WO2020176984A1 - Nanoparticules lipidiques - Google Patents

Nanoparticules lipidiques Download PDF

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WO2020176984A1
WO2020176984A1 PCT/CA2020/050281 CA2020050281W WO2020176984A1 WO 2020176984 A1 WO2020176984 A1 WO 2020176984A1 CA 2020050281 W CA2020050281 W CA 2020050281W WO 2020176984 A1 WO2020176984 A1 WO 2020176984A1
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lipid nanoparticle
nanoparticle composition
lipid
cells
mrna
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Stefano PERSANO
David Stojdl
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University of Ottawa
Childrens Hospital of Eastern Ontario CHEO
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University of Ottawa
Childrens Hospital of Eastern Ontario CHEO
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin

Definitions

  • the present disclosure relates to lipid nanoparticles formulated for the delivery of DNA, RNA or a peptide.
  • Lipid nanoparticles may be used as non-viral vectors to deliver DNA, RNA and proteins.
  • Lipoplexes are one example of lipid nanoparticles, and refer to lipid-based assemblies of non-covalently associated DNA and RNA, where the association is by charge-charge interactions.
  • Cancer immunotherapy is a therapeutic strategy that exploits the natural ability of the immune system to recognize and kill cancer cells, and is one example of a therapeutic strategy that could benefit from the delivery of DNA, RNA or proteins to a cell, such as a cell in a patient.
  • a cancer immunotherapy that delivers the DNA, RNA or protein to a patient in order to stimulate an immune response may be referred to as a vaccine.
  • mRNA a cancer immunotherapy that delivers the DNA, RNA or protein to a patient in order to stimulate an immune response
  • One challenge associated with the use of mRNA for vaccine development is its sensitivity towards catalytic hydrolysis by ribonucleases. Unprotected mRNA may be degraded under physiological conditions, hence rendering it unsuitable for broad therapeutic applications. In some examples, it is desirable to develop a formulation to stabilize mRNA in vivo.
  • viruses can be used to protect DNA and RNA from catalytic hydrolysis, and to deliver DNA or RNA into a cell, the viruses may induce an undesirable immune response in a mammal if one or more proteins forming the virus are
  • non-viral vectors may have lower host immunogenicity.
  • the present disclosure provides lipid nanoparticles that may be used for the delivery of DNA, RNA, or proteins to a cell.
  • the present disclosure provides a lipid nanoparticle composition that includes: a core, and a lipid mixture encapsulating the core.
  • the core includes: (i) a complex of a poly-(beta-amino ester) polymer with DNA or RNA; or (ii) a peptide.
  • the lipid mixture encapsulating the core includes: (a) N1-[2-((1 S)-1-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5); (b) 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and (c) a polyethylene glycol (PEG)-modified lipid.
  • MDL5 N1-[2-((1 S)-1-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide
  • DOPE 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Lipid nanoparticles according to the present disclosure may be used to induce an immune response in a mammal, such as an anti-cancer immune response.
  • Lipid nanoparticles according to the present disclosure may selectively deliver the DNA, RNA or protein to dendritic cells.
  • Figure 3 is a graph illustrating the quantification of EGFP-positive BMDCs by flow cytometry 24 or 48 hours after transfection.
  • Figure 4 are fluorescence microscopic images of the dendritic cells of
  • Figure 3 transfected with EGFP-mRNA.
  • Figure 5 are bioluminescence imaging of mice 6 hours after i.v. injection of multi-LP loaded with LUC-mRNA (20 mg).
  • Figure 6 is a graph illustrating the quantification multi-LP/Cy5-EGFP- mRNA associated APCs in the spleen 24 hours after i.v. or i.d. administration of 20 pg of Cy5-EGFP-mRNA.
  • Figure 7 is a graph illustrating the quantification multi-LP/Cy5-EGFP- mRNA associated APCs in the lymph nodes 24 hours after i.v. or i.d. administration of 20 pg of Cy5-EGFP-mRNA.
  • Figure 8 is a graph illustrating the quantification mono-LP/Cy5-EGFP- mRNA associated APCs in the spleen 24 hours after i.v. or i.d. administration of 20 pg of Cy5-EGFP-mRNA.
  • Figure 9 is a graph illustrating the quantification mono-LP/Cy5-EGFP- mRNA associated APCs in the lymph nodes 24 hours after i.v. or i.d. administration of 20 pg of Cy5-EGFP-mRNA.
