodulatory agent. As used herein, an “immunomodulatory agent” is a molecule or compound that modulates the activity and/or effectiveness of an immune response. “Modulate”, as used herein, means to enhance (upregulate), suppress (downregulate), direct, redirect or reprogram an immune response. The term “modulate” is not intended to mean activate or induce. By this, it is meant that the immunomodulatory agent modulates (enhances, reduces or directs) an immune response that is activated, initiated or induced by a particular antigen, but the immunomodulatory agent is not itself the antigen against which the immune response is directed, nor is the immunomodulatory agent derived from that antigen. An immunomodulatory agent that enhances the immune response may be selected from cytokines (e.g. certain interleukins and interferons), stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, colony stimulating factors, erythropoietins, thrombopoietins, and the like, and synthetic analogs of these molecules. An immunomodulatory agent may include: lymphotoxins, such as tumor necrosis factor (TNF); hematopoietic factors, such as interleukin (IL); colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF); interferon, such as interferons-alpha, -beta or –lamda; and stem cell growth factor, such as that designated "SI factor". Included among the cytokines are hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGFP; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); interleukins (ILs), such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin and tumor necrosis factor. An immunomodulatory agent may be an immune costimulatory molecule agonist. Immune costimulatory molecules are signaling proteins that play a role in regulating immune response. Some immune costimulatory molecules are receptors located on the surface of a cell that respond to extracellular signaling. When activated, immune costimulatory molecules produce a pro-inflammatory response that can include suppression of regulatory T cells and activation of cytotoxic or killer T cells. Exemplary immune costimulatory molecules include CD27, CD28, CD40, CD122, CD137, CD137/4-1BB, ICOS, IL-10, OX40 TGF-beta, TOR receptor, and glucocorticoid-induced TNFR-related protein GITR. An immune costimulatory molecule agonist may therefore be any compound, molecule or substance that is an agonist of a costimulatory immune molecule as described herein. An immunomodulatory agent may be an immunosuppressive agent. By “immunosuppressive agent”, it is meant a molecule or compound that reduces (downregulates) the activity and/or efficacy of the immune response, or directs, redirects or reprograms the immune response in a manner that alleviates an undesired result (e.g. an autoimmune response or allergy). There are many different types of immunosuppressive agent, including calcineurin inhibitors, interleukin inhibitors, selective immunosuppressants and THF-alpha inhibitors. An immunomodulatory agent may be an immunosuppressant selected from 5-fluorouracil, 6-thioguanine, adalimumab, anakinra, Atgam, abatacept, alefacept, azathioprine, basiliximab, belatacept, belimumab, benralizumab, brodalumab, canakinumab, certolizumab, chlorambucil, cyclosporine, daclizumab, dimethyl fumerate, dupilumab, eculizumab, efalizumab, ethanercept, everolimus, fingolimod, golimumab, guselkumab, imiquimod, infliximab, ixekizumab, leflunomide, lenlidomide, mechlorethamine, mepolizumab, methotrexate, muromonab-cd3, mycophenolate mofetil, mycophenolic acid, natallizumab, omalizumab, pomalidomide, pimecrolimus, reslizumab, rilonacept, sarilumab, secukinumab, siltuximab, sirolimus, tacrolimus, teriflunomide, thalidomide, Thymoglobulin, tocilizumab, ustekinumab, and vedolizumab. An immunomodulatory agent may be any molecule or compound that is an immunosuppressive cytotoxic drug. An immunosuppressive cytotoxic drug may include a glucocorticoid, a cytostatic (e.g. alkylating agents, antimetabolites), an antibody, a drug acting on immunophilins, an interferon, an opioid, or a TNF binding protein. Immunosuppressive cytotoxic drugs include nitrogen mustards (e.g. cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs (e.g. methotrexate), purine analogs (e.g. azathioprine and mercaptopurine), pyrimidine analogs (e.g. fluorouracil), protein synthesis inhibitors, cytotoxic antibiotics (e.g. dactinomycin, anthracyclines, mitomycin C, bleomycin and mithramycin), cyclosporine, tacrolimus, sirolimus/rapamycin, everolimus, prednisone, dexamethasone, hydrocortisone, mechlorethamine, clorambucil, mycopholic acid, fingolimod, myriocin, infliximab, etanercept, or adalimumab. An immunomodulatory agent may be an anti- inflammatory agent. An anti-inflammatory agent may be a non-steroidal anti-inflammatory agent (such as a Cox-1 and/or Cox-2 inhibitor), a non-steroidal anti-inflammatory agent, aspirin, salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, a corticosteroid, or celecoxib. An immunomodulatory agent may be an anti-rheumatic agent such as prednisone, dexamethasone, chloroquine, hydroxychloroquine, methotrexate, sulfasalazine, cyclosporine, azathioprine, cyclophosphamide, azathioprine, sulfasalazine, penicillamine, aurothioglucose, gold sodium thiomalate, auranofin, methotrexate, mechlorethamine, cyclophosphamide, chlorambucil, or azathioprine. [0064] In some embodiments, the therapeutic agent is a polynucleotide encoding a polypeptide, or an interfering polynucleotide (such as miRNA, siRNA shRNA, DsiRNA, or a polynucleotide encoding any thereof). A therapeutic agent may be a polynucleotide that contains sequences that correspond largely to the sense or antisense sequence of specific genes or their products, and hence have a direct effect on the expression of these genes and/or their products. For example, the use of polynucleotides that contain gene coding sequences affects the transcription and/or translation of the genes of interest in cells that uptake such polynucleotides. Similarly, the use of RNA interference polynucleotides (miRNA, siRNA, shRNA) affects the expression of specific genes of interest by directly affecting the levels of mRNA in cells that uptake such nucleotides. This differs significantly from other polynucleotide-based molecules such as CpG, and DNA- or RNA- based poly I:C adjuvants, which do not act through the presence of gene specific sequences. Thus, an active agent may be a polynucleotide that is not expressed as a protein in a cell, but rather encodes, for example, an antisense RNA, an interfering RNA, a catalytic RNA, or a ribozyme. RNA interference (RNAi) is a sequence specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA such as small (or short) interference RNA (siRNA), short hairpin RNA (shRNA), and single stranded intracellular RNA such as microRNA (miRNA), all of which can cause degradation of homologous mRNAs in a cell. Interfering RNA may be a naturally occurring or synthetic RNA chain of varying length. Interfering RNA can be duplexes, usually but not always limited to, 20 to 25-nt long that have 19 base pair central double stranded domain with terminal 2-base 3’ overhangs. Interfering RNA can be further modified chemically to enhance its in vivo efficacy, induce nuclease-resistance to prevent degradation and enhance stability. In this regard, the anti-sense strand may have either a free 5’-OH or 5’-phosphate terminus, the latter results in natural Dicer processing and represents the active form of the molecule. Interfering RNA may have phosphorothioate or boranohosphate modification of the internucleoside linkage to improve nuclease stability and prolong life of the duplex when exposed to serum or other nuclease sources. Interfering RNA may have modifications at 2’position, for example, 2’-O-methyl RNA residue incorporation to retain full potency compared with unmodified RNA, retaining stability in serum and significantly reducing the risk of potential IFN responses in the cell. Interfering RNA may also have 2’-fluoro modification, which is usually incorporated selectively at pyrimidine bases, to improve stability and potency. [0065] In some embodiments, the therapeutic agent is a T-helper peptide. As used herein, the term “T-helper peptide” refers to a peptide that is an epitope strongly recognized by T cells when displayed on MHC class I/II molecules. T-helper peptides are recognized by helper T cells (CD4+ T cells), which play an important role in establishing and maximizing the capabilities of the immune system, and are involved in activating and directing other immune cells, such as for example cytotoxic T cells and B cells. Accordingly, T-helper peptides are capable of enhancing or stimulating an immune response to an antigen. Immunodominant T-helper peptides or universal T-helper peptides are known peptides that are broadly reactive in animal and human populations with widely divergent MHC types. T-helper peptides may be comprised in an antigen, or provided with an antigen to boost the immune response to the antigen. In some embodiments, a T helper peptide may include an immunodominant or universal T helper peptide as known in the art such as, for example but not limited to the modified Tetanus toxin peptide A16L (830-844, AQYIKANSKFIGITEL, with an alanine residue added to its amino terminus to enhance stability); PADRE (pan-DR epitope) comprising the peptide sequence AKXVAAWTLKAAA, wherein X may be phenylalanine or cyclohexylalanine; or Tetanus toxin peptide F21E (947-967, FNNFTVSFWLRVPKVSASHLE). [0066] Hydrophobic carrier [0067] The pharmaceutical compositions of the present comprise a hydrophobic carrier. The hydrophobic carrier may be an essentially pure hydrophobic substance or a mixture of hydrophobic substances. Hydrophobic substances that are useful in the compositions described herein are those that are pharmaceutically acceptable. The hydrophobic carrier is typically a liquid but certain hydrophobic carriers that are not liquids at standard room temperature (about 18-25oC) may be liquefied, for example by warming, and may also be useful. [0068] In some embodiments, the hydrophobic carrier is an oil or a mixture of oils. In some embodiments, the hydrophobic carrier is a mineral oil (such as DrakeolTM 6VR or IFA), a vegetable oil (such as soybean oil), a nut oil (such as peanut oil), or a mixture of any thereof. In some embodiments, the hydrophobic carrier is Incomplete Freund’s Adjuvant (IFA), a mineral oil-based hydrophobic carrier. In some embodiments, the hydrophobic carrier is mannide monooleate in mineral oil, such as commercially available MontanideTM ISA 51 (SEPPIC, France). MontanideTM ISA 51 is a mixture of highly purified mineral oil (DrakeolTM 6VR) and mannide monooleate. In another embodiment, the hydrophobic carrier is mannide oleate in non- mineral oil, such as commercially available MontanideTM ISA 720 (SEPPIC, France). In another embodiment, the hydrophobic carrier is MS80 oil which is a mixture of mineral oil (Sigma Aldrich) and a sorbitan monooleate (such as SpanTM 80), the components of which can be purchased separately and mixed prior to use. [0069] In the compositions of the present invention, the amount of hydrophobic carrier used will depend on the desired volume of the final composition and/or the desired concentration of the components suspended and/or solubilized in the hydrophobic carrier. [0070] In some embodiments, the compositions of the present invention are water-free or substantially free of water, meaning that the compositions are not emulsions. By “water-free” it is meant that the compositions contain no water at all. In another embodiment, the compositions may be substantially free of water. The term “substantially free of water” is intended to encompass embodiments where the hydrophobic carrier may still contain small quantities of water, provided that the water is present in the non-continuous phase of the carrier. For example, individual components of the composition may have small quantities of 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. Generally, compositions as disclosed herein that are “substantially free of water” contain, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water on a weight/weight basis of the total weight of the carrier component of the composition. The compositions that still contain small quantities of water do not contain a sufficient amount of water such that