WO2017192470A1 - Polythérapie d'anticorps anti-il-10 et de compositions comprenant des nanoparticules lipidiques et des oligonucléotides cpg agonistes de tlr9 - Google Patents

Polythérapie d'anticorps anti-il-10 et de compositions comprenant des nanoparticules lipidiques et des oligonucléotides cpg agonistes de tlr9 Download PDF

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WO2017192470A1
WO2017192470A1 PCT/US2017/030484 US2017030484W WO2017192470A1 WO 2017192470 A1 WO2017192470 A1 WO 2017192470A1 US 2017030484 W US2017030484 W US 2017030484W WO 2017192470 A1 WO2017192470 A1 WO 2017192470A1
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amine
dien
peg
yloxy
dimethyl
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PCT/US2017/030484
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Marian E. Gindy
Danilo R. Casimiro
David R. Kaufman
Uyen Truong Phan
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Merck Sharp & Dohme Corp.
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    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to combinations of an anti -IL-10 antibody and compositions comprising a TLR9 agonist CpG oligonucleotide and lipid nanoparticles.
  • the lipid nanoparticles are composed of a combination of cationic lipids with other lipid components such as PEG-lipids and optionally non-cationic lipids.
  • interleukin-10 is a potent immunomodulator of hematopoietic cells, particularly immune cells.
  • Cells such as activated Th2 cells, B cells, keratinocytes, monocytes and macrophages produce IL- 10.
  • IL-10 inhibits activation and effector functions of a number of cells that include T cells, monocytes and macrophages.
  • IL-10 inhibits cytokine synthesis, including that of IL-1, IFN- ⁇ , and TNF, by cells such as Thl cells, natural killer cells, monocytes, and macrophages.
  • cells such as Thl cells, natural killer cells, monocytes, and macrophages.
  • IL- 10 in the tumor microenvironment by tumor infiltrating macrophages, dendritic cells, and CD4 + and CD8 + T cells has been shown to inhibit tumor eradication by the immune system (see, e.g., Jarnicki, et al. (2006) J. Immunol. 896-904).
  • Targeting IL-10 with an antagonist of IL-10 could provide potent immunostimulatory activity and tumor eradication.
  • immunostimulatory sequences induces an immune response with a Thl -type bias as indicated by secretion of Thl -associated cytokines.
  • Administration of an immunostimulatory polynucleotide with an antigen results in a Thl-type immune response to the administered antigen.
  • mice injected intradermally (or with a tyne skin scratch applicator) with plasmid DNA (in saline) encoding ⁇ -Gal and containing an immunostimulatory sequence responded by producing IgG2a antibodies and CD4 + cells that secreted IFN- ⁇ , but not IL-4 and IL-5, demonstrating that the T cells were predominantly of the Thl subset.
  • Lipid nanoparticles constitute an alternative to other particulate systems, such as emulsions, liposomes, micelles, microparticles and/or polymeric
  • nanoparticles for the delivery of active ingredients, such as oligonucleotides, peptides, monoclonal antibodies and small molecule pharmaceuticals.
  • active ingredients such as oligonucleotides, peptides, monoclonal antibodies and small molecule pharmaceuticals.
  • LNPs and their use for the delivery of oligonucleotides have been previously disclosed. See U.S. Pat. No. 7,691,405, U.S. Patent Application Publication Nos: US 2006/0083780, US 2006/0240554, US
  • WO2010/054406 See also Semple et al., 2010, Nat. Biotechnol. 25: 172-176. Lipid-based nanoparticles as pharmaceutical drug carriers have also been disclosed. See Puri et al., 2009, Crit. Rev. Ther. Drug Carrier Syst. 26:523-580.
  • the present invention is directed to a composition
  • a composition comprising: a) a lipid nanoparticle (LNP) comprising one or more cationic lipids and a poly(ethyleneglycol)-lipid (PEG-lipid); b) a Toll-like receptor 9 (TLR9) agonist CpG oligonucleotide; and c) an anti-IL- 10 antibody or an antigen-binding fragment thereof.
  • LNP lipid nanoparticle
  • PEG-lipid poly(ethyleneglycol)-lipid
  • TLR9 Toll-like receptor 9
  • the cationic lipid is an ionizable cationic lipid, which may be selected from DLinDMA; DlinKC2DMA; DLin-MC3 -DMA; CLinDMA; S-Octyl CLinDMA; (2 S )-l- ⁇ 7-[(3 p)-cholest-5-en-3-yloxy]heptyloxy ⁇ -3-[(4 Z )-dec-4-en-l-yloxy]- N, N -dimethylpropan-2-amine; (2 R )-l- ⁇ 4-[(3 p)-cholest-5-en-3-yloxy]butoxy ⁇ -3-[(4 Z )- dec-4-en-l-yloxy]- N, N -dimethylpropan-2-amine; l-[(2 R )-l- ⁇ 4-[(3 ⁇ )-cholest-5-en-3- yloxy]butoxy ⁇ -3-(octyloxy)
  • the ionizable cationic lipid is selected from (2S)-l-( ⁇ 6-[(3p))-cholest-5-en-3-yloxy]hexyl ⁇ oxy)-N,N-dimethyl-3-[(9 Z)- octadec-9-en- 1 -yloxy]propan-2-amine; ( 13Z, 16Z)-N,N-dimethyl-3 -nonyldocosa- 13,16-dien- 1-amine; and N,N-dimethyl-l-[(l S,2R)-2-octylcyclopropyl]heptadecan-8-amine; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, or any combination of the foregoing.
  • the LNP comprises 80-99.9 mole % ionizable cationic lipid and 0.1-20 mole % PEG-lipid.
  • the LNP further comprises one or more non-cationic lipids which can be selected from a phospholipid, a phospholipid derivative, a fatty acid, a sterol, or a combination thereof.
  • the sterol may be cholesterol, stigmasterol or stigmastanol.
  • Natural phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), Phosphatidic acid (phosphatidate) (PA),
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PA Phosphatidic acid
  • dipalmitoylphosphatidylcholine dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso PC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), N-Acyl-PE, phosphoinositides, and
  • Phospholipid derivatives include phosphatidic acid (DMPA, DPP A, DSP A), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), phosphatidylserine (DOPS).
  • DMPA phosphatidic acid
  • DPP A DSP A
  • DDPC phosphatidylcholine
  • DDPC DLPC
  • DMPC DPPC
  • DSPC DOPC
  • POPC DEPC
  • phosphatidylglycerol DMPG, DPPG, DSPG, POPG
  • DMPE phosphatidylethanolamine
  • DOPE phosphatidylserine
  • Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18: l), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0 and lethicin.
  • the phospholipid may be phosphatidylserine, l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2- dieicosenoyl-sn-glycero-3-phosphocholine, or l,2-dioleoyl-s «-glycero-3-phosphocholine (DOPC).
  • DSPC Distearoyl-sn-glycero-3- phosphocholine
  • DMPC 1,2-dimyristoyl- sn-glycero-3-phosphocholine
  • DLPC dilauroylphosphatidylcholine
  • DOPC 1,2- dieicosenoyl-sn-gly
  • the PEG-lipid is 1,2-Dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA).
  • the PEG-lipid comprises a polyethylene glycol having an average molecular weight ranging from about 500 daltons to about 10,000 daltons.
  • the LNP comprises 20-99.8 mole % ionizable cationic lipid, 0.1-65 mole % non-cationic lipids, and 0.1-20 mole % PEG-lipid.
  • the non-cationic lipids comprise a mixture of cholesterol and DSPC.
  • the TLR9 agonist is selected from A-class CpG ODN, B-class CpG ODN and C-class CpG ODN.
  • the B-class CpG ODN is 5'TCCATGACGTTCCTGACGTT 3' (SEQ ID NO: 22).
  • the agonist may be physically encapsulated in the LNP before or after LNP preparation.
  • the agonist may be adsorbed, covalently coupled, ionically-interacted or formulated onto the surface of the LNP.
  • the lipid nanoparticle comprises 34- 59 mole % ionizable cationic lipid selected from the group consisting of (2S)-l-( ⁇ 6-[(3P))- cholest-5-en-3-yloxy]hexyl ⁇ oxy)-N,N-dimethyl-3-[(9 Z)-octadec-9-en-l-yloxy]propan-2- amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13, 16-dien-l-amine; and N,N-dimethyl-l- [(l S,2R)-2-octylcyclopropyl]heptadecan-8-amine, 30-48 mole % cholesterol, 10-24% DSPC and 1-2 mole % PEG-DMG.
  • compositions of the invention can be in the form of an aerosol, dispersion, solution, or suspension.
  • the compositions can be formulated for intramuscular, oral, sublingual, buccal, parenteral, nasal, subcutaneous, intradermal, or topical administration.
  • the present invention is also directed to methods of treating cancer in the subject by administering to the subject an effective amount of the compositions of the invention.