  • Figure 12 is an illustration of the flow cytometry analysis of CD69 in spleen
  • Figure 13 is a set of graphs illustrating quantification of serum level of IL-1 .
  • Figure 14 is a set of graphs illustrating quantification of serum level of
  • Figure 15 is a set of flow cytometry plots of CD8+ T cells expressing IFN-y in peripheral blood.
  • Figure 16 is a graph illustrating flow cytometry quantification of
  • Figure 17 is a graph illustrating OVA-specific IgG antibody titer measured in C57BL/6 mice after 4 weeks from the first vaccination.
  • Figure 20 is a graph illustrating the survival of the C57BL/6 mice administered the noted lipid nanoparticle formulations or control formulations.
  • Figure 21 is a graph illustrating the quantification of tumor-infiltrating CD8 and CD4 T-cells by flow cytometry in C57BL/6 mice that were treated by i.v.
  • Figure 23 is a set of flow cytometry plots quantifying intratumoral
  • Figure 24 is a graph illustrating the quantification of CD8+ T cells expressing IFN-g after different prime-boost therapies using TRP2 as the antigen.
  • lipid nanoparticle composition that includes: a core, and a lipid mixture encapsulating the core.
  • the core includes: (i) a complex of a poly-(beta-amino ester) polymer with DNA or RNA; or (ii) a peptide.
  • the lipid mixture encapsulating the core includes: (a) N1-[2-((1 S)-1-[(3-aminopropyl)amino]-4-[di(3- amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); (b) 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and (c) a polyethylene glycol (PEG)-modified lipid.
  • Lipid nanoparticles where the core includes nucleic acids may be referred to as a“Npopolyplex”.
  • DOPE exists in either a lammellar or a hexagonal conformation.
  • the transition from lamellar to hexagonal conformation occurs under acidic conditions, such as the low pH of an endosomal compartment of a cell.
  • acidic conditions such as the low pH of an endosomal compartment of a cell.
  • the transition from lamellar to hexagonal conformation promotes fusion of the lipid nanoparticle with the endosomal membranes, and release of the materials in the core of the lipid nanoparticle into the cytoplasm.
  • Biodistribution and transfection efficacy of a lipid nanoparticle is influenced by the characeristics of the charge of the hydrophilic head of the lipid and by the structure of the hydrophobic tail.
  • MVL5 is a multivalent cationic lipid, whose charge density and hydrophobic tail enable, at least in some examples, endosomal escape, providing dendritic cell-targeting and mRNA delivery, such as after intravenous injection.
  • Size and charge are parameters which may be used to passively target
  • nanoparticles with a size range from ⁇ 25 to 200 nm traffic to the draining lymph node (dLN) where they are rapidly taken up by APCs; and positively charged nanoparticles exhibit a faster uptake rate by phagocytic cells than negatively charged or neutral nanoparticles.
  • DNL draining lymph node
  • the combination of DOPE and MVL5 may be used to form a lipid nanoparticle with a hydrodynamic diameter of 132.3 ⁇ 3.78 nm and a zeta potential of +33.6 mV, due to the cationic lipid included in the external layer.
  • Dendritic cells (DCs) preferentially uptake nanoparticles having a size from 0.1 pm to 0.5 pm. Positively charged nanoparticles are uptaken faster than neutral or anionic nanoparticles.
  • the ratio of polymer : DNA or RNA may be in a range from about 10 : 1 to about 40 : 1 (wt:wt).
  • the ratio of lipid:DNA or RNA molar ratio may be from about 10:1 to about 20: 1 , such as about 15:1 or 16: 1.
  • the ratio of MVL5 : DOPE : PEG- modified may be from about 10 to about 25 : about 75 to about 90 : about 1 to about 5 (wt:wt:wt).
  • the ratio of MVL5 : DOPE : PEG-modified is from about 10 to about 20 : about 80 to about 90 : about 1 to about 2 (wt:wt:wt).
  • the core includes a complex of a poly-(beta-amino ester) polymer with DNA or RNA, where the DNA or RNA encodes an antigenic peptide.
  • the core includes a peptide, where the peptide is antigenic.
  • the antigenic peptide may be a tumor associated antigen.
