US20110020388A1 - Targeted synthetic nanocarriers with ph sensitive release of immunomodulatory agents - Google Patents

Targeted synthetic nanocarriers with ph sensitive release of immunomodulatory agents Download PDF

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Publication number
US20110020388A1
US20110020388A1 US12/788,260 US78826010A US2011020388A1 US 20110020388 A1 US20110020388 A1 US 20110020388A1 US 78826010 A US78826010 A US 78826010A US 2011020388 A1 US2011020388 A1 US 2011020388A1
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United States
Prior art keywords
immunomodulatory agent
composition
synthetic
synthetic nanocarrier
solution
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Abandoned
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US12/788,260
Inventor
Charles Zepp
Yun Gao
Mark J. Keegan
Sam Baldwin
Fen-Ni Fu
Lloyd Johnston
Grayson B. Lipford
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Cartesian Therapeutics Inc
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Selecta Biosciences Inc
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Application filed by Selecta Biosciences Inc filed Critical Selecta Biosciences Inc
Priority to US12/788,260 priority Critical patent/US20110020388A1/en
Assigned to SELECTA BIOSCIENCES, INC. reassignment SELECTA BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BALDWIN, SAM, FU, FEN-NI, GAO, YUN, JOHNSTON, LLOYD, KEEGAN, MARK J., LIPFORD, GRAYSON B., ZEPP, CHARLES
Publication of US20110020388A1 publication Critical patent/US20110020388A1/en
Priority to US13/948,129 priority patent/US20140030344A1/en
Priority to US14/273,099 priority patent/US20140242173A1/en
Abandoned legal-status Critical Current