  • the present invention is also directed to methods of treating cancer in the subject by co-administering to the subject 1) an effective amount of a composition
  • FIGURE 1 A: individual volumes of injected tumors in tc-1 bilateral mouse tumor model.
  • B median volumes (mm 3 ) with 68% confidence intervals (CI) of injected tumors by day in TC-1 bilateral mouse tumor model.
  • C p-values for comparison of volumes of injected tumors between treatments, by day in TC-1 bilateral mouse tumor model.
  • FIGURE 2 A: individual volumes of non-injected tumors in bilateral mouse tumor TC-1 model.
  • B Median volumes (mm 3 ) with 68% confidence intervals (CI) of non- injected tumors by day in TC-1 bilateral mouse tumor model.
  • C p-values for comparison of volumes of non-injected tumors between treatments, by day in TC-1 bilateral mouse tumor model.
  • FIGURE 3 shows amino acid sequences of anti-IL-10 huml2G8, with light chain sequence of SEQ ID NO: 2 and heavy chain sequence of SEQ ID NO: 1. CDR regions are underlined.
  • FIGURE 4 shows amino acid sequences of mouse anti-IL-10 TC40.11D8, with light chain variable region sequence of SEQ ID NO: 3 and heavy chain variable region sequence of SEQ ID NO: 4. CDR regions are underlined.
  • the present invention is directed to a composition
  • a composition comprising: a) a lipid nanoparticle (LNP) comprising one or more cationic lipids and a poly(ethyleneglycol)-lipid (PEG-lipid); b) a Toll-like receptor 9 (TLR9) agonist CpG oligonucleotide; and c) an anti-IL- 10 antibody or an antigen-binding fragment thereof.
  • LNP lipid nanoparticle
  • PEG-lipid poly(ethyleneglycol)-lipid
  • TLR9 Toll-like receptor 9
  • alkyl means a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • alkeny means a straight chain, cyclic or branched aliphatic hydrocarbon having the specified number of carbon atoms and one or more double bonds including but not limited to diene, triene and tetraene unsaturated aliphatic hydrocarbons.
  • cyclic “alkyl” or “alkenyl” examples include:
  • aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydro-naphthyl, indanyl and biphenyl.
  • administering refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • administering and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
  • subject includes any organism, preferably an animal, more preferably a mammal ⁇ e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.
  • co-administration refers to administration of the L P of the invention and a TLR9 agonist or antibody concurrently, i.e., simultaneously in time, or sequentially, i.e., administration of an LNP of the invention, followed by administration of the agonist or antibody. That is, after administration of the LNP, the agonist or antibody can be administered substantially immediately after the LNP or the agonist or antibody can be administered after an effective time period after the LNP; the effective time period is the amount of time given for realization of maximum benefit from the administration of the LNP.
  • An effective time period can be determined experimentally and can be generally within 1, 2, 3, 5, 10, 15, 20, 25, 30, 45 or 60 minutes.
  • antibody refers to any form of antibody that exhibits the desired biological or binding activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies), humanized, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • Monoclonal antibodies including full length monoclonal antibodies
  • polyclonal antibodies include full length monoclonal antibodies, polyclonal antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies
  • humanized fully human antibodies
  • chimeric antibodies camelized single domain antibodies.
  • camelized single domain antibodies camelized single domain antibodies.
  • Parental antibodies are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as humanization of an antibody for use as a human therapeutic.
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).
  • variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the two binding sites are, in general, the same.
  • variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al; National Institutes of Health, Bethesda, Md. ; 5 th ed.; NIH Publ. No.
  • antibody fragment or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • An antibody that "specifically binds to" a specified target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity.
  • An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g. without producing undesired results such as false positives.
  • Antibodies, or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins.
  • Chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to
  • Human antibody refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine
  • mouse antibody or rat antibody refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.
  • Humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the prefix "hum”, "hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies.
  • the humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.
  • Anti-tumor response when referring to a cancer patient treated with a therapeutic regimen, such as a combination therapy described herein, means at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, reduced rate of tumor metastasis or tumor growth, or progression free survival. Positive therapeutic effects in cancer can be measured in a number of ways (See, W. A. Weber, J. Null. Med. 50: 1 S-10S (2009); Eisenhauer et al., supra). In some embodiments, an anti-tumor response to a combination therapy described herein is assessed using RECIST 1.1 criteria, bidimentional irRC or unidimensional irRC.
  • an anti-tumor response is any of SD, PR, CR, PFS, or DFS.
  • Biotherapeutic agent means a biological molecule, such as an antibody or fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports tumor maintenance and/or growth or suppresses the anti-tumor immune response.
  • Classes of biotherapeutic agents include, but are not limited to, antibodies to VEGF, EGFR, Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, and ICOS.
  • cationic lipid refers to any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • CpG ODN or "CpG oligonucleotide” is an oligonucleotide comprising Cytosine nucleotide followed by a Guanine nucleotide.
  • A-class CpG ODNs or "A-class CpG oligonucleotides” are oligonucleotides that comprise a central palindromic phosphodiester sequence containing at least one CpG sequence, flanked on one or both sides by phosphorothioate polyguanosine sequences.
  • the A-class CpG oligonucleotides induce strong plasmacytoid dendritic cell (PDC) IFN-a secretion and moderate expression of costimulatory molecules, but little B-cell activation.
  • PDC plasmacytoid dendritic cell
  • B-class CpG ODNs or "B-class CpG oligonucleotides” are oligonucleotides that comprise at least one CpG sequence. In one embodiment, the B-class CpG
  • oligonucleotides induce strong B-cell proliferation and differentiation, and induces plasmacytoid dendritic cell (PDC) expression of costimulatory molecules and modest IFN-a secretion.
  • PDC plasmacytoid dendritic cell
  • C-class CpG ODNs or "C-class CpG oligonucleotides” are oligonucleotides from 12 to 40 bases in length, which have one to two 5'-TCG trinucleotides wherein the 5'-T is positioned 0, 1, 2, or 3 bases from the 5'-end of the oligonucleotide, and at least one palindromic sequence of at least ten bases in length comprising at least two unmethylated CG dinucleotides.
  • the oligonucleotide is an oligodeoxynucleotide (ODN).
  • ODN oligodeoxynucleotide
  • the oligonucleotide is a 2'-oligodeoxynucleotide.
  • C- class CpG ODNs induce strong B-cell proliferation and differentation, induce plasmacytoid dendritic cell (PDC) maturation and cause secretion of high levels of type I interferons (e.g., IFN-a, IFN- ⁇ , etc.) and expression of costimulatory molecules.
  • the oligonucleotide is an oligodeoxynucleotide.
  • one or more of the internucleotide linkages of the C-class CpG ODN are modified linkages.
  • one or more of the internucleotide linkages of C-class CpG ODN are phosphorothioate (PS) linkages.
  • all of the internucleotide linkages of CpG-C ODN are phosphorothioate (PS) linkages.
  • PS phosphorothioate
  • the term "palindromic sequence” or “palindrome” refers to a nucleic acid sequence that is an inverted repeat, e.g., ABCDD'C'B'A', where the bases, e.g., A, and A', B and B', C and C, D and D', are capable of forming Watson-Crick base pairs.
  • CDR or “CDRs” as used herein means complementarity determining region(s) in a immunoglobulin variable region, defined using the Kabat numbering system, unless otherwise indicated.
  • “Chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytoxic/anti tumor antibiotics, topisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), anti-progesterones, estrogen receptor down-regulators (ERDs), estrogen receptor antagonists, leutinizing hormone-releasing hormone agonists, anti- androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and anti-sense oligonucleotides that inhibit expression of genes implicated in abnormal cell proliferation or tumor growth.
  • Chemotherapeutic agents useful in the treatment methods of the present invention include cytostatic and/or cytotoxic agents.
  • Chothia as used herein means an antibody numbering system described in Al-Lazikani et al, JMB 273:927-948 (1997).
  • Constantly modified variants or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity or other desired property of the protein, such as antigen affinity and/or specificity.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity ⁇ see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)).
  • substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 1 below.
  • an anti-IL-10 antibody that consists essentially of recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which do not materially affect the properties of the binding compound.
  • Framework region or "FR” as used herein means the immunoglobulin variable regions excluding the CDR regions.
  • Kabat as used herein means an immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).
  • conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods ⁇ see, e.g., U.S. Pat. No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.
  • Patient refers to any single subject for which therapy is desired or that is participating in a clinical trial, epidemiological study or used as a control, including humans and mammalian veterinary patients such as cattle, horses, dogs, and cats.
  • RECIST 1.1 Response Criteria means the definitions set forth in Eisenhauer et al., E.A. et al., Eur. J Cancer 45:228-247 (2009) for target lesions or nontarget lesions, as appropriate based on the context in which response is being measured.