  • the lipid:peptide molar ratio (mol:mol) may be from about 20:1 to about 80:1 , such as about 50:1.
  • the lipidnanoparticles may be formulated to deliver from about 100 pg to about 1 ,000 pg of peptide per kilogram of body weight.
  • Nucleic acid-based lipid nanoparticles i.e. lipid nanoparticles where the core comprises DNA or RNA
  • the DNA or RNA may encode both an antigenic peptide sequence and a costimulatory signal, such as toll-like receptor TLR3, TLR7, or TLR8.
  • a costimulatory signal such as toll-like receptor TLR3, TLR7, or TLR8.
  • costimulatory signal may act as an adjuvant to the antigenic peptide.
  • mRNA based lipid nanoparticles provide additional benefits over DNA based lipid nanoparticles since mRNA only needs to gain entry into the cytoplasm of a cell in order to have translation of the encoded peptide occur, and/or mRNA has a reduced risk of integration into the host genome (and therefore has a lower oncogenic potential).
  • the lipid nanoparticles have the DNA or RNA complexed with a poly-(beta-amino ester) (PbAE) polymer.
  • PbAE poly-(beta-amino ester)
  • Such lipid nanoparticles may have a greater transfection efficacy than lipid nanoparticles whose core include uncomplexed DNA or RNA.
  • Such lipopolyplexes may escape from a cell’s endosome through ion-pair formation, the proton sponge effect, or both.
  • Precomplexation of DNA or RNA with PbAE may facilitate and enhance loading of the nucleic acid into the lipid-shell.
  • PbAEs that may be used in lipid nanoparticles of the present disclosure are discussed in Guerrero-Cazares, Hugo et al.“Biodegradable polymeric nanoparticles show high efficacy and specificity at DNA delivery to human glioblastoma in vitro and in vivo” ACS nano vol. 8,5 (2014): 5141-53, which is incorporated herein by reference.
  • a PbAE that may be used is the polymeric product of the reaction between 1 ,4-butanediol diacrylate and 5-amino-1-pentanol, and end-capped with 1-(3- aminopropyl)-4-methylpiperazine.
  • PBAE 447 This specific PbAE may be referred to as“PBAE 447”, “PBAE 447e”, or“PBAE 447e, 1.1 :1” based on the nomenclature convention outlined in the Guerrero-Cazares reference.
  • the PBAE 447 polymer has a number average molar mass (MN) of about 10,700 g/mol, a mass average molar mass (MW) of about 38,200 g/mol, and a polydispersity of about 3.58 as measured using gel permeation
  • Including pegylated lipids in the encapsulating lipid mixture may provide the lipid nanoparticle with one or more beneficial properties over lipid nanoparticles without pegylated lipids.
  • lipid nanoparticles that include pegylated lipids may bypass reticulo-endothelial system and have longer circulation times, resulting in an increased accumulation of the lipid nanoparticles in a tumor.
  • the PEG-modified lipid may be 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (DSPE-2000PEG, or DSPE-PEG); 1 ,2-dimyristoyl-rac-glycero-3- methylpolyoxyethylene (DMG-PEG); 1 ,2-dipalmitoryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated polyethylene glycol (DPPE-PEG); 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE) conjugated polyethylene glycol (DMPE-PEG); 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG); C8 or C16 ceramide conjugated polyethylene glycol (ceramide-PEG); or 1 ,2-dioleoyl-sn-
  • the lipid mixture encapsulating the core may additionally include an immune adjuvanting amount of a-galactosylceramide (a-GalCer), whose poor hydrophilicity makes it difficult to deliver if it is not incoporated into a lipid delivery vehicle.
  • a-GalCer a-galactosylceramide
  • An immune adjuvanting amount of a-GalCer may be an amount from about 50 ng to about 50 pg per kilogram of body weight.
  • the a-GalCer may be present in an amount such that the ratio of a-GalCer
  • DNA, RNA or peptide is in a range of about 1 : about 10 to about 1 : about 75 (wt:wt).
  • a-GalCer may generate more antigen-specific CD8+ T cells compared to TLR-ligands; and a-GalCer may induce iNKT cells to produce immunostimulatory cytokines, particularly IFN-g, and may elicit the induction of one or more costimulatory molecules.