an emulsion would be formed. As used herein, an “emulsion” refers to a mixture of two or more liquids that are normally immiscible wherein droplets of one liquid are dispersed in the other. As an example, a hydrophobic substance (e.g. oil) and an aqueous substance (e.g. water) are immiscible liquids that may form an emulsion when droplets of one are dispersed in the other. A dispersion of water droplets in oil is a water-in-oil (W/O) emulsion in which the water (aqueous phase) forms a discontinuous phase and the oil (hydrophobic phase) forms a continuous phase. “Water-in-oil emulsion” or “W/O”, as used herein, refers to an emulsion of a hydrophobic phase in an aqueous phase. A dispersion of oil droplets in water is an oil-in-water (O/W) emulsion in which the oil (hydrophobic phase) forms a discontinuous phase and the water (aqueous phase) forms a continuous phase. “Oil-in-water emulsion” or “O/W”, as used herein, refers to an emulsion of a hydrophobic phase in an aqueous phase. “Conventional emulsion”, as used herein, refers to emulsions are composed of numerous emulsifier-coated fluid droplets dispersed within another immiscible fluid medium, wherein the emulsifier forms an interfacial thin layer surrounding individual droplets that create interactions with hydrophilic and hydrophobic phases. [0071] Lipids [0072] The pharmaceutical compositions and dried preparations of the present invention comprise at least lipid. In some embodiments, the lipid is an amphipathic lipid or a mixture of amphipathic lipids. Although any amphipathic lipid may be used, particularly suitable lipids may include those with at least one fatty acid chain containing at least 4 carbons, and typically about 4 to 28 carbons in length. The fatty acid chain may contain any number of saturated and/or unsaturated bonds. There are numerous amphipathic lipids which may be used in the pharmaceutical compositions of the present invention, and the pharmaceutical compositions of the present invention may contain a single type of amphipathic lipid or a mixture of different types of amphipathic lipids. The amphipathic lipid may be a natural lipid or a synthetic lipid. Non-limiting examples of amphipathic lipids for use in the present invention include phospholipids, sphingolipids, sphingomyelin, cerobrocides, gangliosides, ether lipids, sterols, cholesterol, glycerophospholipid, cardiolipin, cationic lipids, anionic lipids, and lipids modified with poly (ethylene glycol) and other polymers. Synthetic lipids may include, without limitation, the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids. An “amphipathic lipid” is a lipid having both hydrophilic and hydrophobic parts or characteristics. The term “amphipathic” may be used interchangeably with “amphiphile” or “amphiphilic”. The hydrophobic portion of an amphipathic lipid may be a large hydrocarbon moiety, either linear or cyclic, such as a long chain of the form CH3(CH2)n, with n > 4. The hydrophilic portion of an amphipathic lipid may be either a charged group or a polar uncharged group. Charged groups include anionic and cationic groups. Examples of anionic charged groups include the following (wherein the hydrophobic part of the molecule is represented by "R"): carboxylates: RCO2 ̄ ; sulfates: RSO4 ̄ ; sulfonates: RSO3 ̄ ; and phosphates (the charged functionality in phospholipids). Cationic charged groups include, for example, amines: RNH3+ ("R" again representing the hydrophobic part of the molecule). Uncharged polar groups include, for example, alcohols with large R groups, such as diacyl glycerol (DAG). Amphipathic lipids may have several hydrophobic parts, several hydrophilic parts, or several of both. Cholesterol is also an amphiphilic lipid. [0073] In some embodiments, the lipid is a phospholipid or a mixture of phospholipids. Broadly defined, 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 include, for example and without limitation, those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine (for example DOPC; 1,2-Dioleoyl-sn-glycero-3-phosphocholine) and phosphoinositol. In some embodiments, the phospholipid may be phosphatidylcholine or a mixture of lipids comprising phosphatidylcholine. In some embodiments, the phospholipid may be DOPC or a mixture of phospholipids such as a lecithin (for example Lipoid S100 lecitihin). Lecithin is a mixture of phospholipids typically derived from biological sources such as eggs, soybean, and other vegetable sources. In some embodiments, the phospholipid is sphingomyelin. 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. [0074] In some embodiments, the at least one lipid is present in the pharmaceutical composition at a concentration of 60-160 mg/mL such as, for example 132 mg/mL. In some embodiments, the at least one lipid is present in the pharmaceutical composition at a concentration of at least 60 mg/mL, at least 70 mg/mL, at least 80 mg/mL, at least 90 mg/mL, at least 100 mg/mL, at least 110 mg/mL, at least 120 mg/mL, at least 132 mg/mL, at least 140 mg/mL, at least 150 mg/mL, or at least 160 mg/mL. [0075] In some embodiments, the at least one lipid is a mixture of a phospholipid and a sterol such as cholesterol. In some embodiments, the at least one lipid is a mixture of DOPC and cholesterol. In some embodiments, the at least one lipid is a mixture of lecithin and cholesterol. In some embodiments, the cholesterol or other sterol is used in an amount equivalent to about 2%, 5%, 10%, 12%, 15%, or 20% of the weight of phospholipid or mixture of phospholipids. In some embodiments, the amount of cholesterol or other sterol used is determined as the amount to sufficiently stabilize the formation of lipid-based structures formed by the phospholipid when suspended in the hydrophobic carrier. [0076] Lipid vesicle particles [0077] As used herein, the term “lipid vesicle particle” may be used interchangeably with “lipid vesicle”. A lipid vesicle particle refers to a complex or structure having an internal environment separated from the external environment by a continuous layer of enveloping lipids. In the context of the present disclosure, the expression “layer of enveloping lipids” can mean a single layer lipid membrane (e.g. as found on a micelle or reverse micelle), a bilayer lipid membrane (e.g. as found on a liposome) or any multilayer membrane formed from single and/or bilayer lipid membranes. The layer of enveloping lipids is typically a single layer, bilayer or multilayer throughout its circumference, but it is contemplated that other conformations may be possible such that the layer has different configurations over its circumference. The lipid vesicle particle may contain, within its internal environment, other vesicle structures (i.e. it may be multivesicular). The term “lipid vesicle particle” encompasses many different types of structures, including without limitation micelles, reverse micelles, unilamellar liposomes, multilamellar liposomes and multivesicular liposomes. [0078] The lipid vesicle particles may take on various different shapes, and the shape may change at any given time (e.g. upon drying, sizing, or mixing with therapeutic agents). Typically, lipid vesicle particles are spherical or substantially spherical structures. By “substantially spherical” it is meant that the lipid vesicles are close to spherical, but may not be a perfect sphere. Other shapes of the lipid vesicle particles include, without limitation, oval, oblong, square, rectangular, triangular, cuboid, crescent, diamond, cylinder or hemisphere shapes. Any regular or irregular shape may be formed. Further, a single lipid vesicle particle may comprise different shapes if it is multivesicular. For example, the outer vesicle shape may be oblong or rectangular while an inner vesicle may be spherical. Exemplary embodiments of lipid vesicle particles include, without limitation, single layer vesicular structures (e.g. micelles or reverse micelles) and bilayer vesicular structures (e.g. unilamellar or multilamellar vesicles), or various combinations thereof. [0079] By “single layer” it is meant that the lipids do not form a bilayer, but rather remain in a layer with the hydrophobic part oriented on one side and the hydrophilic part oriented on the opposite side. By “bilayer” it is meant that the lipids form a two-layered sheet, such as with the hydrophobic part of each layer internally oriented toward the center of the bilayer with the hydrophilic part externally oriented. Alternatively, the opposite configuration is also possible, i.e. with the hydrophilic part of each layer internally oriented toward the center of the bilayer with the hydrophobic part externally oriented. The term “multilayer” is meant to encompass any combination of single and bilayer structures. The form adopted may depend upon the specific lipid that is used. [0080] The lipid vesicle particles may be formed from single layer lipid membranes, bilayer lipid membranes and/or multilayer lipid membranes. The lipid membranes are predominantly comprised of and formed by lipids, but may also comprise additional components. For example, and without limitation, the lipid membrane may include stabilizing molecules to aid in maintaining the integrity of the structure. Any available stabilizing molecule may be used such as, for example, a sterol such as cholesterol. [0081] The lipid vesicle particles may be formed by lipids, such as amphipathic lipids, as disclosed herein. [0082] Lipid vesicle particles may be comprised of a single layer lipid assembly. There are various types of these lipid vesicle particles which may form, and the compositions disclosed herein may comprise a single type of lipid vesicle particle having a single layer lipid assembly or comprise a mixture of different such lipid vesicle particles. Lipid vesicle particles having a single layer lipid assembly may comprise aggregates of lipids with the hydrophobic part of the lipids oriented outwards toward the hydrophobic carrier and the hydrophilic part of the lipids aggregating as a core. These structures do not necessarily form a continuous lipid layer membrane. Lipid vesicle particles having a single layer lipid assembly comprise reverse micelles. A typical micelle in aqueous solution forms an aggregate with the hydrophilic parts in contact with the surrounding aqueous solution, sequestering the hydrophobic parts in the micelle center. In contrast, in a hydrophobic carrier, an inverse/reverse micelle forms with the hydrophobic parts in contact with the surrounding hydrophobic solution, sequestering the hydrophilic parts in the micelle center. The size of the lipid-based structures having a single layer lipid assembly is in the range of from 2 nm (20 A) to 20 nm (200 A) in diameter. [0083] Lipid vesicle particles may be bilayer vesicular structures, such as for example, a liposome. Liposomes are completely closed lipid bilayer membranes. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane), multilamellar vesicles (characterized by multimembrane bilayers whereby each bilayer may or may not be separated from the next by an aqueous layer) or multivesicular vesicles (possessing one or more vesicles within a vesicle). Liposomes form in aqueous or partially aqueous environments, and may form when the compositions herein are not water-free or may form in the aqueous solvents and solutions used in the preparation of pharmaceutical compositions. [0084] In some embodiments, the lipid vesicle particles are liposomes. In an embodiment, the liposomes are unilamellar, multilamellar, multivesicular or a mixture thereof. In an embodiment, the mean particle size of the liposomes is ≥80 nm. Thus, in an embodiment, the mean particle size of the liposomes used in the methods disclosed herein is in the range of 80 nm to 120 nm, with a PDI of ≤0.1. As used herein, “mean” refers to the arithmetic mean of the particle size of the lipid vesicle particles in a given population. It is a synonym for average. As such, “mean particle size” is intended to refer to the sum of the diameters of each lipid vesicle particle of a population, divided by the total number of lipid vesicle particles in the population (e.g. in a population with 4 lipid vesicle particles with particle sizes of 95 nm, 98 nm, 102 nm and 99 nm, the mean particle size is (95+98+102+99)/4 = 98.5 nm). However, as the skilled person will appreciate, lipid vesicle particles may not be perfectly spherical, and therefore the “particle size” of a given vesicle particle may not be an exact measure of its diameter. Rather, the particle size may be defined by other means known in the art, including for example: the diameter of the sphere of equal area or the largest perpendicular distance between parallel tangents touching opposite sides of the particle (Feret’s statistical diameter). [0085] In some embodiments, the sized lipid vesicle particles have a mean particle size of less than or equal to 120 nanometers (i.e. ≤120 nm) and a polydispersity index (PDI) of ≤0.1. In an embodiment, the sized lipid vesicle particles have a mean particle size of ≤115 nm, more particularly still ≤110 nm and more particularly still ≤100 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is between 50 nm and 120 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is between 80 nm and 120 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is between about 80 nm and about 115 nm, about 85 nm and about 115 nm, about 90 nm and about 115 nm, about 95 nm and about 115 nm, about 100 nm and about 115 nm or about 105 nm and about 115 nm. In an embodiment, the mean particle size of the sized lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm, about 117 nm, about 118 nm or about 119 nm. In an embodiment, the mean particle size is 120 nm. [0086] As used herein, polydispersity index (PDI) is a measure of the size distribution of the lipid vesicle particles. It is known in the art that the term “polydispersity” may be used interchangeably with “dispersity”. The PDI can be calculated by determining the mean particle size of the lipid vesicle particles and the standard deviation from that size. There are techniques and instruments available for measuring the PDI of lipid vesicle particles. For example, DLS is a well-established technique for measuring the particle size and size distribution of particles in the submicron size range, with available technology to measure particle sizes of less than 1 nm (LS Instruments, CH; Malvern Instruments, UK). [0087] Lipid vesicle particles having a mean particle size of ≤120 nm and a PDI of ≤0.1 may be prepared and provided by any suitable means. In an embodiment, the lipid vesicle particles are prepared in a manner in which their size is controlled in order to achieve the mean particle size of ≤120 nm and a PDI of ≤0.1. In an embodiment, lipid vesicle particles are subjected to one or more sizing steps or protocols to achieve the mean particle size of ≤120 nm and a PDI of ≤0.1. In an embodiment, the lipid vesicle particles may be prepared and provided by any combination of controlling their size during manufacture, performing sizing steps and/or any other means available in the art. In an embodiment, the lipid vesicle particles must be subjected to one or more active steps of sizing in order to achieve the mean particle size of ≤120 nm and a PDI of ≤0.1. In an embodiment, the sizing is performed by filter-extrusion whereby lipid vesicle particles are passed through a filter membrane or a series of filter membranes (e.g. polycarbonate membranes) of appropriate pore size. As such, as used herein, “sized lipid vesicle particles” refers to lipid vesicle particles that have been prepared by a means in which their size is controlled to attain a mean particle size of ≤120 nm and a PDI of ≤0.1 and/or they are sized to meet the criteria of having a mean particle size of ≤120 nm and a PDI of ≤0.1. The skilled person will be well aware of techniques available for providing lipid vesicle particles having a mean particle size of ≤120 nm and a PDI of ≤0.1. Reference herein to “non- sized lipid vesicle particles” or a “non-sized lipid vesicle particle preparation” means that the lipid vesicle particles have not be subject to procedures that limit their size to meet the defined size criteria, and/or they do not have a mean particle size of ≤120 nm and a PDI of ≤0.1. [0088] In an embodiment, the sized lipid vesicle particles may be prepared from a lipid precursor that naturally forms lipid vesicle particles of the required size. For example, and without limitation, the sized lipid vesicle particles may be prepared using Presome® (Nippon Fine Chemical, Japan). Presome® is a dry powder precursor made up of different lipid combinations. Presome® is supplied ready to be wetted in a suitable buffer to prepare liposomes. The liposomes formed from Presome® have an average particle size of about 93 nm, and sizing procedures (e.g. membrane extrusion, high pressure homogenization, etc.) can be used to achieve the required mean particle size of ≤120 nm and PDI of ≤0.1. In an embodiment, Presome® may for example be wetted in sodium acetate, pH 9.0 ± 0.5 to form liposomes. In an embodiment, the Presome® bulk dry powder may be made from DOPC/cholesterol (10:1 (w/w)) or DOPC alone. [0089] In another embodiment, standard procedures for preparing lipid vesicle particles of any size may be employed. For example, conventional liposome forming processes may be used, such as the hydration of solvent-solubilized lipids. Exemplary methods of preparing liposomes are discussed, for example, in Gregoriadis 1990; and Frezard 1999. After the lipid vesicle particles are prepared, the non-sized lipid vesicle particle preparation is subjected to a sizing procedure to obtain lipid vesicle particles having a mean particle size of ≤120 nm and a PDI of ≤0.1. There are various techniques available for sizing lipid vesicle particles (see e.g. Akbarzadeh 2013). For example, in an embodiment, the non-sized lipid vesicle particle preparation may be sized by high pressure homogenization (microfluidizers), sonication or membrane based extrusion. [0090] In an embodiment, the sizing of the non-sized lipid vesicle particle preparation is performed using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1, and the lipid vesicle particles obtained via high pressure homogenization can then be further sized down using membrane based extrusion. In an embodiment, membrane based extrusion is performed by passing the sized lipid vesicle particle preparation 5-20 times through a 0.1 um polycarbonate membrane or, alternatively, 5-20 times through a 0.08 um polycarbonate membrane, thereby attaining a mean particle size of ≤120 nm and PDI of ≤0.1. [0091] In an embodiment, the sized lipid vesicle particles may be prepared by adding the lipids to a suitable solvent (e.g. sodium phosphate, 50 mM, pH 7.0), shaking and/or stirring the lipid mixture (e.g. at 300 RPM for about 1 hour) and using membrane based extrusion to obtain the sized lipid vesicle particles. Exemplary, non-limiting embodiments of membrane based extrusion include: (i) passing a non-sized lipid vesicle particle preparation 20-40 times through a 0.2 μm polycarbonate membrane, and then 10-20 times through a 0.1 μm polycarbonate membrane; or (ii) passing a non-sized lipid vesicle particle preparation 20-40 times through a 0.2 μm polycarbonate membrane, then 10-20 times through a 0.1 μm polycarbonate membrane, and then 10-20 times through a 0.08 μm polycarbonate membrane. [0092] In a particular embodiment, the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 μm polycarbonate membrane, and then 10 times through a 0.1 μm polycarbonate membrane. In another particular embodiment, the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 μm polycarbonate membrane, then 10 times through a 0.1 μm polycarbonate membrane, and then 15 times through a 0.08 μm polycarbonate membrane. [0093] Dried preparation [0094] As used herein, the term “dried preparation” refers to a mixture of components as described herein that is dried by a technique known in the art in order to remove all or substantially all of a solvent. The term “dried preparation” does not necessarily mean that the preparation is completely dry. For example, depending on the solvent or solvents used in the methods disclosed herein, it is possible that a small component of volatile and/or non-volatile material will remain in the dried preparation. In an embodiment, the non-volatile material will remain. By “dried preparation”, it is meant that the preparation no longer contains substantial quantities of water and/or organic solvent. The process used to dry the preparation should be capable of removing substantially all water and/or organic solvent. Thus, in an embodiment, the dried preparation is completely free of water and/or organic solvent. In another embodiment, the dried preparation may contain a residual moisture content based on the limitations of the drying process (e.g. lyophilization). This residual moisture content will typically be less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.05% or less by weight of the dried preparation. This residual moisture content will not be more than 5% by weight of the dried preparation as this would result in a product that is not clear. [0095] Various methods may be used to dry compositions are known in the art. In an embodiment, the drying is performed by lyophilization, spray freeze-drying, or spray drying. The skilled person is well-aware of these drying techniques and how they may be performed. In an embodiment, the drying is performed by lyophilization. As used herein, “lyophilization”, “lyophilized” and “freeze-drying” are used interchangeably. As is well known in the art, lyophilization works by freezing the material and then reducing the surrounding pressure to allow the volatile solvent (e.g. water) in the material to sublime directly from the solid phase to the gas phase. Any conventional freeze-drying procedure may be used to carry out the drying step of the methods disclosed herein. In an embodiment, the lyophilization is performed by sequential steps of loading, freezing, evacuation and drying (e.g. primary drying and secondary drying). In an embodiment, lyophilization of the sized lipid vesicle particle/therapeutic agent mixture can be performed within a sealed bag in a benchtop freeze dryer. This may be particularly advantageous because it reduces the number of steps that must be performed in a sterile laboratory environment and allows for the rapid manufacture of smaller batch sizes. For example, after sterile filtration of the sized lipid vesicle particle/therapeutic agent mixture, aseptically filled vials containing the mixture can be loaded and sealed within a sterile bag under sterile conditions. These sterile, sealed units can then undergo lyophilization in an open laboratory (i.e. non-sterile environment) using a benchtop freeze dryer. By this method, it is also possible to perform the freeze-drying with multiple different sealed units in a single freeze dryer. This may reduce the cost and time of manufacture by avoiding expensive freeze-drying steps in sterile laboratory environments using large-scale freeze dryers. Also, multiple different small- scale batches of dried lipid/therapeutic agent preparation may be prepared simultaneously in separate sealed sterile bags. The lyophilization may be performed in any suitable freeze dryer. In an embodiment, the freeze dryer is a benchtop freeze dryer. In an embodiment, the freeze dryer is a Virtis benchtop lyophilizer. In an embodiment, the freeze dryer is in an open laboratory (i.e. non-sterile environment). [0096] The methods disclosed herein may further comprise a step of sterilization. Sterilization may be performed by any method known in the art. In an embodiment, the sterilization is performed by sterile filtration, steam heat sterilization, irradiation (e.g. gamma irradiation) or chemical sterilization. In a particular embodiment, the sterilization is performed by sterile filtration. In an embodiment, the sterile filtration is be performed before drying. Any conventional procedure for sterile filtration may be employed so long as it does not affect the solubility and stability of the therapeutic agents, lipid-based adjuvant, and/or polyI:C polynucleotide in the sized lipid vesicle particle/therapeutic agent mixture. In this regard, it may be desirable to perform the sterile filtration under low pressure conditions (e.g. between 30-50 psi). The serial filtration may be performed using commercially available sterile filtration membranes (e.g. MilliporeSigma). In an embodiment, the sterile filtration is performed using a 0.22 μm-rated membrane, a 0.2 μm-rated membrane and/or a 0.1 