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WO2024228167A1 - Compositions liposomales de modulateur de cellules inkt et procédés d'utilisation - Google Patents

Compositions liposomales de modulateur de cellules inkt et procédés d'utilisation Download PDF

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Publication number
WO2024228167A1
WO2024228167A1 PCT/IB2024/054317 IB2024054317W WO2024228167A1 WO 2024228167 A1 WO2024228167 A1 WO 2024228167A1 IB 2024054317 W IB2024054317 W IB 2024054317W WO 2024228167 A1 WO2024228167 A1 WO 2024228167A1
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Prior art keywords
liposome
cancer
mage
composition
glycero
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Russell POE
Ian Walters
Robert Kramer
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Iox Therapeutics Inc
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Iox Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • NKT Natural killer T
  • iNKT invariant NKT
  • TCR invariant ⁇ chain T cell receptor
  • Type II NKT cells have a diverse TCR repertoire and are less well defined, although a subset has been shown to be reactive to sulfatide.
  • the iNKT cell TCR recognizes lipid antigens presented in the context of the non-polymorphic MHC class I-like protein, CD1d.
  • the CD1d molecule has been shown to bind a range of dialkyl lipids and glycolipids and the ensuing iNKT cell TCR recognition of the CD1d-lipid complex leads to the rapid proliferation and release of a plethora of cytokines (both pro-inflammatory and regulatory).
  • the activation of iNKT cells is an important step in ‘boosting’ adaptive immune responses through the activation and maturation of dendritic cells (DC) and B cells through CD40 ⁇ CD40L interactions, and the activation of natural killer (NK) cells following interferon gamma (IFN ⁇ ) release.
  • DC dendritic cells
  • B cells through CD40 ⁇ CD40L interactions
  • IFN ⁇ interferon gamma
  • ⁇ -galactosylceramide ⁇ - GalCer
  • ⁇ -GalCer is a derivative of the agelasphins, which are naturally occurring glycolipids that were isolated from the marine sponge Agelas mauritianus.
  • ⁇ -GalCer ⁇ CD1d complex Recognition of the ⁇ -GalCer ⁇ CD1d complex by the iNKT cell TCR results in the secretion of a range of cytokines, and the initiation of a powerful immune response.
  • ⁇ -GalCer remains one of the most potent iNKT cell agonists and has shown potential in the treatment of various conditions, it may prove difficult to use this molecule widely as a useful therapeutic agent, at least as a direct activator of iNKT cells: not only does ⁇ -GalCer- mediated iNKT cell activation lead to the secretion of both T helper Type 1 (Th1) (e.g. IFN- ⁇ ) and T helper Type 2 (Th2) (e.g.
  • Th1 T helper Type 1
  • Th2 T helper Type 2
  • IL-4 interleukin-4
  • IL-4 interleukin-4
  • iNKT cells which can result in their entering a long-term anergic state, i.e., unresponsiveness to subsequent ⁇ -GalCer stimulation and preferential IL-4 production, which would be deleterious for long-term therapy.
  • Loss of circulating levels of iNKT cells could represent a therapeutically significant limitation with iNKT-cell-based therapies if multi-dosing regimens are required.
  • iNKT cell activators that have effective delivery for clinical applications.
  • Immune checkpoints have a natural role in a healthy immune system to prevent uncontrolled immune responses that can be detrimental to healthy cells in the body. However, such checkpoints also can inhibit or prevent the immune system from destroying cancer cells in the body.
  • Immune checkpoint inhibitors represent a relatively new class of drugs that operate by interfering with checkpoint proteins from binding their partner ligand proteins. By interfering with the “off” signal of a checkpoint, these drugs potentiate T cell activity against cancer cells.
  • checkpoint inhibitors are neither effective against all types of cancer nor effective in all patients with a given type of cancer.
  • compositions such as pharmaceutical compositions
  • methods of making the liposomes and compositions methods of evaluating and optimizing liposome compositions
  • methods of using the compositions including therapeutic and prophylactic methods of preventing, ameliorating, treating, and curing diseases; and methods of modulating an immune response in a mammalian subject.
  • Exemplary embodiments of the invention are summarized or defined by the following numbered paragraphs: [0010] 1.
  • a liposome comprised of Compound A: (A) wherein n is 1 (IMM60), 2 (IMM70), or 3 (IMM80), or a salt, ester, solvate, or hydrate thereof, and two or more lipids or salts thereof; wherein the two or more lipids comprise: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG); (b) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and dimethyldio
  • the liposome of any one of Embodiments 1 to 5 wherein the mass ratio of Compound A to the two or more lipids is about 1:5 to about 1:20, or about 1:7 to about 1:15, or about 1:7 to about 1:12, or about 1:9.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol
  • the CHOL is present in an amount of about 5 wt% to about 15 wt%.
  • POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • DC-Chol 3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • DDAB dimethyldioctadecylammonium bromide
  • the liposome of Embodiment 16 wherein the POPC is present in an amount of about 40 wt.% to 80 wt.%, or about 45 wt.% to about 65 wt.%, or about 50 wt.% to about 60 wt.%, based on the total weight of the liposome.
  • EPG L- ⁇ -phosphatidylglycerol
  • EPC 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
  • POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • DMPG 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol
  • CHOL cholest-5-en-3 ⁇ -ol
  • the tumor antigen is selected from the group consisting of: (a) P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE- A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1,
  • composition of Embodiment 38, wherein the mean diameter of the liposomes is within a range of about 50 nm to 200 nm, or about 50 nm to about 170 nm, or about 75 nm to about 145 nm, or about 90 nm to about 130 nm.
  • the composition according to Embodiment 38, wherein the mean diameter of the liposomes is 110 nm ⁇ 60 nm or 110 nm ⁇ 40 nm or 110 nm ⁇ 20 nm.
  • PdI polydispersity index
  • the composition further comprises at least one antigen.
  • the at least one antigen comprises a member selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a tumor antigen, and mixtures thereof.
  • composition of Embodiment 43 or 44, wherein the at least one antigen comprises at least one tumor antigen.
  • the tumor antigen is selected from the group consisting of: [0056] (a) P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C
  • composition of Embodiment 48 wherein the at least one therapeutic agent is selected from the group of an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and combinations thereof.
  • an immune modulator a Toll-like receptor agonist
  • a Nod ligand an anti-viral agent
  • an antifungal agent an antibiotic
  • an antiviral antibody e.g., an antifungal agent
  • a cancer immune therapeutic e.g., a cancer immune therapeutic
  • chemotherapy agent e.g., a kinase inhibitor, a cytotoxic
  • a method of stimulating an immune response in a mammalian subject comprising administering to the subject the composition of any one of Embodiments 38 to 49.
  • composition according to any one of Embodiments 38 to 49 for stimulating an immune response in a mammalian subject.
  • 55 The liposome according to any one of Embodiments 1 to 37 or the composition according to any one of Embodiments 38 to 49 for use in the manufacture of a medicament for stimulating an immune response in a mammalian subject.
  • 56 The composition or use according to any one of Embodiments 53 to 55, wherein the composition is subjected to a heating step prior to its use in stimulating the immune response.
  • 57 The method or use of any one of Embodiments 50-56, wherein the mammalian subject is human.
  • 58 The method or use of any one of Embodiments 50-56, wherein the mammalian subject is human.
  • Embodiments 50 to 57 wherein the mammalian subject has a cancer.
  • 59 A method of treating a mammalian subject with cancer, comprising administering to the subject a therapeutically effective amount of the composition of any one of Embodiments 38 to 49.
  • 60 The method or use according to Embodiment 58 or 59, wherein the composition is administered by subcutaneous, intravenous, or intratumoral injection.
  • 61 The method according to Embodiment 50 or 60, further comprising a step, prior to the administering step, of heating the composition for a time and at a temperature sufficient to reduce average particle size or PdI of the liposomal composition.
  • 62 A method of treating a mammalian subject with cancer, comprising administering to the subject a therapeutically effective amount of the composition of any one of Embodiments 38 to 49.
  • 60 The method or use according to Embodiment 58 or 59, wherein the composition is administered by subcutaneous, intravenous, or intratumoral injection.
  • Embodiments 58 to 64 wherein the cancer is selected from the group of basal cell carcinoma, breast cancer leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell cancer, melanoma, and combinations thereof.
  • the cancer is selected from the group of basal cell carcinoma, breast cancer leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testi
  • Embodiment 69 wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor comprises at least one PD-1 inhibitor selected from Pembrolizumab, Nivolumab, Cemiplimab, and Spartalizumab.
  • 72 The method or use of Embodiment 70, wherein the immune checkpoint inhibitor comprises at least one PD-L1 inhibitor selected from Atezolizumab, Avelumab, and Durvalumab.
  • 73 73.
  • Embodiment 70 wherein the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab.
  • the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab.
  • 74 The method or use of any one of Embodiments 68-73, wherein the composition and the further therapeutic or therapy are administered concurrently.
  • 75 The method or use of any one of Embodiments 68-73, wherein the composition and the further therapeutic or therapy are administered separately.
  • 76 The method or use of Embodiment 70, wherein the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab.
  • the method according to Embodiment 78 that comprises filtering with a filter that has a pore size of 400 nm, or less than about 350 nm, or less than about 300 nm, or less than about 250 nm, or less than about 220 nm, or less than about 200 nm, or less than about 150 nm. [0093] 81. The method according to Embodiment 78 that comprises filtering with a filter that has a pore size of about 50 nm to about 250 nm, or about 50 nm to about 150 nm, or about 60 nm to about 100 nm, or about 80 nm. [0094] 82.
  • Embodiment 78 that comprises filtering with a filter that has a molecular weight cutoff (MWCO) of 1000 kD or 800 kD or 600 kD or 500 kD or 400 kD or 300 kD or 250 kD.
  • MWCO molecular weight cutoff
  • Embodiments 7– 86 The method of any one of Embodiments 7– 86, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol
  • Embodiment 87 or 88 wherein the DSPG is present in an amount of about 20 wt.% to 60 wt.%, or about 30 wt.% to about 50 wt.%, or about 35 wt.% to about 45 wt.%, based on the total weight of the liposome.
  • step (A) further includes admixing at least one antigen with the Compound A and the lipids.
  • step (A) further includes admixing at least one antigen with the Compound A and the lipids.
  • Embodiment 91 wherein the at least one antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, and/or a tumor antigen.
  • the at least one antigen comprises at least one tumor antigen.
  • 94 The method of Embodiment 91 or 92, wherein the at least one antigen comprises at least one tumor antigen.
  • the liposome of Embodiment 93 wherein the tumor antigen is selected from the group consisting of: (a) P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE- A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX- 2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP
  • step (A) further comprises admixing at least one therapeutic agent with the lipids and the Compound A.
  • step (A) further comprises admixing at least one therapeutic agent with the lipids and the Compound A.
  • step (A) further comprises admixing at least one therapeutic agent with the lipids and the Compound A.
  • step (A) further comprises admixing at least one therapeutic agent with the lipids and the Compound A.
  • 96 A liposome prepared by the method of any one of the Embodiments 76-95.
  • 97 A method of treating a liposomal formulation, the method comprising: (a) providing a liposomal composition according to any one of Embodiments 38-49; and (b) heating the composition for a time and at temperature sufficient to reduce average particle size or PdI of the liposomal composition.
  • 98 98.
  • a method of treating a liposomal formulation comprising: (a) providing a liposomal composition according to any one of Embodiments 38-49; and (b) heating the composition for 5-15 minutes at a temperature of about 55 to about 65 °C. [00111] 99.
  • DSPC 1,2-distearoyl-sn-glycero-3- phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3-phospho
  • 106 The liposome of any one of embodiments 99-105 that is free of lipids or phospholipids having a chain length shorter than 16 carbon atoms.
  • 107 The liposome of any one of embodiments 100-106, wherein the at least two phospholipids comprise about 75 wt.% to about 99.9 wt.%, or about 80 wt.% to about 98 wt.%, or about 80 wt.% to about 95 wt.%, or about 85 wt.%, to about 95 wt.% based on the total weight of the phospholipid bilayer.
  • 108. The liposome of any one of embodiments 100-107, further comprising a therapeutic agent.
  • RNA ribonucleic acid
  • the RNA is an siRNA or an mRNA.
  • the at least one therapeutic agent comprises an anti-cancer therapeutic.
  • 116 The liposome of any one of embodiments 100-115, wherein the at least one therapeutic agent comprises an immunostimulant or encodes an immunostimulant. [00129] 117.
  • 119 The liposome of any one of claims 100-119, comprising one or more therapeutic agent.
  • 121. A composition comprising liposomes according to any one of embodiments 100- 120 and a pharmaceutically acceptable carrier.
  • 126 The composition according to embodiment 121 or 122, wherein the liposomes have a mean diameter of about 100 nm ⁇ 60 nm, or about 100 nm ⁇ 40 nm, or about 100 nm ⁇ 20 nm.
  • 127. The composition of any one of embodiments 121-126, wherein the liposome has a steady state volume of distribution in humans between about 2 liters to about 12 liters, or about 2 liters to about 10 liters, or about 2 liters to about 8 liters, after the composition is administered to humans intravenously.
  • 128 The composition of any one of claims 121-127, wherein the liposome has a terminal volume of distribution in a mammalian subject of about 3 liters to about 10 liters after the composition is administered intravenously.
  • 129 The composition of any one of claims 121-128, wherein the composition provides a steady plasma concentration of therapeutic agent between about 2 hours to about 10 hours, or between about 3 hours to about 8 hours, or between about 4 hours to about 6 hours, after administration to a mammalian subject.
  • 130 The composition of any one of embodiments 121-129, wherein the composition provides a steady plasma concentration of therapeutic agent between about 4 hours and about 6 hours after administration to a mammalian subject.
  • 131 The composition of any one of embodiments 121-130, wherein the therapeutic agent is not substantially cleared from plasma between about 4 hours and about 6 hours after administration to a mammalian subject.
  • 132 132.
  • composition of any one of embodiments 128-131, wherein the mammalian subject is human [00145] 133. A method of stimulating an immune response in a mammalian subject comprising administering to the subject the composition of any one of embodiments 121-132. [00146] 134. Use of the composition of any one of embodiments 121-132 for stimulating an immune response in a mammalian subject. [00147] 135. The liposome of any one of embodiments 100-120 or the composition of any one of embodiments 121-132 for use in the manufacture of a medicament for stimulating an immune response in a mammalian subject. [00148] 136. The method or use of any one of embodiments 133-135, wherein the mammalian subject is human.
  • the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor comprises at least one PD-1 inhibitor selected from Pembrolizumab, Nivolumab, Cemiplimab, and Spartalizumab.
  • 150 is a PD-1 inhibitor selected from Pembrolizumab, Nivolumab, Cemiplimab, and Spartalizumab.
  • the immune checkpoint inhibitor comprises at least one PD-L1 inhibitor selected from Atezolizumab, Avelumab, and Durvalumab. [00163] 151.
  • the method or use of embodiment 148, wherein the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab.
  • 152. The method or use of any one of embodiments 146-151, wherein the composition and the further therapeutic or therapy are administered concurrently.
  • 153 The method or use of any one of embodiments 146-151, wherein the composition and the further therapeutic or therapy are administered separately.
  • 155 The method of embodiment 154, further comprising purifying the liposomes.
  • 156 The method of embodiment 154 or 155, wherein purifying the liposomes comprises filtration.
  • 157 The method of embodiment 156, wherein purifying comprises ultrafiltration, centrifugation, dialysis, diafiltration, or tangential flow filtration.
  • 158 The method of embodiment 156, wherein purifying comprises ultrafiltration, centrifugation, dialysis, diafiltration, or tangential flow filtration.
  • the method according to any one of embodiments 154-157 comprising filtering with a filter that has a pore size of about 450 nm or less, or about 400 nm or less, or about 350 nm or less, or about 300 nm or less, or about 250 nm or less, or about 220 nm or less, or about 200 nm or less, or about 150 nm or less.
  • a filter that has a pore size of about 450 nm or less, or about 400 nm or less, or about 350 nm or less, or about 300 nm or less, or about 250 nm or less, or about 220 nm or less, or about 200 nm or less, or about 150 nm or less.
  • 159 The method according to any one of embodiments 154-158, wherein the membrane has a pore size of about 50 nm to about 250 nm, or about 50 nm to about 150 nm, or about 60 nm to about 100 nm
  • the method according to any one of embodiments 154-159 comprising filtering with a filter that has a molecular weight cutoff (MWCO) of about 500 kD, or about 450 kD, or about 400 kD, or about 350 kD, or about 300 kD, or about 250 kD. [00173] 161.
  • any one of embodiments 154-163, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol
  • step 1) further includes admixing at least one antigen with the therapeutic agent and the lipids.
  • step 1) further includes admixing at least one antigen with the therapeutic agent and the lipids.
  • step 2) further includes admixing at least one antigen with the therapeutic agent and the lipids.
  • step 1) further includes admixing at least one antigen with the therapeutic agent and the lipids.
  • step 1) further includes admixing at least one antigen with the therapeutic agent and the lipids.
  • the at least one antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, and/or a tumor antigen.
  • invention 167 or 168 wherein the at least one antigen comprises at least one tumor antigen.
  • the at least one antigen comprises at least one tumor antigen.
  • embodiments relating to the use of checkpoint inhibitors for therapy including caneer therapy.
  • checkpoint inhibitors have not been effective for treating all types of cancer or for treating all patients with a particular type of cancer.
  • the iNKT modulator compositions and methods described herein are believed to have the property of expanding the range and/or efficacy of checkpoint inhibitor therapy, and/or expanding the number of cancer patients who should be deemed sensitive to checkpoint inhibitor therapy for purposes of guiding treatment.
  • the invention includes a method of treating a mammalian subject with an iNKT cell modulator as part of a cancer therapy to increase the sensitivity of the subject’s cancer to a checkpoint inhibitor.
  • the cancer/subject would be scored as insensitive to treatment with the checkpoint inhibitor.
  • the term “insensitive” is intended to define a clinical judgment of expected ineffectiveness at treating the cancer, based on parameters used to classify treatment of cancer patients as a group, and is not intended as a definitive statement about whether any particular individual cancer patient ultimately would or would not benefit from the checkpoint therapy (if administered).
  • a subject/cancer is scored as “insensitive” based on clinical experience a checkpoint inhibitor was ineffective against a cancer in pre-clinical animal experiments and/or human clinical trials and/or published post-approval evidence from the clinic involving similar cancers, and similar subjects, which may optionally take into consideration previous treatments that the subject has received for the cancer.
  • a subject is scored as insensitive by virtue of failing to satisfy an established prescribing guideline for a particular checkpoint inhibitor drug. Prescribing guidelines are exemplified herein with reference to some specific guidelines from the U.S. Food and Drug Administration. However, guidelines from regulatory bodies from other jurisdictions are also contemplated and intended as suitable tools for scoring subjects as sensitive or insensitive.
  • a subject is scored as “insensitive” because the subject has previously received the checkpoint inhibitor, and did not respond to the treatment, or stopped responding to the treatment.
  • PD-L1 expression in the cancer is used as an indicator of sensitivity of the cancer, with a percentage of cancer cells and/or immue cells (especially tumor- associated or tumor-inflitrating immune cells) is used as a cutoff for scoring sensitivity. A cancer with PD-L1 expression on a percentage of cells below the cutoff is scored as insenstitive.
  • the INKT cell modulators described herein are contemplated for sensitizing cancer patients to effective treatment with any checkpoint inhibitor, and several exemplary checkpoint inhibitors are described herein.
  • the checkpoint inhibitor targets the Programmed Cell Death Protein 1 (PD-1) pathway.
  • the method involves first identifying a mammalian subject as having a cancer that is insensitive to treatment with an immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway; administering to the subject a composition comprising an INKT cell modulator, such as IMM60, in an amount effective to increase sensitivity of the cancer to the immune checkpoint inhibitor; and then administering to the subject a composition comprising the immune checkpoint inhibitor following the administration of the composition comprising IMM60.
  • an INKT cell modulator such as IMM60
  • the subject may be tested/evaluated following the administration of the INKT modulator, to determine whether the subject should no longer be scored as “insensitive.” For example, a subject may no longer be scored as insensitive because a biomarker, such as PD-L1 expression, has changed, and the subject now qualifies for treatment under prescribing guidelines for the checkpoint inhibitor. For the subject identified as sensitive, the checkpoint therapy is initiated (or resumed) – either alone or in combination with other standard of care therapy for the cancer. In some variation, the subject continues to be treated with INKT cell modulator, in combination with the checkpoint inhibitor.
  • a biomarker such as PD-L1 expression
  • a threshold or cut-off value is used to score subjects as sensitive or insensitive.
  • the cut-off may be a single number for a single biomarker, such as a percentage of cells that are positive for a marker such as PD-1, PD-L1, or PD-L2.
  • the cut-off may be derived or calculated from multiple measurements, e.g., when multiple biomarkers or multiple cell types are considered.
  • the cut-off may be derived from clinical trial patient information or extrapolated from pre-clinical animal models.
  • the threshold or cut-off value is from prescribing information for a particular drug.
  • a statistical analysis from multiple patient outcomes such as a receiver operating characteristic (ROC) curve, is used to optimize a threshold or cutoff.
  • the INKT cell modulator such as IMM60
  • a liposomal composition such as the liposomal compositions described herein.
  • the IMM60 is formulated in a nanoparticle formulation.
  • a method of treating a mammalian subject with an immune checkpoint inhibitor comprising: (a) identifying a mammalian subject as having a cancer that is insensitive to treatment with an immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway; (b) administering to the subject a composition comprising IMM60, in an amount effective to increase sensitivity of the cancer to the immune checkpoint inhibitor; and (c) administering to the subject a composition comprising the immune checkpoint inhibitor following the administration of the composition comprising IMM60.
  • PD-1 Programmed Cell Death Protein 1
  • the method according to paragraph 201 that comprises identifying the subject as insensitive to the checkpoint inhibitor by: (a1) measuring expression of Programmed Cell Death Ligand 1 (PD-L1) in cancer cells, immune cells, or tumor stroma cells from a tumor sample from the subject; and (a2) identifying the subject as having a cancer that is insensitive to treatment with the immune checkpoint inhibitor by determining that the target of the checkpoint inhibitor is expressed on a percentage of the cancer cells that is lower than a threshold or cut-off level.