  • the authors of the present disclosure believe that these events may promote the activation of antigen presenting cells (APCs), which may release key Th1 cytokines (e.g. IL-12), and may promote the downstream activation of CD4+ and CD8+ T lymphocytes, as well as NK cells and B cells, with important effects on the magnitude and effectiveness of the immune responses.
  • APCs antigen presenting cells
  • Th1 cytokines e.g. IL-12
  • the lipid mixture encapsulating the core may additionally include an immune adjuvanting amount of: monophosphoryl lipid A (MLP-A) or an analog thereof, such as phosphorylated hexaacyl disaccharide (PHAD®), monophosphoryl 3-deacyl lipid A (3D-PHAD®), or monophosphoryl hexa-acyl lipid A, 3-deacyl (3D(6A)-PHAD®); a natural Cd1 ligand, such as phosphatidylinositol (PI), isoglobotriosylceramide
  • MLP-A monophosphoryl lipid A
  • PAD® phosphorylated hexaacyl disaccharide
  • 3D-PHAD® monophosphoryl 3-deacyl lipid A
  • 3D(6A)-PHAD® monophosphoryl hexa-acyl lipid A, 3-deacyl
  • a natural Cd1 ligand such as phosphatidylinositol (PI), isoglobotriosy
  • Gal(a1 3)Gal Glcp(1 1)Cer iGb3), GD3, Gg3Cer, sulfatide, phosphatidylcholine (PC), or phosphatidylethanolamine (PE); an a-GalCer analog, such as a-C-GalCer, b-GalCer, (2S,3S,4R)-1-0-(a-D-galactopyranosyl)-N-tetracosanoyl-2-amino-1 ,3,4-nonanetriol) (OCH), or alpha-GalCer C20:2 (PubChem CID: 1 1707590); a pathogen-derived Cd1 ligan, such as GSL1 , GSL-4, LPG, PIM4, or PPBF; 3M-052, a TLR7/8 agonist; or a methyl-lysophosphatidic acid, such as mLPAs, or leukemia antigen
  • the present disclosure provides a lipid nanoparticle composition that includes: a core that includes (i) a complex of PbAE 447 polymer with DNA or RNA; or (ii) a peptide; and a lipid mixture encapsulating the core, wherein the lipid mixture includes: (a) MVL5, (b) DOPE, (c) DSPE-PEG, and (d) an immune adjuvanting amount of a-GalCer; where the ratio of MVL5 : DOPE : DSPE-PEG is from about 10 to about 20 : about 80 to about 90 : about 1 to about 2.
  • the core includes a complex of PbAE 447 polymer with RNA.
  • Lipid nanoparticle composition according to the present disclosure may be used to induce an immune response in a mammal.
  • the immune response may include activation of natural killer cells (NK cells), T-cells, or any combination thereof.
  • the T-cells may include natural killer T-cells (NKT-cells).
  • the immune response may be an anti-cancer immune response.
  • the immune response may be induced using the lipid nanoparticle composition in a prime-boost format, such as a heterologous prime-boost format.
  • the lipid nanoparticle composition may be used as a priming vector.
  • Lipid nanoparticle composition according to the present disclosure may be used to deliver the DNA, the RNA, or the protein to a dendritic cell.
  • the dendritic cell may express a protein antigen encoded by the DNA or RNA.
  • Lipid nanoparticle compositions according to the present disclosure may be formulated for intradermal or intravenous administration.
  • Lipid nanoparticle compositions according to the present disclosure may be prepared using a thin-film/rehydration technique where the lipids are dissolved in a suitable solvent and then dried to form a thin film, and then the thin film is subsequently rehydrated in an aqueous solution that includes the DNA, RNA or protein to be encapsulated in the core of the lipid nanoparticle.
  • Alternative techniques known known in the art, such as alcohol-dilution/lyophilization or microfluidic nanoparticle synthesis, may also be used.
  • Sodium acetate buffer solution (3M, pH 5.2), nuclease-free water and BSA were purchased from Sigma-Aldrich.
  • TRP-2 Tyrosinase-related protein 2
  • SVYDFFVWL peptide
  • SEQ ID NO: 1) was purchased from GenScript.
  • Anti-CD8 (Clone KT15) and iTAg Tetramer H-2 Kb TRP2 (SVYDFFVWL,
  • SEQ ID NO: 1 were purchased from MBL international.
  • Ovalbumin (OVA)-mRNA and Luciferase (LUC)-mRNA were purchased from TriLink Biotechnologies.