μm-rated membrane. In an embodiment, the sterile filtration is performed by a single passage of the mixture through a single filtration membrane. In another embodiment, the sterile filtration is performed by serially passing the mixture sequentially through a combination of the same or different sized filtration membranes. [0097] The methods disclosed herein may further comprise a step of confirming that the sized lipid vesicle particles have retained a mean particle size of ≤120 nm and PDI of ≤0.1. As described elsewhere herein, there are several techniques, instruments and services that are available to measure the mean particle size and PDI of lipid vesicle particles, such as for example and without limitation TEM, SEM, AFM, FTIR, XPS, XRD, MALDI-TOF-MS, NMR and DLS. In an embodiment, the step of confirming the size and PDI of the lipid vesicle particles is performed using a DLS ZETASIZER NANO-S particle size analyzer. [0098] The methods disclosed herein may further comprise a step of evaluating the stability of the lipids, therapeutic agent(s), lipid-based adjuvant, and/or polyI:C polynucleotide before and/or after the drying step. The stability of the components may be measured by any known means or method. For example and without limitation, stability of the dried preparation may be determined by the appearance of the dried preparation (lyophilisate) or measurement of the content of the components over time (e.g. by HPLC, RP-HPLC, IEX-HPLC, etc.). HPLC is a technique which can be used to separate, identify and quantify each component in a mixture. Thus, by using HPLC, RP-HPLC or IEX-HPLC it is possible to determine the approximate quantity of the lipids, therapeutic agents and other components, as well as characterize the components qualitatively (e.g. observe impurities, degradation products, etc.). In other embodiments, stability may evaluated upon solubilization in a hydrophobic carrier by various methods, such as for example: appearance of the solubilized product; identification and quantification of lipids, therapeutic agent(s), lipid-based adjuvant, and/or polyI:C polynucleotide, impurities or degradants (e.g. by RP-HPLC, IEX-HPLC, etc.); particle size of the lipid vesicle particles (e.g. by SAXS); optical density; viscosity (e.g. as per Ph.Eur.2.2.9); pH; extractable volume, such as from a syringe (e.g. as per Ph.Eur.2.9.17), and immunogenicity assays (e.g. ELISpot). [0099] In embodiments of the methods disclosed herein, lipids may first be dissolved and mixed in an organic solvent. In embodiments where different types of lipid are used, this step will allow a homogenous mixture of the lipids to be formed. In an embodiment, these steps may be carried out in chloroform, chloroform:methanol mixtures, tertiary butanol or cyclohexane. In an embodiment, the lipids are prepared at 10-20mg lipid/mL organic solvent; however, higher or lower concentrations may also be used. In some embodiments, after mixing, the organic solvent is removed (e.g. by evaporation) to yield a lipid film. The lipid film may then be frozen and lyophilized to yield a dry lipid film. The dry lipid film may then be hydrated with an aqueous solution containing therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant to provide a non-sized lipid vesicle particle preparation. The step of hydration may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour). In yet another embodiment of the methods disclosed herein, an aqueous solution of lipids may be combined with at least one solution containing therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant. In another embodiment, one or more dry therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be added to, and solubilized in, the aqueous solution of lipids or sized lipid vesicle preparation. These embodiments may be performed with shaking and/or mixing (e.g. at 300 RPM for about 1 hour). The above procedures are exemplary methods for providing a non-sized lipid vesicle particle preparation comprising non-sized lipid vesicles and one or more first therapeutic agents and/or polyI:C polylnucleotide. [0100] In some embodiments, therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant are either solubilized in a solvent (e.g. aqueous or organic) prior to mixing with lipid vesicle particles or therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant are solubilized upon being mixed with the lipid vesicle particles. In this latter embodiment, therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be added as a dry powder to a solution containing lipid vesicle particles or both the lipid vesicle particles and therapeutic agents/polyI:C polynucleotide/lipid-based adjuvant may be mixed together in a fresh solvent. [0101] Methods and uses [0102] The pharmaceutical compositions disclosed herein may find application in any instance in which it is desired to administer therapeutic agents to a subject. The subject may be a vertebrate, such as a fish, bird or mammal. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human. In an embodiment, the pharmaceutical compositions may be used in methods for treating, preventing or diagnosing a disease, disorder or condition to which the therapeutic agent is targeted. In an embodiment, the pharmaceutical compositions may be used in methods for delivering a therapeutic agent to a subject. In an embodiment, the methods comprise administering to a subject the pharmaceutical composition as described herein. In an embodiment, the pharmaceutical compositions may be used in methods for modulating an immune response in a subject. As used herein, the term “modulating” is intended to refer to both immunostimulation (e.g. inducing or enhancing an immune response) and immunosuppression (e.g. preventing or decreasing an immune response). Typically, the method would involve one or the other of immunostimulation or immunosuppression, but it is possible that the method could be directed to both. As referred to herein, the “immune response” may either be a cell-mediated (CTL) immune response or an antibody (humoral) immune response. [0103] In some embodiments, the pharmaceutical compositions disclosed herein may be used for inducing a cell-mediated immune response to the therapeutic agents (e.g. antigens). As used herein, to “induce” an immune response is to elicit and/or potentiate an immune response. Inducing an immune response encompasses instances where the immune response is initiated, enhanced, elevated, improved or strengthened to the benefit of the host relative to the prior immune response status, for example, before the administration of a composition disclosed herein. [0104] In an embodiment, the pharmaceutical compositions disclosed herein may be used for inducing an antibody immune response to the therapeutic agents (e.g. antigens). An “antibody immune response” or “humoral immune response” (used interchangeably herein), as opposed to cell-mediated immunity, is mediated by secreted antibodies which are produced in the cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as for example those on the surfaces of foreign substances, pathogens (e.g. viruses, bacteria, etc.) and/or cancer cells, and flag them for destruction. [0105] The pharmaceutical compositions disclosed herein may be useful for treating or preventing diseases and/or disorders ameliorated by a cell-mediated immune response or a humoral immune response. The pharmaceutical compositions disclosed herein may find application in any instance in which it is desired to administer therapeutic agents (e.g. antigens) to a subject to induce a cell-mediated immune response or a humoral immune response. In an embodiment, the pharmaceutical compositions is for inducing an antibody immune response and/or cell-mediated immune response to the therapeutic agents (e.g. antigens) in said subject. In an embodiment, pharmaceutical compositions is for the treatment and/or prevention of an infectious disease or cancer. [0106] “Treating” or “treatment of”, or “preventing” or “prevention of”, as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired 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 (e.g. suppression), delay or slowing of disease onset, conferring protective immunity against a disease-causing agent and amelioration or palliation of the disease state. “Treating” or “preventing” 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 or preventing the occurrence of disease, such as by preventing infection in a subject. “Treating” or “preventing” may also refer to a reduction in the size of a tumor mass, reduction in tumor aggressiveness, etc. Treating” may be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, or is known to have already been exposed to an infectious agent, whereas “preventing” typically occurs in a subject who does not have a disease or disorder, or is not known to have been exposed to an infectious agent. As will be appreciated, there may be overlap in treatment and prevention. For example, it is possible to be “treating” a disease in a subject, while at same time “preventing” symptoms or progression of the disease. Moreover, at least in the context of vaccination, “treating” and “preventing” may overlap in that the treatment of a subject is to induce an immune response that may have the subsequent effect of preventing infection by a pathogen or preventing the underlying disease or symptoms caused by infection with the pathogen. These preventive aspects are encompassed herein by expressions such as “treatment of an infectious disease” or “treatment of cancer”. [0107] In an embodiment, the pharmaceutical compositions disclosed herein may be used for treating and/or preventing an infectious disease, such as caused by a viral infection, in a subject in need thereof. The subject may be infected with a virus or may be at risk of developing a viral infection. Viral infections that may be treated and/or prevented by the use or administration of a pharmaceutical composition as disclosed herein, without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus 1 , Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human papillomavirus (HPV), Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Orthoreovirus, Rotavirus, Ebola virus, parainfluenza virus, influenza A virus, influenza B virus, influenza C virus, Measles virus, Mumps virus, Rubella virus, Pneumovirus, respiratory syncytial virus (RSV), Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Coronavirus (e.g. Sars-Cov-2), Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella. [0108] In an embodiment, the compositions disclosed herein may be used for treating and/or preventing an infectious disease, such as caused by a non-viral pathogen (such as a bacterium or protozoan) in a subject in need thereof. The subject may be infected with the pathogen or may be at risk of developing an infection by the pathogen. Without limitation, exemplary bacterial pathogens may include Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli O157: 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. In a particular embodiment, the bacterial infection is Anthrax. Without limitation, exemplary protozoan pathogens may include those of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi), which cause malaria. [0109] In an embodiment, the pharmaceutical compositions disclosed herein may be for use in treating and/or preventing cancer in a subject in need thereof. The subject may have cancer or may be at risk of developing cancer. As used herein, the terms “cancer”, “cancer cells”, “tumor” and “tumor cells”, (used interchangeably) refer to cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized. The term “cancer” or “tumor” includes metastatic as well as non-metastatic cancer or tumors. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor. [0110] Without limitation, cancers that may be capable of being treated and/or prevented by the use or administration of a pharmaceutical composition as disclosed herein include carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors. Without limitation, particularly suitable embodiments may include glioblastoma, multiple myeloma, ovarian cancer, breast cancer, fallopian tube cancer, prostate cancer or peritoneal cancer. In one embodiment, the cancer may be caused by a pathogen, such as a virus. Viruses linked to the development of cancer are known to the skilled person and include, but are not limited to, human papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukaemia virus-1. In an embodiment, the cancer is one that expresses one or more tumor- specific neoantigens. In a particular embodiment, the cancer is breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, peritoneal cancer, glioblastoma or diffuse large B cell lymphoma. [0111] The pharmaceutical compositions disclosed herein may be useful for either the treatment or prophylaxis of cancer; for example, a reduction of the severity of cancer (e.g. size of the tumor, aggressiveness and/or invasiveness, malignancy, etc.) or the prevention of cancer recurrences. [0112] The pharmaceutical composition as disclosed herein may be administered by any suitable route. In an embodiment, the route of administration is injection, such as subcutaneous injection. [0113] Kits [0114] The pharmaceutical compositions disclosed herein are optionally provided to a user as a kit comprising the individual components which may be assembled to produce the pharmaceutical composition. In an embodiment, the kit is for preparing a composition for the treatment, prevention and/or diagnosis of a disease, disorder or condition or for the delivery of a therapeutic agent. [0115] The kits can further comprise one or more additional reagents, packaging materials, and an instruction set or user manual detailing preferred methods of using the kit components. In an embodiment, the containers are vials. [0116] The invention is further illustrated by the following non-limiting examples. EXAMPLES [0117] Example 1 [0118] First, a vial containing DPX-FP (IMV-Inc, Dartmouth, Canada) was reconstituted in 0.45 mL of Montanide ISA 51 oil diluent (SEPPIC, France) by soaking for 5 minutes and shaking for 2 minuntes, then vortexing for one minute. Next, 0.45 mL of this oil reconstituted DPX-FP product was then added to a vial containing DPX-PAM3CSK4 (IMV-Inc, Dartmouth, Canada) and reconstitution was completed by soaking for 5 minutes, shaking for 2 minutes, and vortexing for one minute to obtain the final concentrations of: FP peptide 9 ug/50 uL dose; DNA based polyI:C polynucleotide (dIdC) 18 ug/50 uL dose; PAM3CSK42 ug/50 uL dose; and Lipid 12.5 mg/50 uL dose. [0119] The DPX-FP product vial was previously prepared by adding FP (NeoMPS) and DNA based polyI:C polynucleotide (dIdC) stock (Biospring) to a lipid-mixture solution, mixing well and freeze-drying. A lipid-mixture (132 mg/mL) containing DOPC and cholesterol in a 10:1 ratio (w:w) (Lipoid GmBH, Germany) was dissolved in 40% tertiary-butanol by shaking well at 300 RPM at room temperature for 1 hour or until dissolved. Next, FP stock (10 mg/mL) was prepared in DMSO and DNA based polyI:C polynucleotide adjuvant stock (10 mg/mL) was prepared in sterile water. To a 0.5 mL aliquot of lipid-mixture solution, 10 µL of FP stock was added to obtain 0.1 mg/mL final fill concentration, shaken well at 300 RPM for 5 minutes. To the formed FP-lipid-mixture solution, 20 µL of DNA based polyI:C polynucleotide (dIdC) stock was added to obtain 0.2 mg/mL final fill concentration, shaken well at 300 RPM for 5 minutes. The volume was adjusted to 1.0 mL with 40% tertiary-butanol, freeze-dried, and stored at -20 C. The dried preparation is reconstituted in 0.45 mL oil diluent when needed. [0120] The DPX-PAM3CSK4 vial was prepared as follows: Briefly, a homogenous lipid- mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture and was added to sodium phosphate, 100 mM, pH 6.0 with shaking at 300 RPM for about 1 hour. The mixture was then sized by passing the material 25 times through a 0.2 µm polycarbonate membrane, then 10 times through a 0.1 µm polycarbonate membrane, and then 15 times through a 0.08 µm polycarbonate membrane to attain a particle size of ≤100 nm with a polydispersity index (PDI) of ≤0.1. The lipopeptide adjuvant PAM3CSK4 (Polypeptide group, San Diego, USA) was then added at 0.02 mg/mL to the sized lipid vesicles with shaking at 300 RPM for about 15 minutes. The mixture was then sterile filtered, aseptically filled in 1.0 mL aliquots into sterile glass vials, freeze-dried, and stored at -20°C. The dried preparation is reconstituted in 0.45 mL oil diluent when needed. [0121] Example 2 [0122] First, a vial containing DPX-MVP-S (IMV-Inc, Dartmouth, Canada) was reconstituted in 0.7 mL of Montanide ISA 51 oil (SEPPIC, France) by soaking for 5 minutes, shaking for 2 mins, and vortexing for one minute. Next, 0.45 mL of this oil reconstituted DPX- MVP-S product was then added to a vial containing DPX-PAM3CSK4 (IMV-Inc, Dartmouth, Canada), and reconstitution was completed by soaking for 5 minutes, shaking for 2 minutes, and vortexing for one minute to obtain the final concentrations of: MVP-S peptide each at 45 ug/50 uL dose; T-helper peptide A16L at 22.5 ug/50 uL dose; DNA based polyI:C polynucleotide (dIdC) 18 ug/50 uL dose; PAM3CSK42 ug/50 uL dose; and Lipid 12.5 mg/50 uL dose. [0123] The DPX-MVP-S vial was prepared as follows: Briefly, DNA based polyI:C polynucleotide (dIdC) (BioSpring GmbH (Frankfurt, Germany) and peptide antigens (PolyPeptide Laboratories (San Diego, CA, USA) stock solutions were prepared as listed below: Stock# Component Solvent 5)
Figure imgf000041_0001
in the following order: (4), (2), (3), (5) and then (1). The pH was adjusted to 10.0 ± 0.5. A 10:1 (w:w), homogenous lipid-mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture and added to the peptide/polynucleotide solution to form an intermediate bulk (non-sized) and mixed using a Silverson high speed mixer. The pH was adjusted to 10.0 ± 0.5, if required. The intermediate bulk was then sized using an Emulsiflex C5 or C55 extruder by passing the material 35 times through a 0.2 µm polycarbonate membrane and then 10 times through a 0.1 µm polycarbonate membrane to attain a particle size of ≤120 nm with a PDI of ≤0.1. The peptide stock solutions (6) and (7) were then added to the sized lipid vesicle particle bulk immediately after preparation. The final pH of the solution was adjusted to 7.0 ± 0.5. The final preparation was then sterile filtered, aseptically filled (1.6 mL aliquots) into sterile glass vials, freeze-dried, and stored at -20°C. The dried preparation is reconstituted in 0.7 mL oil diluent when needed. [0125] The DPX-PAM3CSK4 vial was prepared as follows: Briefly, homogenous lipid- mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was weighed to obtain 132 mg/mL of the lipid-mixture was added to sodium phosphate, 100 mM, pH 6.0 with shaking at 300 RPM for about 1 hour. The mixture was then sized by passing the material 25 times through a 0.2 µm polycarbonate membrane, then 10 times through a 0.1 µm polycarbonate membrane, and then 15 times through a 0.08 µm polycarbonate membrane to attain a particle size of ≤100 nm with a PDI of ≤0.1. The lipopeptide adjuvant PAM3CSK4 (Polypeptide group, San Diego, USA) was then added at 0.02 mg/mL to the sized lipid vesicles with shaking at 300 RPM for about 15 minutes. The mixture was then sterile filtered, aseptically filled to 1.0 mL aliquots into sterile glass vials, freeze-dried, and stored at -20°C. The dried preparation is reconstituted in 0.45 mL oil diluent when needed.

Claims

CLAIMS: 1. A method for preparing a pharmaceutical composition, comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the first dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the second dried preparation to produce a pharmaceutical composition.
2. A method for preparing a pharmaceutical composition, comprising: (a) providing: (i) a first dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide, and (ii) a second dried preparation comprising a lipid-based adjuvant and a lipid; (b) combining the second dried preparation with a hydrophobic carrier to produce a reconstituted preparation; and (c) combining the reconstituted preparation with the first dried preparation to produce a pharmaceutical composition.
3. The method of claim 1 or 2, wherein providing the first dried preparation comprises: (i) providing lipid vesicle particles having a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1; (ii) mixing the lipid vesicle particles with at least one solubilized therapeutic agent and a polyI:C polynucleotide to form a mixture; and (iii) drying the mixture formed in step (ii) to form a first dried preparation comprising a therapeutic agent, a lipid, and a polyI:C polynucleotide.
4. The method of claim 3, wherein step (i) comprises sizing lipid vesicle particles to provide the lipid vesicle particles having a mean particle size of ≤120 nm and a PDI of ≤0.1.
5. The method of claim 4, wherein the sizing is by filter-extrusion.
6. The method of claim 4 or 5, wherein the sizing is by extrusion through one or more polycarbonate membranes, such as a 0.2 µm polycarbonate membrane, a 0.1 µm polycarbonate membrane and/or a 0.08 µm polycarbonate membrane.
7. The method of any one of claims 4 to 6, wherein the sizing is by extrusion (i) 20-40 times through a 0.2 μm polycarbonate membrane, and then 10-20 times through a 0.1 μm polycarbonate membrane; or (ii) 20-40 times through a 0.2 μm polycarbonate membrane, then 10-20 times through a 0.1 μm polycarbonate membrane, and then 10-20 times through a 0.08 μm polycarbonate membrane.
8. The method of any one of claims 4 to 7, wherein the sizing is by extrusion (i) 25 times through a 0.2 μm polycarbonate membrane, and then 10 times through a 0.1 μm polycarbonate membrane or (ii) 25 times through a 0.2 μm polycarbonate membrane, then 10 times through a 0.1 μm polycarbonate membrane, and then 15 times through a 0.08 μm polycarbonate membrane.
9. The method of claim 4, wherein the sizing is performed by first using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1 and then sizing by extrusion (i) 5-20 times through a 0.1 μm polycarbonate membrane or (ii) 5-20 times through a 0.08 μm polycarbonate membrane.
10. The method of any one of claims 3 to 9, wherein the mean particle size of the lipid vesicle particles is between about 80 nm and about 120 nm.
11. The method of any one of claims 3 to 10, wherein the mean particle size of the lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm or about 115 nm.
12. The method of any one of claims 3 to 11, wherein the mean particle size of the lipid vesicle particles is ≤100 nm.
13. The method of any one of claims 3 to 12, wherein the lipid vesicle particles comprise a synthetic lipid, a phospholipid, or a mixture of phospholipids.
14. The method of claim 13, wherein the lipid vesicles particles comprise dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
15. The method of claim 13 or 14, wherein the lipid vesicle particles further comprise cholesterol.
16. The method of claim 15, wherein the lipid vesicle particles comprise DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
17. The method of any one of claims 3 to 16, wherein the lipid vesicle particles of step (i) are prepared from a liposome precursor.
18. The method of claim 17, wherein the liposome precursor is Presome®.
19. The method of any one of claims 3 to 18, wherein the lipid vesicle particles are liposomes.
20. The method of claim 19, wherein the liposomes are unilamellar, multilamellar, multivesicular, or a mixture thereof.
21. The method of any one of claims 3 to 20, wherein the at least one therapeutic agent is solubilized in one or more of sodium acetate, sodium phosphate or sodium hydroxide.
22. The method of any one of claims 3 to 21, wherein the at least one therapeutic agent is solubilized in one or more of 0.1 M sodium hydroxide, 100 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0 or 100 mM sodium phosphate having a pH of 6.0 ± 1.0.
23. The method of any one of claims 3 to 22, wherein the mixing of step (ii) is performed in a sodium acetate or sodium phosphate solution.
24. The method of claim 23, wherein the mixing of step (ii) is performed in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
25. The method of claim 23, wherein the mixing of step (ii) is performed in 50 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0 or 100 mM sodium phosphate having a pH of 6.0 ± 1.0.
26. The method of claim 23, wherein the mixing of step (ii) is performed in 50 mM sodium phosphate having a pH of 7.0, 100 mM sodium phosphate having a pH of 6.0, 50 mM sodium acetate having a pH of 6.0, or 100 mM sodium acetate having a pH of 9.5.
