Classifications

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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
• • A—HUMAN NECESSITIES
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  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • A—HUMAN NECESSITIES
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
  • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to the field of vaccine technology, and more specifically to albumin-binding lipids conjugated to cargo and which efficiently target the cargo to the lymph nodes.
• Subunit vaccines present an antigen to the immune system without introducing viral particles in an effort to generate an immune response that is effective against that antigen.
• Such subunit vaccines are often poorly immunogenic and require co-administration of one or more adjuvants to generate an effective immune response (Perrie,Y., et al., Int. J. Pharm. 364,
• Immunostimulatory oligonucleotides such as those containing unmethylated cytosine-phosphate-guanine ("CG” or "CpG”) motifs, can be used as an adjuvant to stimulate both cellular and humoral immune responses (Vollmer,
• oligonucleotides as a vaccine adjuvant is the lack of an efficient system with which to target the oligonucleotides in vivo to the immune cells of the lymphatic system (Von Beust, B. R., et al. Eur. J.
• Antigens/adjuvants introduced into the body may be taken up by immune dendritic cells (DCs) at the injection site and then carried to lymph node through DC trafficking (e.g., cell associated antigen or larger particles, >200 nm).
• DCs immune dendritic cells
• Soluble antigen/adjuvant compounds flush through lymph nodes within hours (Pape, et al., Immunity 26, 491-502 (2007)), providing only a brief exposure to the vaccine.
• Attempts to enhance the delivery of antigens adjuvants to lymph nodes following parenteral injection have included the use of depot-forming adjuvants or particulate carriers that are preferentially internalized by antigen presenting cells (Johansena, et al., Journal of Controlled Release, 148, 56-62 (2010), Moon, et al., Adv. Mater., 24, 3724-3746 (2012), Bachmann and Jennings, Nat. Rev. Immunol.
• CD8+ DCs cytotoxic T lymphocyte
• albumin-binding lipids can be conjugated to cargo and efficiently target the cargo to the lymph nodes in vivo. It is believed that upon in vivo introduction, the lipid conjugates bind to endogenous albumin, which prevents the conjugates from rapidly flushing into the bloodstream and instead re-targets them to lymphatics and draining lymph nodes where they accumulate due to filtering of albumin by antigen presenting cells.
• the lipid conjugate includes an immunostimulant such as an immunostimulatory oligonucleotide or antigenic peptide
• the conjugates can induce or enhance a robust immune response.
• the amphiphilic albumin-binding conjugates include
• an immunomodulatory compound or molecular adjuvant wherein the immunomodulatory compound or molecular adjuvant is bound directly to the lipid or is bound to the lipid via a linker, wherein the conjugate is sufficiently soluble such that the lipid binds to albumin under physiological conditions, and wherein a plurality of the conjugates can spontaneously form micelles in aqueous solution.
• Lipid conjugates including lipid-oligonucleotide conjugates and lipid- peptide conjugates and their use for stimulating immune response are disclosed.
• amphiphilic oligonucleotide conjugates for targeting the lymph nodes can include an immunostimulatory oligonucleotide which (i) is conjugated directly to a lipid, or (ii) is linked to a linker which is conjugated to a lipid.
• the lipid binds to albumin under
• a plurality of the oligonucleotide conjugates can spontaneously form micelles in aqueous solution which can be disrupted by the addition of an albumin containing agent.
• an albumin containing agent In a specific embodiment, 64% or more of the micelles are disrupted in the presence of 20% fetal bovine serum.
• the lymph nodes In some embodiments for targeting the lymph nodes, the lymph nodes
• oligonucleotide includes an oligonucleotide linker including 0, 1, or 2 consecutive guanines.
• the conjugate can have the structure L- 5'-Gêt-ON-3 ⁇ wherein "L” the lipid, "G” is a guanine, "n” is 0-2, and "ON” is the immunostimulatory oligonucleotide.
• the lipid of the conjugate typically binds to albumin.
• An exemplary lipid is a diacyl lipid, such a diacyl lipid wherein the chains include CI 2 or more hydrocarbon units.
• the immunostimulatory oligonucleotide can be a ligand for a pattern recognition receptor such as CpG, and have a modified backbone such as a phosphorothioate (PS) backbone.
• the oligonucleotide includes 20 or more nucleic acids.
• Conjugates for retention at sites at or near the site of administration are also disclosed.
• the cargo and the lipid are typically linked by an oligonucleotide linker including at least three consecutive guanines.
• the conjugates spontaneously form micelles in aqueous solution that are resistant to disruption by albumin. In a particular embodiment, more than 36% of the micelles are intact in the presence of 20% fetal bovine serum.
• the oligonucleotide linker including at least three consecutive guanines.
• the conjugates spontaneously form micelles in aqueous solution that are resistant to disruption by albumin. In a particular embodiment, more than 36% of the micelles are intact in the presence of 20% feta
• oligonucleotide conjugate has the structure L-5'-G n -ON-3', wherein "L” the lipid, “G” is a guanine, “n” is 3-10, and “ON” is the immunostimulatory oligonucleotide.
• Lipid-peptide conjugates are also disclosed.
• the conjugate includes a peptide antigen which (i) is conjugated directly to a lipid, or (ii) is linked to a linker which is conjugated to a lipid.
• the lipid typically binds to albumin under physiological conditions.
• the peptide antigen, the linker, or the peptide antigen and linker in combination are sufficiently polar to reduced or inhibit insertion of the lipid into a cell's plasma membrane.
• Immunogenic compositions including lipid-oligonucleotide conjugates, lipid-peptide conjugates, and combinations thereof are also disclosed.
• the immunogenic compositions can be used to increase an immune response in a subject.
• the subject is administered an effective amount of the immunogenic composition to increase an effector immune cell response, for example, increase the number of CD8+ T cell expressing TNF-a or INF- ⁇ compared to a control.
• the methods can be used to treat subjects with cancer or infectious diseases.
• Figure 1 A is a schematic illustrating three domains of a lipid conjugate: cargo conjugated to a polar block which promotes solublization conjugated to a lipophilic tail.
• Figure IB is a schematic illustrating an exemplary lipid-oligonucleotide conjugate including an immunostimulatory oligonucleotide (CpG) cargo conjugated to a lipophilic tail.
• Figure 1C is a schematic illustrating an exemplary lipid-peptide conjugate including an antigenic peptide cargo conjugated to polar block which is conjugated to a lipophilic tail.
• Figure ID is an exemplary lipid-oligonucleotide conjugate including a diacyl lipid tail conjugated to an oligo-guanine linker which is conjugated to an oligonucleotide cargo.
• Figure IE is an exemplary lipid- peptide conjugate including a diacyl lipid tail conjugated to polyethylene glycol (PEG) linker which is conjugated to a peptide cargo.
• Figure IF is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein labeled free CpG alone (left), fluorescein labeled free CpG mixed with rhodamine labeled bovine serum albumin (BSA) (center), and rhodamine labeled alone (right).
• FRET fluorescence resonance energy transfer
• Figure 1G is a series of plots showing fluorescence resonance energy transfer (FRET) of fluorescein labeled Lipo- CpG alone (left), fluorescein labeled Lipo-CpG mixed with rhodamine labeled bovine serum albumin (BSA) (center), and rhodamine labeled alone (right).
• FRET fluorescence resonance energy transfer
• Figure 2 A is a schematic showing the design of exemplary lymph node targeting amphiphiles: the hydrophobic lipid-like tail (L) is conjugated to the 5'-end of the CpG ODN, CpG sequence has fully phosphorothioated backbone. Three alternative lipids: cholesterol, acyl (C18), and diacyl (C18) are depicted.
• Figure 2B is a line graph showing the results of size exclusion HPLC fluorescein-labeled lipid-conjugated CpGs after incubation with fetal bovine serum (FBS) for 2 hours at 37°C.
• FBS fetal bovine serum
• Figure 2C is two bar graphs showing in vivo LN (inguinal nodes in the left graph, and auxiliary nodes in the right graph) accumulation of CpGs in different fluorescein-labeled formulations (CpG-F, C18-CpG-F, Cho-CpG-F, Lipo-CpG-F, CpG-F in IF A, CpG-F in liposome) 24 hours after subcutaneous injection of 3.3 nmol fluorescein labeled CpGs.
• fluorescein-labeled formulations CpG-F, C18-CpG-F, Cho-CpG-F, Lipo-CpG-F, CpG-F in IF A, CpG-F in liposome
• Figure 2D is line graph showing the kinetics of CpG fluorescence (normalized to the injection dose) in LNs after injection with CpG-F (inguinal nodes (- ⁇ -) and auxiliary nodes (- ⁇ -)) or Lipo-CpG-F (inguinal nodes (- ⁇ -) and auxiliary nodes (- ⁇ -)).
• Figure 2E is a schematic showing a generalized design of a lymph node targeting amphiphile containing an albumin binding domain, a polar spacer and a cargo linked at the end of the spacer.
• Figure 2F is an illustration showing that the length of the polar block controls the balance of three-way equilibrium: intact micelles, albumin bound amhiphiles and cell membrane inserted amphiphiles.
• Figures 2G and H show the effect of varying the length of a poly(ethylene glycol) (PEG) linker of lipo-(PEG)n-FITC conjugates on cell membrane insertion and lymph node targeting, where n is the number of 4- unit oliogethylene glycol repeats in the PEG block.
• Figure 2G is a bar graph showing the quantification of cell insertion of amphiphiles with different PEG length.
• Figure 21 is a line graph showing LNs uptake of amphophilic fluorescein labeled PEG 2000 as a function of lipid molecular weight (i.e., length).
• Figure 2 J is a line graph showing LNs uptake of lipid-oligonucleotide conjugates as a function of oligonucleotide length.