  • P-L1 Programmed Cell Death Ligand 1
  • the method according to paragraph 201 that comprises identifying the subject as insensitive to the checkpoint inhibitor by: (a1) measuring microsatellite instability (MSI), mismatch repair deficiency (MMRD), or tumor mutational burden (TMB) in cancer cells from the subject, and (a2) identifying the mammalian subject as having a cancer that is insensitive to treatment with the immune checkpoint inhibitor by determining that the cancer cells have an MSI, MMRD, or TMB that is lower than a threshold or cut-off level.
  • MSI microsatellite instability
  • MMRD mismatch repair deficiency
  • TMB tumor mutational burden
  • the method according to paragraph 201 that comprises identifying the subject as insensitive to the checkpoint inhibitor by: (a1) comparing a medical diagnosis and history of the subject with a government- approved prescribing guideline for the immune checkpoint inhibitor, and (a2) identifying the mammalian subject as having a cancer that is insensitive to treatment with the immune checkpoint inhibitor by determining that that the subject does not satisfy the indication and usage criteria of the guideline.
  • the prescribing guideline specifies a minimum expression level of PD-L1 in cells from the subject, and (a2) comprises determining that the cells from the subject fail to meet the minimum expression level.
  • the immune checkpoint inhibitor was a European Medicines influence (EMA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the EMA- approved guideline in existence on December 31, 2022; or wherein the immune checkpoint inhibitor was a Japanese Pharmaceuticals and Medical Devices Agency (PDMA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the PDM-approved guideline in existence on December 31, 2022; or wherein the immune checkpoint inhibitor was a Chinese National Medical Products Administration (NMPA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the NMPA-approved guideline in existence on December 31, 2022. [00199] 208.
  • EMA European Medicines influence
  • PDMA Japanese Pharmaceuticals and Medical Devices Agency
  • NMPA Chinese National Medical Products Administration
  • the method according to paragraph 201 that comprises identifying a patient as insensitive to the checkpoint inhibitor by: (a1) comparing a medical diagnosis and history of the subject with a standard-of-care guideline for use of the immune checkpoint inhibitor; and (a2) identifying the subject as having a cancer that is insensitive to treatment with the immune checkpoint inhibitor by determining that the subject does not meet the standard-of-care guideline.
  • the standard-of-care guideline specifies a minimum expression level of PD-L1 in cells from the subject, and (a2) comprises determining that the cells from the subject fail to meet the minimum expression level.
  • step (b) further comprises: (b1) re-evaluating a sample from the subject after one or more administrations of the IMM60; and (b2) determining from the sample that the cancer is no longer classified as insensitive to the treatment with the immune checkpoint inhibitor.
  • step (c) comprises combination therapy, the combination comprising administration of the checkpoint inhibitor and continued administration of IMM60 to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • step (c) comprises repeated administrations of the composition comprising IMM60 and the composition comprising the immune checkpoint inhibitor to the subject.
  • a method of sensitizing a cancer in a mammalian subject to treatment with an immune checkpoint inhibitor comprising: identifying a subject as having a cancer that is insensitive to treatment with an immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway; and administering to the subject a composition comprising IMM60 in an amount and for a duration effective to sensitize the cancer to treatment with the immune checkpoint inhibitor.
  • PD-1 Programmed Cell Death Protein 1
  • the identifying comprises: measuring expression of Programmed Cell Death Ligand 1 (PD-L1) in cancer cells, immune cells, or tumor stroma cells from a tumor sample from the subject; and identifying the mammalian subject as having a cancer that is insensitive to treatment with the immune checkpoint inhibitor by determining that the PD-L1 is expressed on a percentage of the cells that is lower than a threshold or cut-off level.
  • PD-L1 Programmed Cell Death Ligand 1
  • the threshold or cut-off level comprises a level specified in a government-approved prescribing guideline for the immune checkpoint inhibitor.
  • the immune checkpoint inhibitor was a European Medicines Agency (EMA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the EMA- approved guideline in existence on December 31, 2022; or wherein the immune checkpoint inhibitor was a Japanese Pharmaceuticals and Medical Devices Agency (PDMA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the PDM-approved guideline in existence on December 31, 2022; or wherein the immune checkpoint inhibitor was a Chinese National Medical Products Administration (NMPA)-approved immune checkpoint inhibitor before or in 2022, and the prescribing guideline is the NMPA-approved guideline in existence on December 31, 2022. [00211] 220.
  • EMA European Medicines Agency
  • PDMA Japanese Pharmaceuticals and Medical Devices Agency
  • NMPA Chinese National Medical Products Administration
  • the threshold or cut-off level comprises a level specified in a standard-of-care guideline for use of the immune checkpoint inhibitor for the subject’s cancer.
  • the determining comprises: measuring expression of PD-L1 in cancer cells, immune cells, or tumor stroma cells from a tumor sample of the subject subsequent to at least one administration of the composition comprising the IMM60; and identifying the mammalian subject as having a cancer that has become a candidate for treatment with the immune checkpoint inhibitor from a measurement of PD-L1 on a percentage of the cells that is equal to or greater than the threshold or cut-off level.
  • the threshold or cut-off level is expression of PD-L1 on 1% of the cancer cells, or 5% of the cancer cells, or 10% of the cancer cells, or 25% of the cancer cells, or 50% of the cancer cells assayed for PD-L1 expression.
  • the mammalian subject is identified as a candidate from an increase in expression is an increase of at least 10%, or at least 20%, or at least 25%, or at least 50%, or at least 100%, or at least 200%, or at least 500% in the percentage of cells that express PD-L1 subsequent to the administration of the composition comprising the IMM60, compared the percentage of cells that expressed PD-L1 prior to the administration.
  • a method of cancer treatment comprising: administering, to a mammalian subject having a PD-L1-negative cancer, a composition comprising IMM60 in an amount effective to stimulate PD-L1 expression in cancer cells; and administering to the subject a composition comprising an immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway.
  • PD-1 Programmed Cell Death Protein 1
  • the method of paragraph 227 that comprises: administering, to a mammalian subject having a PD-L1-negative cancer, a composition comprising IMM60; determining that cancer in the subject has become PD-L1-positive following at least one administration of the IMM60; and administering to the subject the composition comprising the immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway after determining that cancer in the subject has become PD-L1 positive.
  • PD-1 Programmed Cell Death Protein 1
  • paragraph 227 or 228, further comprising measuring PD-L1 in cancer cells from the subject prior to initiating the administering of the composition comprising IMM60, and determining that PD-L1 expression in the cancer cells is below a threshold for classifying the cancer cells as PD-L1-negative.
  • 230 The method according to any one of paragraphs 226 - 229, further comprising measuring PD-L1 in cancer cells from the subject subsequent to initiating the administering of the composition comprising IMM60, and determining that PD-L1 expression is above a threshold or cut-off for classifying the cancer cells as PD-L1-negative.
  • a composition comprising IMM60 for use in increasing the expression of PD-L1 in cancer cells of a subject identified as having a PD-L1-negative cancer.
  • 232. The composition of paragraph 231 for use in an ordered combination treatment regimen for cancer, said treatment regimen further comprising subsequent use of a composition comprising an immune checkpoint inhibitor that targets the Programmed Cell Death Protein 1 (PD-1) pathway.
  • PD-1 Programmed Cell Death Protein 1
  • the threshold or cut-off for determining that a cancer is insensitive to PD-L1 therapy is PD-L1 expression on less than 1 % of cancer cells assayed for PD-L1 expression, or less than 5 % of cancer cells assayed for PD-L1 expression, or less than 10 % of cancer cells assayed for PD-L1 expression, or less than 25 % of cancer cells assayed for PD-L1 expression or less than 50% of cancer cells assayed for PD-L1 expression.
  • composition comprising IMM60 is a liposomal composition.
  • IMM60 is administered at a dose of 3-9 mg/m 2 .
  • 239B The method according to paragraph 238, wherein the IMM60 is administered at a dose of 30-36 mg.
  • 240 The method according to any of paragraphs 238-239B, wherein IMM60 is administered every three weeks for six cycles.
  • the immune checkpoint inhibitor is an antibody that binds to PD-1 or an antibody that binds to PD-L1.
  • the immune checkpoint inhibitor comprises Pembrolizumab, Nivolumab, or Cemiplimab.
  • the immune checkpoint inhibitor comprises Atezolizumab, Avelumab, or Durvalumab. [00237] 244.
  • the cancer comprises melanoma, non-small cell lung cancer, colorectal cancer, head and neck cancer, renal cell carcinoma, Hodgkin’s lymphoma, squamous cell carcinoma, Merkel cell cancer, cervical cancer, endometrial cancer, Primary mediastinal large B-cell lymphoma (PMBCL), basal cell carcinoma, biliary track cancer, bladder cancer, breast cancer, esophageal cancer, and mesothelioma.
  • PMBCL Primary mediastinal large B-cell lymphoma
  • the cancer is non-small cell lung cancer or melanoma.
  • the disclosure further includes a composition comprising the two or more compounds or agents co-formulated or in admixture with each other; and the disclosure further includes a kit or unit dose containing the two or more compounds/agents packaged together, but not in admixture.
  • such compositions, kits or doses further include one or more carriers in admixture with one or both agents or co-packaged for formulation prior to administration to a subject.
  • compositions useful for therapy and containing two or more therapeutic agents are described herein as compositions useful for therapy and containing two or more therapeutic agents. Equivalent methods and uses are specifically contemplated.
  • the disclosure also pertains to materials and method for improving cancer therapy with checkpoint inhibitors, including materials and methods that expand the patients or cancers that are treatable with checkpoint inhibitors and/or that improve the efficacy of treatment.
  • Figure 1A shows results of an experiment adminstering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein injected intravenously into wildtype C57 BL/or C57 BL/CD1d KO -/- (NKT cell-deficient) mice, wherein the IFN ⁇ levels were measured in serum 18 hours post-injection.
  • Figure 1B shows the results of an experiment administering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein injected intravenously into wildtype C57 BL/or C57 BL/CD1d KO -/- (NKT cell-deficient) mice, wherein the CD8levels on dendritic cells (CD11c + MHC-class II + ) were measured.
  • Figure 1C shows the results of an experiment administering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein, and in soluble form (5 ng), injected intravenously into wildtype C57 BL/or C57 BL/CD1d KO -/- (NKT cell-deficient) mice, wherein the PD-L1 levels on dendritic cells (CD11c + MHC-class II + ) were measured.
  • Figure 1D shows the results of an experiment administering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein, injected intravenously into wildtype C57 BL/(ten bars on left) or C57 BL/CD1d KO -/- (NKT cell-deficient) mice (four bars on right), wherein the CD40 levels on dendritic cells (CD11c + MHC-class II + ) were measured.
  • the CD40 levels of mice that were administered the compositions disclosed herein are compared to the CD40 levels of mice that were administered solubilized IMM60 (sol IMM60) not formulated in liposomes.
  • Figure 1E shows the results of an experiment administering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein intravenously injected into wildtype C57 BL/(ten bars on left) or C57 BL/CD1d KO -/- (NKT cell-deficient) mice (four bars on right), wherein the CD8levels on B cells were measured.
  • the CD8levels of mice that were administered the compositions disclosed herein are compared to the CD8levels of mice that were administered solubilized IMM60 (sol IMM60) not formulated in liposomes.
  • Figure 1F shows the results of an experiment administering 0.1 ng, 1 ng, and 5 ng of IMM60 formulated in liposomal compositions disclosed herein intravenously injected into wildtype C57 BL/(ten bars on left) or C57 BL/CD1d KO -/- (NKT cell-deficient) mice (four bars on right), wherein the PDL-1 levels on B cells were measured.
  • Figures 2 – 4 summarize the results of experiments involving the administering of 0.01 ng, 0.1 ng, and 1 ng of IMM60 formulated in liposomal compositions disclosed herein injected intravenously into wildtype C57 BL/or C57 BL/CD1d KO -/- (NKT cell-deficient) mice, wherein the levels of IFN ⁇ , IL-12p70, IL-2, IL-1 ⁇ , IL6, KC/GRO, TNF- ⁇ , IL-10, IL-4, and IL-5 were measured at 4 hours, 18 hours, and 72 hours post-injection.
  • FIG. 1 summarizes data from experiments performed with IMM60 formulated in POPC/DDAB liposomes.
  • Figure 3 summarizes data from experiments performed with IMM60 formulated in DSPC/DSPG liposomes.
  • Figure 4 summarizes data from experiments performed with IMM60 formulated in POPC/DC-Chol liposomes.
  • Figure 5 shows the results of an experiment of (from left to right for each formulation) 10 ng, 100 ng, 1000 nm and 5000 ng of IMM60 formulated in liposomal compositions disclosed herein intravenously injected into wildtype C57 BL/6, wherein the levels of IFN ⁇ were measured in serum 18 hours post-injection.
  • Figure shows the results of an experiment in which mice were treated with soluble IMM60 or a liposomal IMM60 formulation as disclosed herein. IFN- ⁇ was measured 18 hours post-treatment.
  • Figure 7 shows measurements of CD8on dendritic cells in mice that were treated with a liposomal IMM60 formualtion as disclosed herein compared to mice treated with soluble (free) IMM60 or vehicle-treated or untreated controls.
  • Figure 8 shows measurements of antigen-specific T cells in mice seven days after treatment with an IMM60 liposomal (lip) formulation as disclosed herein with an ovalbumin peptide antigen compared to soluble IMM60 with the antigen.
  • Figure 9A shows the results of a prophylactic dose-response experiment to evaluate the ability of DPSC/DSPG/IMM60 liposomes to inhibit metastatic nodules in the lungs.
  • mice were injected with the liposomal IMM60 formulation at the specified concentrations three days before injection with cells from a melanoma tumor cell line.
  • Figure 9B shows the results of a similar, therapeutic dose-response experiment. Mice were injected with the liposomal IMM60 formulation at the specified concentrations three days after injection with the cells from the melanoma tumor cell line.
  • FIG 10 shows the results of a dose-response experiment in mice treated with an IMM60 liposomal formulation disclosed herein, alone or in combination with anti-PD-1 antibodies, in a seperate subcutaneous melanoma mouse model.
  • IFN- ⁇ was measured 18 hours following the first treatment dose.
  • Ab antibody
  • IFN ⁇ interferon-gamma
  • PD- 1 programmed cell death protein-1.
  • Figure 11 shows the results of tumor growth experiment in mice treated with the liposomal formulation disclosed herein, anti-PD-1 blocking antibodies, or both, in a subcutaneous melanoma mouse model.
  • Figures 12A and 12B show the results of longitudinal tumor growth kinetics in the subcutaneous CT-2tumor model in mice treated with liposomal formulation disclosed herein, anti- PD-1 blocking antibodies, or both.
  • Figure 12C shows the results at day 14 of longitudinal tumor growth kinetics in the subcutaneous CT-2tumor model in mice treated with liposomal formulation disclosed herein, anti-PD-1 blocking antibodies, or both.
  • IFN interferon
  • PD-1 programmed cell death protein-1.
  • Figure 13 depicts the results of a study (Example 9) in which PDL-1 expression was measured on Melanoma B16f10 cells following exposure of the cells to single agents and agent combinations at different concentrations.
  • Figure 14 depicts the effect of IMM60 at different concentrations, alone or in combination with anti-PD-1 antibody, on T cells in B16f10:splenocyte cocultures.
  • Figures 15A and 15B depict the schema for Phase 1 and Phase 2 clinical protocols in Example 13.
  • Figures 16-25 depict preliminary results from the ascending dose arm of the clinical protocol in Example 12 described herein.
  • Figure 16 shows the 24 h exposure results from the human pharmacokinetics study described in Example 12.
  • Figure 17 shows the exposure data from Figure 16 normalized for dose group.
  • Figure 18 shows the total exposure results in AUC ⁇ from the human pharmacokinetics study described in Example 12.
  • Figure 19 shows the total exposure results in AUC ⁇ normalized for actual dose from the human pharmacokinetics study described in Example 12.
  • Figure 20 shows the relative exposure in AUC ⁇ by dose group from the human pharmacokinetics study described in Example 12.
  • Figure 21 shows the C max by dose group from the human pharmacokinetics study described in Example 12.
  • Figure 22 shows the terminal half-life by dose group from the human pharmacokinetics study described in Example 12.
  • FIG. 23 shows the total exposure in AUC ⁇ by dose group normalized by actual dose from the human pharmacokinetics study described in Example 12.
  • Figure 24 shows the Cmax by dose group normalized by actual dose from the human pharmacokinetics study described in Example 12.
  • Figure 25 shows the mean residence time (MRT) by dose group from the human pharmacokinetics study described in Example 12.
  • MRT mean residence time
  • compositions that comprise the liposomes and a pharmaceutically acceptable diluent, excipient, adjuvant, or carrier.
  • Liposomes provided herein provide for a more concentrated and potent delivery form for Compound A; and provide for stability of the compound. Additionally, the liposomal formulation is contemplated to improve absorption, distribution, metabolism and/or elimination (ADME) of Compound A, especially for treatment of cancer tumors.
  • ADME absorption, distribution, metabolism and/or elimination
  • some of the advantages of liposomal formulation described herein include an EPR effect (enhanced permeability and retention), a longer in vivo half-life, a lower volume of distribution, and better toleration (reduction in adverse events).
  • Compound A iNKT modulators
  • a salt, ester, solvate, or hydrate thereof iNKT modulators
  • the compounds categorized herein as Compound A and methods of making them are described in International Patent Application No. PCT/EP2012/074140, filed 30 November 2012, published as International Publication No. WO 2013/079687, “INKT Cell Modulators and Methods of Using the Same,” incorporated herein by reference in its entirety. See also U.S. Patent No.9,365,496, incorporated herein by reference.
  • the salts, e.g., pharmaceutically acceptable salts, of the disclosed therapeutics may be prepared by reacting the appropriate base or acid with a stoichiometric equivalent of the therapeutic.
  • Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids.
  • inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid
  • Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate
  • pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid.
  • Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • Pharmaceutically acceptable salts of Compound A may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible. Examples of metals used as cations are sodium, potassium, magnesium, ammonium, calcium, or ferric, and the like.
  • Suitable amines include isopropylamine, trimethylamine, histidine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.
  • pharmaceutically acceptable derivatives e.g., esters
  • metabolites, hydrates, solvates and prodrugs of the therapeutic may be prepared by methods generally known to those skilled in the art.
  • another embodiment provides compositions that include prodrugs of active Compound A.
  • a prodrug is a compound which is metabolized in vivo (e.g., by a metabolic transformation such as deamination, dealkylation, de-esterification, and the like) to provide an active compound.
  • a “pharmaceutically acceptable prodrug” means a compound which is, within the scope of sound medical judgment, suitable for pharmaceutical use in a patient without undue toxicity, irritation, allergic response, and the like, and effective for the intended use, including a pharmaceutically acceptable ester as well as a zwitterionic form, where possible, of the therapeutic.
  • ester refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than carbon atoms.
  • Representative examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
  • compositions including Compound A described herein may also include metabolites.
  • the term “metabolite” means a product of metabolism of Compound A or a pharmaceutically acceptable salt, analog, or derivative thereof, that exhibits a similar activity in vitro or in vivo to a disclosed therapeutic.
  • the compositions described herein may also include hydrates and solvates.
  • solvate refers to a complex formed by a solute (herein, the therapeutic) and a solvent.
  • solvents for the purpose of the embodiments preferably should not negatively interfere with the biological activity of the solute.
  • Solvents may be, by way of example, water, ethanol, or acetic acid.
  • the compositions disclosed herein can stimulate an immune response in a subject, e.g., a mammalian subject. In various cases, the compositions stimulate iNKT cells in a subject. In various cases, the compositions stimulate an immune response, as measured by an in vitro assay. In various cases, the compositions stimulate an immune response in a subject in vivo.
  • liposomes and Liposomal Formluations Described herein are liposomal formulations of iNKT modulators such as those referred to herein as Compound A. Also described herein are liposomes comprised of a phospholipid bilayer and a lumen, the phospholipid bilayer comprising (a) at least two phospholipids selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl- sn-glycero-3-phospho-rac-glycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1-glycerol)] (DPPG); and (b) at least one therapeutic agent, wherein the phospholipid bilayer is substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (P
  • polyethylene glyocolated lipid refers to a lipid that has been chemically modified (e.g., via conjugation) to comprise a PEG moiety or PEG functional group(s).
  • Liposomes are lipid-containing vesicles that include one, two, or several concentric lipid bilayers (typically comprised of phospholipids) enclosing an aqueous core, and can be used to deliver both hydrophilic and hydrophobic active pharmaceutical ingredients (APIs) to the body. Small, hydrophilic active pharmaceutical ingredients can be encapsulated within the aqueous core (cavity) of a liposome, e.g., in solution or suspension.
  • Both charged, hydrophilic active pharmaceutical ingredients and uncharged, hydrophobic active pharmaceutical ingredients can be associated with the membrane of a liposome through electrostatic or hydrophobic interactions, respectively, or through a covalent bond.
  • Liposomes can deliver active pharmaceutical ingredients to the body by, for example, fusion of the liposome with other bilayers, such as the cell membrane, diffusion of the compound out of the liposome, or digestion of the liposome within macrophages.
  • Numerous methods for liposomal manufacture are known, typically involving applying some form of energy to a dispersion of phospholipids in a polar solvent. Exemplary methods include or involve sonication, extrusion, microfluidization, and heating.
  • the disclosure provides a method or preparing a liposome comprising a phospholipid bilayer and a lumen, the phospholipid bilayer comprising (a) at least two phospholipids selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1-glycerol)] (DPPG); and (b) at least one therapeutic agent, wherein the phospholipid bilayer is substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids), wherein the method comprises 1) admixing the therapeutic agent and the at least two phospholipids to form multi- lamellar ve
  • the disclosed methods comprise extruding the multi-lammellar vesicles through a membrane to form the liposomes.
  • the membrane has a suitable pore size (e.g., about 50 nm to about 250 nm, about 50 nm to about 150 nm, or about 60 nm to about 100 nm, or about 80 nm).
  • the disclosed methods further comprise purifying the liposomes.
  • the liposomes can be purified using any suitable technique.