  • Responder patient when referring to a specific anti-tumor response to treatment with a combination therapy described herein, means the patient exhibited the antitumor response.
  • sustained response means a sustained therapeutic effect after cessation of treatment with a therapeutic agent, or a combination therapy described herein.
  • the sustained response has a duration that is at least the same as the treatment duration, or at least 1.5, 2.0, 2.5 or 3 times longer than the treatment duration.
  • tissue Section refers to a single part or piece of a tissue sample, e.g., a thin slice of tissue cut from a sample of a normal tissue or of a tumor.
  • Tuat or “treating” cancer means to administer a combination therapy of an anti-IL-10 antibody, LNP of the invention and CpG oligonucleotide to a subject having cancer, or diagnosed with cancer, to achieve at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth.
  • Positive therapeutic effects in cancer can be measured in a number of ways (See, W. A. Weber, J. Nucl. Med. 50: 1 S-10S (2009)).
  • a T/C ⁇ 42% is the minimum level of anti-tumor activity.
  • response to a combination therapy described herein is assessed using RECIST 1.1 criteria or irRC (bidimensional or uni dimensional) and the treatment achieved by a combination of the invention is any of PR, CR, OR, PFS, DFS and OS.
  • PFS also referred to as "Time to Tumor Progression" indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a CR or PR, as well as the amount of time patients have experienced SD.
  • DFS refers to the length of time during and after treatment that the patient remains free of disease.
  • OS refers to a prolongation in life expectancy as compared to naive or untreated individuals or patients.
  • response to a combination of the invention is any of PR, CR, PFS, DFS, OR and OS that is assessed using RECIST 1.1 response criteria.
  • the treatment regimen for a combination of the invention that is effective to treat a cancer patient may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject.
  • treatment regimen “dosing protocol” and “dosing regimen” are used interchangeably to refer to the dose and timing of administration of each therapeutic agent in a combination of the invention.
  • Tumor as it applies to a subject diagnosed with, or suspected of having, cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms.
  • a solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).
  • Tumor burden also referred to as “tumor load” refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor(s), throughout the body, including lymph nodes and bone marrow. Tumor burden can be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scans.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • tumor size refers to the total size of the tumor which can be measured as the length and width of a tumor. Tumor size may be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., bone scan, ultrasound, CT or MRI scans.
  • imaging techniques e.g., bone scan, ultrasound, CT or MRI scans.
  • Unidimensional irRC refers to the set of criteria described in Nishino M, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya NH, Hodi FS. Developing a Common Language for Tumor Response to Immunotherapy: Immune-related Response Criteria using Unidimensional measurements. Clin Cancer Res. 2013; 19(14):3936-3943). These criteria utilize the longest diameter (cm) of each lesion.
  • V region means the segment of IgG chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.
  • Anti-IL-10 antibody means an antagonist antibody that binds IL-10 to inhibit the activity of IL-10.
  • Alternative names or synonyms for IL-10 include: Interleukin- 10, cytokine synthesis inhibitor factor or CSIF. Human IL-10 amino acid sequences can be found in US patent 6217857.
  • amino acid sequence of the mature human IL-10 protein is SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFK GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRELPC ENKSKAVEQ VKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO : 21 )
  • the anti-IL-10 antibodies to be used are the ones described in US 8,226,947 and US 7,662,379, the disclosure of which is hereby incorporated by reference in its entirety.
  • the anti-IL-10 antibody is anti-IL-10 huml2G8, which comprises two identical light chains with the sequence of SEQ ID NO: 2 and two identical heavy chains with the sequence of SEQ ID NO: 1. Plasmids containing nucleic acids encoding both the heavy and light chains of huml2G8 were deposited with the ATCC on May 6, 2004, as PTA- 5922 and PTA-5923, respectively.
  • Anti-IL-10 antibodies useful in any of the treatment method, medicaments and uses of the present invention include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to IL-10.
  • the mAb may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgGl or IgG4 constant region.
  • the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab') 2 , scFv and Fv fragments.
  • the anti-IL-10 antibody is a monoclonal antibody, or antigen binding fragment thereof, which comprises: (a) light chain CDRs of SEQ ID NOs: 5, 6 and 7 and heavy chain CDRs SEQ ID NOs: 8, 9 and 10 of anti-IL-10 huml2G8.
  • the anti-IL-10 antibody is a monoclonal antibody, or antigen binding fragment thereof, which comprises: (a) light chain CDRs of SEQ ID NOs: 15, 16 and 17 and heavy chain CDRs SEQ ID NOs: 18, 19 and 20 of anti-IL-10 huml lD8.
  • the anti-IL-10 antibody is a monoclonal antibody, or antigen binding fragment thereof, which specifically binds to human IL-10 and comprises (a) a heavy chain variable region comprising SEQ ID NO: 11 or a variant thereof, and (b) a light chain variable region comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof.
  • a variant of a heavy chain variable region sequence is identical to the reference sequence except having up to 17 conservative amino acid substitutions in the framework region (i.e., outside of the CDRs), and preferably has less than ten, nine, eight, seven, six or five conservative amino acid substitutions in the framework region.
  • a variant of a light chain variable region sequence is identical to the reference sequence except having up to five conservative amino acid substitutions in the framework region (i.e., outside of the CDRs), and preferably has less than four, three or two conservative amino acid substitution in the framework region.
  • Table 2 below provides a list of the amino acid sequences of exemplary anti- IL-10 mAbs for use in the treatment method, medicaments and uses of the present invention, and the sequences are shown in Figures 3-4.
  • DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK
  • CDRL1 SEQ ID NO: 15 RASESVDDYGHSFMH
  • an "anti-IL-10 hum 12G8 variant” means a monoclonal antibody which comprises heavy chain and light chain sequences that are identical to those in anti-IL-10 hum 12G8, except for having three, two or one conservative amino acid substitutions at positions that are located outside of the light chain CDRs and six, five, four, three, two or one conservative amino acid substitutions that are located outside of the heavy chain CDRs, e.g, the variant positions are located in the FR regions or the constant region.
  • anti-IL-10 hum 12G8 and an anti-IL-10 hum 12G8 variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at no more than three or six other positions in their full length light and heavy chain sequences, respectively.
  • An anti-IL-10 hum 12G8 variant is substantially the same as anti-IL-10 hum 12G8 with respect to the following properties: binding affinity to IL-10 and neutralizing effect in vivo.
  • halogen means Br, CI, F and I.
  • heterocyclyl or “heterocycle” means a 4- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • Heterocyclyl therefore includes, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyri
  • tetrahydropyranyl tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piped dinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,
  • dihydrooxazolyl dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl,
  • lipid nanoparticle or “L P” refers to any lipid composition that can be used to deliver a product, including, but not limited to, liposomes or vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single;
  • lipid nanoparticle does not need to have the TLR9 agonist incorporated therein and may be used to deliver a product when in the same formulation.
  • poly amine means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.
  • mole % refers to a mole percent of total lipids.
  • the L Ps of the compositions of the invention are composed of one or more cationic lipids (including ionizable cationic lipids) and one or more
  • the LNPs further comprise one or more non-cationic lipids.
  • the one or more non-cationic lipids can include a phospholipid, phospholipid derivative, a sterol, a fatty acid, or a combination thereof.
  • Ionizable cationic lipids are characterized by the weak basicity of their lipid head groups, which affects the surface charge of the lipid in a pH-dependent manner, rendering them positively charged at acidic pH but close to charge-neutral at physiologic pH.
  • Cationic lipids are characterized by monovalent or multivalent cationic charge on their headgroups, which renders them positively charged at neutral pH.
  • the cationic and ionizable lipid is capable of complexing with hydrophilic bioactive molecules to produce a hydrophobic complex that partitions into the organic phase of a two- phase aqueous/organic system. It is contemplated that both monovalent and polyvalent cationic lipids may be utilized to form hydrophobic complexes with bioactive molecules.
  • Preferred cationic and ionizable cationic lipids for use in forming the LNPs include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (“DOTMA”);
  • DODAC N,N-dioleyl-N,N-dimethylammonium chloride
  • DOTMA N-(2,3dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride
  • N,NdistearylN,N-dimethylammonium bromide (“DDAB”); N-(2,3dioleoyloxy)propyl)- ⁇ , ⁇ , ⁇ -trimethylamntonium chloride (“DODAP”); 1,2 bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP); 3-(N-(N,N-dimethylaminoethane)-carbam-oyl)cholesterol ('DC-ChoP'); diheptadecylamidoglycylspermidine (“DHGS”) and N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydoxyethyl ammonium bromide (“DMRIE”).