• FIG. 3 A is a schematic showing the generalized construction and characterizations of G-quadruplex stabilized CpG adjuvants.
• the ODN self- assemble into three-dimensional spherical micelles with a CpG corona and a lipid core.
• guanine repeats form G-quadruplex structures via Hoogsteen hydrogen bonds and stabilize the micelle structure.
• FIG. 3B is a schematic (top) and a bar graph (bottom) showing pyrene excimer fluorescent constructs used to assay the stabilities of G-quadruplex micelles in the presence of albumin.
• Figure 3C is a line graph showing the stability profiles of G-quadruplex CpG micelles as measured by size-exclusion chromatography in the presence of fetal bovine serum (FBS).
• FBS fetal bovine serum
• Figure 2D is a bar graph showing the percentage of B220+ cells, F4/80+ cells, and CD1 lc+ cells that were CpG positive as determined by flow cytometry. ***,p ⁇ 0.001; **,p ⁇ 0.01; *,p ⁇ 0.05.
• Figure 4A is a bar graph showing the percentage of peripheral blood lymphocytes isolated from C57B1/6 mice that are H-2K b /SIINFEKL tetramer positive by flow cytometry 6 days after completion of an immunization protocol including s.c. injections on day 0 and day 14, with 10 ⁇ g OVA and in combinations of 1.24 nmol CpG formulations as indicated.
• Figure 4B is a bar graph showing quantifications of INF- ⁇ and TNF- ⁇ positive CD8 T-Cells after 6 hrs. antigen-specific restimulation.
• Figure 4C is a line graph showing the correlation between LN CpG fluorescence and immune response measured by SIINFEKL tetramer staining.
• Figure 4D is a bar graph showing the spleen weight (mg g body weight) of CpG, Lipo-G 2 -CpG, and PBS as an indicator of relative systemic toxicity of the different treatments.
• Figure 4E is a schematic showing the assay design.
• Figure 4F is a dot plot showing the impact of LN targeting (Anti-OV A serum IgG titre 20 days after
• Figure 5A is a bar graph showing the percentage of CD8+ cells isolated from C57B1/6 mice that were HPV-16 E7 49-57 positive by flow cytometry 6 days after completion of an immunization protocol including s.c. injections on day 0 and day 14, with HPV-16 E7 minimal peptide (E7 49-57 ) and in combinations with 1.24 nmol CpG as indicated.
• Figure 5B is a bar graph showing quantifications of INF- ⁇ and TNF- ⁇ positive CD8 T-Cells after 6 hrs. antigen-specific restimulation as a measure of the magnitude of antigen-specific CD8 + T cell responses for minimal peptides (All 1 , Trp2, and HPV-16 E7).
• Figure 5C is a bar graph showing direct lipid conjugate to peptide (lipopeptide) does not elicit potent antigen-specific immune response as measured by the frequency of INF- ⁇ and TNF-a positive CD8 T-Cells after restimulation.
• Figure 5D is a bar graph showing the potency of amphiphilic vaccine as assayed by in vivo cytotoxicity experiment on day 7 after the final immunization of Trp2 peptide vaccines.
• Figure 5E is a Kaplan-Meier curve and Figure 5F is a line graph showing tumor area for mice over time following treatment with subcutaneous (s.c.) TC-1 tumors treated by amphiphilic HPV-16 E7 peptide vaccine, soluble vaccine or no vaccine on day 6, 13 and 19 after challenge with 3x10 5 TC-1 cells.
• Figure 6A is a bar graph showing the %OVA-specific CD8+ T cells following treatment with free CpG and MPLA, Lipo-G 6 -CpG-MPLA, or Lipo-CpG-MPLA.
• Figure 6B is a bar graph showing the % TNF-ot and INF- ⁇ positive CD8+ T cells following treatment with free CpG and MPLA, Lipo-Ge-CpG-MPLA, or Lipo-CpG-MPLA.
• Figure 6C is a line graph showing the %OVA-specific CD8+ T cells following treatment with free CpG and MPLA, Lipo-G 6 -CpG-MPLA, or Lipo-CpG-MPLA over time.
• Figure 6D is a bar graph showing the %OVA-specific CD8+ T cells following treatment with free CpG and MPLA, Lipo-G 6 -CpG-MPLA, or Lipo-CpG-MPLA in the blood, spleen, and lymph node.
• Figure 7A is a schematic representation of an exemplary micelle formed by self-assembly of immunostimulatory conjugates, showing G- quadruplex structure formed by Hoogsteen hydrogen bonding.
• Figure 7B is a graph showing size profile of the self-assembled micelles (diameter (nm)).
• Figure 7C is a line graph showing the results circular dichroism analysis (CD (mdeg)) of G-quadruplex stabilized micelles in lxPBS/20mM K + .
• CD circular dichroism analysis
• Figure 8A is a bar graph showing quantitative accumulation (Radiant Efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 24 hours post injection.
• Figure 8B is a bar graph showing quantitative accumulation (Radiant Efficiency) of various CpG-based micelles in the inguinal lymph nodes (left half of the graph) and axillary lymph nodes (right half of the graph) 72 hours post injection.
• Figure 9 is a schematic representation of a lipid-peptide conjugate.
• Figures 1 OA- 10D are bar graphs showing the results of milliplex analyses of proinflammatory cytokines (INF- ⁇ in Fig. 10A; TNF-a in Fig. 10B; IL6 in Fig. IOC; IL12p40 in Fig. 10D) elicited in peripheral blood of mice immunized with a single dose (6.2 nmol) of CpG formulations, blood samples were collect at different time interval (2 hrs. and 24 hrs.) and analyzed per manufacturer's instructions.
• An immunostimulatory oligonucleotide is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response.
• CG ODNs are short single-stranded synthetic DNA molecules that contain a cytosine nucleotide (C) followed by a guanine nucleotide (G).
• Immune cell is meant a cell of hematopoietic origin and that plays a role in the immune response.
• Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and
• T cell refers to a CD4+ T cell or a CD8+ T cell.
• T cell includes TH1 cells, TH2 cells and TH17 cells.
• T cell cytoxicity includes any immune response that is mediated by CD8+ T cell activation.
• exemplary immune responses include cytokine production, CD8+ T cell proliferation, granzyme or perforin production, and clearance of an infectious agent.
• pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
• the terms "subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions.
• the subject can be a vertebrate, for example, a mammal.
• the subject can be a human.
• the subjects can be symptomatic or asymptomatic.
• the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
• a subject can include a control subject or a test subject.
• polypeptide refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation).
• an effective amount or “therapeutically effective amount” means a dosage sufficient to provide treatment for a disorder, disease, or condition being treated, to induce or enhance an immune response, or to otherwise provide a desired pharmacologic and/or physiologic effect.
• the precise dosage will vary according to a variety of factors such as subject- dependent variables (e.g., age, immune system health, etc.), the disease, the disease stage, and the treatment being effected.
• mice and rats refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.
• oligonucleotide or a “polynucleotide” are synthetic or isolated nucleic acid polymers including a plurality of nucleotide subunits.
• lipid conjugates that control targeting of the conjugate to the lymph nodes have been discovered.
• amphiphilic lipid conjugates exist in a 3-way equilibrium depicted in Figure 2F. In pure water certain lipid conjugates form micelles, but in the presence of serum and cells these amphiphiles equilibrate between binding to albumin and insertion of their lipophilic tails into cell membranes.
• lipid conjugates that effectively target the lymph nodes typically include three domains: a lipophilic domain that binds to albumin, a polar block domain, and a cargo such as a molecular adjuvant or immunostimulatory compound (such as oligonucleotide) or antigenic peptide.
• a cargo such as a molecular adjuvant or immunostimulatory compound (such as oligonucleotide) or antigenic peptide.
• the length and compositions of the polar block can be tailored to push the equilibrium toward albumin binding, stable micelle formation, or cell insertion.
• the design guidelines and compositions disclosed below can be used to induce or enhance robust immune responses with low systemic toxicity because the
• immunostimulating compounds are localized to the lymph node (i.e., lymph node-targeting conjugates) or the tissue at the local site of administration (i.e., micelle-stabilizing conjugate).
• the effectiveness of any particular lipid conjugate to target the lymph nodes can be assayed based on the ability of albumin to disrupt micelles formed by a plurality of the conjugates in aqueous solution. For example, if an albumin containing agent such fetal bovine serum can disrupt 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 percent or more of the micelles formed in aqueous solution, the conjugate can be selected to target the lymph nodes. However, if 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 percent or more of the micelles formed in aqueous solution remain intact in the presence of albumin, the conjugate can be selected as a micelle-stabilizing conjugate.
• Lipid conjugates such as lipid-oligonucleotide and lipid-peptide conjugates for use in immunogenic compositions are disclosed. Lymph node- targeting conjugates can be trafficked from the site of administration through the lymph to the lymph node where they accumulate and activate immune cells. It is believed that efficient lymph node accumulation of lipid conjugates dependents on the ability of the amphiphile to partition from micelles into a serum protein-bound state.
• Lymph node-targeting conjugates typically include three domains: a highly lipophilic, albumin-binding domain (e.g., an albumin-binding lipid), a cargo such as a molecular adjuvant or a peptide antigen, and a polar block linker, which promotes solubility of the conjugate and reduces the ability of the lipid to insert into cellular plasma membranes.
• a highly lipophilic albumin-binding domain
• a cargo such as a molecular adjuvant or a peptide antigen
• a polar block linker which promotes solubility of the conjugate and reduces the ability of the lipid to insert into cellular plasma membranes.
• the general structure of the conjugate is L-P-C, where "L” is an albumin-binding lipid, "P” is a polar block, and "C” is a cargo such as a molecular adjuvant or a polypeptide.