27. The method of any one of claims 3 to 26, wherein the at least one therapeutic agent comprises an antigen, a small molecule drug, an antibody or a functional fragment thereof, an antibody mimetic or a functional fragment thereof, an immunomodulatory agent, a polynucleotide encoding a polypeptide, or an interfering polynucleotide.
28. The method of any one of claims 3 to 2265, wherein the at least one therapeutic agent comprises one or more peptide antigens.
29. The method of claim 28, wherein the one or more peptide antigens are 20-30 amino acids in length.
30. The method of claim 28 or 29, wherein the one or more peptide antigens comprise neoantigens.
31. The method of claim 28, wherein the one or more peptide antigens are derived from human papillomavirus (HPV), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), bacillus anthracis, Plasmodium, or a survivin polypeptide.
32. The method of claim 31, wherein the one or more peptide antigens are FTELTLGEF, LMLGEFLKL, RISTFKNWPK, STFKNWPFL or LPPAWQPFL; or any combination thereof.
33. The method of claim 31, wherein the one or more peptide antigens are NKLCEYNVFHNKTFELPRARVNT and/or NKLSEHKTFCNKTLEQGQMYQINT.
34. The method of any one of claims 28 to 31, wherein step (ii) comprises mixing five or more different solubilized peptide antigens with the lipid vesicle particles.
35. The method of claim 34, wherein step (ii) comprises mixing up to 30 different solubilized peptide antigens with the lipid vesicle particles.
36. The method of claim 34, wherein step (ii) comprises mixing 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different solubilized peptide antigens with the lipid vesicle particles.
37. The method of any one of claims 34 to 36, wherein, after step (ii), each of the different solubilized peptide antigens is at a concentration of at least about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml or 1.0 mg/ml.
38. The method of any one of claims 34 to 37, wherein, after step (ii), each of the different solubilized peptide antigens is at a concentration of about 0.5 mg/ml.
39. The method of any one of claims 34 to 38, wherein the different solubilized peptide antigens are not pre-selected based on any characteristic relating to isoelectric point, solubility, stability and/or immunogenicity.
40. The method of any one of claims 34 to 39, wherein the different solubilized peptide antigens have one or more different characteristics relating to isoelectric point, solubility, stability and/or immunogenicity.
41. The method of any one of claims 34 to 40, wherein the different solubilized peptide antigens have a different length, sequence, molecular weight, charge, polarity, hydrophobicity and/or hydrophilicity.
42. The method of any one of claims 28 to 41, wherein step (ii) further comprises mixing a solubilized T-helper epitope with the lipid vesicle particles, the one or more peptide antigens, and the polyI:C polynucleotide.
43. The method of any one of claims 28 to 41, wherein step (ii) comprises mixing 10-15 neoantigens and a polyI:C polynucleotide with one solubilized T-helper epitope
44. The method of claim 42 or 43, wherein the T-helper epitope comprises the amino acid sequence AQYIKANSKFIGITEL.
45. The method of claim any one of claims 42 to 44, wherein step (ii) comprises: (ii-1) providing an antigen stock comprising the one or more peptide antigens, the polyI:C polynucleotide, and the solubilized T-helper epitope; and (ii-2) mixing the antigen stock with the lipid vesicle particles to form the mixture.
46. The method of claim 45, wherein, in step (ii-1), the antigen stock is prepared in 100 mM sodium hydroxide with each solubilized peptide antigen having a concentration of about 2.0 mg/ml.
47. The method of claim 46, wherein the antigen stock is diluted 1:1 with 50 mM sodium acetate having a pH of 6.0 ± 0.5 to provide each solubilized peptide antigen at a concentration of about 1.0 mg/ml.
48. The method of any one of claims 45 to 47, wherein after the mixing in step (ii-2) and prior to drying, the pH of the mixture is adjusted to 10 ± 1.0.
49. The method of any one of claims 3 to 48 further comprising a step of sterile filtration of the mixture formed in step (ii) prior to drying.
50. The method of any one of claims 3 to 49 further comprising, between steps (ii) and (iii), a step of confirming that the lipid vesicle particles still have a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1.
51. The method of any one of claims 3 to 50, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
52. The method of claim 51, wherein the drying is performed by lyophilization.
53. The method of claim 52, wherein the lyophilization is performed by loading one or more containers comprising the mixture of step (ii) into a bag, sealing the bag to form a sealed unit, and lyophilizing the sealed unit in a freeze-dryer.
54. The method of claim 53, wherein the bag is a sterile, autoclaved bag.
55. The method of claim 53 or 54, wherein the freeze-dryer is a benchtop freeze dryer.
56. The method of any one of claims 53 to 55, wherein the freeze-dryer contains more than one sealed unit during the lyophilization.
57. The method of claim 56, wherein each sealed unit contains a different mixture prepared by steps (i) and (ii).
58. The method of any one of claims 3 to 57, further comprising a step of evaluating the stability of the at least one solubilized therapeutic agent before and/or after the drying of step (iii).
59. The method of claim 58, wherein the stability of the therapeutic agents is evaluated by HPLC analysis.
60. The method of claim 58 or 59, wherein the therapeutic agents are peptide antigens and at least 80% of the original peptide concentration of each peptide antigen is retained in undegraded form when evaluated before drying.
61. The method of claim 60, wherein at least 75% of the original peptide concentration of each peptide antigen is retained in undegraded form when evaluated immediately after drying.
62. The method of claim 60 or 61, wherein at least 70% of the original peptide concentration of each peptide antigen is retained in undegraded form when evaluated three months after drying.
63. The method of any one of claims 60 to 62, wherein one or more of the peptide antigens shows no degradation for up to 3 months after drying.
64. The method of claim 1 or 2, wherein providing the first dried preparation comprises: (i) providing a lipid vesicle particle preparation comprising lipid vesicle particles and at least one solubilized first therapeutic agent; (ii) sizing the lipid vesicle particle preparation to form a sized lipid vesicle particle preparation comprising sized lipid vesicle particles and said at least one solubilized first therapeutic agent, said sized lipid vesicle particles having a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1; (iii) mixing the sized lipid vesicle particle preparation with at least one second therapeutic agent to form a mixture, wherein said at least one second therapeutic agent is solubilized in the mixture and is different from said at least one solubilized first therapeutic agent; and (iv) drying the mixture formed in step (iii) to form a first dried preparation comprising the therapeutic agents, a lipid, and a polyI:C polynucleotide, wherein the polyI:C polynucleotide is comprised in the lipid vesicle particle preparation of step (i) or wherein the polyI:C polynucleotide is mixed with the sized lipid vesicle particle preparation of step (iii) or wherein the polyI:C polynucleotide is mixed with the mixture of step (iii).
65. The method of claim 64, wherein prior to step (ii) the lipid vesicle particles are not sized.
66. The method of claim 64 or 65, wherein, in step (i), the lipid vesicle particle preparation is in sodium acetate solution or in sodium phosphate solution.
67. The method of any one of claims 64 to 66, wherein, in step (i), the lipid vesicle particle preparation is in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
68. The method of any one of claims 64 to 67, wherein, in step (i), the lipid vesicle particle preparation is in 50 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0, or 100 mM sodium phosphate having a pH of 6.0 ± 1.0.
69. The method of any one of claims 64 to 68, wherein, in step (i), the lipid vesicle particle preparation is in 100 mM sodium acetate having a pH of 9.5 ± 0.5.
70. The method of any one of claims 64 to 69, wherein step (i) comprises: (i-1) providing a therapeutic agent stock comprising the at least one solubilized first therapeutic agent and the polyI:C polynucleotide; and (i-2) mixing the therapeutic agent stock with a lipid mixture to form the lipid vesicle preparation.
71. The method of claim 70, wherein the polyI:C polynucleotide is encapsulated in the lipid vesicle particles.
72. The method of any one of claims 64 to 71, wherein, in step (i), the at least one solubilized first therapeutic agent is encapsulated in the lipid vesicle particles.
73. The method of any one of claims 64 to 72, wherein each of the at least one first and at least one second therapeutic agents is independently selected from the group consisting of an antigen, a small molecule drug, an antibody or a functional fragment thereof, an antibody mimetic or a functional fragment thereof, an immunomodulatory agent, a polynucleotide encoding a polypeptide, or an interfering polynucleotide.
74. The method of any one of claims 64 to 73, wherein each of the at least one first and at least one second therapeutic agents comprises a peptide antigen.
75. The method of any one of claims 64 to 74, wherein, in step (i), one, two, three, four or five different solubilized first therapeutic agents are in the lipid vesicle particle preparation.
76. The method of any one of claims 64 to 75, wherein, in step (i), four different solubilized first therapeutic agents are in the lipid vesicle particle preparation.
77. The method of claim 76, wherein the four different solubilized first therapeutic agents are peptide antigens, wherein the first peptide antigen comprises the amino acid sequence FTELTLGEF; the second peptide antigen comprises the amino acid sequence LMLGEFLKL; the third peptide antigen comprises the amino acid sequence STFKNWPFL; and the fourth peptide antigen comprises the amino acid sequence LPPAWQPFL.
78. The method of any one of claims 64 to 77, wherein, in step (iii), the sized lipid vesicle particle preparation is mixed with one, two, three, four or five different second therapeutic agents.
79. The method of any one of claims 73 to 78, wherein the at least one second therapeutic agent comprises a peptide antigen comprising the amino acid sequence RISTFKNWPK.
80. The method of any one of claims 74 to 79, wherein step (iii) further comprises mixing at least one T-helper epitope with the sized lipid vesicle particle preparation, wherein the at least one T-helper epitope is solubilized in the mixture.
81. The method of claim 80, wherein the T-helper epitope comprises the amino acid sequence AQYIKANSKFIGITEL.
82. The method of claim 80 or 81, wherein step (iii) comprises: (iii-1) providing one or more peptide antigen stocks comprising a solubilized peptide antigen, and a stock comprising the T-helper epitope; and (iii-2) mixing the stocks with the sized lipid vesicle particles to form the mixture.
83. The method of claim 82, wherein the one or more therapeutic agent stocks are prepared in mild acetic acid.
84. The method of any one of claims 64 to 83, wherein the at least one second therapeutic agent is solubilized in mild acetic acid prior to mixing with the sized lipid vesicle particle preparation in step (iii).
85. The method of any one of claims 64 to 84, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by high pressure homogenization, sonication and/or membrane extrusion.
86. The method of claim 85, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by extrusion through a 0.2 µm polycarbonate membrane followed by extrusion through a 0.1 µm polycarbonate membrane.
87. The method of claim 86, wherein the lipid vesicle particle preparation is sized by extrusion through the 0.2 µm polycarbonate membrane 20 to 40 times and extrusion through the 0.1 µm polycarbonate membrane 10 to 20 times.