  • the liposomes are purified using filtration techniques. Suitable filtration techniques include, for example, ultrafiltration, centrifugation, dialysis, diafiltration, or tangential flow filtration (TFF).
  • the liposomes are filtered using a suitable pore size.
  • suitable pore sizes include, for example, a pore size of about 450 nm or less, or about 400 nm or less, about 350 nm or less, or about 300 nm or less, or about 250 nm or less, or about 220 nm or less, or about 200 nm or less, or about 150 nm or less.
  • the filter has a molecular weight cut off (MWCO) of about 500 kD or less (e.g., about 450 kD, about 400 kD, about 350 kD, or about 300 kD, or about 250 kD).
  • MWCO molecular weight cut off
  • liposomes can have a variety of sizes, e.g., an average diameter as low as 25 nm or as high as 10,000 nm or more. Size is affected by a number of factors, such as, for example, lipid composition and method of preparation, and is determined by a number of techniques, such as dynamic light scattering (DLS). Various methodologies can be used to prepare liposomes of a smaller size from larger liposomes, such as sonication, homogenization, French Press application, and milling. Extrusion (see, e.g., U.S. Pat.
  • the liposomal lipid composition can be tailored to deliver specific APIs or therapeutic agents to targeted locations, which limits the potential toxicity of the ingredients. Further, liposomes allow the delivery of known amounts of APIs because they minimize loss of the API from, for example, degradation, removal by non-target organs, and precipitation, as seen with detergents. Liposomes also provide a method for co-delivering adjuvants and antigens in particular ratios that otherwise cannot occur by, for example, coinjection, as described in U.S. Patent Nos.7,850,990 and 7,842,676, each incorporated herein by reference.
  • a composition including a liposome, wherein the liposome comprises Compound A or a salt, ester, solvate, or hydrate thereof and two or more lipids, and a pharmaceutically acceptable diluent, excipient, adjuvant, or carrier.
  • the liposome includes a lipid bilayer membrane surrounding an aqueous core, wherein the lipid bilayer membrane comprising two or more lipids (e.g., 2, 3, 4, 5, 6, 7, or 8 lipids).
  • liposomes comprised of Compound A and two or more lipids; exemplary pairs of lipids for making the liposomes include: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG); (b) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB); (d) L- ⁇ -phosphatidylglycerol (DSPC) and 1,2-distea
  • the disclosure also contemplates liposomes comprising at least one therapeutic agent and at least two phospholipids selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); and 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1-glycerol)] (DPPG), wherein the phospholipid biylater is substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol
  • DPPC 1,2-dipalmitoyl
  • the disclosed liposomes may comprise at least two (e.g., 3) phospholipids selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl- sn-glycero-3-phospho-rac-glycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1-glycerol)] (DPPG).
  • the phospholipids of the liposomes disclosed herein consist essesentially of DSPC and DSPG.
  • the liposome comprises a phospholipid bilayer that is substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipds (PEG lipids).
  • the term “substantially” refers to little or none of the recited components being present (e.g., 2 wt% or less present, 1.5 wt% or less, 1.0 wt.% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, 0.2 wt% or less, or 0.1 wt% or less present).
  • the disclosed liposomes comprise a phospholipid bilayer having a phase transition temperature above about 37 °C. In some embodiments, the disclosed liposomes are free of of phospholipids having a phase transition temperature below about 37 °C. Moreover, in some embodiments, the disclosed liposomes are free of unsaturated lipids or unsaturated phospholipids. [00299] In some embodiments, the disclosed liposomes are free of lipids or phospholipids having a chain length shorter than 16 carbon atoms. For example, in these embodiments, the liposomes are free of lipids or phospholipids comprising C14, C12, C10 fatty acids, and the like.
  • the disclosed liposomes substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids) provide many advantages over conventional delivery systems, including other conventional liposomal delivery systems.
  • the disclosed liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids provide a delayed release of the payload (e.g., at least one therapeutic agent) and thereby are capable of providing a modified release (e.g., delayed-release, extended-release, sustained-releae) of the payload via intravenous infusion.
  • the pharmacokinetic properties when administered to humans demonstrate that the disclosed liposomes provide a C max over a period of about 1-6 hours (e.g., about 4-6 hours).
  • the disclosed liposomes substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids) advantageously exhibit a long half-life (e.g., about 6-12 hours) and have relative long residence times in the blood compartment (e.g., 5- 10 times longer than other non-PEGylated liposomes) thereby providing improved delivery of the payload and long exposure times to the payload (e.g., higher exposure per dose) to the intended target site (e.g., tumor site).
  • PEG lipids polyethylene glycolated lipids
  • the disclosed liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids have a terminal half-life of about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In some embodiments, the liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids have a terminal half-life of about 8 hours.
  • the liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids have a steady state volume of distribution in a mammalian subject (e.g., human) of about 2 liters to about 12 liters, or about 2 liters to about 10 liters, or about 2 liters to about 8 liters, after being administered to the mammalian subject intravenously.
  • a mammalian subject e.g., human
  • the liposomes SCD and/or compositions comprising said liposomes have a steady state volume of distribution in a mammalian subject of about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, about 10 liters, about 11 liters, or about 12 liters.
  • the liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids have a terminal volume of distribution in a mammalian subject (e.g., a human) of about 3 liters to about 10 liters (e.g., about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters).
  • the liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids and/or compositions comprising said liposomes have a terminal volume of distribution of about 2 liters to about 9 liters, about 3 liters to about 8 liters, about 4 liters to about 7 liters, or about 5 liters to about 6 liters.
  • the liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids provide a steady state plasma concentration of therapeutic agent between about 2 hours and about 10 hours (e.g., about 3 h to about 8 h or about 4 h to about 6 h), after administration to a mammalian subject (e.g., human).
  • the therapeutic or active agent delivered by liposomes substantially devoid of cholesterol, plant sterols, and PEG lipids disclosed herein is not substantially cleared from plasma between about 2 hours and about 10 hours, or between about 3 hours to about 8 hours, or between about 4 to about 6 hours, after administration to a mammalian subject (e.g., human).
  • substantially cleared refers to [00305]
  • the disclosed liposomes further provide an improved safety profile compared to conventional liposomes.
  • the disclosed liposomes advantageously exhibit substantially less off-target toxicity even when administered at relative high doses.
  • the disclosed liposomes do not exhibit any off-target toxicity.
  • the liposomes disclosed herein substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids), advantageously do not invoke an immune response that can be observed with liposomes comprising (PEG)lipids thereby providing yet a further improved safety profile.
  • PEG lipids polyethylene glycolated lipids
  • the disclosed liposomes have a size and rigidity that allow them to preferentially pass through “leaky” vasculatures of tumor cells thereby providing greater localization of payload (e.g., anticancer agent) compared to conventional liposomes.
  • the phenotype of the liposome is “squishy”, e.g., compressible and high(er) membrane fluidity.
  • the disclosed liposomes have a higher melting temperature (T m ) than conventional liposomes (e.g., about 55 °C) due at least in part to the liposomes comprising saturated lipids.
  • T m melting temperature
  • conventional liposomes e.g., about 55 °C
  • the liposomes in the composition have a mean diameter of less than or equal to about 500 nm, or less than about 400 nm, or less than about 300 nm, or less than about 200 nm, or less than about 150 nm, or less than about 125 nm, wherein the diasmeter is measured as a Z-average at 900. Accordingly, in some embodiments the liposomes have a mean diameter within a size range of about 125 nm to about 500 nm, or about 150 nm to about 400 nm, or about 200 nm to about 300 nm.
  • the liposomes in the composition have a mean diameter of less than about 200 nm. In some embodiments, the liposomes in the composition have a mean diameter within a size range of about 50 nm to about 200 nm, or about 50 nm to about 170 nm, or about 70 nm to about 130 nm, or about 60 nm to about 160 nm, or about 70 nm to about 150 nm, or about 75 nm to about 145 nm, or about 90 nm to about 130 nm, or about 75 nm to about 145 nm.
  • the liposomes in the composition have a mean diameter of about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm.
  • the liposomes in the composition can have a mean diameter standard deviation of about 60 nm or less, or about 40 nm, for example, the composition has a mean liposome diameter of about 100 nm ⁇ 60 nm , of about 100 nm ⁇ 40 nm, or about 110 nm ⁇ 40 nm.
  • the mean diameter of the liposomes in the composition is determined by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the liposomes in the composition have a mean diameter falling within a size range, with the lower end of the size range being any size selected from about 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 5nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm,63 nm, 64 nm, 65 nm, 6nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 7nm, 77 nm, 78 nm, 79 nm, 80 nm , 81 nm, 82 nm, 83 nm, 84 n
  • the mean diameter of the liposomes in the composition is within a range of about 50 nm to about 200 nm. In embodiments, the mean diameter of the liposomes in the composition is within a range of about 50 nm to about 170 nm, or about 70 nm to about 150 nm, or about 90 to about 130 nm, or about 110 nm ⁇ 60 nm, or about 110 nm ⁇ 40 nm, or about 110 nm ⁇ 20 nm.
  • the composition exhibits a measure of uniformity in size/mass of the liposomes in the formulation, which can be expressed by measurement (or derivation) of polydispersity index (PdI).
  • PdI is defined as the square of the standard deviation of the particle diameter distribution divided by the mean particle diameter, as set forth in the following formula, in which “a” is the radius of the particle: PdI is used to estimate the uniformity of a particle distribution, and larger PdI values correponsd to a larger size distrubtion in the particles of the sample.
  • the composition has a PdI less than or equal to about 0.4, or less than or equal to about 0.35, or less than or equal to about 0.3, or less than or equal to about 0.25, or less than or equal to about 0.23, or less than or equal to about 0.22, or less than or equal to about 0.21, or less than or equal to about 0.20, or less than or equal to about 0.19, or less than or equal to about 0..18, or less than or equal to about 0.17, or less than or equal to about 0.16, or less than or equal to about 0.15, or less than or equal to about 0.14 or less than or equal to about 0.13 or less than or equal to about 0.12, or less than or equal to about 0.11 or less than or equal to about 0.10.
  • the PdI is in the range of 0.01 – 0.40; or in the range of 0.02 – 0.35; or in the range of 0.03 – 0.30; or in the range of 0.04 – 0.25; or in the range of 0.05 – 0.20; or in the range of 0.0– 0.15.
  • particle diameter (or radius) obtained by particle size measurements by Dynamic Light Scattering at 90° is used.
  • Compound A can be present in the liposome in any amount that provides one or more of: a liposome having a similar efficacy as a corresponding soluble, Compound A that is not incorporated within a liposome, or provides a superior efficacy; or provides a therapeutic window (between minimum effective concentration and toxic concentration). In some embodiments, Compound A is present in the liposome in an amount of about 0.1 wt.% to about 20 wt.%, based on the total weight of the liposome.
  • Compound A is present in an amount of about 0.5 wt.% to about 20 wt.%, or about 3 wt.% to about 8 wt.%, or about 4 wt.% to about 13 wt.%, based on the total weight of the liposome.
  • Compound A can be present in an amount of about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, about 12 wt.%, about 13 wt.%, about 14 wt.%, about 15 wt.%, about 1wt.%, about 17 wt.%, about 18 wt.%, about 19 wt.%, about 20 wt, based on the total weight of the liposome.
  • Compound A is present in an amount of about 0.5 wt.% to about 15 wt.%, or about 1 wt.% to about 12 wt.%, or about 5 wt.% to about 12 wt.%, based on the total weight of the liposome.
  • a weight percentage of Compound A is chosen to produce an effective immunomodulatory response with minimal toxic side effects. If too little Compound A is present relative to total liposome, the liposome will not stimulate a sufficiently robust response. If too much of Compound A is present relative to total liposome, the excess Compound A will compete for binding sites and may cause the liposomes to block each other from binding.
  • the liposomal composition is provided in a concentrated formulation.
  • a formulation is contemplated that includes Compound A in a concentration of 0.01 to 50 mg/ml. Within this range, all subranges are contemplated.
  • subranges with exemplary lower end concentrations of 0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.075 mg/ml, 0.1 mg/ml, 0.125 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.25 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.75 mg/ml, and 1.0 mg/ml are contemplated; and subranges with exemplary upper end concentrations (that are higher than the selected lower end concentration) of 0.5 mg/ml, 0.75 mg/ml, .0 mg/ml, 1.25 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 5.0 mg/ml, 10 mg/ml, 5 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, and 40 mg/ml are contemplated.
  • a lower concentration liposomal composition is contemplated.
  • the lower concentration composition is formulated by dilution of a concentrated formulation with a pharmaceutically acceptable diluent, such as an isotonic saline solution, for purposes of administration.
  • a pharmaceutically acceptable diluent such as an isotonic saline solution
  • the composition includes Compound A in a concentration of about 0.001 ng/mL to about 10,000 ng/mL.
  • compositions are contemplated that include Compound A in a concentration of about 0.01 ng/mL to about 5000 ng/mL, or about 0.1 ng/mL to about 5000 ng/mL, or about 1 ng/mL to about 1000 ng/mL, or about 10 ng/mL to about 100 ng/mL.
  • Exemplary concentrations of Compound A include 0.01 ng/mL, 0.5 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 0.75 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 0.75 ng/mL, 1 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 7.5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1 ⁇ g/ml, 2 ⁇ g/ml, 2.5 ⁇ g/ml, 5 ⁇ g/ml, 7.5 ⁇ g/ml, 10 ⁇ g/ml, 25 ⁇ g/ml, 50 ⁇ g/ml, 75 ⁇ g/ml, and
  • the two or more lipids as disclosed herein can include a lipid including a saturated distearoyl moiety and a lipid including a phosphoglycerol moiety.
  • At least one lipid is selected from phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoylphosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DMPS), distearoyl phosphatidy
  • the lipid including the saturate distearoyl moiety can further include a phophocholine moeity, a phosphoglycerol moiety, a phosphate moiety, a phosphotidylethanolamine, a phosphotidyl acetamide moiety, a polyethylene glycol moiety, or the like.
  • a sterol is contemplated, such as cholesterol, ergosterol, lanosterol, and combinations thereof.
  • the lipid including the phosphoglycerol moiety can further include a saturated distearoyl moiety, a dimyristoyl moiety, or palmitoyl moiety.
  • the two or more lipids disclosed herein can include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).
  • the two or more lipids as disclosed herein can include any of the following lipid pairs: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG); (b) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB); (d) L- ⁇ -phosphatidylglycerol (EPG) and 1,2-dioleoyl-
  • the two or more lipids as disclosed herein can include any of the following lipid pairs: (g) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC); (h)1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero- [phosphor-rac-(1-glycerol)] (DPPG); or (i) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero- [phosphor-rac-(1-glycerol)] (DPPG).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero
  • the two or more lipids comprise 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB).
  • POPC 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine
  • DDAB dimethyldioctadecylammonium bromide
  • the liposome comprises two lipids including 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).
  • the two or more lipids can include (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG); (b) 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and 3ß-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Chol); or (c) 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl-sn-glycero-3-phospho
  • the two or more lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2- distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).
  • the the two or more lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG), and cholest-5-en-3 ⁇ -ol (CHOL).
  • the liposomes are substantially devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids). As used herein, “substantially devoid” refers to less than 10 wt%In some embodiments, the liposomes are completely devoid of cholesterol, plant sterols, and polyethylene glycolated lipids (PEG lipids).
  • the two or more lipids comprised in the liposome can further comprise any lipid that is capable of forming a liposome with Compound A described herein, as long as the resulting liposome has at least a similar or increased efficacy as a corresponding soluble Compound A that is not incorporated within a liposome.
  • one or more lipids as described in U.S. Patent Application Publication No.2014/0050780A1, which is incorporated herein by reference can be included in the liposome.
  • the liposome can include three lipids.
  • the liposome can include four lipids.
  • the two or more lipids each can independently have a net positive charge (i.e., a cationic lipid), a net negative charge (i.e., an anionic lipid), no charges (i.e, a nonionic lipid), or an equal number of positive and negative charges (i.e., a zwitterionic lipid).
  • the lipids can include those lipids that form a bilayer themselves or with Compound A, and those lipids that do not form a bilayer alone, but they can be included as part of a stable bilayer made from one or more other lipids.
  • any lipid described herein is additionally contemplated to be a salt thereof, for example, an ammonium salt, a sodium salt, a sodium/ammonium salt, a lithium salt, a potassium salt, or the like.
  • the amount of lipid that can be present in the liposome is about 75 wt.% to about 99.9 wt.%, or about 80 wt.% to about 98 wt.%, or about 80 wt.% to about 95 wt.%, or about 85 wt.% to about 95 wt.%, based on the total weight of the liposome.
  • the lipid is present in an amount of about 75 wt.% to about 99 wt.%, or about 75 wt.% to about 98 wt.%, or about 75 wt.% to about 95 wt.%, or about 87 wt.% to about 93 wt.%, based on the total weight of the liposome.
  • the lipid can be present in an amount of about 75 wt.%, about 7wt.%, about 77 wt.%, about 78 wt.%, about 79 wt.%, about 80 wt.%, about 81 wt.%, about 82 wt.%, about 83 wt.%, about 84 wt.%, about 85 wt.%, about 8wt.%, about 87 wt.%, about 88 wt.%, about 89 wt.%, about 90 wt.%, about 91 wt.%, about 92 wt.%, about 93 wt.%, about 94 wt.%, about 95 wt.%, about 9wt.%, about 97 wt.%, about 98 wt.%, about 99 wt.%, about 99.5 wt.%, or about 99.9 wt.%, based on the total weight of the lip
  • the mass ratio of Compound A to the two or more lipids can be about 1:5 to about 1:20, or about 1:7 to about 1:15, or about 1:7 to about 1:12. In some embodiments, the mass ratio of Compound A to the two or more lipids can be about 1:8 to about 1:10, or about 1:9. For example, the mass ratio of Compound A to the two or more lipids can be about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20.
  • the two or more lipids includes 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG)
  • the DSPC is present in an amount of about 40 wt.% to 80 wt.%, or about 40 wt.% to about 60 wt.%, or about 45 wt.% to about 55 wt.%, based on the total weight of the liposome.
  • the DSPC is present in an amount of about 40 wt.%, about 45 wt.%, about 50 wt.%, about 55 wt.%, about 60 wt.%, about 65 wt.%, about 70 wt.%, about 75 wt%, or about 80 wt.%, based on the total weight of the liposome.
  • the two or more lipids comprise 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG)
  • the DSPG is present in an amount of about 20 wt.% to 60 wt.%, or about 30 wt.% to about 50 wt.%, or about 35 wt.% to about 45 wt.%, based on the total weight of the liposome.
  • the DSPG is present in an amount of about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.%, about 50 wt.%, about 55 wt.%, or about 60 wt.%, based on the total weight of the liposome.
  • the two or more lipids further includes CHOL.
  • the two or more lipids includes DSPC, DSPG, and CHOL, wherein CHOL is present in an amount of about 1 wt.% to about 20 wt.%.
  • the CHOL can be present in an amount of about 5 wt.% to about 15 wt.%, or about 8 wt.% to about 12 wt.%, such as about 10 wt.%.
  • the two or more lipids include DSPC, DSPG, and CHOL, wherein DSPC is present in an amount of about 40 wt% to about 50 wt%, the DSPG is present in an amount of about 30 wt% to about 40 wt%, and the CHOL is present in an amount of about 5 wt% to about 15 wt%, such as DSPC in an amount of about 45 wt%, DSPG in an amount of about 35 wt%, and CHOL in an amount of about 10 wt%.
  • the two or more lipids include 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 3ß-[N-(N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol).
  • POPC 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine
  • DC-Chol 3ß-[N-(N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • the POPC is present in an amount of about 40 wt.% to 80 wt.%, or about 45 wt.% to about 65 wt.%, or about 50 wt.% to about 60 wt.%, based on the total weight of the liposome.
  • the DC-Chol is present in an amount of about 15 wt.% to 55 wt.%, or about 20 wt.% to about 50 wt.%, or about 25 wt.% to about 35 wt.%, based on the total weight of the liposome.
  • the two or more lipids include 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB).
  • the POPC is present in an amount of about 40 wt.% to 80 wt.%, or about 45 wt.% to about 65 wt.%, or about 50 wt.% to about 60 wt.%, based on the total weight of the liposome.
  • the DC-Chol is present in an amount of about 15 wt.% to 55 wt.%, or about 20 wt.% to about 50 wt.%, or about 30 wt.% to about 40 wt.%, based on the total weight of the liposome.
  • the two or more lipids include L- ⁇ -phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC).
  • EPG L- ⁇ -phosphatidylglycerol
  • EPC 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
  • the EPG is present in an amount of about 40 wt.% to 80 wt.%, or about 55 wt.% to about 75 wt.%, or about 60 wt.% to about 70 wt.%, based on the total weight of the liposome.
  • the EPC is present in an amount of about 10 wt.% to 50 wt.%, or about 15 wt.% to about 40 wt.%, or about 15 wt.% to about 30 wt.%, based on the total weight of the liposome.
  • the two or more lipids comprise 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • the POPC is present in an amount of about 40 wt.% to 80 wt.%, or about 40 wt.% to about 65 wt.%, or about 45 wt.% to about 55 wt.%, based on the total weight of the liposome.
  • the DOTAP is present in an amount of about 20 wt.% to 50 wt.%, or about 25 wt.% to about 45 wt.%, or about 35 wt.% to about 45 wt.%, based on the total weight of the liposome.
  • the two or more lipids include 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG) and cholest-5-en-3 ⁇ -ol (CHOL).
  • DMPG 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol
  • CHOL cholest-5-en-3 ⁇ -ol
  • the DMPG is present in an amount of about 40 wt.% to 80 wt.%, or about 50 wt.% to about 75 wt.%, or about 60 wt.% to about 70 wt.%, based on the total weight of the liposome.
  • the CHOL is present in an amount of about 10 wt.% to 50 wt.%, or about 15 wt.% to about 35 wt.%, or about 15 wt.% to about 30 wt.%, based on the total weight of the liposome.
  • the liposome further comprises a liposomal buffer.
  • the liposomal buffer can include phosphate, sodium chloride, sucrose, or a combination thereof.
  • the liposomal buffer includes sodium phosphate and sodium chloride.
  • the liposomal buffer includes sodium chloride and sucrose.