• the cargo itself can also serve as the polar block domain, and a separate polar block domain is not required. Therefore, in some embodiments the conjugate is only two domains: an albumin-binding lipid and a cargo.
• lipid- oligonucleotide conjugates can include an immunostimulatory
• Lipid-peptide conjugates can include an antigenic peptide which is conjugated directly to a lipid, or is linked to a linker which is conjugated to a lipid.
• Lipid-conjugated peptides are well known (lipopeptides) as vaccine agents, but in our hands lipid conjugated directly to peptide does NOT exhibit lymph node targeting, because the conjugates are not soluble enough to partition preferentially onto albumin in the presence of cells; they instead insert heavily into cell membranes and thus remain trapped at an injection site.
• Lipopeptides Antigenic peptides directly conjugated to lipids (lipopeptides) have been extensively studied as a modality for enhancing vaccine efficacy
• peptide antigens linked directly to albumin-binding diacyl tails elicit barely detectable immune responses in vivo while lipo-PEG-peptides elicit robust T-cell responses (Figure 5C.
• the diacyl tails promoting lipid binding and lymph node targeting here show no direct adjuvant activity on their own, unlike lipopeptides such as pam3cys-peptide conjugates, that are known to have adjuvant activity through binding to TLR-2 and other immunostimulatory receptors.
• the lipid conjugates disclosed herein typically include a hydrophobic lipid.
• the lipid can be linear, branched, or cyclic.
• the lipid is preferably at least 17 to 18 carbons in length, but may be shorter if it shows good albumin binding and adequate targeting to the lymph nodes.
• Lymph node-targeting conjugates include lipid-oligonucleotide conjugates and lipid-peptide conjugates that can be trafficked from the site of delivery through the lymph to the lymph node.
• the activity relies, in-part, on the ability of the conjugate to associate with albumin in the blood of the subject. Therefore, lymph node-targeted conjugates typically include a lipid that can bind to albumin under physiological conditions.
• Lipids suitable for targeting the lymph node can be selected based on the ability of the lipid or a lipid conjugate including the lipid to bind to albumin. Suitable methods for testing the ability of the lipid or lipid conjugate to bind to albumin are known in the art and discussed in the Examples below.
• a plurality of lipid conjugates is allowed to spontaneously form micelles in aqueous solution.
• the micelles are incubated with albumin, or a solution including albumin such Fetal Bovine Serum (FBS).
• Samples can be analyzed, for example, by ELISA, size exclusion chromatography or other methods to determine if binding has occurred, as illustrated in Figure 2B.
• Lipid conjugates can be selected as lymph node-targeting conjugates if in the presence of albumin, or a solution including albumin such Fetal Bovine Serum (FBS), the micelles dissociate and the lipid conjugates bind to albumin as discussed above.
• Examples of preferred lipids for use in lymph node targeting lipid conjugates include, but are not limited to fatty acids with aliphatic tails of 8- 30 carbons including, but not limited to, linear and unsaturatedsaturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids derivatives, such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, Cholesterol, Cholesterol derivatives, and steroid acids such as bile acids; Lipid A or combinations thereof.
• the lipid is a diacyl lipid or two-tailed lipid.
• the tails in the diacyl lipid contain from about 8 to about 30 carbons and can be saturated, unsaturated, or combinations thereof.
• the tails can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof.
• the diacyl lipids are phosphate lipids, glycolipids, sphingolipids, or combinations thereof.
• lymph node-targeting conjugates include a lipid that is 8 or more carbon units in length. It is believed that increasing the number of lipid units can reduce insertion of the lipid into plasma membrane of cells, allowing the lipid conjugate to remain free to bind albumin and traffic to the lymph node.
• the lipid can be a diacyl lipid composed of two C18 hydrocarbon tails.
• the lipid for use in preparing lymph node targeting lipid conjugates is not a single chain hydrocarbon (e.g., C18), or cholesterol. Cholesterol conjugation has been explored to enhance the immunomodulation of molecular adjuvants such as CpG and
• the cargo of the conjugates disclosed herein is a typically a molecular adjuvant such as an immunostimulatory oligonucleotide, or a peptide antigen.
• the cargo can also be other oligonucleotides, peptides, Toll-like receptor agonists or other immunomodulatory
• Lipid-oligonucleotide conjugates are disclosed.
• the oligonucleotide conjugates described herein typically contain an immunostimulatory oligonucleotide.
• the immunostimulatory oligonucleotide can serve as a ligand for pattern recognition receptors (PRRs).
• PRRs pattern recognition receptors
• Examples of PRRs include the Toll-like family of signaling molecules that play a role in the initiation of innate immune responses and also influence the later and more antigen specific adaptive immune responses. Therefore, the oligonucleotide can serve as a ligand for a Toll-like family signaling molecule, such as Toll-Like Receptor 9 (TLR9).
• TLR9 Toll-Like Receptor 9
• oligonucleotide can include one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs.
• CG cytosine-guanine
• the 'p' refers to the phosphodiester backbone of DNA, as discussed in more detail below, some oligonucleotides including CG can have a modified backbone, for example a phosphorothioate (PS) backbone.
• PS phosphorothioate
• an immunostimulatory oligonucleotide can contain more than one CG dinucleotide, arranged either contiguously or separated by intervening nucleotides).
• the CpG motif(s) can be in the interior of the oligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9 with variations in the number and location of CG
• CG ODNs are classified based on their sequence, secondary structures, and effect on human peripheral blood mononuclear cells (PBMCs).
• the five classes are Class A (Type D), Class B (Type K), Class C, Class P, and Class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3): 195-204 (2009), incorporated herein by reference).
• CG ODNs can stimulate the production of Type I interferons (e.g., IFNa) and induce the maturation of dendritic cells (DCs).
• Type IFNa Type IFNa
• DCs dendritic cells
• Some classes of ODNs are also strong activators of natural killer (NK) cells through indirect cytokine signaling.
• Some classes are strong stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20): 10833-7 (1997); Dalpke, AH, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).
• PRR Toll-like receptors include TLR3, and TLR7 which may recognize double-stranded RNA, single-stranded and short double-stranded RNAs, respectively, and retinoic acid-inducible gene I (RIG-I)-like receptors, namely RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are best known as RNA-sensing receptors in the cytosol. Therefore, in some embodiments, the oligonucleotide contains a functional ligand for TLR3, TLR7, or RIG-I-like receptors, or combinations thereof. Examples of immunostimulatory oligonucleotides, and methods of making them are known in the art, see for example, Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(l):87-93 (2011), incorporated herein by reference.
• the oligonucleotide cargo includes two or more immunostimulatory sequences.
• the oligonucleotide can be between 2-100 nucleotide bases in length, including for example, 5 nucleotide bases in length, 10 nucleotide bases in length, IS nucleotide bases in length, 20 nucleotide bases in length, 25 nucleotide bases in length, 30 nucleotide bases in length, 35 nucleotide bases in length, 40 nucleotide bases in length, 45 nucleotide bases in length, 50 nucleotide bases in length, 60 nucleotide bases in length, 70 nucleotide bases in length, 80 nucleotide bases in length, 90 nucleotide bases in length, 95 nucleotide bases in length, 98 nucleotide bases in length, 100 nucleotide bases in length or more.
• the 3' end or the 5' end of the oligonucleotides can be conjugated to the polar block or the lipid.
• the 5' end of the oligonucleotide is linked to the polar block or the lipid.
• the oligonucleotides can be DNA or RNA nucleotides which typically include a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxy 1 function of the sugar moiety.
• the principal naturally-occurring nucleotides comprise uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases, and ribose or deoxyribose sugar linked by phosphodiester bonds.
• the oligonucleotides are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target receptor, relative to a DNA or RNA counterpart.
• the chemical modifications include chemical
• nucleobases modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof.
• 'modified nucleotide or "chemically modified nucleotide” defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents.
• the charge of the modified nucleotide is reduced compared to DNA or RNA oligonucleotides of the same nucleobase sequence.
• the oligonucleotide can have low negative charge, no charge, or positive charge.
• nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the
• oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
• a standard polynucleotide e.g., single-stranded RNA or single-stranded DNA.
• the analogs have a substantially uncharged, phosphorus containing backbone.
• the principal naturally-occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases.
• oligonucleotides can include chemical modifications to their nucleobase constituents. Chemical modifications of heterocyclic bases or heterocyclic base analogs may be effective to increase the binding affinity or stability in binding a target sequence. Chemically-modified heterocyclic bases include, but are not limited to, inosine, 5-(l-propynyl) uracil (pU), 5-(l-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine,
• pseudoisocytosine 5 and 2-ammo-5-(2'-deoxy-.beta.-D- ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.
• Cyclic dinucleotides known to trigger cytosolic danger sensors such as STING could be used.
• Oligonucleotides can also contain nucleotides with modified sugar moieties or sugar moiety analogs.
• Sugar moiety modifications include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl (2'-OAE), 2'-O- methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and 2'-O-Wmethy ⁇ acetamido) (2'- OMA).
• 2'-O-aminoethyl sugar moiety substitutions are especially preferred because they are protonated at neutral pH and thus suppress the charge repulsion between the TFO and the target duplex.
• This modification stabilizes the C3'-endo conformation of the ribose or dexyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.
• the oligonucleotide is a morpholino oligonucleotide.
• Morpholino oligonucleotides are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, which are linked together by phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer.
• the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine.
• Important properties of the morpholino-based subunits typically include: the ability to be linked in a oligomeric form by stable, uncharged backbone linkages; the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil or inosine) such that the polymer formed can hybridize with a complementary-base target nucleic acid, including target RNA, with high T m , even with oligomers as short as 10-14 bases; the ability of the oligomer to be actively transported into mammalian cells; and the ability of an oligomer:RNA heteroduplex to resist RNAse degradation.