  • the reaction was maintained at 90 °C and 1 ,000 rpm of stirring speed for the first 4 hours. After 4 hours, the stirring speed was slowed to 300 rpm and the reaction was maintained at 90 °C for further 12-16 hr.
  • the B4S4 polymer was dissolved in 10 mL of anhydrous tetrahydrofuran (THF) at a final concentration of 100 mg/mL.
  • THF anhydrous tetrahydrofuran
  • 1- (3-Ami nopropyl)-4- methylpiperazine (“E7”) Alfa Aesar
  • the end-chain capping reaction was performed by mixing the two solutions and leaving it at room temperature, stirring at 400 rpm for 24 hours protected from light.
  • the final product is termed PbAE 447.
  • a PbAE 447 / mRNA polyplex was prepared by mixing one volume of the
  • PbAE 447 polymer with one volume of mRNA molecules in acetate buffer using a PbAE 447 / mRNA (w/w) mass ratio 30. After a 20 min incubation at room temperature, the polyplex was analyzed for size distribution and zeta-potential using a Zetasizer NanoZS dynamic light scattering instrument (Malvern Instruments). The PbAE 447 / mRNA polyplex was also analyzed in a gel retardation assay. Briefly, a polyplex sample containing 250 ng mRNA was loaded into each well and separated by electrophoresis in a 0.7% agarose gel with 1 x TBE buffer (BioRad, Hercules, CA). RNA bands were stained with Gelred nucleic acid gel stain (Biotium) and visualized with a GelDoc system
  • Lipopolyplexes were prepared using EDOPC, DOPE and DSPE-PEG to form a monovalent cationic lipid nanoparticle.
  • a monovalent cationic lipid nanoparticle may also be referred to as a“mono-LP” or“mono-LP nanoparticles” or“mono-LP formulation” or“mono-LP vector”.
  • Lipopolyplexes were also prepared using MVL5, DOPE and DSPE-PEG to form a multivalent cationic lipid nanoparticle.
  • a multivalent cationic lipid nanoparticle may also be referred to as“multi-LP” or“multi-LP nanoparticles” or “multi-LP formulation” or“multi-LP vector”.
  • the lipid nanoparticle formed using MVL5, DOPE, and DSPE-PEG is one example of a lipid nanoparticle composition according the present disclosure.
  • BMDCs bone marrow-derived dendritic cells
  • BMDCs were prepared from C57BL/6 mice as described in Xia X, Mai J,
  • GM- CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 interleukin-4
  • BMDCs were seeded in a 96-well plate at a seeding density of 3 x 10 4 cells/well and treated with 0.1 pg mRNA loaded into mono-LP or multi-LP. Cell viability was measured 24 h later with a tetrazolium-based Cell Titer 96®Aqueous One Solution Cell Proliferation (MTS) assay (Promega) following the manufacturer’s instruction.
  • MTS tetrazolium-based Cell Titer 96®Aqueous One Solution Cell Proliferation
  • Enhanced GFP-mRNA was used as a reporter to test the transfection efficiency in BMDCs. 3 x 10 5 cells/well at day 5 were seeded in 24-well plates and treated in complete RPMI-1640 with mono-LP and multi-LP loaded with 0.5 pg of EGFP-mRNA. The expression of EGFP in cells was determined with a fluorescence microscope (Carl Zeiss) and the percentage of EGFP expressing cells was measured by flow cytometry (BD Fortessa X-20).
  • mice were injected with multi-LP/LUC-mRNA (20 pg of mRNA) following an intravenous administration route. After 6 hours, the mice were injected intraperitoneally with 200 pi D-Luciferin (15 mg/mL) (Gold Biotechnology) and bioluminescence was measured in a PerkinElmer MS® Spectrum imaging system.
  • dLNs spleen and draining lymph nodes
  • the cells were stained with anti-B220, anti-CD19, anti-CD1 1 c and anti-F4/80 in order to gate dendritic cells (CD1 1c+ and F4/80-), Macrophages (CD1 1 c- and F4/80+) and B-cells (CD1 1 c-, B220+ and CD19+) populations.
  • the samples were analyzed by flow cytometry (BD Fortessa X-20). Animal experiments in this study were carried out in accordance with guidelines evaluated and approved by the ethics committee of University of Ottawa to ensure the humane animal care and use.