  • DMRIE N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydoxyethyl ammonium bromide
  • cationic lipid nanoparticles comprising DOTMA and l,2dioleoyl-sn- 3-phosphoethanolamine ("DOPE"), from GIBCOBRL, Grand Island, N.Y., USA); and
  • LIPOFECTAMINE® commercially available cationic lipid nanoparticles comprising N-(l- (2,3dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA') and (“DOPE”), from (GIBCOBRL).
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9, 12- dienyl-[l,3]dioxolan-4-ylmethyl)-dimethylamine, DLinKDMA (WO 2009/132131 Al), DLin-K-C2-DMA (WO2010/042877), DLin-M-C3 -DMA (WO2010/146740 and/or
  • DLin-MC3 -DMA heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate; Jayaraman et al., 2012, Angew. Chem. Int. Ed. Engl. 51 :8529- 8533), 2- ⁇ 4-[(3p)-cholest-5-en-3-yloxy]butoxy ⁇ -N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12- dienlyloxyl]propan-l -amine) (CLinDMA), and the like.
  • cationic lipids suitable for use in the invention include, e.g., the cationic lipids described in U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185, and U.S. Patent Application Publication Nos.
  • cationic lipids suitable for use in the invention include, e.g., Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages 112 - 139) of International Patent Application Publication No. WO2011/076807 (which also discloses methods of making, and methods of using these cationic lipids).
  • the LNPs comprise one or more of the following ionizable cationic lipids: DLinDMA, DlinKC2DMA DLin-MC3- DMA, CLinDMA, or S-Octyl CLinDMA (See International Patent Application Publication No. WO2010/021865).
  • LNPs comprise one or more ionizable cationic lipids described in International Patent Application Publication No. WO2011/022460 Al, or any pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds or salts therein.
  • p 1 to 8;
  • R1 and R2 are independently selected from H, (Ci-C io)alkyl, heterocyclyl, and a polyamine, wherein said heterocyclyl and polyamine are optionally substituted with one to three substituents selected from R4, or Rl and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle optionally substituted with one to three substituents selected from R4;
  • R3 is selected from H and (Ci -C6)alkyl, said alkyl optionally substituted with one to three substituents selected from R4;
  • R4 is independently selected from halogen, OR5, SR5, CN, C0 2 R5 and
  • R5 is independently selected from H, (Ci-Cio)alkyl and aryl;
  • Y is a (C4-C22)alkyl, (C4-C22)perfluoroalkyl, or a (C4-C22)alkenyl;
  • Exemplary ionizable cationic lipids include compounds 4-11 and 13-20 described in International Patent Application Publication No. WO2011/022460 Al, as shown in Table 3 (preceded by or any pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds or salts.
  • the ionizable cationic lipid is compound 9 described in International Patent Application Publication No. WO2011/022460 Al (designated herein as compound 1-9 in Table 3 herewith), or a pharmaceutically acceptable salt thereof, or a stereoisomer of the compound or its salts.
  • LNPs comprise one or more low molecular weight ionizable cationic lipids described in International Patent
  • Rl and R2 are independently selected from H, (Ci-C6)alkyl, heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one to three substituents selected from R', or Rl and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one to three substituents selected from R';
  • R3 is independently selected from H and (Ci-C6)alkyl, said alkyl optionally substituted with one to three substituents selected from R';
  • R' is independently selected from halogen, R", OR", SR", CN, CO 2 R" or
  • R" is independently selected from H and (Ci-C6)alkyl, wherein said alkyl is optionally substituted with halogen and OH;
  • n O, 1, 2, 3, 4 or 5;
  • Li is selected from C4-C24 alkyl and C4-C24 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R'; and
  • L2 is selected from C3-C9 alkyl and C3-C9 alkenyl, said alkyl and alkenyl are optionally substituted with one or more substituents selected from R';
  • the ionizable cationic lipid is a compound having Formula A2, wherein:
  • R1 and R2 are each methyl
  • R3 is H
  • n 0;
  • Li is selected from C4-C24 alkyl and C4-C24 alkenyl
  • L2 is selected from C3-C9 alkyl and C3-C9 alkenyl
  • Exemplary ionizable cationic lipids are compounds 1-44 described in
  • N,N-dimethylnonadecan- 10-amine N,N-dimethyl-l-[(l S,2R)-2--35 octylcyclopropyl]nonadecan- 10- amine
  • the ionizable cationic lipids are compounds 32 and 33 described in International Patent Application Publication No. WO2012/040184 (designated herein as compounds 2-32 and 2-33, respectively), or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds or its salts.
  • stereoisomers When structures of the same constitution differ in respect to the spatial arrangement of certain atoms or groups, they are stereoisomers, and the considerations that are significant in analyzing their interrelationships are topological. If the relationship between two stereoisomers is that of an object and its nonsuperimposable mirror image, the two structures are enantiomeric, and each structure is said to be chiral. Stereoisomers also include diastereomers, cis-trans isomers and conformational isomers. Diastereoisomers can be chiral or achiral, and are not mirror images of one another.
  • Cis-trans isomers differ only in the positions of atoms relative to a specified plane in cases where these atoms are, or are considered as if they were, parts of a rigid structure.
  • Conformational isomers are isomers that can be interconverted by rotations about formally single bonds. Examples of such
  • conformational isomers include cyclohexane conformations with chair and boat conformers, carbohydrates, linear alkane conformations with staggered, eclipsed and gauche conformers, etc. See J. Org. Chem. 35, 2849 (1970).
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture.
  • Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms.
  • a chiral carbon can be designated with an asterisk (*).
  • bonds to the chiral carbon are depicted as straight lines in the Formulas of the invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the Formula.
  • one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines (bonds to atoms below the plane).
  • the Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.
  • the compounds of the present invention contain one chiral center, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixtures.
  • the enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt
  • Designation of a specific absolute configuration at a chiral carbon of the compounds of the invention is understood to mean that the designated enantiomeric form of the compounds is in enantiomeric excess (ee) or in other words is substantially free from the other enantiomer.
  • the "R” forms of the compounds are substantially free from the "S” forms of the compounds and are, thus, in enantiomeric excess of the "S” forms.
  • S forms of the compounds are substantially free of “R” forms of the compounds and are, thus, in enantiomeric excess of the "R” forms.
  • Enantiomeric excess is the presence of a particular enantiomer at greater than 50%. In a particular embodiment when a specific absolute configuration is designated, the enantiomeric excess of depicted compounds is at least about 90%.
  • a compound of the present invention When a compound of the present invention has two or more chiral carbons it can have more than two optical isomers and can exist in diastereoisomeric forms.
  • the compound when there are two chiral carbons, the compound can have up to 4 optical isomers and 2 pairs of enantiomers ((S,S)/(R,R) and (R,S)/(S,R)).
  • the pairs of enantiomers e.g., (S,S)/(R,R)
  • the stereoisomers that are not mirror-images e.g., (S,S) and (R,S) are diastereomers.
  • the diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above.
  • the present invention includes each diastereoisomer of such compounds and mixtures thereof.
  • the L Ps may also comprise any combination of two or more of the cationic lipids described herein.
  • the cationic lipid typically comprises from about 0.1 to about 99.9 mole % of the total lipid present in said particle.
  • the cationic lipid can comprise from about 80 to about 99.9% mole %.
  • the cationic lipid comprises from about 2% to about 70%, from about 5% to about 50%, from about 10% to about 45%, from about 20% to about 99.8%, from about 30% to about 70%, from about 34% to about 59%, from about 20% to about 40%, or from about 30% to about 40% (mole %) of the total lipid present in said particle.
  • the LNPs described herein can further comprise a noncationic lipid, which can be any of a variety of neutral uncharged, zwitterionic or anionic lipids capable of producing a stable complex. They are preferably neutral, although they can be negatively charged. Examples of noncationic lipids useful in the present invention include
  • phospholipid-related materials such as natural phospholipids, synthetic phospholipid derivatives, fatty acids, sterols, and combinations thereof.
  • Natural phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidyl serine (PS), phosphatidylinositol (PI), Phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso PC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), N-Acyl-PE, phosphoinositides, and
  • Phospholipid derivatives include phosphatidic acid (DMPA, DPP A, DSP A), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), and phosphatidylserine (DOPS).
  • DMPA phosphatidic acid
  • DPP A DSP A
  • DDPC phosphatidylcholine
  • DDPC DLPC
  • DMPC DPPC
  • DSPC DOPC
  • POPC DEPC
  • phosphatidylglycerol DMPG, DPPG, DSPG, POPG
  • DMPE phosphatidylethanolamine
  • DOPE phosphatidylserine
  • Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18: l), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0 and lethicin.
  • the non-cationic lipid is selected from lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,
  • phosphatidylserine phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC),
  • Noncationic lipids also include sterols such as cholesterol, stigmasterol or stigmastanol. Cholesterol is known in the art. See U.S. Patent Application Publication Nos: U.S. 2006/0240554 and U.S.