88. The method of claim 85, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by first using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1 and then sizing by extrusion (i) 5-20 times through a 0.1 μm polycarbonate membrane or (ii) 5-20 times through a 0.08 μm polycarbonate membrane.
89. The method of any one of claims 85 to 88, wherein the membrane extrusion is performed at 1000 to 5000 psi back pressure.
90. The method of any one of claims 85 to 89, wherein the at least one solubilized first therapeutic agent is soluble at alkaline pH during high pressure membrane extrusion at about 5000 psi.
91. The method of any one of claims 64 to 90, wherein the mean particle size of the sized lipid vesicle particles is between about 80 nm and about 120 nm.
92. The method of any one of claims 64 to 91, wherein the mean particle size of the sized lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm or about 115 nm.
93. The method of any one of claims 64 to 92, wherein the mean particle size of the sized lipid vesicle particles is ≤100 nm.
94. The method of any one of claims 64 to 93, wherein the lipid vesicle particles comprise a synthetic lipid, a phospholipid, or a mixture of phospholipids.
95. The method of claim 94, wherein the lipid vesicle particles comprise dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
96. The method of claim 94 or 95, wherein the lipid vesicle particles further comprise cholesterol.
97. The method of claim 96, wherein the lipid vesicle particles comprise DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
98. The method of any one of claims 64 to 97, wherein the lipid vesicle particles are liposomes.
99. The method of claim 98, wherein the liposomes are unilamellar, multilamellar, or a mixture thereof.
100. The method of any one of claims 64 to 99 further comprising a step of sterile filtration of the mixture formed in step (iii) prior to drying.
101. The method of any one of claims 64 to 100 further comprising, between steps (iii) and (iv), a step of confirming that the sized lipid vesicle particles still have a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1.
102. The method of any one of claims 64 to 101, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
103. The method of claim 102, wherein the drying is performed by lyophilization.
104. The method of claim 103, wherein the lyophilization is performed by loading one or more containers comprising the mixture of step (iii) into a bag, sealing the bag to form a sealed unit, and lyophilizing the sealed unit in a freeze-dryer.
105. The method of claim 104, wherein the bag is a sterile, autoclaved bag.
106. The method of claim 104 or 105, wherein the freeze-dryer is a benchtop freeze dryer.
107. The method of any one of claims 104 to 106, wherein the freeze-dryer contains more than one sealed unit during the lyophilization.
108. The method of claim 107, wherein each sealed unit contains a different mixture prepared by steps (i) to (iii).
109. The method of claim 1 or 2, wherein providing the first dried preparation comprises: (i) solubilizing at least one lipid in an organic solvent to produce a lipid solution; (ii) combining the lipid solution with at least one therapeutic agent and a polyI:C polynucleotide to produce a mixture; and (iii) drying the mixture formed in step (ii) to form a dried preparation comprising at least one therapeutic agent, a lipid, and a polyI:C polynucleotide.
110. The method of claim 109, wherein the organic solvent comprises tertiary-butanol, optionally a 40% tertiary butanol solution.
111. The method of claim 109 or 110, wherein the at least one therapeutic agent is solubilized in DMSO or an aqueous solution prior to combining with the lipid solution.
112. The method of any one of claims 109 to 111, wherein the polyI:C polynucleotide is solubilized in water or an aqueous solution prior to combining with the lipid solution.
113. The method of any one of claims 109 to 112, wherein the at least one lipid comprises a synthetic lipid, a phospholipid, or a mixture of phospholipids.
114. The method of claim 113, wherein the at least one lipid comprises dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
115. The method of claim 113 or 114, wherein the at least one lipid further comprise cholesterol.
116. The method of claim 115, wherein the at least one lipid comprises DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
117. The method of any one of claims 109 to 116, wherein the at least one therapeutic agent comprises an antigen, a small molecule drug, an antibody or a functional fragment thereof, an antibody mimetic or a functional fragment thereof, an immunomodulatory agent, a polynucleotide encoding a polypeptide, or an interfering polynucleotide.
118. The method of any one of claims 109 to 116, wherein the at least one therapeutic agent comprises a peptide antigen comprising the amino acid sequence AKXVAAWTLKAAARAHYNIVTF wherein ‘X’ is phenylalanine or cyclohexylalanine.
119. The method of any one of claims 109 to 118, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
120. The method of claim 119, wherein the drying is performed by lyophilization.
121. The method of any one of claims 1 to 120, wherein providing the second dried preparation comprises: (i) providing lipid vesicle particles having a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1; (ii) mixing the lipid vesicle particles with a lipid-based adjuvant to form a mixture; and (iii) drying the mixture formed in step (ii) to form a second dried preparation comprising a lipid-based adjuvant and a lipid.
122. The method of claim 121, wherein step (i) comprises sizing lipid vesicle particles to provide the lipid vesicle particles having a mean particle size of ≤120 nm and a PDI of ≤0.1.
123. The method of claim 122, wherein the sizing is by filter-extrusion.
124. The method of claim 122 or 123, wherein the sizing is by extrusion through one or more polycarbonate membranes, such as a 0.2 µm polycarbonate membrane, a 0.1 µm polycarbonate membrane and/or a 0.08 µm polycarbonate membrane.
125. The method of any one of claims 122 to 124, wherein the sizing is by extrusion (i) 20-40 times through a 0.2 μm polycarbonate membrane, and then 10-20 times through a 0.1 μm polycarbonate membrane; or (ii) 20-40 times through a 0.2 μm polycarbonate membrane, then 10-20 times through a 0.1 μm polycarbonate membrane, and then 10-20 times through a 0.08 μm polycarbonate membrane.
126. The method of any one of claims 122 to 125, wherein the sizing is by extrusion (i) 25 times through a 0.2 μm polycarbonate membrane, and then 10 times through a 0.1 μm polycarbonate membrane or (ii) 25 times through a 0.2 μm polycarbonate membrane, then 10 times through a 0.1 μm polycarbonate membrane, and then 15 times through a 0.08 μm polycarbonate membrane.
127. The method of claim 122, wherein the sizing is performed by first using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1 and then sizing by extrusion (i) 5-20 times through a 0.1 μm polycarbonate membrane or (ii) 5-20 times through a 0.08 μm polycarbonate membrane.
128. The method of any one of claims 121 to 127, wherein the mean particle size of the lipid vesicle particles is between about 80 nm and about 120 nm.
129. The method of any one of claims 121 to 128, wherein the mean particle size of the lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm or about 115 nm.
130. The method of any one of claims 121 to 129, wherein the mean particle size of the lipid vesicle particles is ≤100 nm.
131. The method of any one of claims 121 to 130, wherein the lipid vesicle particles comprise a synthetic lipid, a phospholipid, or a mixture of phospholipids.
132. The method of claim 131, wherein the lipid vesicles particles comprise dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
133. The method of claim 131 or 132, wherein the lipid vesicle particles further comprise cholesterol.
134. The method of claim 133, wherein the lipid vesicle particles comprise DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
135. The method of any one of claims 121 to 134, wherein the lipid vesicle particles of step (i) are prepared from a liposome precursor.
136. The method of claim 135, wherein the liposome precursor is Presome®.
137. The method of any one of claims 121 to 136, wherein the lipid vesicle particles are liposomes.
138. The method of claim 137, wherein the liposomes are unilamellar, multilamellar, multivesicular, or a mixture thereof.
139. The method of any one of claims 121 to 138, wherein the lipid-based adjuvant is solubilized in an organic solvent or an aqueous solution prior to mixing with the lipid vesicle particles.
140. The method of any one of claims 121 to 139, wherein the lipid-based adjuvant is solubilized in 0.1 M sodium hydroxide, 100 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0 or 100 mM sodium phosphate having a pH of 6.0 ± 1.0 prior to mixing with the lipid vesicle particles.
141. The method of any one of claims 121 to 140, wherein the mixing of step (ii) is performed in a sodium acetate or sodium phosphate solution.
142. The method of claim 141, wherein the mixing of step (ii) is performed in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
143. The method of claim 141, wherein the mixing of step (ii) is performed in 50 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0 or 100 mM sodium phosphate having a pH of 6.0 ± 1.0.
144. The method of claim 141, wherein the mixing of step (ii) is performed in 50 mM sodium phosphate having a pH of 7.0, 100 mM sodium phosphate having a pH of 6.0, 50 mM sodium acetate having a pH of 6.0, or 100 mM sodium acetate having a pH of 9.5.
145. The method of any one of claims 121 to 144 further comprising a step of sterile filtration of the mixture formed in step (ii) prior to drying.
146. The method of any one of claims 121 to 145 further comprising, between steps (ii) and (iii), a step of confirming that the lipid vesicle particles still have a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1.
147. The method of any one of claims 121 to 146, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
148. The method of claim 147, wherein the drying is performed by lyophilization.
149. The method of claim 148, wherein the lyophilization is performed by loading one or more containers comprising the mixture of step (ii) into a bag, sealing the bag to form a sealed unit, and lyophilizing the sealed unit in a freeze-dryer.
150. The method of claim 149, wherein the bag is a sterile, autoclaved bag.
151. The method of claim 149 or 148, wherein the freeze-dryer is a benchtop freeze dryer.
152. The method of any one of claims 149 to 151, wherein the freeze-dryer contains more than one sealed unit during the lyophilization.
153. The method of claim 152, wherein each sealed unit contains a different mixture prepared by steps (i) and (ii).
154. The method of any one of claims 1 to 120, wherein providing the second dried preparation comprises: (i) providing a lipid vesicle particle preparation comprising lipid vesicle particles and a lipid-based adjuvant; (ii) sizing the lipid vesicle particle preparation to form a sized lipid vesicle particle preparation comprising sized lipid vesicle particles and said lipid-based adjuvant, said sized lipid vesicle particles having a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1; and (iii) drying the sized lipid vesicle particle preparation formed in step (ii) to form a second dried preparation comprising the lipid-based adjuvant.
155. The method of claim 154, wherein prior to step (ii) the lipid vesicle particles are not sized.
156. The method of claim 154 or 155, wherein, in step (i), the lipid vesicle particle preparation is in sodium acetate solution or in sodium phosphate solution.
157. The method of any one of claims 154 to 156, wherein, in step (i), the lipid vesicle particle preparation is in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
158. The method of any one of claims 154 to 157, wherein, in step (i), the lipid vesicle particle preparation is in 50 mM sodium acetate having a pH of 6.0 ± 1.0, 100 mM sodium acetate having a pH of 9.5 ± 1.0, 50 mM sodium phosphate having a pH of 7.0 ± 1.0, or 100 mM sodium phosphate having a pH of 6.0 ± 1.0.
159. The method of any one of claims 154 to 158, wherein, in step (i), the lipid vesicle particle preparation is in 100 mM sodium acetate having a pH of 9.5 ± 0.5.