  • Lymphocytes were stained for TCRp, NK1.1 and CD69 to gate NK cells
  • TCRp- and NK1.1 + NKT cells
  • TCRp+ and NK1.1 + T-cells
  • the samples were analyzed by flow cytometry (BD Fortessa X-20).
  • Formulations that included mRNA had 10 pg of TRP-2 mRNA.
  • Formulations that included a-GalCer had 1 pg of a-GalCer.
  • Serum TNF-a, IFN- a, IFN-b, IFN-g, I L- 1 b , IL-6 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience).
  • TRP2-mRNA 10 pg of TRP2-peptide, and/or 0.5 pg of a-GalCer.
  • PBMCs were stimulated with 1 pg/mL of TRP2-peptide (SVYDFFVWL, SEQ ID NO: 1) for 6 hours in the presence of protein transport inhibitor, Brefeldin A (BD GolgiPlugTM). After incubation, cells were washed and stained with Abs for surface markers-TCRp, CD8 and
  • V450/viability dye Cells were then washed, fixed and permeabilized (BD Fixation/Permeabilization Solution Kit), and stained with anti-IFN-g. Cells were washed, re-suspended in FACS buffer (1 x PBS and 0.5% BSA) and analyzed by flow cytometry (BD Fortessa X-20).
  • SVYDFFVWL-specific tetramer (MBL International) staining was carried out by incubation at room temperature for 10 minutes in FACS buffer. After washing, the PBMCs were stained with anti-TCRp, anti-CD8 clone KT15, and V450/viability for 30 min at 4°C in FACS buffer. Then, the samples were analyzed by flow cytometry (BD Fortessa X-20).
  • Plates were blocked with 1 % bovine serum albumin in PBS for 2 h, and serial two-fold dilutions of serum samples in PBS were added to the wells. After a 2-h incubation, plates were washed with PBS containing 0.05% Tween 20 and incubated for 1 h at room temperature with HRP- conjugated goat anti-mouse immunoglobulin G (IgG), lgG1 and lgG2c antibodies (Southern Biotechnology Associates). The plates were incubated with
  • TMB tetramethylbenzidine
  • mice were intravenously administered with different lipid nanoparicle formulations according to the present disclosure (10 pg of TRP2-mRNA with or without 1 pg of a-GalCer) and boosted at days 9 and 13 post-tumor inoculation.
  • TIL tumor infiltrating lymphocytes
  • mice were intravenously administered with different lipid nanoparicle formulations according to the present disclosure (10 pg of TRP2-mRNA with or without 1 pg of a-GalCer) and boosted 15 days post-tumor inoculation.
  • Tumor cell suspensions were prepared from solid tumors by mechanical disaggregation as described in Pachynski, R. K., Scholz, A., Monnier, J., Butcher, E. C., Zabel, B. A. Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model. J. Vis. Exp. (98), e52657, doi: 10.3791/52657 (2015). Erythrocytes were removed by the addition of ACK lysis buffer (Quality Biological). The resulting suspension was passed through a 40-pm cell strainer, washed once with PBS and resuspended in FACS buffer for flow cytometric analysis.
  • TIL evaluation cells were stained with viability dye, anti-CD45, anti- TCRp, anti-CD8, anti-CD4 and anti-NK1.1 and analyzed by flow cytometry (BD Fortessa X-20).
  • viability dye anti-CD45, anti- TCRp, anti-CD8, anti-CD4 and anti-NK1.1
  • flow cytometry BD Fortessa X-20.
  • SVYDFFVWL-specific tetramer (MBL International) staining was carried out by incubation at room temperature for 10 minutes in FACS buffer, followed by surface staining with anti-CD45, anti-TCRp and anti-CD8 clone KT15 for 30 min at 4°C in FACS buffer.
  • the samples were analyzed by flow cytometry (BD Fortessa X-20).
  • GC Germinal center
  • TCM central memory CD8+ T-cells
  • a multi-LP loaded with a-GalCer/mRNA was characterized for physical properties, such as size and zeta-potential ( Figures 1 and 2, respectively), exhibiting a hydrodynamic diameter of 132.3 ⁇ 3.78 nm and a strongly positive zeta potential of +33.6 mV, due to the cationic lipid included in the external layer.