  • the LNP comprise a combination of a phospholipid and a sterol.
  • the non-cationic lipid typically comprises from about 0.1% to about 65%, about 2% to about 65%, about 10% to about 65%, or about 25% to about 65% expressed as mole percent of the total lipid present in the LNP.
  • the LNPs described herein further include a polyethyleneglycol (PEG) lipid conjugate ("PEG-lipid") which may aid as a bilayer stabilizing component.
  • PEG-lipid polyethyleneglycol
  • the lipid component of the PEG lipid may be any noncationic lipid described above including natural phospholipids, synthetic phospholipid derivatives, fatty acids, sterols, and combinations thereof.
  • the PEG-lipids include, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., International Patent Application Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos.
  • PEG-DAA dialkyloxypropyls
  • PEG-DAG PEG coupled to diacylglycerol
  • PEG coupled to phosphatidylethanolamine PEG-PE
  • PEG conjugated to 1,2-Di-O-hexadecyl-sn-glyceride PEG-DSG
  • the PEG-DAG conjugate is a dilaurylglycerol (C 12)-PEG conjugate, a PEG dimyristylglycerol (C14)conjugate, a PEG-dipalmitoylglycerol (C16) conjugate, a PEG-dilaurylglycamide (C12) conjugate, a PEG-dimyristylglycamide (C14) conjugate, a PEG-dipalmitoylglycamide (CI 6) conjugate, or a PEG-disterylglycamide (CI 8).
  • a dilaurylglycerol (C 12)-PEG conjugate a PEG dimyristylglycerol (C14)conjugate
  • a PEG-dipalmitoylglycerol (C16) conjugate conjugate
  • PEG-dilaurylglycamide C12
  • PEG-lipids include, but are not limited to, PEG- dimyristolglycerol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c- DMA).
  • PEG-DMG PEG- dimyristolglycerol
  • PEG-DSG PEG-disteryl glycerol
  • PEG-dipalmetoleyl PEG-dioleyl
  • PEG-distearyl PEG-diacylglycamide
  • PEG-DPPE PEG-dipalmitoyl phosphatidylethanolamine
  • the PEG-lipid is PEG coupled to dimyristoylglycerol (PEG-DMG), e.g., as described in Abrams et al., 2010, Molecular Therapy 18(1): 171, and U.S. Patent Application Publication Nos. US 2006/0240554 and US 2008/0020058.
  • PEG-DMG dimyristoylglycerol
  • the PEG-lipid such as a PEG-DAG, PEG-cholesterol, PEG-DMB, comprises a polyethylene glycol having an average molecular weight ranging of about 500 daltons to about 10,000 daltons, of about 750 daltons to about 5,000 daltons, of about 1,000 daltons to about 5,000 daltons, of about 1,500 daltons to about 3,000 daltons or of about 2,000 daltons.
  • the PEG-lipid comprises PEG400,
  • PEG1500 PEG2000 or PEG5000.
  • acyl groups in any of the lipids described above are preferably acyl groups derived from fatty acids having about CIO to about C24 carbon chains.
  • the acyl group is lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.
  • the PEG-lipid conjugate typically comprises from about 0.1% to about 15%, from about 0.5% to about 20%, from about 1.5% to about 18%, from about 4% to about 15%, from about 5% to about 12%, from about 1% to about 4%, or about 2% expressed as a mole % of the total lipid present in said particle.
  • the LNPs comprise one or more cationic lipids, cholesterol and 1,2-Dimyristoyl-sn-glycerol methoxypoly ethylene glycol (PEG-DMG).
  • the LNPs comprise one or more cationic lipids, cholesterol, l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2- Dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG).
  • DSPC l,2-Distearoyl-sn-glycero-3-phosphocholine
  • PEG-DMG 1,2- Dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • the LNPs comprise lipid compounds assembled within the following molar ratios:
  • Non-cationic lipid 0.1-65 mole %)
  • the LNPs comprise lipid compounds assembled within the following molar ratios:
  • Non-cationic lipid (20-65 mole %)
  • the non-cationic lipid is cholesterol.
  • Exemplary LNPs may include cationic lipid/cholesterol/PEG-DMG at about the following molar ratios: 58/30/10.
  • the non-cationic lipid is cholesterol and DSPC.
  • Exemplary LNPs may include cationic lipid/cholesterol/DSPC/PEG-DMG at about the following molar ratios: 59/30/10/1; 58/30/10/2; 43/41/15/1; 42/41/15/2; 40/48/10/2;
  • the TLR9 agonist is selected from A-class CpG ODN, B-class CpG ODN and C-class CpG ODN.
  • the oligonucleotide is an oligodeoxynucleotide.
  • one or more of the intemucleotide linkages of the CpG ODN are modified linkages.
  • one or more of the intemucleotide linkages of CpG ODN are phosphorothioate (PS) linkages.
  • all of the intemucleotide linkages of C-class and B-class CpG ODNs are phosphorothioate (PS) linkages.
  • oligonucleotides with mixed intemucleotide linkages See Krieg et al, 1995, Nature 374:546-549; Om et al, 1997, J. Exp. Med. 186: 1623-1631; Lipford et al, 1997, Eur. J. Immunol. 27:2340-2344; Roman et al, 1997, Nat. Med. 3 :849-854; Davis et al, 1998, J. Immunol. 160:870-876; Lipford et al, 1998, Trends Microbiol. 6:496-500; and U.S. Pat. Nos. 6,207,646; 7,223,398; 7,250,403; or 7,566,703. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in U.S. Pat.
  • the invention formulation features LNP compositions formulated or complexed with TLR9 agonist compounds, and assembled within the following molar ratios:
  • Non-cationic lipid (0.1-65 mole %)
  • TLR9 agonists (0.1-50 mole %).
  • the TLR9 agonist is provided at a wt/wt% of agonist to total lipids in a range of 1% to 20% or 4% to 15%.
  • the one or more TLR9 agonist is physically encapsulated in the LNP before or after LNP preparation.
  • TLR9 agonists physically encapsulated in the LNP can be prepared via confined-volume ethanol desolvation method as described, or via alternative techniques known in the art, including, but not limited to thin-film hydration, emulsion diffusion, or homogenization.
  • one or more TLR9 agonist is adsorbed, covalently coupled, ionically-interacted or formulated onto surfaces of the LNP. See, e.g., Li et al., 2002, Vaccine 20: 148-157; Wilson et al., 2009, J. Gene Med. 11 : 14-25; Goldinger et al, 2012, Eur. J. Immunol. 42:3049-3061; Gursel et al, 2001, J. Immunol. 167:3324-3328; and Chikh et al, 2009, Int. Immunol. 7:757-767.
  • LNPs can be formed, for example, by a rapid precipitation process which entails micro-mixing the lipid components dissolved in ethanol with an aqueous solution using a confined volume mixing apparatus such as a confined volume T-mixer, a multi-inlet vortex mixer (MIVM), or a microfluidics mixer device as described below.
  • the lipid solution contains one or more cationic lipids, one or more noncationic lipids (e.g., DSPC), PEG-DMG, and optionally cholesterol, at specific molar ratios in ethanol.
  • the aqueous solution consists of a sodium citrate or sodium acetate buffered salt solution with pH in the range of 2-6, preferably 3.5-5.5.
  • the two solutions are heated to a temperature in the range of 25°C-45°C, preferably 30°C-40°C, and then mixed in a confined volume mixer thereby instantly forming the LNP.
  • the T-mixer has an internal diameter (ID) range from 0.25 to 1.0 mm.
  • ID internal diameter
  • the alcohol and aqueous solutions are delivered to the inlet of the T-mixer using programmable syringe pumps, and with a total flow rate from 10-600 mL/minute.
  • the alcohol and aqueous solutions are combined in the confined-volume mixer with a ratio in the range of 1 : 1 to 1 :3 vol: vol, but targeting 1 : 1.1 to 1 :2.3.
  • the combination of ethanol volume fraction, reagent solution flow rates and t-mixer tubing ID utilized at this mixing stage has the effect of controlling the particle size of the
  • LNPs between 30 and 300 nm.
  • the resulting LNP suspension is twice diluted into higher pH buffers in the range of 6-8 in a sequential, multi-stage in-line mixing process.
  • the LNP suspension is mixed with a buffered solution at a higher pH (pH 6-7.5) with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol, but targeting 1 :2 vol: vol.
  • This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C.
  • the resulting LNP suspension is further mixed with a buffered solution at a higher pH, e.g., 6-8 and with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol, but targeting 1 :2 vol: vol.
  • This later buffered solution is at a temperature in the range of 15-40°C, targeting 16-25°C.
  • the mixed LNPs are held from 30 minutes to 2 hours prior to an anion exchange filtration step.
  • the temperature during incubation period is in the range of 15-40°C, targeting 30-40°C.