  • the liposomal buffer includes sodium chloride, phosphate and sucrose.
  • the liposomal buffer is pH of about to 7, or about 6.3 to about 6.7, such as 6.5.
  • Antigen-Carrying Liposomes [00342]
  • the liposomes described herein comprise at least one antigen, such as, for example, a viral antigen, a bacterial antigen, a fungal antigen, a tumor antigen, or mixtures thereof. The exact amount of the antigen with respect to the liposome depends on the composition and purpose of the antigen, and can be determined by one skilled in the art.
  • the liposomes described herein can be used as immunostimulants or adjuvants to produce a protective immune response, such as a B-cell response, an IgG antibody response, a T- cell response, or a CTL response to the administered antigen.
  • the antigen can be any antigenic material that is suitable for treatment of a particular disease.
  • the liposome described herein comprises an antigen and is used to treat cancer.
  • the antigen can be a full-length protein antigen, a long peptide antigen (i.e., a peptide that comprises at least 25 amino acids, such as 27-75, 25-50, 25-40, or 25-30 amino acids), or a short peptide antigen (i.e., a peptide that comprises 6-25 amino acids, such as 6-25, 8- 25, 10-25, or 15-20 amino acids).
  • a long peptide antigen i.e., a peptide that comprises at least 25 amino acids, such as 27-75, 25-50, 25-40, or 25-30 amino acids
  • a short peptide antigen i.e., a peptide that comprises 6-25 amino acids, such as 6-25, 8- 25, 10-25, or 15-20 amino acids.
  • the antigen can be a tumor associated peptide or protein that induces or enhances immune response and is derived from tumor associated genes and encoded proteins including, for example, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1,
  • fusion protein PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, Lü-1, Mage-C2, NA-88, /Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens Eand E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c- met, nm-23H1, PS
  • antigenic peptides characteristic of tumors include those listed in International Patent Application Publication No. WO 20000/020581 and U.S. Patent Application Publication No.2010/0284965, which are each incorporated herein by reference.
  • the antigen is a tumor antigen selected from the group consisting of MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, and mixtures thereof.
  • the tumor antigen is selected from the group consisting of P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE- A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE- C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA
  • the tumor antigen includes NY-ESO-1 or an antigenic fragment thereof.
  • the tumor antigen is a mammalian protein.
  • the tumor antigen is a human protein.
  • the full-length protein is employed as the antigen.
  • peptides comprising an antigenic fragment of these proteins is used as the tumor antigen.
  • antigenic fragments of these antigens are used as the tumor antigen. [00344] Small, hydrophilic antigens can be encapsulated within the core of the liposome.
  • Small or large hydrophobic antigens can be noncovalently associated (e.g., through hydrophobic interactions) with the nonpolar portion of the lipid bilayer, small or large charged antigens can be attached (e.g., through electrostatic interactions) to a charged portion on the outside of the lipid bilayer, and small or large hydrophobic or hydrophilic antigens also can be covalently linked to any portion of the liposomal membrane.
  • the liposome described herein is carrying an antigen
  • the lipid used to form the liposome is dictated by the size and charge of the antigen (e.g., small, hydrophilic, hydrophobic, positively charged, or negatively charged antigens).
  • At least one lipid of the two or more lipids preferably has a net negative charge and is selected from the group consisting of an anionic sphingosine (e.g., a dimethyl sphingosine-1-phosphate, a ceramide phosphate, a dihydroceramide phosphate, a ganglioside, and a sulfatide), an anionic phospholipid (e.g., a phosphatidic acid, a phosphatidylglycerol, a phosphatidylinositol, an inositol phosphate, a cardiolipin, a bis(monoacylglycero)phosphate, an anionic detergent that is not a sphingolipid or a phospholipid, and an anionic bioactive lipid, such as, for example, an adjuvant, a liponucle
  • an anionic sphingosine e.g., a dimethyl sphingosine-1-phosphat
  • the cation lipid can be cationic sphingosine (e.g., a trimethyl sphingosine, a trimethyl phytosphingosine, and a pyridinium ceramide), a cationic lipid that is not a sphingosine (e.g., 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol ⁇ HCl), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (18:1 TAP, DOTAP), 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate salt) (18:1 T
  • Lipids that can be used to produce a liposome having a bilayer that can non-covalently associate with a hydrophobic antigen include, but are not limited to, phosphatodylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin and phosphatidic acid (PA).
  • Therapeutic agents [00349] In some embodiments, the liposome further comprises at least one therapeutic agent in the core of the liposome.
  • the at least one therapeutic agent is selected from the group of an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and combinations thereof.
  • the composition further comprises at least one therapeutic agent.
  • the at least one therapeutic agent is selected from the group of an immune modulator, a Toll-like receptor agonist, a Nod ligand, an anti-viral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immune therapeutic, a chemotherapy agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine agent, an anti- inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and combinations thereof.
  • the therapeutic agent comprises a hydrophobic or amphiphilic agent that intercalates or integrates into the phospholipid bilayer.
  • the at least one therapeutic agent includes a hydrophilic agent contained in the lumen of the liposome.
  • the therapeutic agent is selected from small molecules, nucleic acids, RNA, DNA, aptamers, proteins, peptides, and a mixture thereof.
  • the therapeutic agent comprises an RNA.
  • the therapeutic agent comprises a short interfering RNA (siRNA) useful for modulating gene expression in a target cell.
  • the therapeutic agent comprises a messenger RNA (mRNA) for expression in a cell.
  • the RNA is a micro RNA (miRNA).
  • the therapeutic agent comprises an anti-cancer therapeutic.
  • the therapeutic agent comprises an immunostimulant or encodes an immunostimulant.
  • the therapeutic agent when present, is present in a suitable amount.
  • the therapeutic agent(s) can be present in the liposome in any amount that provides one or more of: a liposome having a similar efficacy as the therapeutic agent(s) that is not incorporated within a liposome, or provides a superior efficacy; or provides a therapeutic window (between minimum effective concentration and toxic concentration).
  • the therapeutic agent is present in the liposome in an amount of about 0.1 wt.% to about 20 wt.%, based on the total weight of the liposome.
  • the therapeutic agent is present in an amount of about 0.5 wt.% to about 20 wt.%, or about 3 wt.% to about 8 wt.%, or about 4 wt.% to about 13 wt.%, based on the total weight of the liposome.
  • the therapeutic agent can be present in an amount of about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, about 12 wt.%, about 13 wt.%, about 14 wt.%, about 15 wt.%, about 1wt.%, about 17 wt.%, about 18 wt.%, about 19 wt.%, about 20 wt, based on the total weight of the liposome.
  • the therapeutic agent is present in an amount of about 0.5 wt.% to about 15 wt.%, or about 1 wt.% to about 12 wt.%, or about 5 wt.% to about 12 wt.%, based on the total weight of the liposome.
  • a weight percentage of therapeutic agent is chosen to produce an effective therapeutic response with minimal toxic side effects. If too little therapeutic agent is present relative to total liposome, the liposome will not stimulate a sufficiently robust response. If too much of the therapeutic agent is present relative to total liposome, the excess therapeutic agent may compete for binding sites and may cause the liposomes to block each other from binding.
  • compositions described herein include a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier.
  • the disclosed compositions comprising Compound A and/or therapeutic agent(s) is administered by any route that permits treatment of the disease or condition.
  • composition comprising Compound A may be delivered to a patient using any standard route of administration, including parenterally, such as intravenously (IV), intraarterially (IA), perivascularly, intraperitoneally (IP), intrapulmonary, subcutaneously (SC) or intramuscularly (IM), intrathecally, topically, transdermally, rectally, orally, nasally or by inhalation.
  • parenterally such as intravenously (IV), intraarterially (IA), perivascularly, intraperitoneally (IP), intrapulmonary, subcutaneously (SC) or intramuscularly (IM), intrathecally, topically, transdermally, rectally, orally, nasally or by inhalation.
  • IV intravenously
  • IA intraarterially
  • IP intraperitoneally
  • IP intrapulmonary
  • SC subcutaneously
  • IM intramuscularly
  • intrathecally topically, transdermally, rectally, orally, nasally or by inhalation.
  • the composition as disclosed herein is
  • Slow-release formulations may also be prepared from the agents described herein in order to achieve a controlled release of the active agent (Compound A) in contact with the body fluids in the gastrointestinal tract, and to provide a substantial constant and effective level of the active agent in the blood plasma.
  • Administration may take the form of single dose administration, or the compositions as disclosed herein can be administered over a period of time, either in divided doses or in a continuous-release formulation or administration method (e.g., a pump).
  • the compositions of the embodiments administered to the subject, the amounts of the composition administered, and the route of administration chosen is selected to permit efficacious treatment of the disease condition.
  • the compositions are formulated with one or more pharmaceutically acceptable excipient, carriers, solvents, stabilizers, adjuvants, diluents, or the like, depending upon the particular mode of administration and dosage form.
  • the compositions should generally be formulated to achieve a physiologically compatible pH, and may range from a pH of about 3 to a pH of about 11, preferably about pH 3 to about pH 7, depending on the formulation and route of administration.
  • the pH is adjusted to a range from about pH 5.0 to about pH 8.
  • the compositions may comprise a therapeutically or prophylactically effective amount of Compound A as described herein, together with one or more pharmaceutically acceptable excipients.
  • compositions disclosed hereins are most typically solids, liquid solutions, suspensions, while inhalable formulations for pulmonary administration are generally liquids or powders.
  • Composition disclosed herien can also be formulated as a lyophilized solid that is reconstituted with a physiologically compatible solvent prior to administration. Lyophilization (freeze-drying) removes water by sublimation at low temperature and pressure, and preserves sensitive materials such as drugs, vaccines and probiotic cultures.
  • compositions disclosed herein can be formulated as a solution that is reconstituted with a physiologically compatible solvent prior to administration for intravenous or intramuscular injection. Parenteral injection or extended infusion, e.g., over a period of 1, 2, 3, 4, 5, 6, 7, 8, 12 or 24 hours is possible.
  • the composition described herein comprise a lipid-bilayer coated particle, in place of, or in addition to, a liposome.
  • pharmaceutically acceptable excipient refers to an excipient for administration of a pharmaceutical agent, such as the compounds described herein. The term refers to any pharmaceutical excipient that may be administered without undue toxicity.
  • Suitable excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there exists a wide variety of suitable formulations of pharmaceutical compositions (see, e.g., Remington's Pharmaceutical Sciences). [00363] Suitable excipients may be carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • compositions disclosed herein may be formulated as suspensions comprising Compound A and two or more lipids of the embodiments in admixture with at least one pharmaceutically acceptable excipient suitable for the manufacture of a suspension.
  • composition disclosed herein may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle, such as water or isotonic saline or dextrose solution.
  • an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
  • compounds which have been modified by substitutions or additions of chemical or biochemical moieties which make them more suitable for delivery e.g., increase solubility, bioactivity, palatability, decrease adverse reactions, etc.
  • PEGylation for example by PEGylation.
  • the compounds described herein may be formulated for oral administration in a lipid-based formulation suitable for low solubility compounds.
  • An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response.
  • Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes.
  • Adjuvants of many kinds are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella Minnesota Re 595 lipopolysaccharide; saponins including QS21 (SmithKlineBeecham), a pure QA-21 saponin purified from Quillja saponaria extract; DQS21, described in PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18, and QS-Ll (So et al., MoI Cells (1997) 7:178-186); ISCOMATRIX adjuvant, a cage-like structure composed of saponin, phospholipid, and cholesterol (see, e.g., Maraskovsky et
  • the disclosed liposomes can be suitable for stimulating an immune response.
  • the disclosure provides methods of stimulating an immune response in a mammalian subject comprising administering to the subject the disclosed compositions.
  • a subject e.g., a mammal
  • methods of treating different types of cancer in a subject including administering to the subject the composition as described herein in a therapeutically effective amount.
  • the subject is a mammalian subject.
  • the mammalian subject is a human subject.
  • Practice of methods described herein in other mammalian subjects, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans is also contemplated.
  • mice that were used in pre-clinical studies may be 1000-fold or more sensitive to Compound A than humans, which provides guidance for adjusting dosing. Standard dose-response studies are used to optimize dose and dosing schedule.
  • compositions disclosed herein in the manufacture of a medicament, such as those described above, for treatment of cancer in a mammalian subject.
  • the disclosed methods are useful for treating cancer, for example, inhibiting cancer growth, including complete cancer remission, for inhibiting cancer metastasis, and for promoting cancer resistance.
  • cancer growth generally refers to any one of a number of indices that suggest change within the cancer to a more developed form.
  • indices for measuring an inhibition of cancer growth include but are not limited to a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of cytolytic T-lymphocytes, and a decrease in levels of tumor-specific antigens.
  • CT computed tomographic
  • sonography sonography
  • indices for measuring an inhibition of cancer growth include but are not limited to a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of cytolytic T-lymphocytes, and a decrease in levels of tumor-specific antigens.
  • cancer resistance refers to an improved capacity of
  • the cancer comprises a solid tumor, for example, a carcinoma and a sarcoma.
  • Carcinomas include malignant neoplasms derived from epithelial cells which infiltrate, for example, invade, surrounding tissues and give rise to metastases.
  • Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures.
  • Another broad category of cancers includes sarcomas and fibrosarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance, such as embryonic connective tissue.
  • the invention also provides methods of treatment of cancers of myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically are not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. Further contemplated are methods for treatment of adult and pediatric oncology, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, cancer metastases, including lymphatic metastases.
  • the cancers listed herein are not intended to be limiting. Age (child and adult), sex (male and female), primary and secondary, pre- and post- metastatic, acute and chronic, benign and malignant, anatomical location cancer embodiments and variations are contemplated targets.
  • Cancers are grouped by embryonic origin (e.g., carcinoma, lymphomas, and sarcomas), by organ or physiological system, and by miscellaneous grouping. Particular cancers may overlap in their classification, and their listing in one group does not exclude them from another.
  • Carcinomas that may targeted include adrenocortical, acinar, acinic cell, acinous, adenocystic, adenoid cystic, adenoid squamous cell, cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex, adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar, alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroid gland, apocrine, basal cell, basal cell, alveolar, comedo basal cell, cystic basal cell, morphea-like basal cell, multicentric basal cell, nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basal cell, superficial basal cell, basaloid, basosquamous cell, bile duct, extrahepatic bile duct, intrahepati
  • Sarcomas that may be targeted include adipose, alveolar soft part, ameloblastic, avian, botryoid, sarcoma botryoides, chicken, chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal, endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl, giant cell, granulocytic, hemangioendothelial, Hodgkin's, idiopathic multiple pigmented hemorrhagic, immunoblastic sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen's, Kaposi's, kupffer cell, leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio, idiopathic hemorrhagic, multipotential primary sarcoma of bone, osteoblastic, osteogenic, parosteal, polymorph
  • Lymphomas that may be targeted include AIDS-related, non-Hodgkin's, Hodgkin's, T- cell, T-cell leukemia/lymphoma, African, B-cell, B-cell monocytoid, bovine malignant, Burkitt's, centrocytic, lymphoma cutis, diffuse, diffuse, large cell, diffuse, mixed small and large cell, diffuse, small cleaved cell, follicular, follicular center cell, follicular, mixed small cleaved and large cell,follicular, predominantly large cell,follicular, predominantly small cleaved cell,giant follicle, giant follicular, granulomatous, histiocytic, large cell, immunoblastic, large cleaved cell, large nocleaved cell, Lennert's, lymphoblastic, lymphocytic, intermediate; lymphocytic, intermediately differentiated, plasmacytoid; poorly differentiated lymphocy
  • Leukemias and other blood cell malignancies that may be targeted include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukemia, chronic myelogenous, hairy cell, erythroleukemia, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, hemoblastic, histiocytic, leukopenic, lymphatic, Schilling's, stem cell, myelomonocytic, monocytic, prolymphocytic, promyelocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic, rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic, multiple myeloma, nonlymphoc
  • Brain and central nervous system (CNS) cancers and tumors that may be targeted include astrocytomas (including cerebellar and cerebral), brain stem glioma, brain tumors, malignant gliomas, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumor, medulloblastoma, myelodysplastic syndromes, oligodendroglioma, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative disorders, neuroblastoma, plasma cell neoplasm/multiple myeloma,
  • Gastrointestimal cancers that may be targeted include extrahepatic bile duct cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastronintestinal carcinoid tumors, gastrointestinal stromal tumors, bladder cancers, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia.
  • gastric (stomach) cancer gastric (stomach) cancer
  • gastrointestinal carcinoid tumor gastronintestinal carcinoid tumors
  • gastrointestinal stromal tumors gastrointestinal stromal tumors
  • bladder cancers islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and
  • Lung and respiratory cancers that may be targeted include bronchial adenomas/carcinoids, esophagus cancer esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumor, non-small cell lung cancer, small cell lung cancer, small cell carcinoma of the lungs, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer; paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.
  • bronchial adenomas/carcinoids esophagus cancer esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumor, non-small cell lung cancer
  • Urinary tract and reproductive cancers that may be targeted include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, penile cancer, renal cell cancer (including carcinomas), renal cell cancer, renal pelvis and ureter (transitional cell cancer), transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumor, testicular cancer, ureter and renal pelvis, transitional cell cancer, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine cancer and solid tumors in the
  • Skin cancers and melanomas (as well as non-melanomas) that may be targeted include cutaneous T-cell lymphoma, intraocular melanoma, tumor progression of human skin keratinocytes, basal cell carcinoma, and squamous cell cancer.
  • Liver cancers that may be targeted include extrahepatic bile duct cancer, and hepatocellular cancers.
  • Eye cancers that may be targeted include intraocular melanoma, retinoblastoma, and intraocular melanoma.
  • Hormonal cancers that may be targeted include parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumors, pituitary tumor, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, and ACTH-producing tumors.
  • Miscellaneous other cancers that may be targeted include advanced cancers, AIDS- related, anal cancer adrenal cortical, aplastic anemia, aniline, betel, buyo cheek, cerebriform, chimney-sweeps, clay pipe, colloid, contact, cystic, dendritic, cancer àdes, duct, dye workers, encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial, glandular, cancer in situ, kang, kangri, latent, medullary, melanotic, mule-spinners', non-small cell lung, occult cancer , paraffin, pitch workers', scar, schistosomal bladder, scirrhous, lymph node, small cell lung, soft, soot, spindle cell, swamp, tar, and tubular cancers.
  • Miscellaneous other cancers that may be targeted also include carcinoid (gastrointestinal and bronchal) Castleman's disease chronic myeloproliferative disorders, clear cell sarcoma of tendon sheaths, Ewing's family of tumors, head and neck cancer, lip and oral cavity cancer, Waldenström's macroglobulinemia, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, Wilms' tumor, mycosis fungoides, pheochromocytoma, sezary syndrome, supratentorial primitive neuroectodermal tumors, unknown primary site, peritoneal effusion, malignant pleural effusion, trophoblastic neo-plasms, and hemangiopericytoma.
  • carcinoid gastrointestinal and bronchal
  • Castleman's disease chronic myeloproliferative disorders clear cell sarcoma of tend
  • the cancer can be selected from the group of basal cell carcinoma, breast cancer leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell cancer, melanoma, and combinations thereof.
  • the cancer includes melanoma.
  • the cancer includes non-small cell lung cancer.
  • compositions described herein are useful for the induction of an immune response to a tumor antigen, one or more pathogenic organisms, or other antigen as described herein.
  • dosesing refer to an amount of a composition sufficient to treat, ameliorate, or prevent the identified disease or condition, or to exhibit a detectable therapeutic, prophylactic, or inhibitory effect.
  • the effect can be detected by, for example, an improvement in clinical condition, reduction in symptoms, or by any of the assays or clinical diagnostic tests described herein.
  • the precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically and prophylactically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of one or more symptoms associated with a specific disorder or condition and/or preventing or eliminating one or more symptoms, and/or reducing the severity of the condition and/or slowing the progression of the condition.
  • prophylaxis refers to a preventative therapy and its effectiveness can be shown by comparison between a population that receives the prophylaxis and a population that only receives a negative control. Effective prophylaxis results in reduced incidence and/or delayed onset and/or reduced severity, compared to the control population.
  • treating refers to slowing the spread of the infection and/or stopping the spread of the infection and/or reducing or eliminating the amount of the infective agent in a host.
  • an effective treatment slows the growth of the cancer and/or reduces its harmful effect on a patient and/or increases the life span of a cancer patient and/or increases the span of quality life of the patient; or causes shrinkage of a tumor or reduction in the number of cancer cells, or results in elimination of the cancer.
  • treatment causes the body to produce an immune response that inhibits or prevents future infection by an infective agent or reduces the infection’s severity or duration or negative effects, if infection occurs.
  • Dosages of the therapeutic can be administered as a dose measured in micrograms or milligrams.
  • “therapeutic” refers to the amount of Compound A in the dosage.
  • Contemplated dosages of the disclosed therapeutics include doses in the range of about 0.1 ⁇ g to about 50000 ⁇ g (50 mg). [00390] Within the above broad guidelines, dose subranges are specifically contemplated. For example, dose ranges are contemplated with a lower end dose of Compound A of any of about 0.5 ⁇ g, or about 1 ⁇ g or about 0.5 ⁇ g, or about 1 ⁇ g, or about 5 ⁇ g, or about 10 ⁇ g, or about 20 ⁇ g, or about 30 ⁇ g, or about 40 ⁇ g, or about 50 ⁇ g, or about 60 ⁇ g, or about 70 ⁇ g, or about 80 ⁇ g, or about 90 ⁇ g, or about 100 ⁇ g, or about 110 ⁇ g, or about 120 ⁇ g, or about 130 ⁇ g, or about 140 ⁇ g, or about 150 ⁇ g, or about 160 ⁇ g, or about 170 ⁇ g, vabout 180 ⁇ g, or about 190 ⁇ g, or about 200 ⁇
  • Doses and dosage ranges at the higher end of this spectrum e.g., with a minimum dose of about 1 mg, or about 5 mg, or about 10 mg, or about 15 mg, or about 20 mg, or about 25 mg, or about 30 mg, are contemplated for oncology indications; doses and dosage ranges at the lower end of the above spectrum, e.g., with a maximum dose of about 10 mg, are contemplated for non- oncology indications.