  • the polyplex core alone prepared using a PbAE 447/mRNA (w/w) ratio of 20, showed a hydrodynamic size of 1 13 ⁇ 3.16 nm and a zeta-potential of +35.8 mV.
  • a mono-LP prepared using the lipids described above resulted in a nanoparticle with a size of 150.9 ⁇ 3.62 nm and zeta- potential of 38 mV.
  • the exemplary multi-LP and mono-LP nanoparticles used in the experiments discussed herein were prepared using two different lipidic formulations, MVL5/DOPE/DSPE-PEG (16:82:2) and EDOPC/DOPE/DSPE-PEG (49:49:2), respectively.
  • the mono- LP and multi-LP were loaded with a mRNA encoding for EGFP and the expression of the reporter gene was detected by flow cytometry ( Figures 3 and 4) and fluorescence microscopy ( Figure 5). The transfection efficiency was quantified at 24 and 48 hours posttransfection and peak expression was observed at 48 hours.
  • Multi-LP nanoparticles loaded with mRNA encoding for luciferase reporter were tested in vivo, since transfection systems that work efficiently in vitro may fail to function or have serious toxicity in vivo. Interestingly, 6 hours after administration a significant bioluminescence signal was detected at the abdominal region, demonstrating successful transfection by mRNA-loaded multi-LP particles (Figure 5).
  • Multi-LP nanoparticles did not elicit signs of toxicity in the mice. No changes in the body mass or behavior of mice were detected for up to 4 weeks following a relative high administration dose.
  • APCs are responsible for driving the induction of CD8+ T-cell mediated immune responses.
  • mice were administered once i.d. or i.v. with mono-LP and multi-LP nanoparticles loaded with fluorescently labelled mRNA (Cy5-mRNA). 24 hours post treatment, single cell suspensions obtained from spleens and dLNs were stained with an antibody panel directed against common APC markers; DCs (CD1 1 c+ and F4/80-), macrophages (CD1 1 c- and F4/80+) and B cells (CD1 1 c-, B220+ and CD19+).
  • administration route in terms of its ability to target APC populations.
  • Naive mice were injected either i.v. or i.d. with multi-LP nanoparticles loaded with antigen-mRNA with or without a-GalCer.
  • single cell suspensions obtained from spleens and dLNs were analyzed by flow cytometry in order to measure the up-regulation of the lymphocyte activation marker CD69 on NKT, NK and T cells.
  • IFN-g The induction of IFN-g was only attributable to the activation of iNKT.
  • multi-LP vector loaded with TRP2-mRNA alone did not induce a significant release of IFN-g while both a-GalCer/TRP2-mRNA and a-GalCer loaded multi-LP nanoparticles induced a comparable level of IFN-g without a significant difference.
  • the levels of Type-I IFNs were higher in the group receiving multi-LP/aGalCer+mRNA compared to the group treated with multi-LP/mRNA, the difference was not significant.
  • the formulation without lipid nanoparticles did not induce detectable levels of Type IFNs.
  • the introduction of a-GalCer in the mRNA lipid nanoparticle formulation may be used to stimulate or enhance the release of key cytokines over a corresponding non-adjuvanted mRNA lipid nanoparticle formulation.
  • mice were intravenously administered with multi-LP/a-GalCer+TRP2- mRNA, multi-LP/a-GalCer+TRP2-peptide, multi-LP/TRP2-mRNA, free a-GalCer+TRP2- mRNA and free a-GalCer+peptide on days 0 and 7.
  • TRP2-specific CD8+ T cells was evaluated by intracellular staining for IFN-g and TRP2-specific tetramer staining.
  • CD8+ T cells in spleen and dLNs from treated mice were analyzed for the presence of memory T cells (CD62L+ and CD44+ T cells).
  • T cells from mice administered multi-LP/a-GalCer+TRP2-mRNA nanoparticles showed the higher percentage of TCM compared to the groups administered multi-LP/TRP2-mRNA and multi-LP/a-GalCer.
  • Multi-LP nanoparticles were prepared using mRNA encoding for ovalbumin protein (OVA-mRNA).
  • OVA-mRNA ovalbumin protein
  • the level of OVA- specific IgG in the serum of mice immunized with multi-LP/a-GalCer+OVA-mRNA was higher than that in the multi-LP/OVA-mRNA and multi-LP/a-GalCer groups ( Figure 17).