  • the LNP suspension is filtered through a 0.8 ⁇ filter containing an anion exchange separation step.
  • This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/minute.
  • the LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the buffer is exchanged for the final buffer solution such as phosphate buffered saline or a buffer system suitable for cryopreservation (for example containing sucrose, trehalose or combinations thereof).
  • the ultrafiltration process uses a tangential flow filtration format (TFF).
  • TMF tangential flow filtration format
  • This process uses a membrane nominal molecular weight cutoff range from 30-500 KD, targeting 100 KD.
  • the membrane format can be hollow fiber or flat sheet cassette.
  • the TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol and final buffer wastes.
  • the TFF process is a multiple step process with an initial concentration to a lipid concentration of 20-30 mg/mL. Following concentration, the LNP suspension is diafiltered against the final buffer (for example, phosphate buffered saline
  • the material is then concentrated an additional 1-3 fold via ultrafiltration.
  • the final steps of the LNP manufacturing process are to sterile filter the concentrated LNP solution into a suitable container under aseptic conditions. Sterile filtration is accomplished by passing the LNP solution through a pre-filter (Acropak 500 PES 0.45/0.8 ⁇ capsule) and a bioburden reduction filter (Acropak 500 PES 0.2/0.8 ⁇ capsule). Following filtration, the vialed LNP product is stored under suitable storage conditions (2°C-8°C, or -20°C if frozen formulation).
  • the LNPs of the compositions provided herein have a mean geometric diameter that is less than 1000 nm. In some embodiments, the LNPs have mean geometric diameter that is greater than 50 nm but less than 500 nm. In some embodiments, the mean geometric diameter of a population of LNPs is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
  • the mean geometric diameter is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometric diameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the mean geometric diameter is between 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter that is less than 500 nm.
  • 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter that is greater than 50 nm but less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm.
  • 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), or more of the LNPs of a population of LNPs have a diameter that is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter that is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm.
  • the size of the LNPs ranges between about 1 and 1000 nm, preferably between about 10 and 500 nm, more preferably between about 100 to 300 nm, and preferably 100 nm.
  • the present invention is also directed to methods of treating cancer in the subject by co-administering to the subject 1) an effective amount of a composition
  • the invention provides a medicament comprising the LNP of the invention and a TLR9 agonist for use in combination with an anti-IL-10 antibody for treating cancer in the subject by co-administering to the subject 1) an effective amount of a composition comprising the LNP of the invention and TLR9 agonist; and 2) an anti-IL-10 antibody.
  • the invention provides a medicament comprising the
  • the invention provides a medicament comprising the LNP of the invention and a TLR9 agonist for use in combination with an anti-IL-10 antibody for treating cancer in the subject by intratum orally administering to the subject 1) an effective amount of a composition comprising the LNP of the invention and TLR9 agonist; and 2) intratumorally administering an anti-IL-10 antibody.
  • the invention provides a medicament comprising the LNP of the invention and a TLR9 agonist for use in combination with an anti-IL-10 antibody for treating cancer in the subject by intratum orally administering to the subject 1) an effective amount of a composition comprising the L P of the invention and TLR9 agonist; and 2) intravenously administering an anti-IL-10 antibody.
  • cancers include, but are not limited to breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and meduUoblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia;
  • the combination therapy may also comprise one or more additional therapeutic agents.
  • the additional therapeutic agent may be, e.g., a chemotherapeutic other than a CpG oligonucleotide, a biotherapeutic agent, immunotherapeutic agent, an
  • the immunogenic agent for example, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor derived antigen or nucleic acids, immune stimulating cytokines (for example, IL-2, IFNa2, GM-CSF), cells transfected with genes encoding immune stimulating cytokines such as but not limited to GM-CSF), and radiation.
  • the immunotherapeutic agent comprises one or more of a cytokine, a small molecule adjuvant, and an antibody.
  • the cytokine comprises one or more of a chemokine, an interferon, an interleukin, a lymphokine, and a tumour necrosis factor.
  • the specific dosage and dosage schedule of the additional therapeutic agent can further vary, and the optimal dose, dosing schedule and route of administration will be determined based upon the specific therapeutic agent that is being used.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine;
  • acetogenins especially bullatacin and bullatacinone
  • a camptothecin including the synthetic analogue topotecan
  • bryostatin cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
  • calicheamicin phill see, e.g., Agnew, Chem. Intl. Ed. Engl., 33 : 183-186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic
  • chromomophores aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, uben
  • etoglucid gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
  • pentostatin phenamet
  • pirarubicin losoxantrone
  • podophyllinic acid 2-ethylhydrazide
  • procarbazine razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
  • pipobroman gacytosine; arabinoside ("Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
  • methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum;
  • etoposide VP-16
  • ifosfamide mitoxantrone
  • vincristine vinorelbine
  • novantrone VP-16
  • teniposide edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
  • topoisomerase inhibitor RFS 2000 difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozo
  • Each therapeutic agent in a combination therapy of the invention may be administered either alone or in a medicament (also referred to herein as a pharmaceutical composition) which comprises the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients and diluents, according to standard pharmaceutical practice.
  • Each therapeutic agent in a combination therapy of the invention may be administered simultaneously (i.e., in the same medicament), concurrently (i.e., in separate medicaments administered one right after the other in any order) or sequentially in any order. Sequential administration is particularly useful when the therapeutic agents in the
  • combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and/or are administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.
  • a chemotherapeutic that is administered at least daily
  • a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.
  • the CpG oligonucleotide and LNP of the invention is administered before administration of the anti-IL-10 antibody, while in other embodiments, the CpG oligonucleotide and LNP of the invention is administered after administration of the anti-IL-10 antibody. In another embodiment, the CpG oligonucleotide is administered concurrently with the anti-IL-10 antibody.
  • the CpG oligonucleotide and LNP of the invention is administered intratumorally or intravenously.
  • the anti-IL-10 antibody is administered intratumorally or intravenously.
  • At least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer.
  • the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and/or shorter treatment duration.
  • Each small molecule therapeutic agent in a combination therapy of the invention can be administered orally or parenterally, including the intravenous,
  • intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration are intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration.
  • a combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during or after radiation therapy.
  • a combination therapy of the invention is administered to a patient who has not been previously treated with a biotherapeutic or chemotherapeutic agent, i.e., is treatment-naive.
  • the combination therapy is administered to a patient who failed to achieve a sustained response after prior therapy with a biotherapeutic or chemotherapeutic agent
  • biotherapeutic or chemotherapeutic agent i.e., is treatment-experienced.
  • a combination therapy of the invention is typically used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan.
  • a dosage regimen for a combination therapy of the invention depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated.
  • a dosage regimen maximizes the amount of each therapeutic agent delivered to the patient consistent with an acceptable level of side effects.
  • the dose amount and dosing frequency of each biotherapeutic and chemotherapeutic agent in the combination depends in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available.
  • Determination of the appropriate dosage regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, on the patient's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.
  • Biotherapeutic agents in a combination therapy of the invention may be administered by continuous infusion, or by doses at intervals of, e.g., daily, every other day, three times per week, or one time each week, two weeks, three weeks, monthly, bimonthly, etc.
  • a total weekly dose is generally at least 0.05 ⁇ g/kg, 0.2 ⁇ g/kg, 0.5 ⁇ g/kg, 1 ⁇ g/kg, 10 ⁇ g/kg, 100 ⁇ g/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang et al. (2003) New Engl. J. Med.
  • the anti-IL-10 antibody in the first embodiment in the second embodiment of the invention, the anti-IL-10 antibody in the first embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the first embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the second embodiment, the anti-IL-10 antibody in the
  • combination therapy is anti-IL-10 hum 12G8, which is administered intravenously at a dose selected from the group consisting of: 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 4 mg/kg Q3W, 5 mg/kg Q3W, 6 mg/kg Q3W, 7 mg/kg Q3W, 8 mg/kg Q3W, 9 mg/kg Q3W, 10 mg/kg Q3W, 11 mg/kg Q3W, 12 mg/kg Q3W, 13 mg/kg Q3W, 14 mg/kg Q3W and 15 mg/kg Q3W.
  • a dose selected from the group consisting of: 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 4 mg/kg Q3W, 5 mg/kg Q3W, 6 mg/kg Q3W, 7 mg/kg Q3W, 8 mg/kg Q3W, 9 mg/kg Q3W, 10 mg/kg Q3W, 11 mg/kg Q3W, 12 mg/
  • the anti-IL-10 antibody in the combination therapy is anti-IL-10 hum 12G8, which is administered intravenously at a dose of 1 mg/kg Q3W.
  • the anti-IL-10 antibody in the combination therapy is anti-IL-10 hum 12G8, which is administered intravenously at a dose of 3 mg/kg Q3W.