• a nucleotide base e.g. adenine, cytosine, guanine, thymidine, uracil or inosine
• oligonucleotides employ morpholino-based subunits bearing base-pairing moieties, joined by uncharged linkages, as described above.
• Oligonucleotides connected by an internucleotide bond that refers to a chemical linkage between two nucleoside moieties may increase the binding affinity or stability oligonucleotides, or reduce the suseptability of oligonucleotides nuclease digestion.
• Cationic modifications including, but not limited to, diethyl-ethylenediamide (DEED) or dimethyl- aminopropylamine (DMAP) may be especially useful due to decrease electrostatic repulsion between the oligonucleotide and a target.
• DEED diethyl-ethylenediamide
• DMAP dimethyl- aminopropylamine
• Modifications of the phosphate backbone may also include the substitution of a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate internucleoside linkage in place of the phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.
• modified nucleotides with reduced charge examples include modified internucleotide linkages such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic Chem., 52:4202, ( 1987)), and uncharged morpholino-based polymers having achiral intersubunit linkages (see, e.g., U.S. Pat. No. 5;034,S06), as discussed above.
• Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles.
• the oligonucleotides are composed of locked nucleic acids.
• Locked nucleic acids are modified RNA nucleotides (see, for example, Braasch, et al., Chem. Biol., 8(1): 1-7 (2001)).
• LNAs form hybrids with DNA which are more stable than DNA DNA hybrids, a property similar to that of peptide nucleic acid (PNA)/DNA hybrids.
• LNA can be used just as PNA molecules would be.
• LNA binding efficiency can be increased in some embodiments by adding positive charges to it.
• Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs.
• the oligonucleotides are composed of peptide nucleic acids.
• Peptide nucleic acids are synthetic DNA mimics in which the phosphate backbone of the oligonucleotide is replaced in its entirety by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are typically replaced by peptide bonds.
• the various heterocyclic bases are linked to the backbone by methylene carbonyl bonds.
• PNAs maintain spacing of heterocyclic bases that is similar to conventional DNA oligonucleotides, but are achiral and neutrally charged molecules.
• Peptide nucleic acids are comprised of peptide nucleic acid monomers.
• oligonucleotides such as PNA may be peptide linkages, or alternatively, they may be non-peptide peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O- linkers), amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like.
• O- linkers amino spacers
• amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like.
• Methods for the chemical assembly of PNAs are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.
• Oligonucleotides optionally include one or more terminal residues or modifications at either or both termini to increase stability, and/or affinity of the oligonucleotide for its target.
• Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful.
• Oligonucleotides may further be modified to be end capped to prevent degradation using a propylamine group. Procedures for 3' or 5' capping oligonucleotides are well known in the art.
• the oligonucleotide is single-stranded DNA, single-stranded RNA, or double-stranded RNA.
• Lipid-peptide conjugates are disclosed.
• the peptide conjugates described herein typically include an antigenic protein or polypeptide.
• the peptide can be 2-100 amino acids (aa), including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be > 100 amino acids.
• a protein/peptide can be linear, branched or cyclic.
• the peptide can include D amino acids, L amino acids, or a combination thereof.
• the peptide or protein can be conjugated to the polar block or lipid at the N-terminus or the C-terminus of the peptide or protein.
• the protein or polypeptide can be any protein or peptide that can induce or increase the ability of the immune system to develop antibodies and T-cell responses to the protein or peptide.
• Examples of specific peptide and protein antigens that can be used in the lipid-peptide conjugates disclosed herein are discussed in more detail below with respect to preferred antigens that can be used in vaccine formulations.
• Lipid-protein-based micelles can be formed in an aqueous solution by self-assembly of conjugates containing a peptide antigen linked (attached) to a polyethylene glycol (PEG) moiety or derivative or analog thereof, which is linked to hydrophobic lipid.
• PEG polyethylene glycol
• the cargo can include therapeutic, prophylactic or diagnostic agents.
• chemotherapy drugs are of interest for targeting tumors as albumin is known to accumulate in tumors by the EPR effect and also by fast metabolism in tumors.
• the lipid conjugates disclosed herein include a detection label, for example, a fluorophore such as fluorescein or rhodamine, Alexa Fluor dyes, DyLight Fluor dyes, Quasar and Cal Fluor dyes, cyanine dyes (Cy3, Cy5, Cy5.5, Cy7) or other fluorescent dyes.
• a fluorophore such as fluorescein or rhodamine
• Alexa Fluor dyes Alexa Fluor dyes
• DyLight Fluor dyes DyLight Fluor dyes
• Quasar and Cal Fluor dyes Quasar and Cal Fluor dyes
• cyanine dyes Cy3, Cy5, Cy5.5, Cy7 or other fluorescent dyes.
• the label can be the cargo, or can be in addition to a cargo.
• a polar block linker can be included between the cargo and the lipid to increase solubility of the conjugate.
• the polar block reduces or prevents the ability of the lipid to insert into the plasma membrane of cells, such as cells in the tissue adjacent to the injection site.
• the polar block can also reduce or prevent the ability of cargo, such as synthetic oligonucleotides containing a PS backbone, from non-specifically associating with extracellular matrix proteins at the site of administration.
• the polar block increases the solubility of the conjugate without preventing its ability to bind to albumin. It is believed that this combination of characteristics allows the conjugate to bind to albumin present in the serum or interstitial fluid, and remain in circulation until the albumin is trafficked to, and retained in a lymph node.
• the length and composition of the polar block can be adjusted based on the lipid and cargo selected.
• the oligonucleotide itself may be polar enough to insure solubility of the conjugate, for example, oligonucleotides that are 10, 15, 20 or more nucleotides in length. Therefore, in some embodiments, no additional polar block linker is required.
• some lipidated peptides can be essentially insoluble. In these cases, it can be desirable to include a polar block that mimics the effect of a polar oligonucleotide.
• a polar block can be used as part of any of lipid conjugates described herein, for example, lipid-oligonucleotide conjugates and lipid-peptide conjugates, which reduce cell membrane insertion/preferential portioning ont albumin.
• Suitable polar blocks include, but are not limited to,
• oligonucleotides such as those discussed above, a hydrophilic polymer including but not limited to polyethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a string of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; polysaccharides, including but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da), or combinations thereof.
• a hydrophilic polymer including but not limited to polyethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a string of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,
• the hydrophobic lipid and the linker/cargo are covalently linked.
• the covalent bond may be a non-cleavable linkage or a cleavable linkage.
• the non-cleavable linkage can include an amide bond or phosphate bond
• the cleavable linkage can include a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme- cleavable linkage.
• the polar block is one or more ethylene glycol (EG) units, more preferably 2 or more EG units (i.e., polyethylene glycol (PEG)).
• EG ethylene glycol
• PEG polyethylene glycol
• a peptide conjugate includes a protein or peptide (e.g., peptide antigen) and a hydrophobic lipid linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.
• protein conjugates described herein contain protein antigen linked to PEG which is in turn linked to a hydrophobic lipid, or lipid-Gn-ON conjugates, either covalently or via formation of protein- oligo conjugates that hybridize to oligo micelles.
• a polar block can have between about 1 and about 100, between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 EG units. In some embodiments, the polar block has between about 45 and 55 EG, units. For example, in one preferred embodiment, the polar block has 48 EG units.
• the polar block is an oligonucleotide.
• the polar block liner can be have any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar).
• the polar block linker includes one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.
• the polar block linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.
• the linker is one or more guanines, for example between 1-10 guanines. It has been discovered that altering the number of guanines between a cargo such as a CpG oligonucleotide, and a lipid tail controls micelle stability in the presence of serum proteins. Therefore, the number of guanines in the linker can be selected based on the desired affinity of the conjugate for serum proteins such as albumin. As illustrated in the Examples below, when the cargo is a CpG immunostimulatory
• the number of guanines affects the ability of micelles formed in aqueous solution to dissociate in the presence of serum: 20% of the non-stabilized micelles (lipo-GoTio-CG) were intact, while the remaining 80% were disrupted and bonded with FBS components. In the presence of guanines, the percentage of intact micelles increased from 36% (lipo-G 2 T 8 -CG) to 73% (lipo-G 4 T 6 -CG), and finally reached 90% (lipo-GeT-i-CG). Increasing the number of guanines to eight (lipo-G 8 T 2 -CG) and ten (lipo-G 10 T 0 -CG) did not further enhance micelle stability.
• the linker in a lymph node- targeting conjugate can include 0, 1, or 2 guanines.
• linkers that include 3 or more consecutive guanines can be used to form micelle-stabilizing conjugates with properties that are well suited for local applications at or near the site of administration.
• Micelle-stabilizing conjugates include conjugates such as lipid- oligonucleotide conjugates and lipid-peptide conjugates that accumulate in the tissue surrounding the site of delivery.
• the conjugates typically do not bind to albumin.
• the lipid used to prepare a micelle- stabilizing lipid conjugate is the same as the lipid used in the lymph node targeting lipid conjugates discussed above, and the ability to resist binding to albumin is controlled by the molecular or biochemical properties of the cargo, the linker, or a combination thereof.
• lipids that would not be effective for use in lymph node targeted conjugates are useful in micelle-stabilizing conjugates because the micelle-stabilizing conjugates do not necessarily have to bind to albumin.