  • the level of lgG2c isotype in the serum of mice in the multi-LP/a- GalCer+OVA-mRNA group was higher than the IgG 1 isotype levels ( Figure 18).
  • Germinal center (GC) B cells formation is essential for the production of high affinity antibodies. GC structures support somatic hypermutation, selection of high affinity B cells and their differentiation into plasma and memory cells. Enhanced expression of germinal center (GC) markers (GL7 and FAS) was seen following immunization with multi-LP/a-GalCer+TRP2-mRNA nanoparticles compared with multi- LP/TRP2-mRNA and multi-LP/a-GalCer vaccinations.
  • B16-F10 melanoma-bearing mice were i.v. administered with exemplary lipid nanoparticles according ot the present disclosure, as well as control formulations, at days 5, 9 and 13 post-tumor inoculation and the therapeutic efficacy of the combined treatments was evaluated in terms of tumor growth inhibition and survival.
  • Figure 24 is a graph quantifying CD8+ T cells expressing IFN-g after different prime-boost therapies.
  • the mice were treated with: adenovirus (Ad) as a prime and FMT as a boost (each expressing TRP2); an exemplary lipid nanoparticle of the present disclosure where the core encapsulated TRP2-peptide as a prime and FMT expressing TRP2 as a boost; and an exemplary lipid nanoparticle of the present disclosure where the core encapsulated TRP2-mRNA as a prime and FMT expressing TRP2 as a boost.
  • Ad adenovirus

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Abstract

L'invention concerne une composition de nanoparticules lipidiques. La composition de nanoparticules lipidiques comprend : un noyau et un mélange lipidique encapsulant le noyau. Le noyau comprend : (i) un complexe d'un polymère poly-(bêta-amino ester) avec de l'ADN ou de l'ARN ; ou (ii) un peptide. Le mélange de lipides encapsulant le noyau comprend : (A) N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)éthyl]-3,4-di[oléyloxy]-benzamide (MVL5) ; (b) 1,2-dioléoyl-sn-glycéro-3-phosphoéthanolamine (DOPE) ; et (c) un lipide modifié par le polyéthylène glycol (PEG). L'invention concerne également des utilisations et des procédés associés à la composition de nanoparticules lipidiques.
PCT/CA2020/050281 2019-03-04 2020-03-02 Nanoparticules lipidiques Ceased WO2020176984A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031392A2 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques comprenant de la phosphatidylsérine
WO2024184500A1 (fr) 2023-03-08 2024-09-12 CureVac SE Nouvelles formulations de nanoparticules lipidiques pour l'administration d'acides nucléiques
CN119792544A (zh) * 2024-12-27 2025-04-11 山东大学 一种纳米载体及其制备方法与应用
EP4514329A4 (fr) * 2022-04-26 2026-04-22 Univ Johns Hopkins Compositions de nanoparticules cinétiques contenant des acides nucléiques, des polycations et des lipides

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GUEVARA ET AL.: "Codelivery of mRNA with a-Galactosylceramide Using a New Lipopolyplex Formulation Induces a Strong Antitumor Response upon Intravenous Administration", ACS OMEGA, vol. 4, 7 August 2019 (2019-08-07), pages 13 015 - 13026, XP055735837 *
PERSANO ET AL.: "Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination", BIOMATERIALS, vol. 125, 2017, pages 81 - 89, XP085095041 *
WONDER ET AL.: "Competition of Charge-Mediated and Specific Binding by Peptide-Tagged Cationic Liposome-DNA Nanoparticles In Vitro and In Vivo", BIOMATERIALS, vol. 166, 2018, pages 52 - 63, XP055735859 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031392A2 (fr) 2021-09-03 2023-03-09 CureVac SE Nouvelles nanoparticules lipidiques pour l'administration d'acides nucléiques comprenant de la phosphatidylsérine
EP4514329A4 (fr) * 2022-04-26 2026-04-22 Univ Johns Hopkins Compositions de nanoparticules cinétiques contenant des acides nucléiques, des polycations et des lipides
WO2024184500A1 (fr) 2023-03-08 2024-09-12 CureVac SE Nouvelles formulations de nanoparticules lipidiques pour l'administration d'acides nucléiques
CN119792544A (zh) * 2024-12-27 2025-04-11 山东大学 一种纳米载体及其制备方法与应用

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