  • the anti-IL-10 antibody in the combination therapy is anti-IL-10 hum 12G8, which is administered intravenously at a dose of 10 mg/kg Q3W.
  • the anti-IL-10 antibody in the combination therapy is anti-IL-10 hum 12G8, an anti-IL-10 hum 12G8 variant, which is administered in a liquid medicament at a dose selected from the group consisting of 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 4 mg/kg Q3W, 5 mg/kg Q3W, 6 mg/kg Q3W, 7 mg/kg Q3W, 8 mg/kg Q3W, 9 mg/kg Q3W, 10 mg/kg Q3W, 11 mg/kg Q3W, 12 mg/kg Q3W, 13 mg/kg Q3W, 14 mg/kg Q3W and 15 mg/kg Q3W.
  • one or more of the TLR9 agonist is adsorbed, covalently coupled, ionically-interacted, or formulated onto surfaces of the L P.
  • the LNP may be co-administered with one or more TLR9 agonists.
  • compositions of the invention can be administered to cells by a variety of methods known to those of skill in the art.
  • delivery systems of the invention include, for example, aqueous and nonaqueous gels, multiple emulsions, microemulsions, aqueous and nonaqueous solutions, aerosols, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
  • the pharmaceutically acceptable carrier is a transdermal enhancer.
  • delivery systems of the invention include patches, suppositories, and gels, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as
  • the invention features a composition comprising one or more formulated TLR9 agonists in an acceptable carrier, such as a stabilizer, buffer, and the like.
  • an acceptable carrier such as a stabilizer, buffer, and the like.
  • the compositions of the invention can be administered and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a composition.
  • the compositions of the present invention can also be formulated and used as gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.
  • compositions of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation.
  • systemic administration is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and
  • compositions or formulation suitable for administration in the physical location most suitable for their desired activity include: P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired lipid nanoparticles in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the formulated composition.
  • Aqueous suspensions contain the lipid nanoparticles in a mixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as
  • polyoxyethylene sorbitol monooleate or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the lipid nanoparticles in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the lipid nanoparticles in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol,
  • compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Formulated compositions of the invention can be administered parenterally in a sterile medium.
  • the lipid nanoparticles depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the composition can also be added to water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • LNP lipid nanoparticle
  • LNP A comprised compound 1-9 in Table 3 (amino lipid 9 from WO2011/022460), cholesterol, polyethylene glycol-lipid (PEG-DMG), and phospholipid (DSPC) at a specified molar composition of 58:30: 10:2 amino lipid:cholesterol:DSPC:PEG-DMG.
  • the LNP composition was prepared by a rapid precipitation process which entailed micromixing lipids dissolved in ethanol with an aqueous solution using a confined volume mixing apparatus (Gindy et al., Molecular Pharmaceutics, 2014, 11 (11), pp 4143- 4153).
  • the lipid solution contained amino lipid, cholesterol, PEG-DMG, and DSPC at the specified molar ratios in ethanol.
  • the aqueous solution consisted of a sodium citrate buffered salt solution [20 mM] with pH in the range of 5-5.5. The two solutions were heated to a temperature in the range of 35°C-40°C and then mixed in a confined volume mixer (T-mixer with ID of 0.5 mm) instantly forming the LNP.
  • the alcohol and aqueous solutions were delivered to the inlet of the T-mixer using programmable syringe pumps, and with a total flow rate from 100-150 mL/minute.
  • the alcohol and aqueous solutions were combined in the confined-volume mixer with a ratio in the range of 11 : 1.5 to 2.1 :3.8 to produce 40-55 vol:vol % alcohol in the mixed solution.
  • the combination of ethanol volume fraction, reagent solution flow rates and t-mixer tubing ID utilized at this mixing stage had the effect of controlling the particle size of the LNPs between 30 and 300 nm.
  • the resulting LNP suspension was twice diluted into higher pH buffers [pH range 6-8] in a sequential, multistage in-line mixing process.
  • the LNP suspension was mixed with a 20 mM sodium citrate, 300 mM sodium chloride buffered solution with pH 6 with a mixing ratio of 1 : 1 vol: vol. This buffered solution was at a temperature in the range of 35-40°C.
  • the resulting LNP suspension was further mixed with a buffered solution (phosphate buffered saline (PBS), pH of 7.5) and with a mixing ratio of 1 : 1 vol: vol.
  • PBS phosphate buffered saline
  • This later buffered solution was at a temperature in the range 16-25°C.
  • the mixed LNPs were held from 30 minutes prior to an anion exchange filtration step. The temperature during incubation period was in the range of 30-40°C.
  • the LNPs were concentrated and diafiltered via an ultrafiltration process where the alcohol was removed and the buffer was exchanged for the final buffer solution.
  • the ultrafiltration process used a tangential flow filtration format (TFF). This process used a PES membrane nominal molecular weight cutoff of 100 KD.
  • the membrane format was hollow fiber or flat sheet cassette. Ultrafiltration with a 100 kDa PES membrane is first used to concentrate the LNP solution 8-fold by volume, targeting a total lipids concentration of 20-30 mg/mL. Ethanol removal is effected by subsequent diafiltration using lOmM Tris, 140 mM sodium chloride, pH 7-7.5 (5-10 diavolumes).
  • a final buffer exchange into a buffer solution comprising 10 mM Tris, 70 mM NaCl and 5 wt% sucrose is performed.
  • the LNP solution is then sterile filtered into sterile vials under asceptic conditions via Pall 0.45 ⁇ PES, and a Pall 0.2 PES ⁇ syringe filters.
  • the LNP solution is stored under refrigeration (2-8°C) or as a frozen image (-20°C).
  • Particle size and polydispersity LNPs were diluted to a final volume of 3 ml with 1 x phosphate buffered saline (PBS).
  • the particle size and polydispersity of the samples was measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instalments Corporation, Holtsville, NY). The scattered intensity was measured with He-Ne laser at 25°C with a scattering angle of 90°.
  • LNPs were diluted to a final volume of 2 ml with 1 mM Tris buffer (pH 7.4). Electrophoretic mobility of samples was determined using ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY) with a disposable Zeta cell DTS 1060C (Malvern Instruments Ltd, Worcestershire, UK) and He-Ne laser as a light source. The Smoluchowski limit (Z. Phys. Chem., 93 (1918), p. 129) was assumed in the calculation of zeta potentials.
  • lipid concentrations were determined by Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a Corona charged aerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, MA). Individual lipids in RDVs were analyzed using an Agilent Zorbax SBC 18 (50 x 4.6 mm, 1.8 ⁇ particle size) column with CAD at 60°C. The mobile phase was composed of A: 0.1% TFA in H20 and B: 0.1% TFA in IPA.
  • the individual lipid concentration was determined by comparing to the standard curve with all the lipid components in the RDVs with a quadratic curve fit. The molar percentage of each lipid was calculated based on its molecular weight. Analytic analysis confirmed the correct molar ratios.
  • Lipid nanoparticles (LNP) formulations encapsulating immunostimulatory oligonucleotides were prepared as described in Example 1 with the following modifications.
  • the LNP composition, LNP B comprised compound 1-9, cholesterol, DSPC and PEG-DMG at a molar composition of 58:30: 10:2, respectively, and encapsulated Cytidine phospho-guanosine (CpG)-based phosphorothioate oligodeoxynucleotide, ODN 1826, (InvivoGen, San Diego, CA).
  • ODN 1826 (5'-tccatgacgttcctgacgtt-3') (SEQ ID NO: 22) is a mouse TLR9 specific agonist and is a class B type sequence.
  • the LNP composition, LNP C, comprised compound 1-9, cholesterol, DSPC and PEG-DMG at a molar composition of 58:30: 10:2, respectively, and encapsulated a negative control ODN 1826 sequence.
  • ODN 1826 control sequence contains GpC dinucleotides instead of CpGs and is used as a negative control ODN.
  • LNPs encapsulating immunostimulatory molecules were formed by micro-mixing lipids dissolved in ethanol with an aqueous solution containing the immunostimulatory molecule using a confined volume mixing apparatus.
  • the aqueous solution containing ODN consisted of a sodium citrate solution [20 mM sodium citrate] with pH in the range of 5-5.5.
  • the ODN 1826 concentrations were determined by Strong Ani on-Exchange High-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector.
  • SAX-HPLC Strong Ani on-Exchange High-Performance Liquid Chromatography
  • the LNPs were treated with 0.5% Triton X-100 to free total ODN 1826 and analyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4 ⁇ 250 mm) column with UV detection at 254 nm.
  • Mobile phase was composed of A: 25 mM NaC104, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaC104, 10 mM Tris, 20% EtOH, pH 7.0 with a linear gradient from 0-15 min and a flow rate of 1 ml/minute.
  • the ODN 1826 amount was determined by comparing to the ODN 1826 standard curve.