  • Specific ranges of doses in ⁇ g include about 1 ⁇ g to about 20000 ⁇ g, about 2 ⁇ g to about 10000 ⁇ g, about 5 ⁇ g to about 2000 ⁇ g, about 5 ⁇ g to about 1000 ⁇ g, about 10 ⁇ g to about 1000 ⁇ g, about 20 ⁇ g to about 500 ⁇ g, about 30 ⁇ g to about 200 ⁇ g, and about 50 ⁇ g to about 100 ⁇ g.
  • the doses can be total daily amounts given to a subject or the dose given at any single time.
  • the dose can be administered as a single dose or in divided doses throughout the day (e.g., in two, three, four, or five doses over the course of a day).
  • the dose of the liposomes that is administered will vary with the exact composition of the liposome. In some embodiments, it is contemplated that the liposomes are administered to result in a daily dosage of Compound A of about 0.5 ⁇ g to about 50 mg, or about 1 ⁇ g to about 45 mg, or about 10 ⁇ g to about 40 mg, or about 50 ⁇ g to about 3mg, or about 1 ⁇ g to about 20 mg, e.g., about 0.5 ⁇ g, about 1 ⁇ g, about 10 ⁇ g, about 20 ⁇ g, about 30 ⁇ g, about 40 ⁇ g, about 50 ⁇ g, about 60 ⁇ g, about 70 ⁇ g, about 80 ⁇ g, about 90 ⁇ g, about 100 ⁇ g, about 110 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about 140 ⁇ g, about 150 ⁇ g, about 160 ⁇ g, about 170 ⁇ g, about 180 ⁇ g, about 190 ⁇ g, about 200 ⁇ g,
  • the liposomes are administered to result in a daily dosage of the nonglycosidic ceramide in an amount of about 1 ⁇ g to about 200 ⁇ g, or about 1 mg to about 10 mg, or about 700 ⁇ g to about 2 mg, or about 800 ⁇ g to about 28 mg, or about 10 ⁇ g to about 40 mg, about 10 ⁇ g to about 5 mg.
  • the liposomes are administered in divided doses 1 to 4 times daily or in a sustained release form.
  • Dosages of the therapeutic can alternately be administered as a dose measured in mg/kg (mg compound per kilogram of body weight for the treated subject).
  • “therapeutic” refers to the amount of Compound A in the dosage.
  • Contemplated mg/kg doses of the disclosed therapeutics include about 0.00001 mg/kg to about 1 mg/kg. Specific ranges of doses in mg/kg include about 0.0001 mg/kg to about 0.5 mg/kg, about 0.0005 mg/kg to about 0.2 mg/kg, about 0.001 mg/kg to about 0.1 mg/kg, C.
  • Dosages of the therapeutic can alternately be administered as a dose measured in mg/m 2 (mg compound per m 2 surface area for the treated subject).
  • an average or typical body surface are for an adult female is 1.m 2 and for an adult male is 1.9 m 2 , and these numbers can be used to convert/express any of the foregoing doses or dosage ranges into doses or dosages expressed in mg/m 2 .
  • Exemplary contemplated mg/m 2 doses of the disclosed therapeutics include about 0.5 mg/m 2 to about 20 mg/m 2 ; about 1 mg/m 2 to about 15 mg/m 2 ; about 2 mg/m 2 to about 15 mg/m 2 ; about 3 mg/m 2 to about 10 mg/kg; or about 4 mg/m 2 to about 10 mg/m 2 .
  • Combination therapy can also include the use of compositions as described herein together with one or more additional therapeutic agents for the treatment of disease conditions.
  • the combination of active ingredients may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art.
  • the methods described herein may comprise administering or delivering the active ingredients sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in simultaneous therapy, effective dosages of two or more active ingredients are administered together, which can be co-formulation or separate administration at essentially the same time.
  • a composition disclosed herein is administered and/or formulated with a second therapeutic.
  • the composition as disclosed herein and the further therapeutic or therapy are administered concurrently.
  • the composition as disclosed herein and the further therapeutic or therapy are administered separately.
  • the second therapeutic can be one or more of a chemotherapeutic or an immunotherapeutic agent.
  • the second therapeutic is a cytokine, an anti- inflammatory agent, a cancer vaccine, a cancer antigen, or a polynuecleotide encoding a cancer antigen. In some cases, the second therapeutic is radiation.
  • Contemplated chemotherapeutics for use in combination therapies as disclosed herein include aspirin, sulindac, curcumin, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine
  • Contemplated biological response modifying agents for use in combination therapies as disclosed herein include, but are not limited to, interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p ⁇ -DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and
  • Contemplated immunotherapeutic agents such as an immune checkpoint inhibitor, for use in the combination therapies disclosed herein.
  • the immunotherapeutic agents include, but are not limited to, a Her2/neu receptor antibody such as trastuzumab (marketed as Herceptin®), an anti-CD52 antibody such as alemtuzumab (marketed as Campath®.
  • an anti-CD33 antibody such as gemtuzumab linked to calicheamicin (marketed as Mylotarg®), an anti-CD20 antibody such as rituximab (marketed as Rituxan® and MabThera®), Ibritumomab tiuxetan (marketed as Zevalin®), anti-TNF ⁇ antibodies such as infliximab (marketed as Remicade®) or adalimmumab (marketed as Humira®), a soluble TNFR2 molscule such as etanercept (marketed as Enbrel®), an antibody to the CD25 chain of the IL-2 receptor such as basiliximab (marketed as Simulect®), an anti CD40/CD40L antibody such as humanized IgG1 anti-human CD40 antibody (SGN-40), an anti-CTLA-4 blocking antibody such as iplimumab (marketed as MDX-101 or MDX-010) or tremelimum
  • the methods or use disclosed herein further comprise administering to the subject or using at least one checkpoint inhibitor.
  • the immune checkpoint inhibitor includes PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor can include at least one PD-1 inhibitor.
  • the at least one PD-1 inhibitor can be selected from Pembrolizumab, Nivolumab, Cemiplimab, and Spartalizumab.
  • the at least one PD-L1 inhibitor can be selected from Atezolizumab, Avelumab, and Durvalumab.
  • the at least one CTLA-4 inhibitor can be selected from Ipilimumab and Tremelimumab.
  • the immune checkpoint inhibitor is an agent that targets the Programmed Cell Death Protein 1 (PD-1) pathway.
  • PD-1 Programmed Cell death protein 1
  • CD279 cluster of differentiation 279
  • CD279 is a cell surface co-inhibitory receptor expressed on activated T cells, B cells and macrophages, and is a component of immune checkpoint blockade (Shinohara et al., Genomics., 23(3):704, (1994); Francisco et al., Immunol Rev., 236: 219, (2010)).
  • PD-1 limits the activity of T cells upon interaction with its two ligands PD-L1 (also known as B7-H1; CD274) and PD-L2 (B7-DC; CD273) (Postow et al., J Clin Oncol., 33: 9, (2015)). Interaction of PD-1 with PD-L1 and PD-L2, reduces T-cell proliferation, cytokine production, and cytotoxic activity (Freeman GJ et al., J Exp Med., 192:1027–34, (2000); Brown JA et al., J Immunol., 170:1257–66, (2003)).
  • Inhibitors of PD-1/PD-L1 include antibodies directed to PD-1, PD-L1 and PD-L2, PD-1/PD-L1 peptides, PD-1/PD-L1 small molecule chemical compounds, for example. (Jiang et al., Front Immunol.2020, 12;11:339; Guo et al. Front. Immunol., 2020;11:1508).
  • PD-1 antibodies [00405] Antibodies to PD-1 have been described in, for example, US Patent Nos.8,735,553; 8,617,546; 8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869; 8,287,856; 8,927,697; 8,088,905; 7,595,048; 8,168,179; 6,808,710; 7,943,743; 8,246,955; 8,217,149; 8,952,136; 9,102,727; 10,428,146; and 10,752,687 (incorporated by reference in their entirety). [00406] It is contemplated that any known antibody can be used in the present methods.
  • a murine monoclonal anti-target antibody for human PD-1 is used in the present methods.
  • InVivo MAb anti h PD-1 BioXCell, Clone: RMP1-14, Cat. no.: BE0146.
  • Anti-PD-1 antibodies have been shown to be effective in human therapy, see e.g., pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp.), nivolumab (Opdivo®, Bristol- Myers Squibb) and cemiplimab (Libtayo®, Regeneron Pharmaceuticals) which are anti-PD-1 antibodies approved for use in human therapy.
  • pidilizumab CureTech Ltd.
  • SSI-361 Livgen
  • Prolgolimab BCD-100
  • Spartalizumab PDR001
  • GLS-010 Long et al., Blood (2020) 13(Supplement 1): 17
  • camrelizumab SHR1210
  • sintilimab IBI308
  • IBI308 Innovent and Eli Lilly
  • PD-L1 antibodies [00408] Antibodies to PD-L1 have been described, for example, in US Patent Nos.
  • anti-PD-L1 antibodies have been shown to be effective in human therapy. See, for e.g., atezolizumab (Tecentriq®, Genentech/Roche); avelumab (Bavencio®, Merck and Pfizer); and durvalumab (Imfinzi®, AstraZeneca) which are anti-PD-L1 antibodies approved for use in human therapy of certain cancers.
  • Additional PD-L1 antibodies in clinical development also are specifically contemplated.
  • KN035 Zhang et al., Cell Discovery.3(1): 17004
  • CK-301 Checkpoint Therapeutics
  • Source Checkpoint Therapeutics, Inc.
  • BMS-936559 MDX 1105) (Bristol-Myers Squibb/ Medarex Inc)
  • Pacmilimab CX-072
  • CytomX Therapeutics (Guo et al. Front.
  • PD-L2 antibodies [00411] Antibodies to PD-L2 have been described, for example, in US Patent Nos.9,845,356; 10,370,448; and 10,647,771(incorporated by reference in their entirety). [00412] Table PD-A: FDA-approved anti-PD-1/L1 antibodies (adapted from Guo et al. Front.
  • IgG 4 ⁇ (PDR-001) Cemiplimab Regeneron US20150203579 Papadopoulos IgG 4 (Libtayo) Pharmaceuticals et al. Camrelizumab Incyte Biosciences US20160376367A1 Yuan et al. IgG 4 (SHR-1210) and Jiangsu Hengrui Medicine Tislelizumab BeiGene US8735553B1 Li et al. IgG4 (BGB-A317) Dostarlimab Tesaro/AnaptysBio US9815897B2 King et al.
  • IgG4 (TSR-042) MEDI-0680 MedImmune LLC US8609089B2 Langermann IgG4 (AMP-514) et al. SSI-361 Lyvgen US20180346569A1 Wang et al. IgG4 AMP-224 Amplimmune Inc US20130017199 Langermann IgG4 et al. PD-L1 CX-072 CytomX US20160311903A1 West et al. protease activatable prodrug BMS-936559 Medarex Inc US7943743 Korman et al. IgG4 (MDX 1105) KN035 Jiangsu Alphamab US20180327494A1 Xu et al.
  • PD-1/ PD-L1 peptide and small molecule inhibitors are contemplated as embodiments.
  • AUNP12 PD-1/PD-L1 inhibitor
  • JTX-4014 Jounce Therapeutics
  • CA-170 PD-L1 and VISTA antagonist
  • BMS-986189 microcyclic peptide
  • Bristol-Myers Squibb Bristol-Myers Squibb
  • the anti-CTLA-4 antibody is a monoclonal antibody directed against the T-cell receptor protein cytotoxic T-lymphocyte –associated protein 4 (CTLA-4).
  • CTLA-4 cytotoxic T-lymphocyte –associated protein 4
  • An example of an anti-CTLA-4 antibody is human IgG2 monoclonal antibody tremelimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, resulting in inhibition of B7-CTLA4-mediated downregulation of T-cell activation.
  • an anti-CTLA-4 antibody is human IgG1 monoclonal antibody ipilimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, resulting in inhibition of B7-CTLA4-mediated downregulation of T-cell activation.
  • Ipilimumab is undergoing clinical trials for the treatment of non-small cell lung carcinoma, small cell lung cancer and metastatic hormone-refractory prostate cancer.
  • the anti-GITR antibody is a monoclonal antibody directed against glucocorticoid-induced tumor necrosis factor receptor (GITR) which blocks the interaction of GITR with its ligand, enhances cytotoxicity of natural human killer cells and/or downmodulates GITR expression on peripheral blood lymphocytes.
  • GITR glucocorticoid-induced tumor necrosis factor receptor
  • the anti-OX40 antibody is an agonistic monoclonal antibody that mimicks the natural OX40 ligand and selectively binds to and activates the OX40 receptor. Receptor activation induces proliferation of memory and effector T cells.
  • Exemplary Immune Checkpoint Inhibitor Approved Indications [00419] A summary of indications for various checkpoint inhibitors is provided by Vaddepally RK, et al., “Review of Indications of FDA-Approved Immune Checkpoint Inhibitors per NCCN Guidelines with the Level of Evidence.” Cancers (Basel).2020 Mar 20;12(3):738. doi: 10.3390/cancers12030738. PMID: 32245016; PMCID: PMC7140028, incoporatated herein by reference in its entirety.
  • cancer patients that do not meet prescribing information are scored as “insensitive” to a checkpoint inhibitor for purposes of methods of treatment described herein.
  • a product label such as a government-approved product label
  • Exemplary prescribing information for nivolumab is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125554s058lbl.pdf, incorporated herein by reference in its entirety.
  • Exemplary prescribing information for Pembrolizumab (marketed as KEYTRUDA ® ) is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/125514s096lbl.pdf, incorporated herein by reference in its entirety.
  • Exemplary prescribing information for Cemiplimab (marketed as LIBTAYO TM ) is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/761097s007lbl.pdf, incorporated herein by reference in its entirety.
  • Exemplary prescribing information for Avelumab (marketed as BAVENCIO ® ) is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761049s009lbl.pdf, incorporated herein by reference in its entirety.
  • Exemplary prescribing information for Atezolizumab (marketed as TECENTRIQ ® ) is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/761034s042lbl.pdf, incorporated herein by reference in its entirety.
  • Exemplary prescribing information for Durvalumab (marketed as IMFINZI ® ) is provided at https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761069s018lbl.pdf, and at https://den8dhaj6zs0e.cloudfront.net/50fd68b9-106b-4550-b5d0-12b045f8b184/9496217c- 08b3-432b-ab4f-538d795820bd/9496217c-08b3-432b-ab4f- 538d795820bd_viewable_rendition__v.pdf, incorporated herein by reference in its entirety.
  • Pembrolizumab is considered: ⁇ as a single agent for the first-line treatment of patients with NSCLC expressing PD-L1 [Tumor Proportion Score (TPS) ⁇ 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations; ⁇ as a single agent for the first-line treatment of patients with metastatic or with unresectable, recurrent (head and neck squamous cell cancer) HNSCC whose tumors express PD-L1 [Combined Positive Score (CPS) ⁇ 1] as determined by an FDA-approved test; ⁇ for the treatment of patients with locally advanced or metastatic urothelial carcinoma who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 [Combined Positive Score (CPS) ⁇ 10] as determined by an FDA-
  • Cemiplimab is considered for the first-line treatment of patients with NSCLC whose tumors have high PD-L1 expression [Tumor Proportion Score (TPS) ⁇ 50%] as determined by an FDA-approved test, with no EGFR, ALK or ROS1 aberrations.
  • TPS Tumor Proportion Score
  • Atezolizumab is considered: ⁇ for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD- L1 (PD-L1 stained tumor-infiltrating immune cells [IC] covering ⁇ 5% of the tumor area), as determined by an FDA-approved test; ⁇ as adjuvant treatment following resection and platinum-based chemotherapy for adult patients with Stage II to IIIA NSCLC whose tumors have PD-L1 expression on ⁇ 1% of tumor cells, as determined by an FDA-approved test; and ⁇ for the first-line treatment of adult patients with metastatic NSCLC whose tumors have high PD-L1 expression (PD-L1 stained ⁇ 50% of tumor cells [TC ⁇ 50%] or PD-L1 stained tumor-infiltrating immune cells [IC] covering ⁇ 10% of the tumor area [IC ⁇ 10%] ), as determined by an FDAapproved test, with no EGFR or ALK genomic tumor aberrations [
  • the increased PD-L1 expression converts the patient from ineligible/insensitive (under the prescribing guideline) to a patient classifyable as sensitive (and appropriate for treatment) under the prescribing guideline.
  • Checkpoint inhibitor biomarker assays [00431]
  • the measurement of PD-L1, e.g., for the purpose of evaluating whether a patient satisfies a prescribing guideline is performed with an immunohistochemistry assay, such as an assay with FDA Premarket Approval (PMA, see https://www.accessdata.fda.gov/) or approval.
  • Exemplary suitable assays are set forth in the following Table: P roduct Manufacturer Antibody Original PMA Currently Associated N umber Drugs VENTANA PD-L1 (SP263) Assay Ventana SP263 P160046 TECENTRIQ, LIBTAYO VENTANA PD-L1 (SP142) Assay Ventana SP142 P160002 TECENTRIQ PD-L1 IHC 28-8 pharmDx Agilent 28-8 P150025/P150027 OPDIVO PD-L1 IHC 22C3 pharmDx Agilent 22C3 P150013 KEYTRUDA, LIBTAYO [00432] There are several scoring methods used across multiple indications and drugs which are summarized from the intended use statements of the products: Table 2 Name Assay Scoring Method Cutoff Drug Indication TC SP263 Percentage of tumor cells (% TC) with any ⁇ 1% TECENTRIQ NSCLC m embrane staining above background ⁇ 50% LIBAYTO
  • IC is a measure of only immune cell PD-L1 expression.
  • CPS Combined Positive Score
  • VENTANA PD-L1 (SP263) Assay is a qualitative immunohistochemistry assay using rabbit monoclonal anti-PDL1 clone SP263 intended for use in the assessment of the programmed death ligand-1 (PD-L1) protein in formalin fixed, paraffin-embedded (FFPE) nonsmall cell lung carcinoma (NSCLC) tissue specimens by light microscopy.
  • FFPE programmed death ligand-1
  • the VENTANA PD-L1 (SP263) Assay is used with the OptiView DAB IHC Detection Kit for staining on the BenchMark ULTRA instrument.
  • PD-L1 protein expression in NSCLC is determined by the percentage of tumor cells (% TC) with any membrane staining above background.
  • VENTANA PD-L1 (SP263) Assay is indicated as an aid in identifying patients eligible for treatment with the therapies listed in Ventana Table 1 for the indication and PD-L1 status in accordance with the approved therapeutic product labeling.
  • VENTANA PD-L1 (SP142) Assay is a qualitative immunohistochemical assay using rabbit monoclonal anti-PD-L1 clone SP142 intended for use in the assessment of the programmed deathligand 1 (PD-L1) protein in tumor cells and tumor-infiltrating immune cells in the formalin- fixed, paraffin-embedded (FFPE) tissues indicated below stained with OptiView DAB IHC Detection Kit and OptiView Amplification Kit on a BenchMark ULTRA instrument.
  • FFPE formalin- fixed, paraffin-embedded
  • Determination of PD-L1 status is based on either the proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells (% IC) of any intensity or the percentage of PD-L1 expressing tumor cells (% TC) of any intensity.
  • VENTANA PD-L1 (SP142) Assay is indicated as an aid for identifying patients for treatment with the therapy-and cutoff listed in Table 1 in accordance with the approved therapeutic product labeling.
  • PD-L1 IHC 28-8 pharmDx is a qualitative immunohistochemical assay using Monoclonal Rabbit Anti-PD-L1, Clone 28-8 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), and urothelial carcinoma (UC) tissues using EnVision FLEX visualization system on Autostainer Link 48.
  • FFPE paraffin-embedded
  • NSCLC non-small cell lung cancer
  • SCCHN squamous cell carcinoma of the head and neck
  • UC urothelial carcinoma
  • ⁇ PD-L1 expression ( ⁇ 1% or ⁇ 5% or ⁇ 10% tumor cell expression), as detected by PD-L1 IHC 28-8 pharmDx in non-squamous NSCLC (nsNSCLC) may be associated with enhanced survival from OPDIVO®;
  • ⁇ PD-L1 expression ( ⁇ 1% tumor cell expression), as detected by PD-L1 IHC 28-8 pharmDx in SCCHN may be associated with enhanced survival from OPDIVO®;
  • ⁇ PD-L1 expression ( ⁇ 1% tumor cell expression), as detected by PD-L1 IHC 28-8 pharmDx in UC may be associated with enhanced response rate and enhanced disease-free survival from OPDIVO®.
  • PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assay using monoclonal mouse anti-PD-L1, Clone 22C3 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), cervical cancer, head and neck squamous cell carcinoma (HNSCC), and triple-negative breast cancer (TNBC) tissues using EnVision FLEX visualization system on Autostainer Link 48.
  • FFPE paraffin-embedded
  • NSCLC non-small cell lung cancer
  • ESCC esophageal squamous cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • TNBC triple-negative breast cancer
  • PD-L1 protein expression in NSCLC is determined by using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity.
  • TPS Tumor Proportion Score
  • PD-L1 protein expression in ESCC, cervical cancer, HNSCC, and TNBC is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.
  • CPS Combined Positive Score
  • PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying patients for treatment with the therapies for the indications listed in PD-L1 IHC 22C3Table 1.
  • Cytokines that are effective in inhibiting tumor growth/metastasis are contemplated for use in the combination therapy.
  • Such cytokines, lymphokines, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, 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, IFN, TNF ⁇ , TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • An immunotherapeutic agent can be a cancer vaccine.
  • a cancer vaccine is an agent, molecule, or immunogen which stimulates or elicits an endogenous immune response in an individual or subject against one or more tumor antigens.
  • a cancer antigen is broadly defined as an antigen specifically expressed by a tumour or cancer cell: a cancer antigen which is present on the surface of cancer cells in an individual, but which is not present on the surface of normal somatic cells of the individual, i.e., the antigen is exposed to the immune system in cancer cells but not in normal somatic cells.
  • the antigen may be expressed at the cell surface of the tumour cell where it is recognized by components of the humoral immune system such as B lymphocytes (B cells).
  • B cells B lymphocytes
  • Intracellular tumour antigens are processed into shorter peptide fragments which form complexes with major histocompatibility complex (MHC) molecules and are presented on the cell surface of cancer cells, where they are recognized by the T cell receptors (TCR’s) of T lymphocytes (T cells).