• Micelle-stabilizing conjugates can be selected based on the ability to spontaneously form micelles in aqueous solution that are not disrupted by serum components such as albumin, as discussed above. Suitable methods for testing the ability of the lipid or lipid conjugates to bind to albumin are known in the art and discussed in the Examples below. For example, in one embodiment, a plurality of lipid conjugates is allowed to spontaneously form micelles in aqueous solution. The micelles are incubated with albumin, or a solution including albumin such Fetal Bovine Serum (FBS). Samples can be analyzed, for example, by ELIS A, size separation chromatography or other methods to determine if binding has occurred.
• albumin Fetal Bovine Serum
• Lipid conjugates can be selected as micelle stabilized conjugates if in the presence of albumin, or a solution including albumin such Fetal Bovine Serum (FBS), the micelles remain intact and the lipid conjugates do not bind to albumin.
• preferred lipids for use in micelle-stabilizing lipid conjugates include, but are not limited to fatty acids with aliphatic tails of 8- 30 carbons including, but not limited to, linear and unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids derivatives, such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, Cholesterol, Cholesterol derivatives, and steroid acids such as bile acids; Lipid A or combinations thereof.
• the lipid is a diacyl lipid or two-tailed lipid
• the tails in the diacyl lipid contain from about 8 to about 30 carbons and can be saturated, unsaturated, or combinations thereof.
• the tails can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof.
• the diacyl lipids are phosphate lipids, glycolipids, sphingolipids, or combinations thereof.
• the stability of micelles in the presence of albumin is affected by the linker.
• an oligonucleotide such as an immunostimulatory oligonucleotide
• the lipid can be linked by three or more intervening guanine nucleotides.
• the nucleotides can be positioned at the 5' end of the oligonucleotide.
• Guanine- rich DNA sequences can form quadruplex structures via hydrogen bonding, where the oligoguanines molecularly "glue” together four individual guanine-rich DNA sequences.
• the immunostimulatory oligonucleotide conjugates can self-assemble into "G-quadruplexes," which then assemble to form micelles having a hydrophobic lipid core and a nucleic acid corona.
• kinetic stability of a micelle can be controlled by altering the number of guanine nucleotides that link the hydrophobic lipid to the immunostimulatory oligonucleotide.
• the immunostimulatory oligonucleotide and the hydrophobic lipid are linked by a single guanine at the 5' end of the oligonucleotide, while in other embodiments the immunostimulatory oligonucleotide and the hydrophobic lipid are linked by two guanines at the 5' end of the
• the cargo of micelle-stabilizing conjugates can includes any of the cargo discussed above with respect to lymph node targeted conjugates, as well as small molecules, oligonucleotide, or peptide therapeutics (i.e., any cargo that would one of skill in the art would select for accumulation at a site of local delivery).
• Micelle-stabilizing conjugates can form micelles spontaneously in aqueous solution by self-assembly.
• the micelle has a hydrophobic lipid core and a hydrophilic surface. Formation of a micelle in an aqueous
• a cation such as potassium (K + )
• the cation connects two G- quadruplexes and minimizes the electrostatic interactions between the immunostimulatory oligonucleotides.
• Guanine-rich oligonucleotide sequences can fold into various types of structures (e.g., intramolecular, intermolecular, parallel, and antiparallel) (Davis, J. T. Angew. Chem. Int. Ed. Engl. 43, 668-698 (2004)).
• the lipid-oligonucleotide conjugates can be suspended in pure water to permit assembly of the micelle, and then potassium-containing buffer can be added to stabilize the G-quadruplexes.
• micelles of a homogeneous micelle population are substantially uniform in size.
• micelles of a homogeneous micelle population are substantially uniform in size.
• homogeneous population each are similarly composed of the same type of lipid-oligonucleotide conjugate (e.g., a L-5'-G n -CG-ODN-3' conjugate).
• the stability of the micelle can be controlled by altering the number of guanine nucleotides in the polar block.
• the conjugate includes one or more guanine nucleotides at the 5' end of the oligonucleotide and hydrophobic lipid linked to the most 5' guanine.
• Micelle "stability" as used herein refers to resistance to disassembly or changes in micelle size in the presence of serum, albumins, or other proteins or lipids, and/or resistance of the micelles to changes in size or composition in the presence of cells.
• the diameter of a micelle as described herein can be from about 3 nm to about 100 nm.
• the diameter of a micelle is 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52
• compositions including lipid conjugates are provided.
• Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
• compositions are administered
• compositions systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the compositions to targeted cells.
• Other possible routes include trans-dermal or oral.
• the compositions are administered locally, for example by injection directly into a site to be treated.
• the compositions are injected or otherwise administered directly to one or more tumors.
• local injection causes an increased localized concentration of the compositions which is greater than that which can be achieved by systemic administration.
• the compositions are delivered locally to the appropriate cells by using a catheter or syringe.
• Other means of delivering such compositions locally to cells include using infusion pumps (for example, from Alza Corporation, Palo Alto, Calif.) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J. G. Lloyd- Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987), which can effect a sustained release of the nanolipogels to the immediate area of the implant.
• dosage levels for treatment of various conditions in various patients will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing.
• the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally dosage levels of 0.001 to 10 mg/kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.
• the lipid conjugates are administered in an aqueous solution, by parenteral injection.
• the composition includes albumin, or other serum proteins.
• the formulation can be in the form of a suspension or emulsion.
• pharmaceutical compositions are provided including an effective amount of the conjugate and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
• compositions can include diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).
• buffered saline of various buffer content e.g., Tris-HCl, acetate, phosphate
• pH and ionic strength e.g., Tris-HCl, acetate, phosphate
• additives e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80
• non-aqueous solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• the formulations may be lyophilized and redissolved/resuspended immediately before use.
• the formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the
• Topical and Mucosal Administration The lipid conjugates can be applied topically. Topical administration can include application to the lungs (pulmonary), nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In some cases, the conjugates may be transcytosed on albumin across mucosal barriers
• Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.
• nebulizers metered dose inhalers
• powder inhalers all of which are familiar to those skilled in the art.
• Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powder inhaler (Fisons Corp., Bedford, Mass.).
• Ultravent® nebulizer Maligninckrodt Inc., St. Louis, Mo.
• Acorn® II nebulizer Marquest Medical Products, Englewood, Colo.
• Ventolin® metered dose inhaler Gaxo Inc., Research Triangle Park, N.C.
• Spinhaler® powder inhaler Fesons Corp., Bedford, Mass.
• Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator. Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules, or lozenges.
• Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations can include penetration enhancers.
• immunogenic compositions disclosed herein can be used in immunogenic compositions or as components in vaccines.
• immunogenic compositions disclosed herein include an adjuvant, an antigen, or a combination thereof.
• the combination of an adjuvant and an antigen can be referred to as a vaccine.
• the adjuvant and antigen can be administered in separate pharmaceutical compositions, or they can be administered together in the same
• the adjuvant when administered in combination, can be a lipid conjugate, the antigen can be a lipid conjugate, or the adjuvant and the antigen can both be lipid conjugates.
• An immunogenic composition can include a lipid conjugate that is an adjuvant such as an immunostimulatory oligonucleotide-lipid conjugate, administered alone, or in combination with an antigen.
• Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, or combinations thereof.
• the antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components or molecular components thereof.
• Suitable antigens are known in the art and are available from commercial government and scientific sources.
• the antigens are whole inactivated or attenuated organisms. These organisms may be infectious organisms, such as viruses, parasites and bacteria. These organisms may also be tumor cells.
• the antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources.
• the antigens can be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system.
• the antigens can be DNA encoding all or part of an antigenic protein.
• the DNA may be in the form of vector DNA such as plasmid DNA.
• Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids. Exemplary antigens are provided below, a. Viral antigens
• a viral antigen can be isolated from any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue
• Lipothrixviridae Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g.
• Retroviridae e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2
• Rhabdoviridae for example, rabies virus, measles virus, respiratory syncytial virus, etc.
• Togaviridae for example, rubella virus, dengue virus, etc.
• Totiviridae Suitable viral antigens also include all or part of
• Dengue protein M Dengue protein E, Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.
• Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, e.g., herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever,and lymphocytic
• a hepatitis virus for example, hepatitis A virus (HAV), hepatitis B virus (HBV),
• Bacterial antigens can originate from any bacteria including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,
• Leptspirosis Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,
• Phodospirillum Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.
• Parasite antigens can be obtained from parasites such as, but not limited to, an antigen derived rom Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.
• parasites such as, but not limited to, an antigen derived rom Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Ricke
• Sporozoan antigens include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein.
• the antigen can be an allergen or environmental antigen, such as, but not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb,
• birch Betula alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar ⁇ Cryptomeriaand Juniperus), Plane tree (Platanus), the order of Poales including e.g., grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including i.a. herbs of the genera Ambrosia, Artemisia, and Parietaria.
• allergen antigens that may be used include allergens from house dust mites of the genus
• Dermatophagoides and Euroglyphus storage mite e.g Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps
• allergen antigens include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium.
• a cancer antigen is an antigen that is typically expressed
• the cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell.
• the cancer antigen can be MART- 1 /Melan- A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3-zeta chain, and CD20.
• the cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE- A 10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE- Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-1, GAGE-2, GAGE-3, GAGE- 4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE- 1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, a-fetoprotein, E-cadherin, a
• An immunogenic composition can include a lipid conjugate that is an antigen such as an antigenic polypeptide-lipid conjugate, administered alone, or in combination with an adjuvant.
• the adjuvant may be without limitation alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified f om the bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.);
• alum e.g., aluminum hydroxide, aluminum phosphate
• saponins purified f om the bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.);
• PCPP polymer poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS
• Adjuvants may be TLR ligands, such as those discussed above.
• Adjuvants that act through TLR3 include without limitation double-stranded RNA.
• Adjuvants that act through TLR4 include without limitation derivatives of lipopoly saccharides such as monophosphoryl lipid A (MPL A; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma S A, Meyrin, Switzerland).