160. The method of any one of claims 154 to 159, wherein step (i) comprises: (i-1) providing a lipid-based adjuvant stock comprising the lipid-based adjuvant; and (i-2) mixing the lipid-based adjuvant stock with a lipid mixture to form the lipid vesicle preparation.
161. The method of any one of claims 154 to 160, wherein, in step (i), the lipid-based adjuvant is encapsulated in the lipid vesicle particles.
162. The method of any one of claims 154 to 161, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by high pressure homogenization, sonication and/or membrane extrusion.
163. The method of claim 162, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by extrusion through a 0.2 µm polycarbonate membrane followed by extrusion through a 0.1 µm polycarbonate membrane.
164. The method of claim 163, wherein the lipid vesicle particle preparation is sized by extrusion through the 0.2 µm polycarbonate membrane 20 to 40 times and extrusion through the 0.1 µm polycarbonate membrane 10 to 20 times.
165. The method of claim 162, wherein, in step (ii), the lipid vesicle particle preparation of step (i) is sized by first using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1 and then sizing by extrusion (i) 5-20 times through a 0.1 μm polycarbonate membrane or (ii) 5-20 times through a 0.08 μm polycarbonate membrane.
166. The method of any one of claims 162 to 165, wherein the membrane extrusion is performed at 1000 to 5000 psi back pressure.
167. The method of any one of claims 154 to 166, wherein the mean particle size of the sized lipid vesicle particles is between about 80 nm and about 120 nm.
168. The method of any one of claims 154 to 167, wherein the mean particle size of the sized lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm or about 115 nm.
169. The method of any one of claims 154 to 168, wherein the mean particle size of the sized lipid vesicle particles is ≤100 nm.
170. The method of any one of claims 154 to 169, wherein the lipid vesicle particles comprise a synthetic lipid, a phospholipid, or a mixture of phospholipids.
171. The method of claim 170, wherein the lipid vesicle particles comprise dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
172. The method of claim 170 or 171, wherein the lipid vesicle particles further comprise cholesterol.
173. The method of claim 172, wherein the lipid vesicle particles comprise DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
174. The method of any one of claims 154 to 173, wherein the lipid vesicle particles are liposomes.
175. The method of claim 174, wherein the liposomes are unilamellar, multilamellar, or a mixture thereof.
176. The method of any one of claims 154 to 175 further comprising a step of sterile filtration of the sized lipid vesicle particle preparationformed in step (ii) prior to drying.
177. The method of any one of claims 154 to 176 further comprising, between steps (ii) and (iii), a step of confirming that the sized lipid vesicle particles still have a mean particle size of ≤120 nm and a polydispersity index (PDI) of ≤0.1.
178. The method of any one of claims 154 to 177, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
179. The method of claim 178, wherein the drying is performed by lyophilization.
180. The method of claim 179, wherein the lyophilization is performed by loading one or more containers comprising the mixture of step (ii) into a bag, sealing the bag to form a sealed unit, and lyophilizing the sealed unit in a freeze-dryer.
181. The method of claim 180, wherein the bag is a sterile, autoclaved bag.
182. The method of claim 180 or 181, wherein the freeze-dryer is a benchtop freeze dryer.
183. The method of any one of claims 180 to 182, wherein the freeze-dryer contains more than one sealed unit during the lyophilization.
184. The method of claim 183, wherein each sealed unit contains a different mixture prepared by steps (i) to (ii).
185. The method of any one of claims 1 to 120, wherein providing the second dried preparation comprises: (i) solubilizing at least one lipid in an organic solvent to produce a lipid solution; (ii) combining the lipid solution with a lipid-based adjuvant to produce a mixture; and (iii) drying the mixture formed in step (ii) to form a dried preparation comprising a lipid- based adjuvant and a lipid.
186. The method of claim 185, wherein the organic solvent comprises tertiary-butanol, optionally a 40% tertiary butanol solution.
187. The method of claim 185 or 186, wherein the lipid-based adjuvant is solubilized in an organic solvent or an aqueous solution prior to combining with the lipid solution.
188. The method of any one of claims 185 to 187, wherein the at least one lipid comprises a synthetic lipid, a phospholipid, or a mixture of phospholipids.
189. The method of claim 188, wherein the at least one lipid comprises dioleoyl phosphatidylcholine (DOPC) and/or lecithin.
190. The method of claim 188 or 189, wherein the at least one lipid further comprise cholesterol.
191. The method of claim 190, wherein the at least one lipid comprises DOPC and cholesterol at a DOPC:cholesterol ratio of 10:1 (w/w).
192. The method of any one of claims 185 to 191, wherein the drying is performed by lyophilization, spray freeze-drying, or spray drying.
193. The method of claim 192, wherein the drying is performed by lyophilization.
194. The method of any one of claims 1 to 193, wherein the polyI:C polynucleotide is RNA- based polyI:C polynucleotide or DNA-based polyI:C polynucleotide.
195. The method of any one of claims 1 to 194, wherein the polyI:C polynucleotide is double- stranded and each strand is a homopolymer of inosinic or cytidylic residues.
196. The method of any one of claims 1 to 194, wherein the polyI:C polynucleotide is double- stranded and each strand is a heteropolymer comprising both inosinic and cytidylic residues.
197. The method of any one of claims 1 to 194, wherein the polyI:C polynucleotide is a mixture comprising both homopolymeric polyI:C polynucleotides and heteropolymeric polyI:C polynucleotides.
198. The method of any one of claims 1 to 197, wherein the lipid-based adjuvant comprises one or more lipopeptide(s).
199. The method of claim 198, wherein at least one of the lipopeptides comprises palmitic acid as the lipid component.
200. The method of any one of claims 1 to 199, wherein the lipid-based adjuvant comprises dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys).
201. The method of claim 200, wherein the lipid-based adjuvant is PAM2Cys-Ser-(Lys)4 or PAM3Cys-Ser-(Lys)4.
202. The method of claim 201, wherein the lipid-based adjuvant is PAM3Cys-Ser-(Lys)4.
203. The method of any one of claims 1 to 202, wherein the hydrophobic carrier is an oil or a mixture of oils.
204. The method of claim 203, wherein the hydrophobic carrier comprises a vegetable oil, nut oil, or mineral oil.
205. The method of claim 204, wherein the hydrophobic carrier is mineral oil or is a mannide oleate in mineral oil solution.
206. The method of claim 205, wherein the carrier is Montanide® ISA 51 VG.
207. A pharmaceutical composition prepared by the method of any one of claims 1 to 206.
208. A method of inducing an antibody and/or CTL immune response in a subject comprising administering to the subject the pharmaceutical composition of claim 207.
209. The method of claim 208, which is for treating cancer or an infectious disease.
210. Use of the pharmaceutical composition of claim 207 for inducing an antibody and/or CTL immune response in a subject.
211. The use of claim 210, which is for the treatment of cancer.
212. The use of claim 210, which is for the treatment of an infectious disease.
213. A method of delivering a therapeutic agent to a subject comprising administering to the subject the pharmaceutical composition of claim 207.
214. Use of the pharmaceutical composition of claim 207 for delivering a therapeutic agent to a subject.
215. A kit for preparing the pharmaceutical composition of claim 207, the kit comprising: a container comprising a first dried preparation prepared by the method of any one of claims 3 to 120 and 194 to 206; a container comprising second dried preparation prepared by the method of any one of claims 121 to 206; and a container comprising a hydrophobic carrier.
PCT/US2024/018048 2023-03-03 2024-03-01 Methods of making dried pharmaceutical compositions WO2024186623A1 (en)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067023A2 (en) 2003-01-30 2004-08-12 Survac Aps Survivin-derived peptides and use thereof
WO2006081826A2 (en) 2005-02-04 2006-08-10 Survac Aps Survivin peptide vaccine
US20080233143A1 (en) 2005-02-07 2008-09-25 Lipotek Pty Ltd. Adjuvanting Material
US20100129385A1 (en) 2005-02-08 2010-05-27 Jackson David C Immunogenic molecules
WO2012065997A1 (en) 2010-11-15 2012-05-24 Vib Vzw Respiratory syncytial virus vaccine
WO2016176761A1 (en) 2015-05-01 2016-11-10 Immunovaccine Technologies Inc., Methods for potentiating an immune response using depot-forming and non-depot-forming vaccines
WO2017083963A1 (en) * 2015-11-18 2017-05-26 Immunovaccine Technologies Inc. Adjuvanting systems and water-free vaccine compositions comprising a polyi:c polynucleotide adjuvant and a lipid-based adjuvant
WO2019178677A1 (en) * 2018-03-20 2019-09-26 Immunovaccine Technologies Inc. Methods and compositions for targeted delivery of active agents and immunomodulatory agents to lymph nodes

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067023A2 (en) 2003-01-30 2004-08-12 Survac Aps Survivin-derived peptides and use thereof
WO2006081826A2 (en) 2005-02-04 2006-08-10 Survac Aps Survivin peptide vaccine
US20080233143A1 (en) 2005-02-07 2008-09-25 Lipotek Pty Ltd. Adjuvanting Material
US20110200632A1 (en) 2005-02-07 2011-08-18 Lipotek Pty Ltd. Adjuvanting material
US20100129385A1 (en) 2005-02-08 2010-05-27 Jackson David C Immunogenic molecules
WO2012065997A1 (en) 2010-11-15 2012-05-24 Vib Vzw Respiratory syncytial virus vaccine
WO2016176761A1 (en) 2015-05-01 2016-11-10 Immunovaccine Technologies Inc., Methods for potentiating an immune response using depot-forming and non-depot-forming vaccines
WO2017083963A1 (en) * 2015-11-18 2017-05-26 Immunovaccine Technologies Inc. Adjuvanting systems and water-free vaccine compositions comprising a polyi:c polynucleotide adjuvant and a lipid-based adjuvant
WO2019178677A1 (en) * 2018-03-20 2019-09-26 Immunovaccine Technologies Inc. Methods and compositions for targeted delivery of active agents and immunomodulatory agents to lymph nodes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOWEN P: "Particle Size Distribution Measurement from Millimeters to Nanometers and from Rods to Platelets", JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY, TAYLOR AND FRANCIS GROUP, NEW YORK, NY, US, vol. 23, no. 5, 1 January 2002 (2002-01-01), pages 631 - 662, XP009102859, ISSN: 0193-2691, Retrieved from the Internet <URL:https://www.tandfonline.com/doi/full/10.1081/DIS-120015368> DOI: 10.1081/DIS-120015368 *
CASTLE ET AL.: "Exploiting the Mutanome for Tumor Vaccination", CANCER RES, vol. 72, no. 5, 2012, pages 1081 - 1091, XP055231746, DOI: 10.1158/0008-5472.CAN-11-3722

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