  • MHC major histocompatibility complex
  • TCR T cell receptors
  • T cells T lymphocytes
  • the cancer antigen is one which is not expressed by normal cells, or at least not expressed to the same level as in tumour cells.
  • a cancer vaccine may enhance the presentation of one or more cancer antigens to both antigen-presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
  • preparations and/or formulations of cancer vaccines may be used together with one or more adjuvants that are well known in the art, to induce an immune response or to increase an immune response.
  • Cancer antigens may include, for example, cancer-testis antigens encoded by cancer- germ line genes.
  • Cancer-testis (CT) antigens constitute a unique group of genes which are predominantly expressed in human germ line cells such as placenta and testis but become reactivated in various malignancies (Simpson et al., Nature Rev (2005) 5, 615-625). Most of these genes are located as multigene families on the X-chromosome and are also referred to as CT-X antigens (Simpson et al., Nature Rev (2005) 5, 615-625).
  • CT-X antigens are broadly expressed in a wide variety of cancer types including for example bladder cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, Brain cancer, glioma, glioblastoma, hepatocellular carcinoma and melanoma.
  • their expression pattern is closely associated with advanced disease and poor outcome and might thus be of diagnostic and/or prognostic relevance (Gure et al., Clin Cancer Res (2005) 11,8055-8062; Velazquez et al., Cancer Immun.
  • Tumor antigens which may be comprised of the full-length polypeptide sequence of the tumor antigen or an immunogenic fragment, or epitope derived from the full- length polypeptide sequence of the tumor antigen.
  • Tumor antigens include the corresponding nucleotide sequence encoding for the full-length polypeptide, immunogenic fragment, or epitope derived from the full-length polypeptide sequence of the tumor antigen.
  • a fragment of a cancer antigen is a contiguous stretch of amino acid residues from the sequence of the antigen which is shorter than the full-length antigen (i.e., it consists of fewer amino acid residues).
  • a fragment may comprise less than 500, less than 400, less than 300, less than 200, less than 100 amino acids, or less than 50 amino acids.
  • a fragment will generally consist of at least 5 amino acids, for example, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids or at least 35 amino acids.
  • Fragments of cancer antigens may include immunogenic regions or epitopes that bind to MHC class I or class II molecules and are recognized by TCR’s of T lymphocytes. Many such epitopes of cancer antigens are known in the art, such as listed on the webpage www.cancerimmunity.org/peptidedatabase/Tcellepitopes.
  • the cancer antigen can be a tumor associated peptide, or protein that induces or enhances immune response and is derived from tumor associated genes and encoded proteins including, for example, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE- A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX
  • fusion protein PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, Lü-1, Mage-C2, NA-88, /Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens Eand E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PS
  • antigenic peptides characteristic of tumors include those listed in International Patent Application Publication No. WO 2000/020581 and U.S. Patent Application Publication No.2010/0284965, which are each incorporated herein by reference.
  • the antigen is a tumor antigen selected from the group consisting of MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, and mixtures thereof.
  • the cancer antigen is a mammalian protein.
  • the cancer antigen is a human protein.
  • the full-length protein is employed as the antigen.
  • peptides comprising an antigenic fragment of these proteins is used as the tumor antigen.
  • suitable antigens include cancer antigens in the following classes: cancer testis antigens (e.g., HOM-MEL-40), differentiation antigens (e.g., HOM-MEL-55), overexpressed gene products (HOM-MD-21), mutated gene products (NY-COL-2), splice variants (HOM-MD- 397), gene amplification products (HOM-NSCLC-11) and cancer related autoantigens (HOM- MEL-2.4) as reviewed in Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge.
  • MART-1 Melnoma Antigen Recognized by T-cells-1
  • MAGE-A MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A12
  • MAGE B MAGE-B1- MAGE- B24
  • MAGE–C MAGE-C1/CT7, CT10
  • GAGE GAGE-1, GAGE-8, PAGE-1, PAGE-4, XAGE-1, XAGE-3
  • LAGE LAGE-1a(1S), -1b(1L), NY-ESO-1
  • SSX SSX1-SSX-5
  • BAGE SCP-1, PRAME (MAPE), SART-1, SART-3, CTp11, TSP50, CT9/BRDT, gp100, MART-1, TRP-1, TRP-2, MELAN-A/MART-1, Carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), MUCIN
  • the tumor antigen comprises NY-ESO-1 or an antigenic fragment thereof.
  • antigen refers to protein or peptide to be introduced into a subject. As described herein, an antigen may be provided through delivering a peptide or protein or through delivering a nucleic acid encoding a peptide or protein. [00451] By “antigen” in the context of the present disclosure it is also meant to incorporate an antigenic peptide derived from an antigen. In particular, “cancer associated antigen” is intended to encompass a peptide derived from a cancer associated antigen.
  • An antigen such as a cancer-associated antigen can be provided for use as a medicament in several different ways. It can be administered as part of a vector.
  • Any suitable vector may be used to introduce a polynucleotide that encodes a polypeptide of the invention encoding one of the tumor antigen proteins into the host. Additional vectors that have been described in the literature include replication deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al., J. Virol., 72(1): 811-81(1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp.4346.]; adeno-associated viral (AAV) vectors [ U.S.
  • AV adenoviral vectors
  • Patent No.5,856,152 or a vaccinia viral or a herpesviral
  • U.S. Patent No. 5,879,934 U.S. Patent No.5,849,571; U.S. Patent No.5,830,727; U.S. Patent No.5,661,033; U.S. Patent No.5,328,688; Lipofectin mediated gene transfer (BRL); liposomal vectors
  • Suitable cancer vaccines are known in the art and may be produced by any convenient technique.
  • a cancer vaccine may be generated wholly or partly by chemical synthesis.
  • a peptide-based vaccine or immunogen may be synthesised using liquid or solid-phase synthesis methods; in solution; or by any combination of solid-phase, liquid phase and solution chemistry, e.g., by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • Chemical synthesis of peptides is well-known in the art (J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984); M.
  • peptide-based cancer vaccines may be generated wholly or partly by recombinant techniques.
  • a nucleic acid encoding a cancer antigen may be expressed in a host cell and the expressed antigen isolated and/or purified from the cell culture.
  • antigen may be expressed in E. coli either in soluble form or in inclusion bodies, which may be solubilized and refolded. After expression, the antigen may be isolated and/or purified. Cancer antigen may be analyzed by standard techniques, such as mass spectrometry and western blot analysis.
  • the use of tumor antigens to generate immune responses is well-established in the art (see for example: Kakimi K, et al.
  • an immunotherapeutic agent such as a cancer vaccine
  • a method as described herein may comprise the step of identifying a cancer antigen which is displayed by one or more cancer cells in a sample obtained from the individual.
  • a biological sample may be obtained from the subject such as a biopsy, blood or bone marrow sample and tested for the presents of cancer cells which may be identified as displaying the cancer antigen using any standard techniques including but not limited to immunological techniques, such as immunocytochemistry and immunohistochemistry may be employed. Additional techniques include immunological analysis such as serologically determining an autologous immune response to said cancer antigen, see WO2001/007917. Analysis of gene expression can be performed using methods known in the art such as polymerase chain reaction or microarray analysis.
  • the cancer vaccine may be administered in conjunction with an adjuvant, including adjuvants described above.
  • an immunotherapeutic, immunogenic or vaccine formulation may comprise an adjuvant.
  • a formulation may comprise 1-500 ⁇ g, preferably 1-50 ⁇ g, of cancer antigen and 0.5 to 20 mg, preferably 1-10 mg, of adjuvant in a pharmaceutically acceptable carrier or diluent as mentioned above.
  • a vaccine formulation may comprise a Toll-like Receptor (TLR) ligand.
  • TLR Toll-like Receptor
  • TLR ligands include polyinosinicpolycytidylic acid (poly I:C), lipopolysaccharide (LPS), CpG oligodeoxynucleotide, poly LC, poly ICLC, MPL (Corixa Corp) and imidazoquinolines, such as imiquimod and R848.
  • poly I:C polyinosinicpolycytidylic acid
  • LPS lipopolysaccharide
  • CpG oligodeoxynucleotide poly LC
  • poly ICLC poly ICLC
  • MPL Corixa Corp
  • imidazoquinolines such as imiquimod and R848.
  • the use of TLR ligands to modulate immune responses is well-known in the art (see for example, Weiner et al (1997) PNAS USA 9410833-10837; Vabulas et al J. Immunol. (2000) 1642372-2378; Gunzer et al
  • Formulations of immunotherapeutic agents are well-known in the art and include MAGE-A3 ASCI, NY-ESO-1 ASCI and PRAME ASCI (GSK Bio); Provenge (Dendreon), Abogovomab (Meranini), , M-Vax (Avax), Allovectin-7 (Vial) for metastatic melanoma, GSK1572932A (GSK Bio) Belagenpumatucel-L (Novarex) BMP-25 (Merck Serono), BiovaxID (Biovest/Accentia), MDX-1379 (Medarex/BMS) , Ipilimumab (BMS) Trovax (Oxford Biomedical) Oncophage (Antigenics) and PR1 leukemia peptide (The Vaccine company).
  • Antibody co-therapeutics Embodiments that involve use of antibodies as co-therapeutics may include all varieties of antibodies that have been developed for such purposes, including but not limited to monoclonal antibodies, chimeric antibodies, and antibody fragments.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are generally highly specific, and may be directed against a single antigenic site, in contrast to polyclonal antibody preparations that typically include different antibodies directed against the same or different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.
  • Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. (Nature, 256:495-7, 1975) (Harlow & Lane; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1988); Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.4,816,567).
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol.222:581- 597, 1991). Additional methods for producing monoclonal antibodies are well-known to a person of ordinary skill in the art.
  • Monoclonal antibodies such as those produced by the above methods, are suitably separated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.
  • HIC hydrophobic interaction chromatography
  • Chimeric monoclonal antibodies are less immunogenic in humans than the parental non-human (e.g., mouse) monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis.
  • Chimeric monoclonal antibodies in which the variable Ig domains of a non-human (e.g., mouse) monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984); and Boulianne et al, Nature 312, 643-646, (1984)).
  • Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as humanizing through “CDR grafting”), (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”), or, alternatively, (3) substituting human amino acids at positions determined to be unlikely to adversely affect either antigen binding or protein folding, but likely to reduce immunogenicity in a human environment (e.g., HUMAN ENGINEERINGTM).
  • CDRs complementarity determining regions
  • humanized antibodies will include both “humanized,” “veneered” and “HUMAN ENGINEEREDTM” antibodies. These methods are disclosed in, e.g., Jones et al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-153(1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immunol.31:169-217 (1994); Studnicka et al.
  • Human antibodies to target protein can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci.
  • WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci.
  • WO 91/0090 also discloses transgenic non- primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated.
  • 6,091,001 disclose the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule.
  • WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci.
  • U.S. Patent No.5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. See also, U.S.
  • the invention will be more fully understood by reference to the following examples which detail exemplary embodiments of the invention. They should not, however, be construed as limiting the scope of the invention. All citations throughout the disclosure are hereby expressly incorporated by reference.
  • IMM60 is a highly lipophilic molecule (necessary for its tight binding to CD1d) and is practically insoluble in aqueous solutions. Thus, a liposomal formulation was selected so that IMM60 can be delivered in a physically stable and soluble form necessary for the IV route of administration.
  • a related physiochemical consideration is that IMM60 has a high calculated log of the partition coefficient (cLogP) value (17.6), indicating its lipophilicity. Structurally, IMM60 contains 2 straight chain aliphatic tails. IMM60 is more lipophilic based on the cLogP than DSPC.
  • IMM60 – with its long lipid chain – is intercalated within the liposomal bilayer and solubilized as a component of the phospholipid bilayer membrane.
  • Liposomal formulations can specifically target the tumor site through the enhanced permeability and retention (EPR) effect.
  • EPR enhanced permeability and retention
  • Micelles and liposomes containing anticancer drugs are some examples of nanoparticle formulations in which small, colloidal nanoparticles, in the range of 100 to 200 nm, are able to extravasate across tumour vasculature, and penetrate and accumulate in tumor tissue via the EPR effect.
  • the drug concentration in the tumor compared to the blood can be as high as 10 to 30 times when administered in liposomes versus solubilized formulations, and the EPR effect can be evident within 10 minutes after IV injection.
  • IMM60 glycolipid such as IMM60 for use in the treatment of cancer.
  • Lipid Hydration (MLV formation): The aqueous phase is composed of 145 mM sodium chloride and 10 mM sodium phosphate at pH 6.5 or an alternative buffer. Lipids are hydrated and multilamellar vesicles (MLVs) are formed upon dilution of the organic phase containing the API and lipid into the aqueous phase. Hydration is allowed to proceed for a few minutes at temperature with mixing.
  • Extrusion The Lipex extrusion system is set up, connected to a heated water bath to maintain the process temperature, and prepared with several layers of polycarbonate filters. MLVs are put through a LIPEX extruder with polycarbonate filters to perform size reduction to large unilamellar vessicles (LUVs) with a target vesicle size in the range between 90 and 100 nm, in diameter. Nitrogen gas is used to pressurize the system up to 600 psi and to extrude the MLVs through the polycarbonate membrane filters. Several extrusion passes may be required to attain the target particle size range.
  • LUVs large unilamellar vessicles
  • Diafiltration Upon size reduction the LUVs are cooled to near room temperature ( ⁇ 30°C) and is loaded onto diafiltration cartridges. In this process the liposomes are essentially washed using tangential flow with buffer exchange using 145mM sodium chloride and 10mM sodium phosphate at pH 6.5 to remove organic solvent from the formulation. Tangential flow filtration is also used to concentrate the product to adjust the lipid and API concentration to the target specification (to 1mg/mL API and 9 mg/ml lipids). [00479] Clarifying and Sterile Filtration: Upon completion of the diafiltration process the product is filtered through a sterilizing grade 0.2 ⁇ m filter to reduce bioburden.
  • the concentration of the product is slightly at higher value than label claim.
  • the product is stored overnight during in process determination for lipid and API content.
  • the final concentration of the product is diluted to label claim and sterile filtered aseptically by redundant filtration 2 x 0.2 ⁇ m, mPES membrane filters (Sartopore, Sartorius).
  • Particle Size Measurements by Dynamic Light Scattering During manufacturing the particle size was monitored using dynamic light scattering measurements at two angles, 90° and 12.8°.
  • the product was diluted to a lipid concentration range between 0.5 mg/mL and 1 mg/mL in saline (0.9 % sodium chloride) as diluent, pre-equilibrated for 2 minutes at 23°C in disposable sizing cuvettes and read using the Malvern Zetasizer instrument with the following settings: dispersant refractive index (RI) of 1.332, material RI of 1.50.
  • RI dispersant refractive index
  • RI dispersant refractive index
  • 4.5 mL of ethanol/0.5 mL of water were charged with 0.1238 g DSPC, 0.1004 g DSPG, and 0.0272 g IMM60 (API required: 0.0259 g corrected for 95.3 % purity).
  • the alcohol solution was added to a buffer solution (e.g., 250 mM sucrose and 145 mM NaCl).
  • a buffer solution e.g. 250 mM sucrose and 145 mM NaCl.
  • the multi- lamellar vesicles solution was extruded, e.g., through 2 x 80 nm membranes at 65°C, to form the liposomes.
  • the ultrafiltered solution was diafiltered (e.g., Krosflow) with 50 mL reservoir (e.g., 115 cm 2 ) against 10 working volumes (500 mL).
  • the solution was concentrated down to about 1.3 mg/mL of API.
  • the solution was filtered using a clarifying filter at ambient temperature (filter 0.2 ⁇ m Sartopore (mPES membrane).
  • the alcohol solution above can contain two lipids selected from the group of: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3- phospho-rac-glycerol (DSPG); (b) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and di
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero- [phosphor-rac-(1-glycerol)]
  • DPPG 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol
  • DPPG 1,2-dipalmitoyl-sn- glycero-[phosphor-rac-(1-glycerol)]
  • the alcohol solution may comprise at least two (e.g., 3) phospholipids selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl- sn-glycero-3-phospho-rac-glycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1-glycerol)] (DPPG).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPG 1,2-distearoyl- sn-glycero-3-phospho-rac-glycerol
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-[phosphor-rac-(1
  • the alcohol solution consist essesentially of DSPC and DSPG.
  • Table 2 and Table 3 summarize their particle size and API and lipid content after sterile filtration. Initially, a liposomal IMM60 prototype was prepared to assess feasibility of the manufacturing process by incorporating IMM60 into EPC/EPG liposomes (A). The particle size of the MLV from batch A was particularly small with ⁇ 5nm and remained stable throughout the process. API content was below the 1 mg/mL target (refer to Table 3).
  • Batch 05 did exhibit some particle size growth and was difficult to sterile-filter, resulting in a loss of 50% of the API and lipids.
  • Batches 04 and 07 contained DOTAP which appeared to significantly degrade during the solubilisation process, as the DOTAP content was well below expectations in the MLV’s (roughly 0.1-0.2 mg/mL). The degradation may have been due to the high temperature (60°C) that was required in the manufacturing process, as the API solubility is dependent on this temperature.
  • Batch 04 contained POPC/DOTAP and exhibited slight particle size growth.
  • Batch 07 contained only cholesterol as the second lipid and significantly increased in size. Batch 07 also showed flocculation, which lead to the difficulty in filtration and the significant loss of lipids and API.
  • the DOTAP:POPC batch was manufactured a second time under protocol D with a shorter temperature exposure time and a sucrose buffer instead of saline. This repeated batch showed improvements in particle size, as well as increased stability of lipids during each stage of the manufacturing process.
  • Table 1 API and Lipid Composition Batch Composition (wt/wt/wt) A EPC, EPG, IMM60 (2.25/6.75/1) B DSPC, DSPG, IMM60 (4.78/3.88/1) C01 EPC, EPG, IMM60 (2.25/6.75/1) C02 POPC, DDAB, IMM60 (4.58/3.04/1) C03 POPC, DC-Chol, IMM60 (4.60/2.60/1) C04 POPC, DOTAP, IMM60 (4.61/3.40/1) C05 DSPC, DSPG, IMM60 (4.76/3.86/1) C06 DMPG, Chol, IMM60 (4.60/1.64/1) C07 DOTAP, Chol, IMM60 (4.66/1.64/1) D01 POPC, DOTAP, IMM60 (4.61/3.40/1) Table 2: Particle Size Data Batch 90° 12.8° Size (nm) PdI Size (nm) PdI A 52.15
  • Study I confirmed a stable particle size upon extrusion into room temperature buffer (1:1); and improved lipid recovery by ⁇ 9% when a cartridge with smaller pore size, 100 kD versus 500 kD, was used.
  • a 50 mL batch (Lot J) using IMM60 was prepared using the modified procedures including a post-extrusion dilution (1:1) with RT buffer and diafiltration prior to ultrafiltration.
  • the use of 100 KDa pore size cartridge for DF/UF was problematic and significantly increased the processing time. For this reason, the DF/UF was completed with a 115 cm 2 , 500 kDa cartridge instead.
  • Initial stability testing of the first composition included the testing of particle size of multi-lamellar vesicles (MLV), after addition of the liposomes into the the buffer in a 1:1 dilution (Pass 1), after diafiltration (post DF), after ultrafiltration (post UF), and after clarifying filtration (post CF) shown in Table 5.
  • the first composition was then stored in a buffer (250 mM Sucrose, 145 mM NaCl, 10 mM Na-Phosphate, pH 6.5) at 25 °C, 5°C, and -20°C. Table shows the concentrations of the various components in the composition after 9 days and after 1 month.
  • the heat treatment includes heating the liposomes to 55°C for 10 minutes after storage.
  • Table 7 shows the formulation when stored at -20°C, 5°C, and 25°C.
  • Exemplary tools for heating include heat blocks, hot plates, ovens, and water baths.
  • Initial stability testing of the second composition included the testing of particle size after MLV, after addition of the liposomes into the the buffer in a 1:1 dilution (Pass 1), after diafiltration (post DF), after ultrafiltration (post UF), and after clarifying filtration (post CF) shown in Table 9.
  • the second composition was then stored in a buffer (250mM Sucrose, 145 mM NaCl, 10mM Na-Phosphate, pH 6.5) at 25 °C, 5°C, and -20°C.
  • Table 10 shows the concentrations of the various components in the composition after 8 days and after 1 month.
  • the heat treatment includes heating the liposomes to 55 °C for 10 minutes after storage.
  • Table 11 shows the formulation when stored at -20 °C, 5 °C, and 25 °C.
  • compositions comprising heat-treated liposomes are contemplated as an aspect of the invention.
  • Methods of manufacture that include a heat treatment step are intended as an aspect of the invention.
  • Methods of preparing or standarding liposomal compositions that include a heat treatment are intended as an aspect of the invention, especially for a liposomal composition that has been stored under freezing or sub-zero conditions or freeze-dried and reconstituted in an aqueous buffer.
  • the heat treatment is for a time and at a temperature sufficient to reduce average particle size and/or reduce PdI of the composition.
  • the heat treatment is at a temperature in the range of about 55 °C to about 65 °C.
  • the heat treatment is for about 5 minutes to about 15 minutes, e.g., for about ten minutes.
  • Liposomal IMM60 Compared to Soluble IMM60
  • Liposomal IMM60 (DSPC:DSPG:IMM60) was compared to soluble IMM60 for its ability to generate an ovalbumin-specific immune response as well as its ability to mature dendritic cells and to induce accumulation of IFN- ⁇ in the serum.
  • the two IMM60 formulations were each co-injected with the ovalbumin antigen into C57BL/mice.
  • Liposomal IMM60 and soluble IMM60 were injected at 0.1 ng/mouse (approx.4 ng/Kg), they induced similar levels of serum IFN- ⁇ accumulation with no statistically significant different between them.
  • liposomal IMM60 was significantly more potent at maturing splenic dendritic cells 18 hours after administration, and also resulted in significantly more CD8+ T cells that were specific for the ovalbumin peptide epitope SIINFEKL 7 days after administration.
  • the early stimulation capacity of these formulations was tested by measureing serum IFN- ⁇ 18 hours after injection.