• Adjuvants that act through TLR5 include without limitation flagellin.
• Adjuvants that act through TLR7 and/or TLR8 include single- stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)).
• Adjuvants acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN.
• Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.
• the adjuvant can also be oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP- ribosylating toxins and detoxified derivatives; alum; BCG; mineral- containing compositions (e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and/or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g.,
• lysolecithin pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-l, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-.gamma.), macrophage colony stimulating factor, and tumor necrosis factor.
• immunomodulators such as cytokines, interleukins (e.g., IL-l, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-.gamma.), macrophage colony stimulating factor, and tumor necrosis factor.
• the conjugates are administered in
• the agents can be administered in the same pharmaceutical composition as the conjugates or the conjugates and the additional therapeutic agent can be administered in separate pharmaceutical compositions.
• the conjugates are administered in the same pharmaceutical composition as the conjugates or the conjugates and the additional therapeutic agent can be administered in separate pharmaceutical compositions.
• the conjugates are administered in the same pharmaceutical composition as the conjugates or the conjugates and the additional therapeutic agent can be administered in separate pharmaceutical compositions.
• the conjugates are administered in the same pharmaceutical composition as the conjugates or the conjugates and the additional therapeutic agent can be administered in separate pharmaceutical compositions.
• the conjugates are administered in
• the conjugates can be co-administered with a chemotherapeutic drug; or if the disease or condition is a bacterial infection, the conjugates can be co-administered with an antibiotic.
• lymph nodes are oval-shaped organs of the immune system, distributed widely throughout the body including the armpit and stomach and linked by lymphatic vessels. Lymph nodes are bastions of B, T, and other immune cells. Lymph nodes act as filters or traps for foreign particles and are important in the proper functioning of the immune system. They are packed tightly with the white blood cells called lymphocytes and macrophages.
• Lymph node targeting conjugates are typically transported from the injection site to secondary organs of the lymphatic system (e.g., lymph nodes), where interact with immune cells. It is believed that albumin-binding of the conjugates prevents the conjugates from rapidly flushing into the bloodstream and re-targets them to lymphatics and draining lymph nodes, where they are filtered, accumulate, and present their immunostimulatory oligonucleotide, antigenic peptide, or other cargo to immune cells.
• lymphatic system e.g., lymph nodes
• albumin-binding lipids can be conjugated to, for example, an immunostimulatory oligonucleotide or an antigenic peptide which increases the immunostimulatory effect of the oligonucleotide or the antigenic peptide compared to administering non-conjugated oligonucleotide or antigenic peptide.
• conjugation of the oligonucleotide or an antigenic peptide increases the immunostimulatory effect of the oligonucleotide or the antigenic peptide compared to administering non-conjugated oligonucleotide or antigenic peptide.
• immunostimulatory oligonucleotide or peptide antigen to the an albumin- binding lipid increases accumulation of the cargo 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold compare to unconjugated cargo.
• Micelle-stabilizing conjugates can be used to increase delivery and accumulation of the cargo to the tissue at or near a site of administration.
• Micelle-stabilizing conjugates are believed to be resistant to disruption by serum proteins such as albumin. T erefore, they can accumulate at the site of injection, for example, by binding to extracellular matrix proteins, or inserting into the cell membranes of local cells.
• Micelle-stabilizing conjugates can be used to increase local accumulation of immunostimulatory oligonucleotides, antigenic peptides, small molecules, and other targets at the site of administration.
• conjugation of the immunostimulatory oligonucleotide or peptide antigen increases local accumulation of the cargo 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold compare to unconjugated cargo.
• Lipid conjugates including an immunostimulatory oligonucleotide or antigenic peptide cargo can be administered in an effective amount to induce, increase or enhance an immune response.
• the "immune response” refers to responses that induce, increase, induce, or perpetuate the activation or efficiency of innate or adaptive immunity.
• albumin-binding lipid conjugates of polypeptide antigens administered in the absence of other adjuvants may be used to promote tolerance rather than immunity, e.g., to a allergen or autoimmune antigen.
• the conjugates can be delivered
• parenterally by subcutaneous, intradermal, or intramuscular injection
• the lymphatics can filter albumin-bound conjugates. Therefore, in some embodiments parenteral administration does not result in systemic distribution as the conjugates may be preferentially filtered by the closest lymph node(s). This tendency also reduces systemic toxicity such as swelling of the spleen.
• the conjugates are administered at a site adjacent to or leading to one or more lymph nodes which are close to the site in need of an immune response (i.e., close to a tumor or site of infection).
• the conjugates are administered in multiple doses at various locations throughout the body.
• the conjugates, particularly micelle-stabilizing conjugates can also be administered directly to a site in need of an immune response (e.g., a tumor or site of infection).
• the immune response can be induced, increased, or enhanced by the lipid conjugate compared to a control, for example an immune response in a subject induced, increased, or enhanced by the cargo alone, or the cargo delivered using an alternative delivery strategy such as liposomes.
• lipid conjugates reduce inactivation and/or prolong activation of T cells (i.e., increase antigen-specific proliferation of T cells, enhance cytokine production by T cells, stimulate differentiation ad effector functions of T cells and or promote T cell survival) or overcome T cell exhaustion and/or anergy.
• the lipid conjugates can be used, for example, to induce an immune response, when administering the cargo alone, or the cargo in combination with an alternative delivery system, is ineffectual.
• the lipid conjugates can also be used to enhance or improve the immune response compared to administering cargo alone.
• the lipid conjugates may reduce the dosage required to induce, increase, or enhance an immune response; or reduce the time needed for the immune system to respond following administration.
• Lipid conjugates may be administered as part of prophylactic vaccines or immunogenic compositions which confer resistance in a subject to subsequent exposure to infectious agents, or as part of therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a viral antigen in a subject infected with a virus or with cancer.
• a pre-existing antigen such as a viral antigen in a subject infected with a virus or with cancer.
• a prophylactic or therapeutic immune response may vary according to the disease or condition to be treated, or according to principles well known in the art.
• an immune response against an infectious agent may completely prevent colonization and replication of an infectious agent, affecting "sterile immunity" and the absence of any disease symptoms.
• a vaccine against infectious agents may be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or reduces the transmission of an infectious agent.
• immune responses against cancer, allergens or infectious agents may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease.
• the lipid conjugates induce an improved effector cell response such as a CD4 T-cell immune response, against at least one of the component antigen(s) or antigenic compositions compared to the effector cell response obtained with the corresponding composition without the lipid conjugate.
• improved effector cell response refers to a higher effector cell response such as a CD8 or CD4 response obtained in a human patient after administration of the vaccine composition than that obtained after administration of the same composition without a lipid conjugate.
• the improved effector cell response is obtained in an immunocompromised subject.
• the improved effector cell response can be assessed by measuring the number of cells producing any of the following cytokines: (1) cells producing at least two different cytokines (CD40L, IL-2, IFN-gamma, TNF- alpha); (2) cells producing at least CD40L and another cytokine (IL-2, TNF- alpha, IFN-gamma); (3) cells producing at least IL-2 and another cytokine (CD40L, TNF-alpha, IFN-gamma); (4) cells producing at least IFN-gamma and another cytokine (IL-2, TNF-alpha, CD40L); (5) and cells producing at least TNF-alpha and another cytokine (IL-2, CD40L, IFN-gamma).
• the composition increases the number of T cells producing IFN-gamma, TNF-alpha, or a combination thereof, or increases the production of IFN-gamma, TNF-alpha, or a combination thereof in the existing T cells.
• the administration of the immunogenic composition alternatively or additionally induces an improved B-memory cell response in patients administered lipid conjugates compared to a control.
• An improved B-memory cell response is intended to mean an increased frequency of peripheral blood B lymphocytes capable of differentiation into antibody-secreting plasma cells upon antigen encounter as measured by stimulation of in vitro differentiation.
• the immunogenic composition increases the primary immune response as well as the CD8 response.
• the administration of the lipid conjugates induces an improved CD4 T-cell, or CD8 T-cell immune response against a specific antigen compared to a control. This method may allow for inducing a CD4 T cell response which is more persistent in time.
• the CD4 T-cell immune response such as the improved
• CD4 T-cell immune response obtained in an unprimed subject involves the induction of a cross-reactive CD4 T helper response. In particular, the amount of cross-reactive CD4 T cells is increased.
• cross-reactive CD4 response refers to CD4 T-cell targeting shared epitopes for example between influenza strains.
• the disclosed lipid conjugates are useful for stimulating or enhancing an immune response in host for treating cancer.
• the types of cancer that may be treated with the provided compositions and methods include, but are not limited to, the following: bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, uterine, ovarian, testicular and hematologic.
• Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived.
• Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands.
• Sarcomas which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
• the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
• the conjugates can be administered in as an immunogenic composition or as part of vaccine, such as prophylactic vaccines, or therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer.
• vaccine such as prophylactic vaccines, or therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer.
• the desired outcome of a prophylactic or therapeutic immune response may vary according to the disease, according to principles well known in the art.
• immune responses against cancer may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease.
• administration of the lipid conjugates may reduce tumor size, or slow tumor growth compared to a control.
• the stimulation of an immune response against a cancer may be coupled with surgical, chemotherapeutic, radiologic, hormonal and other immunologic approaches in order to affect treatment.
• the lipid conjugates are useful for treating acute or chronic infectious diseases. Because viral infections are cleared primarily by T-cells, an increase in T-cell activity is therapeutically useful in situations where more rapid or thorough clearance of an infective viral agent would be beneficial to an animal or human subject.
• the lipid conjugates antagonists can be administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and common cold (e.g., human rhinovirus) viral infections.