  • Fluorescence reagent SYBR Gold was employed for ODN 1826 quantitation to monitor the encapsulation of oligonucleotide in RDVs.
  • RDVs with or without Triton X- 100 were used to determine the free ODN 1826 and total ODN 1826 amount.
  • the assay was performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples were excited at 485 nm and fluorescence emission was measured at 530 nm. The ODN 1826 amount is determined by comparing to an ODN 1826 standard curve.
  • Encapsulation rate (1- free ODN/total ODN) 100% EXAMPLE 3 : PREPARATION OF LIPID NANOPARTICLE COMPOSITIONS CO- FORMULATED WITH IMMUNOSTIMULATORY OLIGODEOXYNUCLEOTIDES
  • Lipid nanoparticles (LNP) composition co-formulated with immunostimulatory ODN 1826 (InvivoGen, San Diego), LNP D, was prepared by co-mixing LNP composition of Example 1, LNP A, with ODN 1826 (InvivoGen, San Diego, CA), in buffer solution comprising 10 mM Tris, 70 mM NaCl and 5 wt% sucrose. The solution was mixed gently at room temperature for 15 minutes and stored at 4-8°C for further use.
  • Lipid Nanoparticle composition co-formulated with ODN 1826 control sequence, LNP E was similarly prepared.
  • ODN 1826 control sequence (InvivoGen, San Diego) contains GpC dinucleotides instead of CpGs and is used as a negative control ODN.
  • EXAMPLE 4 EFFECTS OF LIPID NANOPARTICLES ON ANTI-TUMOR ACTIVITY OF ANTI-IL-10 AND PERITUMORAL CpG IN ANIMAL MODEL
  • TC40.11D8 is a mouse IgGl/kappa monoclonal antibody targeted against mouse IL- 10.
  • the mouse IgGl isotype control is a mouse monoclonal antibody specific for adenoviral hexon 25. Both antibodies were obtained from internal sources as frozen (-80°C) stocks.
  • the formulation buffer is specific for each antibody to stabilize proteins and prevent precipitation.
  • the formulations for both TC40.11D8 and mouse IgGl isotype control were 75 mM sodium chloride, 10 mM sodium phosphate, 3% sucrose, pH7.3.
  • Cytidine phospho-guanosine (CpG)-based phosphorothioate oligodeoxynucleotide ODN 1826 (InvivoGen, San Diego, CA) is a mouse TLR9 specific agonist. ODN 1826 has CpG class B type sequence. ODN 1826 control contains GpC dinucleotides instead of CpGs and is used as a negative control ODN.
  • Nanoparticle encapsulating ODN 1826 (Example 2, LNP B), Lipid Nanoparticle encapsulating ODN 1826 control (Example 2, LNP C), or free ODN 1826 and ODN 1826 control, is 10 mM Tris, 5 wt% sucrose, pH 7.5.
  • TC-1 cell line provided by Johns Hopkins University (Baltimore, MD) is derived from mouse primary lung epithelial cells that were cotransformed with human papilloma virus (HPV-16) E6 and E7 and c-Ha.ras oncogene (Lin et al., Cancer Res., 1996 Jan 1, 56(l):21-6). TC-1 cells are syngeneic to C57BL6/J strain.
  • TC-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 0.4 mg/mL Geneticin. 1 x 10 5 TC-1 cells were injected subcutaneously (SC) in 0.1 mL of serum-free DMEM in lower dorsal right and left flanks of each animal. Animals were first shaved with electronic clippers in the areas that were used for the implantation. Tumor measurements and body weights
  • Frozen stocks of the antibodies were thawed on ice. To avoid repeated freeze thaw, each vial of stock was thawed once and aliquots made in volumes sufficient for one time use. The aliquots were stored at -80°C. Before each dosing, one aliquot was thawed on ice and diluted to nominal concentration in appropriate formulation buffer.
  • Stocks of ODN 1826 and ODN 1826 control were stored at 4°C.
  • Stocks of empty Lipid Nanoparticles (L P A) and Lipid Nanoparticle-encapsulated ODN 1826 (LNP B) and Lipid Nanoparticle-encapsulated ODN 1826 (LNP C) control were stored at -20°C, and stocks were thawed on ice and diluted to nominal concentration in the appropriate formulation buffer. If vial of thawed stock was not used in entirety, it was stored at 4°C for next dosing.
  • Isotype control mlgGl and anti-IL-10 mlgGl were administered intraperitoneally (IP) at 10 mg/kg on Days 0, 3, 7, and 12.
  • ODN 1826 control 0.5 mg/kg
  • ODN 1826 0.5 mg/kg
  • Lipid Nanoparticle, LNP A 4.2 mg/kg
  • Lipid Nanoparticle (LNP)- encapsulating ODN 1826, LNP B 4.2 mg/kg LNP /0.5 mg/kg ODN 1826
  • Lipid Nanoparticle (LNP)-encapsulating ODN 1826 control, LNP C (3.8 mg/kg LNP /0.5 mg/kg ODN 1826 control) were administered peritumoral (PT) only in right tumors on Days 0, 3, 7 , and 12.
  • PT peritumoral
  • volume for each day and treatment group were summarized by their median.
  • a distribution function for each day and treatment group was estimated by the Kaplan-Meier method, with confidence band obtained using the beta product confidence procedure.
  • the median was estimated as the 50th percentile of the distribution function, with confidence interval obtained by inverting the confidence band.
  • a 68% confidence level was used, to be comparable to the common "mean ⁇ SE" format for summarizing data, since the latter is approximately a 68% confidence interval for the mean.
  • tumor burden right-censor at last measured value
  • tumor ulceration right-censor at last measured value, provided this exceeded a threshold (1000 mm 3 ); otherwise omit animal at later times
  • weight loss/ill omit animal at later times
  • (4) found dead and (5) unrelated to treatment (e.g., accident, administrative termination): right-censor at last measured value, provided this exceeded a threshold (1000 mm 3 ); otherwise omit animal at later times.
  • the threshold for using the last measured volume as a lower bound on later volumes was based on a judgment that once a tumor's volume exceeded that threshold, it was unlikely to shrink.
  • TC-1 tumor-bearing C57BL/6J mice were grouped into 8 treatment groups of ten animals when the mean volume of tumors on right flank reached approximately 98 mm 3 (51 mm 3 - 196 mm 3 ): (1) mlgGl isotype control + ODN 1826 control; (2) anti-IL-10 + ODN 1826; (3) mlgGl isotype control + LNP A/ODN 1826 control; (4) mlgGl isotype control + LNP A/ODN 1826; and (5) anti-IL-10 + LNP A + ODN 1826 control; (6) anti-IL-10 + LNP A + ODN 1826; (7) anti-IL-10 + LNP A/ODN 1826 control; and (8) anti-IL-10 + LNP A/ODN 1826.
  • CR Complete regression

Abstract

La présente invention concerne une composition comprenant : a) une nanoparticule lipidique (LNP) comprenant un ou plusieurs lipides cationiques et un poly(éthylèneglycol)-lipide (PEG-lipide) ; b) un oligonucléotide CpG agoniste du récepteur 9 de type Toll (TLR9) ; et c) un anticorps anti-IL-10 ou un fragment de liaison à l'antigène de ce dernier. La présente invention concerne également des méthodes de traitement du cancer chez le sujet par co-administration au sujet 1) d'une quantité efficace d'une composition comprenant la LNP de l'invention et un agoniste de TLR9 ; et 2) d'un anticorps anti-IL-10 ou d'un fragment de liaison à l'antigène de ce dernier.
PCT/US2017/030484 2016-05-03 2017-05-02 Polythérapie d'anticorps anti-il-10 et de compositions comprenant des nanoparticules lipidiques et des oligonucléotides cpg agonistes de tlr9 WO2017192470A1 (fr)

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US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10266485B2 (en) 2015-09-17 2019-04-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
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US10442756B2 (en) 2015-09-17 2019-10-15 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
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US10195156B2 (en) 2015-12-22 2019-02-05 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US10857105B2 (en) 2017-03-15 2020-12-08 MordernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2018170336A1 (fr) * 2017-03-15 2018-09-20 Modernatx, Inc. Formulation de nanoparticules lipidiques
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
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WO2019226650A1 (fr) * 2018-05-23 2019-11-28 Modernatx, Inc. Administration d'adn
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
WO2021202449A1 (fr) * 2020-03-30 2021-10-07 Unm Rainforest Innovations Cellules silicifiées chargées de nanoparticules, leurs procédés de fabrication et leurs procédés d'utilisation
WO2022119883A3 (fr) * 2020-12-02 2022-09-01 Merck Sharp & Dohme Llc Compositions de nanoparticules lipidiques contenant des lipides cationiques de monoesters
WO2023114889A1 (fr) * 2021-12-16 2023-06-22 Modernatx, Inc. Procédés de préparation de nanoparticules lipidiques

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