  • IFN- ⁇ in the serum at this time point is a well- established standard assay for iNKT cell activation, and it is used as a surrogate assay for the early activation of iNKT cell, which then cascades into amplified production of IFN- ⁇ by NK cells.
  • a second assay assessed the maturation level of splenic dendritic cells 18 hours after administration of the IMM60 formulations. Cognate interaction between iNKT cells and dendritic cells has been shown, in previous studies, to result in maturation of the dendritic cells in a CD40:CD40-ligand dependant manner.
  • SIINFEKL ovalbumin-derived peptide
  • mice are a chosen animal model of in vivo studies described herein since they are the lowest vertebrate group in the evolutionary tree for which suitable models of the immune responses in general, and iNKT biology in particular, are available, including mutant strains that lack either iNKT cells or CD1d moleclules.
  • Soluble IMM60 - Lyophilized material was resuspended in chloroform:methanol:water (10:10:3) to 10 mg/ml (vials of 100 ⁇ g in 10 ⁇ l) and swirled around to capture all the material, and then Vehicle solution (150 mM NaCl, 0.5 % Tween20 in distilled water) was added quickly to a final concentration of 100 ⁇ g/ml (for 100 ⁇ g in 10 ⁇ l , added 990 ⁇ l of Vehicle). This composition is then heated to 80-90 °C for 5 minutes and sonicated with a waterbath sonicator for 5 minutes.
  • Vehicle solution 150 mM NaCl, 0.5 % Tween20 in distilled water
  • Liposomal IMM60 (stock 0.47 mg/ml): 1st dilution: 4.7 ml PBS + 10 ⁇ l of stock DSPC:DSPG:IMM60 2nd dilution 10 ml of PBS + 10 ⁇ l of 1st dilution 3rd dilution 9 ml of PBS + 1 ml of 2nd dilution Soluble IMM60 (stock 100 ⁇ g/ml): 1st dilution 5 ml PBS + 5 ⁇ l stock 2nd dilution 4.95 ml PBS + 50 ⁇ l of 1st dilution [00505] Mice were then warmed up for 20-30 minutes in cages place in a 37 °C heating chamber.
  • Group 1 200 ⁇ l PBS was injected intravenously.
  • Group 2 200 ⁇ l of a mix of 1:1 ovalbumin:PBS.
  • Group 3 200 ⁇ l of a mix of 1:1 ovalbumin:soluble IMM60 (2 nd dilution).
  • Group 4 200 ⁇ l of a mix of 1:1 ovalbumin:Liposomal IMM60 (2 nd dilution).
  • Group 5 200 ⁇ l of a mix of 1:1 ovalbumin:Liposomal IMM60 (3 rd dilution).
  • Animals were injected intravenously in the lateral tail vein. Cages containing 3-mice were warmed in a heating chamer (37 °C) for 20-30 minutes.
  • mice were placed in a restrainer that allowed the tail to protrude out. Using 27G insulin syringe needles, mice were injected with 200 ⁇ l of the recorded dose over approximately 4 seconds. Group size is detailed in Table 12 below, and mice were housed in individually ventilated cages in groups of 4-6: Table 12 Group Injected material C57BL/6 C57BL/6 1 Na ⁇ ve 5 2 2 ovalbumin+ vehicle 6 2 3 ovalbumin+ soluble IMM600.1 n g/mouse 11 2 4 ovalbumin+ liposomal IMM600.1 n g/mouse 11 2 5 ovalbumin+ liposomal IMM600.01 n g/mouse 11 2 44 10 culled at 18 hrs [00507] 18 hours after injection of the doses, blood samples were taken for analysis of serum and mice were euthanized and splenocytes were recovered for analysis of CD11c+ cells’ maturation.
  • Blood sampling – Serum Cages containing 3-mice were warmed in a 37 °C heating chamber for 20-30 minutes after which individual mice were placed in a restrainer leaving the tail exposed. Using a scalpel, a nick in the lateral tail vein was made and blood was collected into serum separation tubes. Blood flow was stopped by applying pressure and the mice returned to their normal housing cages. Blood was allowed to coagulate for 30 min. at room temperature and then serum was separated by centrifugation in a benchtop microcentrifuge for 2 minutes at 13000g. Serum transferred to wells in a 9U bottomed well dish and frozen at -20 °C. Thawed samples were later used without dilution in a standard Sandwich ELISA.
  • Blood Sampling – cells Blood was collected from the lateral tail vein as above but instead of serum separation tubes, drops of blood (about 100 ⁇ l) were collected into a 1.5 ml eppendoff tube containing 200 ⁇ l of 10mM EDTA/PBS solution and mixed by inverting times. Cells were kept on ice until a full cage of mice were bled. 1 ml of Red Blood Cell Lysis Solution (Qiagen) was added to the blood and mixed by inversion. Samples were kept on ice until they turned transluscent. Tubes were then centrifuged on a microcentrifuge 4 KRPM for 4 minutes at room temperature. Supernatant was removed along with platlettes leaving a buffy-coloured pellet.
  • Red Blood Cell Lysis Solution Qiagen
  • the cells in the pellet were washed with 200 ⁇ l of cold PBS and transferred into U- bottomed wells in a 96-well plate and pelleted for 2 minutes at 577g (1800 rpm). Supernatant was removed and cells were treated with Fc Block (anti CD16/CD32 antibody). Pellet was resuspended in 10 ⁇ l of 1:10 dilution of tetrameric pMHC K b /SIINFEKL, and the plate was covered in aluminum foil and incubated at 37 °C for 30 minutes.
  • Splenocytes Mice were euthanised by a Schedule 1 procedure (cervical dislocation) and spleens were collected and kept in 1.5 ml RPMI on ice.
  • Spleens were mashed and squeezed through a 70 ⁇ m sieve into a 50 ml Falcon tube using the flat end of a 1ml syringe plunger, and the cells were peleted by centrifugation at 1500 rpm for 5 minutes in an underbench centrifuge (484g).
  • Splenocytes were resuspended in 2 ml Red Cell Lysis Solution (Quiagen) and after 2 minutes at room temperature were diluted with 8 ml of full media (RPMI, 10%FBS, Pen/strep, Glut). Splenocytes were mixed and then peleted by centrifugation (484g) at room temperature.
  • Splenocyets were then resuspended in 3mls of full media and counted. 2x1ealiquotes of the splenocytes were placed in a well of a plate of 9U bottom wells. Cell were peleted by centrifugation 2 minutes at 1800 rpm (577g) and the supernatant discarded. Each pellet was resuspended in a mixture of fluorescent antibodys and left on ice for 15 minutes then washed twice in PBS + 0.1 % FBS before being diluted with 200 ⁇ l of the same solution and analysed using BD LSR Fortessa FACS machine.
  • non- glycosidic compounds can stimulate both human and mouse iNKT cells); the source of the IFN- ⁇ is NK cells that had been transactivated into producing IFN- ⁇ by iNKT cells which produce the same cytokine at an earlier stage following recognition of the iNKT cell agonist. In contrast, no induction of IFN- ⁇ was observed in the animals that were untreated or treated with Vehicle (and ovalbumin) alone. Injection of liposomal IMM60 (co-injected with ovalbumin) produce intermediate amounts of serum IFN- ⁇ .
  • Maturation of CD11c+ dendritic cells in mice treated with liposomal IMM60 versus soluble IMM60 Maturation of dendritic cells was determined by measuring the levels of the costimulatory molecule CD8on the surface of CD11c+ MHCclass II+ cells. In this assay, a clear difference in the potency of IMM60 was observed when it was delivered as a soluble molecule compared to when it was delivered as a liposomal formulation ( Figure 7). Both treatments contained the same amount of IMM60 (0.1ng/mouse), yet the liposomal formulation induced CD8levels dendritic cells that were 2.5 times higher.
  • Example 4 Intravenous Injection of six different liposomal formulations of IMM60 (POPC/DDAB, DSPC/DSPG, and POPC/DC-Chol) Into Mice [00518]
  • Figure 5 shows results from an experiment to measure INF-gamma 18 hours after administration of six different prototype liposomal formulations of IMM60, compared to soluble ⁇ GalCer. This experiment showed that a DSPC/DSPG prototype liposome had the highest dynamic range (flat and elevated response across the doses) and the response at the lowest dose.
  • the doses are (from left to right) 0.01, 0.1, 1, 5 ng/mL with a 100 uL injection.
  • the positive control used for this experiment was soluble alpha-GalCer at 1.0 ng/mL.
  • the data also showed that, for the second through fifth prototype liposomes, IFN ⁇ measurements for the higher two doses are substantially elevated compared to soluble alpha-GalCer at 1.0 ng/mL.
  • Example 5 Intravenous Injection of three different liposomal formulations of IMM60 (POPC/DDAB, DSPC/DSPG, and POPC/DC-Chol) Into Mice [00519] Three different IMM60 liposomal formulations (POPC/DDAB, DSPC/DSPG, and POPC/DC-Chol) were tested by IV injection in mice. The effect of the different liposomal preparations on IMM60’s ability to induce serum levels of IFN- ⁇ at 18 hours was determined, as well as the degree of iNKT cell-mediated maturation of splenic dendritic cells (DCs) and compensatory upregulation of PD-L1 ( Figure 1A-1C).
  • DCs splenic dendritic cells
  • CD1d knockout mice were used as the control, because CD1 knockout mice do not produce iNKT cells (due to lack of CD1 expression).
  • Example 7 Stability of liposomal IMM60 in vivo following intravenous injection of mice [00523] Synopsis [00524] To estimate the biological half-life of IMM60 following an intravenous injection of liposomal IMM60, levels of IMM60 in the plasma were measured in two strains of mice, injected with two doses (0.5 and 50 ⁇ g/Kg), 10 minutes or 180 minutes after injection of Liposomal IMM60. The two strains of mice that were compared were CD1 mice (not to be confused with CD1d-KO mice) and C57BL/mice. The half-life, volume of distribution, clearance, AUC, and Cmax were calculated using the one-compartmental model with first-order kinetics.
  • Lipid formulation DSPG:DSPC:IMM60 (3.44:4.37:1 mg/ml) in a 250 mM sucrose, 145 mM NaCl, 10 mM sodium phosphate, pH 6.5 buffer.
  • Vials of liposomal IMM60 were thawed at room temperature (21°C-22°C), then heated to 55 °C for 10 minutes using a heated water bath. Vials were mixed by inversion six times, cooled to room temperature, and diluted to either 100 ng/ml or 10,000 ng/ml using injection grade saline (0.9 % NaCl).
  • Soluble IMM60 Lyophilized material was resuspended in Chloroform:methanol:water (10:10:3) to 10 mg/ml (vials of 100 ⁇ g in 10 ⁇ l) swirled around to capture all the material and then Vehicle solution (150 mM NaCl, 0.5% Tween20 in distilled water) added quickly to a final concentration of 100 ⁇ g/ml (for 100 ⁇ g in 10 ⁇ l , added 990 ⁇ l of Vehicle), then then heated to 80-90 °C for 5 minutes, and sonicated with a waterbath sonicator for 5 minutes. Samples were diluted to 10,000 ng/ml using injection grade saline.
  • mice were warmed up for 20-30 minutes in cages place in a 37 °C heating chamber. Mice within each group were injected intravenously, using 27G insulin syringe needles, with 100 ⁇ l (C57BL/mice) or 150 ⁇ l (CD-1 mice) of the recorded dose over approximately 4 seconds.
  • Plasma samples were analyzed by LC/MS/MS at Frontage Laboratories. The assay used to measure IMM60 in the plasma after administration of liposomal IMM60 injection did not distinguish liposome-bound IMM60 from the soluble (unbound) form of IMM60.
  • the method uses alpha GalCer (analog to IMM60) as an internal standard and the mass spectroctoscopic ionization is electrospray in the positive ionization mode.
  • the range of calibration standards is between 1 ng/mL to 1000 ng/mL IMM60 in mouse plasma.
  • Plasma concentrations were measured 10 and 180 minutes after IV administration.
  • the half-life, clearance, and AUC (0 ⁇ ) were calculated using the one-compartmental model with first- order kinetics since only two time points were samples along the PK profile. Using these asumptions allow for the calculation of PK parameters.
  • Vd Dose/C10 min where dose is expressed as is or dose/body weight
  • AUC (0 ⁇ ) C 10 min /k e [00531]
  • SD standard deviation
  • SE standard error of the mean
  • p-value from the t-test was calculated using Microsoft Excel 201(Microsoft Corporation). Equal or unequal variances were chosen based on use of the F-test function. All t-test comparasons were performed with the 1000 ng IMM60 in liposome form as the control.
  • Half-life T(1/2) in Mouse of IMM60 The half-life for mice of IMM60 is calculated using Equation 1 using the one-compartment model with 1 st order kinetics. Table 13. Half-life in Mouse of IMM60 half life 1000ng IMM60 1000ng IMM60 1500ng IMM60 1500ng IMM60 (min) in liposome soluble in liposome soluble C57BL/6 C57BL/6 CD1 CD1 49.00 90.93 63.56 159.41 50.97 98.13 62.36 118.64 42.94 37.80 46.25 46.02 Average 47.29 94.53 52.43 139.03 STD 3.49 5.09 12.62 28.82 SE 1.74 3.60 6.31 20.38 N 4 2 4 2 p-value 0.00 0.46 0.14 [00533] Volume of distribution: The volume of distribution in mice of IMM60 is calculated using Equation 2 using the one-compartment model with 1 st order kinetics.
  • AUC(0- ⁇ ) Exposure (AUC(0- ⁇ )) in Mouse of IMM60 AUC(0- ⁇ )) 1000ng IMM60 1000ng IMM60 1500ng IMM60 1500ng IMM60 (ng/mL/min) in liposome soluble in liposome soluble C57BL/6 C57BL/6 CD1 CD1 8,061 12,032 13,757 14,353 8,311 12,418 30,056 17,976 9,603 8,236 10,144 8,367 Average 9,030 12,225 15,104 16,165 STD 1,004 273 10,294 2,562 SE 502 193 5,147 1,812 N 4 2 4 2 p-value 0.01 0.32 0.01
  • C max of IMM60 The C max in mice of IMM60 is calculated using the T 10Min time point concentration. Table 17.
  • Cmax in Mouse of IMM60 Cmax 1000ng IMM60 1000ng IMM60 10ng IMM60 in 1500ng IMM60 1500ng IMM60 15ng IMM60 in (ng/mL) in liposome soluble liposome in liposome soluble liposome C57BL/6 C57BL/6 C57BL/6 CD1 CD1 CD1 114.0 91.7 2.9 150.0 62.4 1.5 113.0 87.7 1.4 334.0 105.0 2.7 155.0 2.1 151.0 152.0 126.0 Average 133.5 89.7 2.1 190.3 83.7 2.1 STD 23.1 2.8 0.8 96.5 30.1 0.8 SE 11.6 2.0 0.5 48.3 21.3 0.6 N 4 2 3 4 2 2 2 p-value 0.07 0.00 0.33 0.08 0.00 [00537] Pharmacokinetic Parameters for the High Dose (50 ⁇ g/kg) Liposomal and Soluble IMM60 In
  • mice of IMM60 are calculated using the equations above using the one-compartment model with 1 st order kinetics. Table 18.
  • the volume of distribution of the soluble formulation appears larger with V d between 558 to 639 ml/kg.
  • Clearance The clearance of the liposomal IMM60 formulation in both the C57BL/and CD1 strains were considered equivalent with the clearance between 4.3-5.ml/kg/min for the high dose. The low dose could not be calculated due to the 3 hour plasma samples being less than the assay LOQ. The clearance of the soluble formulation appears lower with the values between 3.1 to 4.1 ml/kg/min.
  • Exposure The exposure of the liposomal IMM60 formulation in both the C57BL/and CD1 strains were considered equivalent with AUC (0- ⁇ ) between 9,030 to 15,104 ng/mL/min for the high dose.
  • C max The C max of the liposomal IMM60 formulation in both the C57BL/and CD1 strains were considered equivalent with concentration between 134 to 190 to ng/ml for the high dose.
  • the low dose for each strain was also considered equivalent, when scaled for the dose proportionality using the IMM60 dose in ⁇ g/kg for the low and high dose animals (low dose is 100-fold lower than the high dose).
  • the Cmax for the low dose was approximately 2.1 ng/mL for both mice strains.
  • the invention includes liposomal formulations of the API that, when administered to C57BL/or CD1 mice, provide a C max at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 75%, or at least 100% greater than the C max observed with an equivalent size dose of soluble API. [00545] There were no pharmacokinetic differences detected between the C57BL/and CD1 mouse strains.
  • the invention includes liposomal formulations of the API that, when administered to C57BL/or CD1 mice, provide a Vd (measured in (mL/kg) that is at least 20%, or at least 30%, or at least 40%, or at least 50% less than the Vd observed with an equivalent size dose of soluble API.
  • Example 8 Mouse Melanoma Injections of Lipsomal IMM60 [00547] To evaluate anti-cancer therapeutic properties of liposomal IMM60, the potential of liposomal IMM60 to prevent lung metastatic lesions in a mouse melanoma model was tested in both a prophylactic and therapeutic setting. Three doses of liposomal IMM60 were injected IV either 3 days before (prophylactic) or 3 days after (therapeutic) tumor cell injection. Mice were injected IV with B16-F10 melanoma cells and 15 days later metastatic nodules in the lung were counted.
  • Prophylactic treatment was more effective than therapeutic treatment at preventing tumour formation in the lungs; nevertheless, with both prophylactic and therapeutic treatments, there was significant tumor reduction compared with mock-treated mice even at the lowest liposomal IMM60 dose (0.01 ng/mouse) ( Figure 9A and 9B). In fact, maximal effect was found at 0.01 ng liposomal IMM60/mouse, with no significant additional benefit found at higher liposomal IMM60 doses.
  • Treatment with soluble IMM60 can upregulate the levels of PD-1 on iNKT cells. The same treatment also upregulates expression of PD-L1, the ligand for PD-1, on a variety of immune cells.
  • a nonresponsive B16-F10 mouse melanoma SC model (“cold tumour model”) was utilized to assess the combination treatment in a therapeutic setting where the treatments were administered on Days 3, 6, and 9 after subcutaneous tumour implantations. Two dose levels of liposomal IMM60 were tested together with the immune checkpoint inhibitor anti-PD-1 antibody (10 mg/kg), and compared with either liposomal IMM60 alone, anti-PD-1 antibody alone, or an isotype control antibody.
  • Example 10 Combination Therapy with Anti-PD-1 Blocking Antigen – CT-2tumor model Overview
  • IMM60 liposomes as a stand-alone treatment or in combination with anti-mouse PD-1 on tumor growth and survival in the CT-2tumor model in BALB/c mice was assessed, and IFN- ⁇ levels in the serum of experimental mice were measured 18 hours after the first treatment.
  • Tumor volumes in the mice were measured over 14 days. In the fold- change over time, the interaction showed a trend towards statistical significance and the effect of treatment approached a trend towards significance and the post-hoc analysis detected a significant difference between the IMM60/anti-PD1 and vehicle control groups on Days 11 and 14.
  • Survival curves were not significantly different due to treatment.
  • mice received a subcutaneous inoculation of 0.3 ⁇ 10CT-2cells. Grouping and treatments for 32 mice were initiated when the mean tumor volume reached approximately 100 mm 3 on Day 0, ten days after CT-2cells were inoculated. The four treatment groups received vehicle, IMM60, anti-PD1, or IMM60 in combination with anti-PD1 as outlined in the following Table.
  • Serum was separated from whole blood by clotting for 30’ at room temperature and centrifuging for 15’ at 1000 x g, frozen at -80°C until analysis. IFN- ⁇ levels were determined using Meso Scale Discovery (MSD), according to the manufacturer’s protocol.
  • MSD Meso Scale Discovery
  • Body weights of the animals in different groups closely tracked each other over the study period.
  • Bonferroni’s post-hoc analysis is shown in Figure 12B and detected a significant difference between the IMM60/anti-PD1 and vehicle control groups on Days 11 and 14.
  • Tumor volumes for individual animals are shown in Figure 12C.
  • IFN- ⁇ levels measured at 18 hours are plotted in Figure 12D.
  • a significant effect of treatment was detected in the one-way ANOVA (P ⁇ 0.0001).
  • the levels of IFN- ⁇ were highly elevated in the IMM60 and IMM60/anti-PD1 treatment groups relative to the anti-PD1 group or vehicle controls.
  • the statistical differences in treatment groups versus the vehicle controls are show in the graph.
  • Example 11 - IMM60 induces expression of PD-L1 on melanoma cells co cultured with mouse splenocytes Overview
  • Using an in vitro mouse cell model we measured the effect of IMM60 on levels of PD-L1 expressed on the surface of tumour cells that are co-cultured with splenocytes. More specifically, we examined the PD-L1 expression on mouse melanoma tumor cell line B16f10 co- cultured with splenocytes (as a source of iNKT cells and antigen-presenting cells (APC) such as CD1 + B cells and CD11c + dendritic cells that are present in the ex vivo splenocytes at about 1% of live cells).
  • APC antigen-presenting cells
  • Soluble IMM60 or IMM47 Lyophilized material was resuspended in Chloroform:methanol:water (10:10:3) to 10mg/ml (vials of 100 ⁇ g in 10 ⁇ l), swirled around to capture all the material, and then Vehicle solution (150mM NaCl, 0.5% Tween20 in distilled water) added quickly to a final concentration of 100 ⁇ g/ml (for 100 ⁇ g in 10 ⁇ l , added 990 ⁇ l of Vehicle). This solution is then heated to 80 °C for 5 minutes and sonicated using a water-bath sonicator for 5 minutes.
  • Vehicle solution 150mM NaCl, 0.5% Tween20 in distilled water
  • both IMM60 and IMM47 are stored at 4 °C, and before each use they are sonicated in a water bath sonicator for 5 minutes.
  • the antibodies used were an anti-PD-1 antibody (rat anti-mouse PD-1) clone RMP1-14 (IgG2a, k) from BioXCell InVivoPlus and an Isotype control antibody (rat IgG2a,k) clone 2A3 from BioXCell InVivoPlus.
  • Antibodies were diluted to the required concentration using complete medium (RPMI, 10% FBS, Pen/Strep and Glutamine) before use.