• immunodeficiency e.g., HIV
• papilloma e.g., HPV
• herpes e.g., HSV
• encephalitis e.g., human influenza virus A
• common cold e.g., human rhinovirus
• compositions including the lipid conjugates can be administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts.
• the lipid conjugates can also be administered to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.
• infections that can be treated include but are not limited to infections cause by microoganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,
• Chromatium Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Histoplasma, Hyphomicrobium, Legionella, Leishmania, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yers
• Toxoplasma gondii Trichomonas vaginalis and Schistosoma mansoni.
• the type of disease to be treated or prevented is a chronic infectious disease caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, e.g., by cytotoxic T lymphocytes.
• infections to be treated are chronic infections cause by a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein- Barr virus, or a human papilloma virus.
• HIV human immunodeficiency virus
• HTLV human T-lymphotrophic virus
• herpes virus an Epstein- Barr virus
• Epstein- Barr virus or a human papilloma virus.
• Example 1 Albumin-binding lipo-oligo conjugates accumulate in the lymph nodes
• Oligonucleotides were synthesized in 1.0 micromolar scale on an automated DNA synthesiszer (ABI 394, Applied Biosystems, Inc.)- All DNA synthesis reagents including cholesteryl-TEG phosphoramadite and DMT- PEG-phosphoramadite were purchased from Glenres and Chemgenes and used by following manufacturer's instructions. Immunostimulatory CpG oligos employed were a type B sequence known as 1826. Synthesis of lipid phosphoramidite and solid phase conjugation was followed by previous reports. Particle size was determined by dynamic light scattering (DLS) using a 90Plus/ZetaPals particle size and ⁇ -potential analyzer (Brookhaven Instruments).
• DLS dynamic light scattering
• DSPE-PEG 2000 -Maleimide was purchased from Laysan Bio Inc.
• carboxyfluorescein labeled PEG 2000 -DSPE were purchased from Avanti Polar lipids Inc.
• carboxyfluorescein labeled NHS-PEG2 000 was purchased from nanocs Inc.
• Peptides were purchased from Genscript Corp.
• Incomplete Freund s adjuvant (IF A) and fatty acid free BSA were purchased from Sigma- Aldrich.
• the diacyllipid phosphoramidite was synthesized in two steps as described by Liu, et al.. J. Angew. Chem., Int. Ed. 2011, 50, 7252-7255.
• lipid phosphoramidites were dissolved in dichloromethane and coupled to oligos by using the so-called syringe synthesis technique (Storhoff, et al., J Am. Chem. Soc, 120:1959-1964 (1999)). Briefly, lipid phosphoramidites (200 ⁇ ) were mixed with activator (0.2 mM 5-Ethylthio Tetrazole in 200 ⁇ Acetonitrile), and the mixture were pushed back and forth through the CpG column using 2 syringes for 10 min. Alternatively, lipophilic
• phosphoramidite could also be coupled using the DNA synthesizer (15 min coupling time).
• DNA was cleaved from the CpG and deprotected and purified by reverse phase HPLC using a C4 column (BioBasic-4, 200mm x 4.6mm, Thermo Scientific), 100 mM triethylamine- acetic acid buffer (TEAA, pH 7.5)-acetonitrile (0-30 min, 10-100%) as an eluent.
• TEAA triethylamine- acetic acid buffer
• Lipophilic ODNs typically eluted at 20 min while unconjugated ODNs eluted at 8 min.
• Immunostimulatory CpG oligos employed were a type B sequence known as 1826 (Bellas, et al., J. Immunol., 167, 4878-4886 (2001)).
• Circular Dichroism (CD) spectra were recorded on an Aviv Model 202 Circular Dichroism Spectrometer at 20 °C. Scans from 220 to 320 nm were performed with 100 nm/min scanning speed, 1 nm bandwidth. For each spectrum, an average of three scans was taken, spectral contribution from the buffer was subtracted. Animals and cells
• mice were cared for in the USDA-inspected MIT Animal Facility under federal, state, local and NIH guidelines for animal care. C57BL/6 albino mice (6-8 weeks) were obtained from the Jackson Laboratory. Cells were cultured in complete medium (MEM, 5% fetal bovine serum (Greiner Bio-one), 100 U/ml penicillin G sodium and 100 ug ml streptomycin
• Albumin serves as the main fatty acid transporter in extracellular fluids.
• Antigen/adjuvants modified with a lipophilic albumin-binding domain would accumulate in lymphoid organs following injection via in situ complexation and transport with endogenous albumin.
• model vaccines were developed that include peptide antigens combined with CpG DNAs, single-stranded oligonucleotides containing unmethylated cytosine-guanine motifs that bind Toll-like receptor-9 and serve as potent molecular adjuvants.
• Diacyl lipid- conjugated CpGs (lipo-CpGs) in aq. solution eluted as micelles (3.7 min), but following incubation with 20% FBS for 2 hr, -46% of this amph-CpG co-migated with albumin (Figure 2B).
• CpG and rhodamine-conjugated albumin confirmed molecular association of the diacyl lipid amphiphile and albumin in solution ( Figures IF and 1G).
• CpGs with different affinity for albumin exhibit differential LN targeting
• amph-CpGs were injected s.c. at the tail base of mice, and 24 hr later, draining inguinal and axillary LNs were excised and analyzed intact by IVIS fluorescence imaging.
• C18-CpG and Cho-CpG showed marginally increased uptake in LNs relative to unmodified CpG.
• lipo-CpG showed a dramatic increase in LN accumulation, 8-fold over soluble CpG at 24 hr, and much greater than CpG delivered in two prototypical vaccine vehicles, incomplete Freund's adjuvant or poly(ethylene glycol) (PEG)-coated liposomes.
• Example 2 Stabilized micelles exhibit reduce lymph node targeting Materials and Methods
• Cells were plated in 96-well round-bottomed plates and pulsed with minimum peptides in the presence of brefeldin A for 6 hours in complete media at 37°C. Cells were stained with anti-CD8-APC and then fixed using Cytofix (BD biosciences) according to the manufacturer's instructions. Cells were then washed and permeabilized. Intracellular staining for anti-INF- ⁇ - ⁇ and anti-TNF-a-FITC was then performed according to BD's protocol. FACS data were collected and analyzed as described before.
• Immunofluorescent staining was performed on 10- ⁇ m frozen sections of lymph node biopsy specimens. To reduce fading of FITC, sections were mounted in Vectashield mounting medium. (Vector Laboratories, Inc.
• lymph nodes sections were done directly with an PE-labeled CD1 lc and APC-labeled F4/80, or PE-labeled B220 and APC-labeled CD3 antibodies.
• Example 1 The in vitro analyses in Example 1 indicate that lipo-CpG molecules equilibrate between micellar and albumin-bound forms in the presence of serum. However, enhanced lymph node accumulation achieved by these amphiphiles could have been driven by either species. To distinguish these possibilities, poly-guanine repeats were introduced between the diacyl lipid and CpG sequence. G-quadruplex hydrogen bonding between adjacent oligo strands in lipo-G n -CpG micelles containing 4 or more guanine repeats blocked access of albumin to the lipid tails and rendered the micelles stable against disassembly in the presence of serum (discussed in more detail below).
• Mouse serum albumin (10 mg in 200 uL PBS) was added 0.79mg BMPS (Aldrich) dissolved in 20 uL DMSO. The mixture was agitated at RT for 2 hours. The extra BMPS was removed by passing the mixture through a G-25 column. After which the solution was added 246 ug disulfide labeled fluorescein-CpG (preactivated by 20uL lOOmM TCEP). The mixture was allowed to react overnight and extra CpG was dialysized (50K MWCO) and the absence of free CpG was confirmed by size-exclusion chlomatography. Results
• Example 4 Albumin-binding lipo-oligo conjugates enhance immune responses while minimize systemic toxicity in vivo
• OVA ovalbumin
• Animals were primed on day 0, boosted on day 14, and CD8+ T-cell responses were analyzed on day 20.
• Intracellular cytokine staining on peripheral blood lymphocytes showed qualitatively identical trends, with large frequencies of IFN-g- and TNF-a-producing T-cells expanded by the albumin-binding CpG
• PE lipids (1 ,2-dilauroyl-sn-glycero-3-phosphoethanolamine, DMPE;
• 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine DMPE; 1,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine, DPPE; 1,2- dioctadecanoyl-sn-glycero-3-phosphoethanolamine, DSPE, Avanti polar lipids.
• N-terminal cysteine modified peptides were dissolved in DMF and mixed with 2 equivalents Maleimide-PEG 2 ooo-DSPE (Laysan Bio, Inc.), the mixture was agitated at RT for 24 hours. Bioconjugation was judged to be essentially complete by HPLC analysis. The peptide conjugate was then diluted in lOx ddH 2 0 and lyophilized into powder, redissolved in H 2 0 and stored under -80 °C.
• lipo-CpG is straightforward due to the solubility promoted by the long polar oligonucleotide block, but depending on the amino acid sequence, lipidated peptides can be essentially insoluble.
• lipo-PEG amphiphiles composed of a diacyl lipid tail linked to peptide cargos via a polar PEG block (amph-peptides; e.g., Figure 1C) were generated using ethylene glycol spacers of varying lengths to mimic the long polar block of lipo-CpG.
• the length of the PEG block in this design controls the balance of a 3 -way equilibrium in physiological conditions: amph-peptides and lipo- PEGs in pure water form micelles, but in the presence of serum and cells these amphiphiles equilibrate between binding to albumin and insertion of their diacyl tails into cell membranes (Figure 2F).
• Lipo-PEG-FAM amphiphiles with short PEG blocks showed stable plasma membrane insertion when incubated with cells in the presence of serum in vitro (Figure 2G), which would block transit to LNs on albumin in vivo.