  • mice Two C57BL/6JOlaHsd female, 8-week-old mice, were used in this study. Mice were purchased from Envigo and weighed 19-20gr. One male mutant mouse (Ja18 KO mutation that prevents expression of the invariant natural killer T cell T cell receptor (NKT TCR) ) was used from a colony that is maintained in the Oxford University Biomedical animal facility.
  • NKT TCR natural killer T cell T cell receptor
  • FACS Fluorescence Activated Cell Sorting
  • PBS Phosphate- buffered saline
  • RPMI Full medium: (RPMI with 10% Feotal Bovine Serum (FBS), Penicillin (25,000U) Streptomycin (25mg/L), glutamine (2.1mM), (all Tissue Culture grade products from Gibco); Red Blood Cell Lysis Solution (Qiagen).
  • mice were euthanized by cervical dislocation, and spleens were collected and kept in 1.5 ml RPMI on ice. Spleens were mashed and squeezed through a 70 ⁇ m sieve into a 50ml Falcon tube using the flat end of a 1ml syringe plunger, and the cells were pelleted by centrifugation at 1500rpm for 5 minutes in an under-bench centrifuge (484g). Splenocytes were resuspended in 2 ml Red Cell Lysis Solution (Qiagen), and after 2 minutes at room temperature were diluted with 8 ml of full media (RPMI,10% FBS,Pen/strep, Glut).
  • Splenocytes were washed and then pelleted by centrifugation (484g) at room temperature. Splenocyets were then resuspended in 3mls of full media and counted. Cells were resuspended at 5x1e7/ml and 100 ⁇ l (5x1e6) aliquots of the splenocytes were placed in a well of a plate of 9U bottom wells. Assay [00576] 100 ⁇ l of splenocytes were placed in wells of a 9U-bottom well plate.
  • Antibodies RMPI-14 and 2A3 were diluted to 4-fold of the final concentration (400 ⁇ g/ml) and added as 50 ⁇ l to each well to achieve a final concentration of 100 ⁇ g/ml.
  • Cells were incubated for 10 minutes at room temperature before adding the iNKT cell agonists, to allow the antibodies to bind.
  • An additional 50 ⁇ l of full media containing x4 concentration of final concentration of IMM60, IMM47 or Vehicle were added. Final effective concentrations of IMM60 or IMM47 of 0.0, 0.0001, 0.001, 0.01, 0.1, 1.1, 11, and 110 ng/ml were assayed.
  • Plates were moved to a 37 °C, 5% CO 2 incubator for 48 hours. Plates were then removed, washed, and treated with purified anti-mouse CD16/CD32 (1:50 dilution Biolegend) and then washed and stained with fluorescent antibodies and live stain as summarized in the following table: FACS analysis of splenocytes.
  • FLUORESCENCE PANEL FOR MARKER DILUTION DENDRITIC CELLS FITC CD11b Myeloid cell 1:200 population PE PD-L1 molecule up 1:200 regulated in response to INF-gamma and also involved in inhibition of PD-1+ T cells BV421 CD105 Distinguishes 1:200 B16f10 cells from splenocytes APC CD45.2 Immune cell 1:400 APC-Cy7 TCR pan Beta T cell 1:200 PE-Cy7 CD69 T cell activation 1:200 Aqua(510nm) Live/Dead Viability 1:200 [00580] Cell were pelleted by centrifugation for 2 minutes at 1800 rpm (577g), and the supernatant was discarded.
  • FIG. 13 Treatment with a combination of IMM60 and anti-PD-1 increases the level of surface PD-L1 further.
  • IMM60 and IMM47 co-cultured B16f10 cells and splenocytes were cultured with varying concentrations of IMM60 and IMM47, also supplemented with antibodies (100 ⁇ g/ml), either anti-mouse PD-1 (clone RMPI-14) or an isotype control antibody (clone 2A3).
  • Figure 13 shows a slight increase of PD-L1 expression when anti-PD-1 antibodies where used compared with isotype control antibodies. This increase was only seen in co-cultures at and above 11 ng/ml IMM60 (indicated by the arrow).
  • T cells (identified using a TCRpanBeta antibody) in the same B16f10 cells:splenocytes co-culture also upregulate surface PD-L1 when IMM60 was added. However, unlike in the B16f10 cells, this upregulation was not affected by the presence of an anti-PD-1 antibody ( Figure 14).
  • Figure 14 Conclusions [00585] The results of this experiment show that in vitro co-cultured splenocytes contain a sufficient number of iNKT cells to exert an agonist-specific effect on B16f10 melanoma cells that were co-cultured with them. This effect is likely somewhat muted compared with the effect that can be seen in vivo.
  • Non-glycosidic compounds can stimulate both human and mouse iNKT cells. It is quite possible that such sensitivity, compared with the in vitro assay, is due to efficiency of capture of the IMM60 in the live mouse and the efficient delivery to the iNKT cells. Such efficiency of capture and delivery has been demonstrated in a paper discussing the capture of blood-borne antigens by CD169+ marginal zone macrophages and dendritic cells which can present lipids to iNKT cells.
  • Example 12 Clinical Trial Protocol [00590] Following is a clinical trial protocol for administration if IMM60 alone or as part of a combination therapy.
  • the dose and dosing regimens described herein are contemplated as specific embodiments.
  • the protocol described can be repeated/modified with different liposomal formulations of IMM60; and can be repeated/modified with an alternative secondary therapy, such as a different anti-PD-1 antibody or a different immune checkpoint inhibitor.
  • IMM60 and anti-PD1 [00591]
  • the combination of IMM60 plus anti-PD1 treatment in mice results in enhanced IFN ⁇ ⁇ secretion in a dose dependent manner over IMM60 alone. This results in further activation of NK cells and greater production of IFN- ⁇ secretion.
  • Pembrolizumab is an anti-PD-1 antibody marketed by Merck under the trade name Keytruda®.
  • Pembrolizumab Solution for Infusion is a sterile, non-pyrogenic aqueous solution supplied in a single-use Type I glass vial containing 100 mg/4 ml of pembrolizumab.
  • Pembrolizumab infusion solutions should be prepared by site pharmacy in 0.9% Sodium Chloride (normal saline) and the final concentration of pembrolizumab in the infusion solution should be between 1mg/ml and 10 mg/ml. For intravesical infusions the final volume of infusion instilled will be 40 ml.
  • IMM60 is formulated in liposomes as described herein. IMM60 should be stored frozen, then thawed and diluted by the site pharmacy prior to dispensing to the ward.
  • the recommended dose of liposomal IMM60 is either 1, 3, or 9 mg/m 2 administered by intravenous infusion over 60 minutes in a 250 ml Macopharma saline diluent, every 3 weeks.
  • IMM60 is supplied as a 1mg/ml solution, with 2 ml in each vial and should be stored at -20 °C and will need to be thawed at pharmacy prior to dispensing.
  • vials Prior to use, vials are allowed to thaw for 1 to 2 hours until ambient temperature is reached. Heat vials in a 55°C ⁇ 2°C water bath for 10 minutes. Invert and swirl the vials several times by hand and allow to cool to room temperature prior to use.
  • This clinical trial provides a protocol to demonstrate a synergistic effect of IMM60 and pembrolizumab, compared to pembrolizumab alone in patients with melanoma and non-small cell lung cancer, for which pembrolizumab was already licensed as a standard of care.
  • the trial evaluates if IMM60 can restore sensitivity to patients with melanoma resistant to pembrolizumab as well as ascertain if IMM60 can promote PDL1 expression in PDL1 non-expressing small cell lung cancer.
  • the clinical trial includes two phases, phase 1 and phase 2.
  • the clinical trial is executed following the protocol in Table CT-1.
  • NSCLC Phase 2 To report the progression Progression free survival (PFS) at 12 free survival rate at 12 months for months pembrolizumab alone versus IMM60 + pembrolizumab in patients with advanced PDL1 +ve (PDL1 ⁇ 50%) NSCLC or Melanoma Secondary To characterize the safety of IMM60 Frequency of grade 3 or higher Endpoints: in combination with pembrolizumab, treatment related AEs.
  • PFS progression Progression free survival
  • IMM60 dose escalation arm 12 2.
  • IMM60 + pembrolizumab safety arm 6
  • TMG Trial Management Group
  • MEG Trial Management Group
  • Pembrolizumab alone 7 2.
  • IMM60 + pembrolizumab 14 Additional melanoma cohort
  • IMM60 alone 10 Phase 2 randomized 1:2 in NSCLC, “NSCLC Cohort 1”: 1. pembrolizumab alone in PDL1+ NSCLC: 12 2.
  • NSCLC NSCLC ⁇ Progression through systemic therapy consisting of at least platinum based chemotherapy and immunotherapy (either sequentially or in combination) Melanoma ⁇ Patients progressing through at least one line of immunotherapy.
  • NSCLC Non-small cell lung cancer
  • NSCLC Cohort 1 Metastatic NSLC, PDL1 +ve (PDL1 ⁇ 50%) not previously exposed to a PD-1 inhibitor
  • NSCLC Cohort 2 Metastatic NSLC, PDL1 –ve (PDL1 ⁇ 50%) not previously exposed to a PD-1 inhibitor but pre-treated with at least one line of systemic treatment
  • Patients in NSCLC Cohorts 1 must meet CDF criteria for funding for pembrolizumab, with the exception of pembrolizumab administration as monotherapy
  • Name of drug Formulation, dose, route of administration Investigational IMM60 & pembrolizumab doses and schedule as per institution standard of Medicinal Product(s) care
  • Treatment Duration IMM60 is given in 3-weekly cycles with a maximum of cycles, pembrolizumab as per its licensed indication
  • the STD10 in mice was > 5 mg/kg (15 mg/m 2 ), as there were no deaths, clinical signs, or decreases in body weight at this dose.
  • Humans are expected to be much less sensitive than mice to the toxicological effects of liposomal IMM60 since these are likely mediated through exaggerated pharmacological effects (excessive IFN- ⁇ production). Therefore, the estimated clinical starting dose conservatively based upon nonclinical safety studies in the mouse would be 1/10 the STD10 in mice or 1.5 mg/m 2 .
  • the actual planned clinical dose is set at 1.0 mg/m 2 , a dose that is lower than 1/10 the STD10 in mice (1.5 mg/m 2 ).
  • Trial Design This will be a randomized phase 1/2 trial. Initial safety will be assessed in a multiple ascending dose cohort for IMM60, then for IMM60 + pembrolizumab.
  • the Trial Management Group (TMG) will review the Phase 1 data, the current standard of care and feasibility to make a recommendation on Phase 2 cohorts. Planned arms in the Phase 2 component include: pembrolizumab alone & IMM60+pembrolizumab for NCSLC patients; and pembrolizumab alone, IMM60+pembrolizumab, and additional IMM60 alone cohort for melanoma patients.
  • the Phase 1 trial is a modified 3 + 3 trial design.
  • IMM60 ascending dose safety arm three dose levels of IMM60 were assessed (1/3/9 mg/m 2 ). If one of the first 3 patients experiences a pre-defined dose limiting toxicity (DLT), that cohort is expanded to a total of patients. If a further patient in this cohort experiences a DLT, this dose level is defined as the maximum tolerated dose (MTD). If more than 2 DLTs occur in the patients in the expanded cohort, then MTD will be defined as the dose level below that cohort. Should more than 2 DLTs occur in the lowest IMM60 dose level cohort, then de-escalating this dose level is contemplated. If no patient in the highest dose cohort (9mg/m 2 ) experiences a DLT, this cohort will still be expanded to patients.
  • DLT dose limiting toxicity
  • TMG Trial Management Group
  • available data e.g., safety profile
  • Subsequent safety reviews by the TMG will be conducted prior to enrolment of patients in a new cohort.
  • 32615/55875 For the IMM60 + pembrolizumab safety arm, pembrolizumab will be administered in combination with IMM60. The dose of pembrolizumab will be that administered as per standard of care; the IMM60 dose will be dependent on the IMM60 dose escalation arm.
  • This combination safety arm will also use the modified 3+3 design - the IMM60 starting dose level will be one below IMM60 MTD (referred to as MTD-1), and will dose escalate to a cohort with IMM60 at MTD. For example, if the IMM60 MTD is 9mg/m 2 , the first combination cohort will be with IMM60 at 3mg/m 2 and will escalate to a combination cohort with IMM60 at 9mg/m 2 . Should the IMM60 MTD be 1mg/m 2 (lowest dose level in the ascending dose cohort), then the TMG will consider whether to begin the combination safety arm at MTD, or a de-escalated dose. The DLT rules applied to the IMM60 dose escalation arms will also apply to the combination safety arms.
  • MTD-1 IMM60 MTD
  • An additional comteplated cohort of PDL1- NSCLC is treated with one cycle of IMM60, with a tumour biopsy before and after, to determine any changes in PDL1 expression. After this one cycle, the patients will receive the combination of IMM60 + pembrolizumab and a second biopsy will be taken. Depending on review IMM60 monotherapy activity, a potential group of patients who are receiving pembrolizumab monotherapy will be allowed to receive IMM60 at progression in combination with the pembrolizumab to see if sensitivity can be restored.
  • Dose escalation procedure An evaluable patient is defined as a patient who has received the Cycle 1 dose and has completed the minimum safety evaluation requirements in the 21-day period, or experienced a DLT in the 21-day DLT evaluation period. Dose escalation decisions will be made when 3 evaluable patients in a cohort have completed the 21-day DLT evaluation period; should a cohort need to be expanded to patients due to a DLT, the dose-escalation 32615/55875 decision will be made once evaluable patients have completed their 21-day DLT period. Dose escalation will occur based on the toxicity information obtained from the evaluable patients in a cohort during the initial 21-day treatment cycle.
  • Dose escalation guidelines for the Phase 1 cohorts are indicated in Table CT-3 below.
  • Table CT-3 IMM60 Dose Escalation Safety Arm No additional Dose/Arm No DLT 1x DLT DLT 2x DLT 3+ x DLT 1mg/m 2
  • Dose escalate Expand to Dose escalate to 1mg/m 2 is TMG to to 3mg/m 2 3mg 2
  • IMM60 consider dose IMM60 patients /m MTD de-escalation 3mg/m 2
  • Dose escalate Expand to Dose escalate to 3mg/m2 is 2 IMM 1mg/m is IMM60 to 9mg/m 2 patients 9mg/m 2 60 MTD IMM60 MTD nd to Expand to 9/mg 2 9mg/m 2 Expa /m is 2 IMM60 patients patients IMM60 MTD 3mg/m is IMM60 MTD IMM60 MTD IMM60 + pembrolizumab Combination Safety Arm No additional Dose
  • Tumour Assessment Radiological evaluation of malignancy, in line with the protocol, will be performed before starting the study treatment. The same methods that detect lesions at baseline will be used to follow these lesions throughout the study. To ensure compatibility, the radiological assessments used to assess response will be performed using identical techniques. Imaging based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the anti-tumour effect of a treatment. [00609] Baseline evaluations: These will include radiological measurements of the extent of disease by CT scan, or MRI scan where applicable. All areas of disease present must be mentioned (even if specific lesions are not going to be followed for response) and the measurements of all measurable lesions must be recorded on the scan reports. Any non- measurable lesions must be stated as being present.
  • Tumour response should be classified as “not evaluable” (NE), only when it is not possible to classify it under another response category, e.g., when baseline and/or follow-up assessment is not performed or not performed appropriately.
  • the applicable overall response category for each visit that includes disease assessment must be recorded in the medical record for inclusion in the appropriate CRF in OpenClinica.
  • Best Overall Response is defined as RECIST 1.1 response rate including confirmed CR and PR.
  • the plasma exposure data from the ascending dose arm is summarized in Tables CT5 and CT6 and shows a proportional, linear response to ascending dose. Plasma exposures generally exhibited a plateau from about 1 to about 6 hours. As shown in Table CT6, Patients 2, 3, 4, 6, 7, and 8 exhibited Cmax at about 4-6 h post dosing. In contrast, Patients 1, 5, and 9 exhibited Cmax earlier (2 h, 2 h, and 1 h, respectively). Without wishing to be bound to any particular theory, it is believed that the variance in plasma exposure may be due to RES/plasma protein interaction.
  • the volume distribution during the terminal phase ranged from about 1 to about 3 times the plasma compartment. In mice, this ratio was approximately 3.8 to 5.4 times. Moreover, the exposure by AUC generally was less than 100 times multiple of mid 50 ⁇ g/kg dose used in animal toxicology studies. The exception was Patient 7 which exhibited the highest but dose proportion AUC out of the nine patients. [00621] Without wishing to be bound to any particular theory, it is believed that the plasma profile of Patient 10 is qualitatively unique (monotonically decreasing) and exhibited no plateau on plasma concentration between 1 and 6 hours as compared to the other patients.
  • Example 13 Alternate Clinical Trial Protocol
  • the protocol described can be repeated/modified with different liposomal formulations of IMM60; and can be repeated/modified with an alternative secondary therapy, such as a different anti-PD-1 antibody or a different immune checkpoint inhibitor.
  • a fourth dose level of IMM60 of 24, 27, 3033, or 3mg per patient is administered for cycles.
  • Pembrolizumab MK-3475: 200 mg Q3W administered IV for up to 35 cycles or approximately 2 years
  • Condition/Disease/Target Population Phase 1 (See Figure 15A): [00625] NSCLC - stage IV NSCLC ⁇ IMM60 dose escalation arm: Progression through systemic therapy consisting of at least platinum-based chemotherapy and immunotherapy (either sequentially or in combination) ⁇ IMM60 + pembrolizumab dose safety arm: Previous chemotherapy and/or immunotherapy is permissible [00626] Melanoma - unresectable stage III or IV cutaneous or unknown primary melanoma 32615/55875 ⁇ IMM60 dose escalation arm: Patients progressing through at least one line of immunotherapy.
  • phase 1 9 to 18 participants in the initial dose-escalation part of the study and to 12 participants in the following safety part; phase 2: 70 participants [45 PD-L1 > 50% total NSCLC participants, 15 PD-L1 ⁇ 1% NSCLC participants, 10 PD-1 pretreated melanoma participants]). Should a dose limiting toxicity (DLT) occur in any of the phase 1 cohorts, then an additional 3 participants will be recruited to that dose level cohort.
  • DLT dose limiting toxicity
  • Phase 1 [00634] The phase 1 study will be a standard 3 + 3 trial design in participants with advanced NSCLC or melanoma: [00635] IMM60 dose escalation safety arm: 3 dose levels of IMM60 will be assessed (1/3/9 mg/m 2 administered IV). If one of the first 3 participants experiences a pre-defined DLT, that cohort will be expanded to a total of participants. If a further participant in this cohort experiences a DLT, then the MTD will be considered to be exceeded at this dose. If more than 1 DLT is observed in the participants in the expanded cohort then MTD will be defined as the dose level below that cohort.
  • IMM60 + pembrolizumab combination safety arm pembrolizumab will be administered in combination with IMM60.
  • the dose of pembrolizumab will be that administered as per standard of care (200 mg Q3W administered IV); the IMM60 dose (administered IV) will be dependent on the IMM60 dose escalation arm.
  • the IMM60 + pembrolizumab safety arm will open using the 3 mg/m 2 IMM60 dose level, and the combination safety arm will open in parallel to the 9 mg/m 2 IMM60 dose escalation cohort. If the combination safety arm opens in parallel to the 9 mg/m 2 IMM60 dose escalation cohort, then the combination safety arm will dose escalate to 9 mg/m 2 if ⁇ 2 DLTs occur in the first combination safety cohort in phase 1.
  • the IMM60 starting dose level for the combination safety arm will be one below IMM60 MTD (referred to as MTD-1) and will open after the completion of the 9 mg/m 2 dose escalation cohort and will dose escalate to a cohort with IMM60 at MTD.
  • MTD-1 IMM60 MTD
  • Dose escalation will occur based on the toxicity information obtained from the evaluable participants in a cohort during the initial 21-day treatment cycle. The TMG will review 32615/55875 the phase 1 data, the current standard of care, and feasibility to make a recommendation on phase 2 cohorts.
  • NSCLC Cohort 1 PD-L1 positive (TPS ⁇ 50% PD-L1 IHC 22C3 pharmDx)
  • NSCLC Participants will be randomized 1:2 to either pembrolizumab as per its licensed indication (pembrolizumab alone, 200 mg Q3W administered IV) or the combination of IMM60 + pembrolizumab 200 mg Q3W administered IV.
  • pembrolizumab alone cohort who have radiographic progression with clinical deterioration or confirmed radiographic progression at two time points at least 4 weeks apart can then receive pembrolizumab + IMM60.
  • NSCLC Cohort 2 PD-L1 negative (TPS ⁇ 1% PD-L1 IHC 22C3 pharmDx)
  • NSCLC Participants will be treated with one cycle of IMM60 with a tumor biopsy before and after, to determine any changes in PD-L1 expression. After this one cycle, the participants will receive the combination of IMM60 + pembrolizumab 200 mg Q3W administered IV.
  • Melanoma Cohort Participants with melanoma for whom treatment with a PD-1 inhibitor has failed will receive IMM60 monotherapy at the MTD given Q3W administered IV.

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Abstract

L'invention concerne des compositions comprenant un liposome comprenant le composé A (A), dans lequel n est 1 (IMM60), 2 (IMM70), ou 3 (IMM80), ou un sel, un ester, un solvate ou un hydrate de celui-ci, et au moins deux lipides ou sels de celui-ci ; et l'utilisation de celui-ci comprenant des méthodes de stimulation d'une réponse immunitaire. L'invention concerne également des liposomes comprenant au moins un agent thérapeutique et une bicouche phospholipidique comportant au moins deux phospholipides choisis parmi 1,2-distéaroyl-sn-glycéro-3-phosphocholine (DSPC), 1,2-distéaroyl-sn-glycéro-3-phospho-rac-glycérol (DSPG), 1,2-dipalmitoyl-sn-glycéro-3-phosphocholine (DPPC) et 1,2-dipalmitoyl-sn-glycéro-[phosphor-rac-(1-glycérol)] (DPPG), le phospholipide étant sensiblement dépourvu de cholestérol, de stérols végétaux et de lipides glycolés au polyéthylène (lipides PEGylés).
PCT/IB2024/054317 2023-05-03 2024-05-03 Compositions liposomales de modulateur de cellules inkt et procédés d'utilisation Pending WO2024228167A1 (fr)

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