• increasing the polar block to 48 ethylene glycol units gave lipo-PEG amphiphiles that partitioned into solution while retaining albumin binding (Figure 2G).
• peptide preparation was purchased from Anaspec; ovalbumin was purchased from Worthington Biochemical Corporation; cysteine (Cys) modified peptide HPV-16 E7 49-57 (CRAHYNIVTF), AL-11 (CAAVKNWMTQTL) and Trp-2 (CSVYDFFVWL) were synthesized by GenScript and purified by reverse phase HPLC.
• DSPE- PEG 2000 -Maleimide was purchased from Laysan Bio Inc.
• CpG ODNs were synthesized in house. IFA was purchased from Sigma-Aldrich.
• Vaccine preparation was purchased from Anaspec; ovalbumin was purchased from Worthington Biochemical Corporation; cysteine (Cys) modified peptide HPV-16 E7 49-57 (CRAHYNIVTF), AL-11 (CAAVKNWMTQTL) and Trp-2 (CSVYDFFVWL) were synthesized by GenScript and purified by reverse phase HPLC.
• DSPE- PEG 2000 -Maleimide was purchased from Lay
• mice were vaccinated by a prime-boost regimen, typically, each priming and boost vaccine in experiments consisted of the following ingredients: 10 ⁇ g OVA, 1.24 nmol CpG suspended in lxPBS with 20mM K + , 10mM Mg + .
• CpG/OVA were combined with same volume of IFA and extensively emulsified. The volume of all vaccine injections was 100 ⁇ .
• mice were primed with 10 ug of peptide-PEG 2000 -DSPE conjugate, mixed with 1.24 nmol CpGs suspended in lxPBS with 20 mM K + , lOmM Mg + and boosted with 20 ⁇ g of peptide- PEG2000-DSPE conjugate, mixed with 1.24 nmol CpGs.
• Mice were injected at the base of tail s.c.
• Tissue samples were collected and a single cell suspension (spleens and lymph nodes) was prepared. Blood were collected and red blood cells were depleted by ACK lysing buffer. Cells were then blocked with Fc- blocker (anti-mouse CD16/CD32 monoclonal antibody) and stained with PE labeled tetramers (Beckman Coulter) and anti-CD8-APC for 30 minutes at room temperature. Cells were washed twice and resuspended in FACS buffer. FACS data were collected on a BD FACScanto flow cytometer and analyzed using flowjo software. Analysis typically gated on CD8 + , Tetramer positive live cells.
• Splenocytes from naive mice were pulsed with or without 10 uM SIINFEKL peptide for 30 min, cells were then labeled with either 1 ⁇ (for pulsed cells) or 0.1 ⁇ (control cells) CFSE for 10 min at 37°C and extensively washed. Cells were mixed at a 1:1 ratio and 10x10 6 total cells were injected i.v. into mice challenged previously with vaccine formulations as described above. 18 hours later, splenocytes from each receipt mouse were analyzed by FACS to detect the CFSE labeled cells.
• peptide antigens were conjugated to commercially-available DSPE-PEG(18-carbon diacyl lipid tail, 2KDa PEG block) to generate amph-peptides for use in vaccination studies (Figure 9).
• TC-l which expresses the E7 oncoprotein from human papillomavirus type- 16 (HPV-16). 6- to 8-wk-old C57BL 6 mice were inoculated at the left above flank with TC-l tumor cells (3x10 s cells/mouse) subcutaneously.
• mice were randomized and treated on day 6, day 13 and dayl9 with amph-HPV (DSPE-PEG-E749-57) combined with amph-CpG (Lipo-G 2 -CpG) at mouse tail base, using unconjugated E7 49-57 peptide and CpG as control. Tumor growth was followed every 2-3 days.
• amph-HPV DSPE-PEG-E749-57
• amph-CpG Lipo-G 2 -CpG
• Example 8 G-quadruplex linkers stabilizes oligonucleotide micelles
• Oligonucleotides were synthesized in 1.0 micromolar scale on an automated DNA synthesizer (ABI 394, Applied Biosystems, Inc.). All DNA synthesis reagents including cholesteryl-triethylene glycol (TEG)- phosphoramadite and DMT-polyethylene glycol (PEG)-phosphoramadite were purchased from Glenres and Chemgenes and used according to manufacturer's instructions. Immunostimulatory cytosine-guanine (CG) oligonucleotides were a type B sequence referred to as 1826 (Lipo-G n -CG: 5'-diacyl lipid-G complicat-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO:l).
• lipid phosphoramidite and solid phase conjugation were followed by previous reports.
• Particle size was determined by dynamic light scattering (DLS) using a 90Plus/ZetaPals particle size and ⁇ -potential analyzer (Brookhaven Instruments).
• DLS dynamic light scattering
• carboxyfluorescein labeled PEG2000-DSPE were purchased from Avanti Polar lipids Inc.
• Circular Dichroism (CD) spectra were recorded on an Aviv Model 202 Circular Dichroism Spectrometer at 20C. Scans from 220 to 320 nm were performed with 100 nm/min scanning speed, 1 nm bandwidth. For each spectrum, an average of three scans was taken, and spectral contribution from the buffer was subtracted.
• PBS IX phosphate buffered saline
• CD Circular Dichroism
• HPLC system equipped with a SEC-biosil column (repacked in a
• G-rich nucleic acid sequences can fold into various types of G-quadruplex structures (Davis, J. T. Angew. Chem. Int. Ed Engl 43, 668-698 (2004)) (e.g., intramolecular, intermolecular, parallel, and antiparallel).
• the lipid-oligonucleotide conjugate was first suspended in pure water, and then potassium (K + ) containing buffer was added to stabilize the G-quadruplex. Formation of micelles was confirmed by transmission electronic microscopy, dynamic light scattering measurements, and size-exclusion chromatography.
• Figure 7B illustrates the size profile of the self-assembled micelles, which show uniform size distribution.
• Circular dichroism was conducted to characterize the formation of G-quadruplex.
• the spectrum of Lipo-G 0 T 10 -CG oligonucleotide showed a small negative peak near 245 nm and a positive peak near 278 run, while changing the number of guanines from zero to ten induced a parallel G- quadruplex, as manifested by the shifting of positive peak from 278 nm toward 262 nm (signature bands for parallel G-quadruplex) (Paramasivan, S., et al., Methods 43, 324-331 (2007)), while retaining the negative 245 nm bands (Figure 7C).
• G-quadruplex stabilized CpG adjuvants The design of G-quadruplex stabilized CpG adjuvants is shown in Figure 3 A.
• mice C57BL 6 albino mice (6-8 weeks) were obtained from the Jackson Laboratory. Animals were cared for in the USDA-inspected Massachusetts Institute of Technology ( ⁇ ) Animal Facility under federal, state, local and NIH guidelines for animal care.
• Bone marrow-derived dendritic cells were prepared following a modification of the procedure of Inaba as previously reported. Dendritic cells were activated/matured with 500 nM CG probes for 12 hours and washed three times with PBS before use. Cells were cultured in complete medium (MEM, 5% fetal bovine serum (Greiner Bio-one), 100 units (U)/ml penicillin G sodium and 100 g/ml streptomycin (Pen Strep), MEM sodium pyruvate (1 mM), NaH2C03, MEM vitamins, MEM non-essential amino acids (all from Invitrogen), and 20 uM ⁇ -mercaptoethanol ( ⁇ - ⁇ )).
• MEM complete medium
• MEM 5% fetal bovine serum
• Pen Strep 100 units
• MEM sodium pyruvate 1 mM
• NaH2C03 NaH2C03
• MEM vitamins, MEM non-essential amino acids all from Invitrogen
• the lymphatic system absorbs interstitial fluid from tissue and returns it to the blood via lymph nodes. Animal experiments were conducted to assess micelle targeting to the lymphatic system.
• Example 10 Immunostimulatory micelles induce antigen-specific CD8 + T-cell expansion
• Ovalbumin was used as a model antigen because it has a well- studied H-2 Kb-restricted MHC class I epitope in B6 mice.
• IF A Incomplete Freund's adjuvant
• a volume of soluble CpG oligonucleotides and soluble OVA antigen were combined with an equal volume of IF A and emulsified. The total volume of each vaccine injection was 100 ⁇ .
• Mice were injected subcutaneously at the base of the tail. Post-immunization, blood samples were collected from spleens and lymph nodes, and single-cell suspensions were prepared (red blood cells were depleted by ACK lysing buffer).
• the blood sample preparations were evaluated by MHC class I-restricted OVA 257-264 tetramer staining to track SIINFEKL-specific CD8 + T-cell expansion.
• Cysteine (cys) modified peptide OVA 257-264 (CSIINFEKL) was synthesized by GenScript and purified by reverse phase HPLC. Cells were then blocked with Fc-blocker (anti-mouse CD16/CD32 monoclonal antibody) and stained with PE labeled
• lymphocytes were re-stimulated ex vivo for 6 hours with the OVA-specific peptide, SIINFEKL, and analyzed for the production of cytokines, IFN- ⁇ and TNF-a.
• Cells were plated in 96-well round-bottomed plates and pulsed with minimum peptides in the presence of brefeldin A for 6 hours in complete media at 37 °C.
• Cells were stained with anti-CD8-APC and then fixed using Cytofix (BD biosciences) according to the manufacturer's instructions. Cells were then washed and permeabilized. Intracellular staining for anti-INF- ⁇ - PE and anti-TNF- ⁇ -FITC was then performed according to BD's protocol. FACS data were collected and analyzed as described. Again, the
• CTL cytotoxic lymphocyte
• oligonucleotide lysed an average 54.6% of target cells.

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