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Definitions

  • This invention relates to compositions, and related methods, of synthetic nanocarriers that target sites of action in cells, such as antigen presenting cells (APCs), and comprise immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner.
  • the invention additionally relates to protection of labile immunomodulatory agents by means of their encapsulation in synthetic nanocarriers.
  • Immunomodulatory agents are used to produce immune responses in subjects. Stimulation of the immune system, which includes stimulation of either or both innate immunity and adaptive immunity, is a complex phenomenon that can result in either protective or adverse physiologic outcomes for the host. In recent years there has been increased interest in the mechanisms underlying innate immunity, which is believed to initiate and support adaptive immunity. This interest has been fueled in part by the recent discovery of a family of highly conserved pattern recognition receptor proteins known as Toll-like receptors (TLRs) believed to be involved in innate immunity as receptors for pathogen-associated molecular patterns (PAMPs).
  • TLRs Toll-like receptors believed to be involved in innate immunity as receptors for pathogen-associated molecular patterns (PAMPs).
  • compositions and methods useful for modulating innate immunity are therefore of great interest, as they may affect therapeutic approaches to conditions involving inflammation, allergy, asthma, infection, cancer, and immunodeficiency, etc.
  • the immunomodulatory agent is coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety. In certain embodiments, the immunomodulatory agent is encapsulated within the synthetic nanocarrier. In some embodiments, the immunomodulatory agent comprises a labile immunomodulatory agent such as an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
  • a labile immunomodulatory agent such as an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
  • the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod.
  • the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • the immunomodulatory agent is an adjuvant. In certain embodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist such as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.
  • TLR Toll-like receptor
  • the TLR agonist is an immunostimulatory nucleic acid such as an immunostimulatory DNA or immunostimulatory RNA.
  • the immunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid that comprises one or more stabilizing chemical modifications that function to stabilize the backbone under physiological conditions.
  • the adjuvant comprises a universal T-cell antigen.
  • the synthetic nanocarriers further comprise a B cell antigen and/or a T cell antigen. In certain embodiments, the synthetic nanocarriers further comprise an antigen presenting cell (APC) targeting feature. In some embodiments, the synthetic nanocarriers comprise one or more biodegradable polymers. In some embodiments, the immunomodulatory agent is coupled to the one or more biodegradable polymers via the immunomodulatory agent coupling moiety. In certain embodiments, the biodegradable polymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).
  • the biodegradable polymers have a weight average molecular weight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography.
  • the immunomodulatory agent coupling moiety comprises an amide bond. In some embodiments, the immunomodulatory agent coupling moiety comprises an ester bond.
  • the synthetic nanocarriers comprise lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles, nanoparticles that comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidal nanoparticles.
  • the immunomodulatory agent comprises a labile immunomodulatory agent encapsulated within the synthetic nanocarrier.
  • the labile immunomodulatory agent comprises an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
  • the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod.
  • the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • the immunomodulatory agent comprises a labile immunomodulatory agent encapsulated within the synthetic nanocarrier.
  • the labile immunomodulatory agent comprises an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
  • the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, a imidazoquinoline amine, imiquimod, or resiquimod.
  • the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • the immunomodulatory agent is coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety. In some embodiments, the immunomodulatory agent is encapsulated within the synthetic nanocarrier.
  • the immunomodulatory agent is an adjuvant.
  • the adjuvant comprises a Toll-like receptor (TLR) agonist such as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.
  • TLR agonist is an immunostimulatory nucleic acid such as an immunostimulatory DNA or immunostimulatory RNA.
  • the immunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid that comprises one or more stabilizing chemical modifications that function to stabilize the backbone under physiological conditions.
  • the adjuvant comprises a universal T-cell antigen.
  • the synthetic nanocarriers further comprise a B cell antigen and/or a T cell antigen.
  • the synthetic nanocarriers further comprise an antigen presenting cell (APC) targeting feature.
  • the synthetic nanocarriers comprise one or more biodegradable polymers.
  • the immunomodulatory agent is coupled to the one or more biodegradable polymers via the immunomodulatory agent coupling moiety.
  • the biodegradable polymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).
  • the biodegradable polymers have a weight average molecular weight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography.
  • the immunomodulatory agent coupling moiety comprises an amide bond. In some embodiments, the immunomodulatory agent coupling moiety comprises an ester bond.
  • the synthetic nanocarriers comprise lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles, nanoparticles that comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidal nanoparticles.
  • compositions associated with the invention further comprise a pharmaceutically acceptable excipient.
  • compositions comprising a vaccine comprising any of the compositions associated with the invention.
  • compositions associated with the invention involve methods comprising administering any of the compositions associated with the invention to a subject.
  • the composition is in an amount effective to induce or enhance an immune response.
  • the subject has cancer, an infectious disease, a non-autoimmune metabolic disease, a degenerative disease, or an addiction.
  • FIG. 1 demonstrates the release of resiquimod (R848) from synthetic nanocarrier formulations at pH 7.4, 37° C.
  • FIG. 2 demonstrates the release of R848 from synthetic nanocarrier formulations at pH 4.5, 37° C.
  • FIG. 3 demonstrates the release of R848 from synthetic nanocarrier formulations at pH 7.4 and pH 4.5 at 24 hours.
  • FIG. 4 shows the level of antibody induction by synthetic nanocarriers with a CpG-containing immunostimulatory nucleic acid (Groups 2 and 3) as compared to the level of antibody induction by synthetic nanocarriers without the CpG-containing immunostimulatory nucleic acid (Group 1).
  • FIG. 5 shows the level of antibody induction by synthetic nanocarriers that release a phosphodiester, non-thioated CpG-containing immunostimulatory nucleic acid or a thioated CpG-containing immunostimulatory nucleic acid.
  • FIG. 6 shows the level of antibody induction by synthetic nanocarriers that release R848 at different rates.
  • FIG. 7 shows the level of antibody induction by synthetic nanocarriers carrying entrapped phosphodiester (PO) CpG, designated as NC-Nic/PO-CpG.
  • PO phosphodiester
  • FIG. 8 shows the release of entrapped PO-CpG from nanocarriers at a pH of 4.5 versus pH 7.5.
  • a labile immunomodulatory agent such as PO-CpG
  • Such a labile agent can be released at a desired site of action with a pH of 4.5 (e.g., in the endosome/lysosome) with low levels of release occurring at a pH of 7.4 (e.g., generally the pH outside of the endosome/lysosome).
  • This invention is useful in that it provides a way to release immunomodulatory agents more directly at the sites of action in cells of interest, in particular antigen presenting cells, which would result in beneficial immune response and/or reduce off-target effects and toxicity, as the majority of the release of the immunomodulatory agents would be at a site of action in the cells of interest.
  • This is of particular interest for the delivery of adjuvants.
  • the controlled release properties offer for the first time a controlled way of delivering immunomodulatory agents to the immune cells of interest and allow for a more precise intervention on the immune system, including the ability to release immunomodulatory agents over an extended period. All of this leads to a very tunable system to get the optimum release of immunomodulatory agent such that it will release primarily at a site of action in the desired cells.
  • the inventors have further recognized that coupling labile immunomodulatory agents within the inventive synthetic nanocarriers through encapsulating the labile immunomodulatory agents within the inventive synthetic nanocarriers, and providing a controlled way of delivering labile immunomodulatory agents to immune cells of interest, preferably over an extended period, results in targeted delivery of the labile immunomodulatory agents while minimizing off-target effects of the immunomodulatory agents, especially off-target effects associated with systemic administration of the immunomodulatory agents. Additionally, this approach can enhance the performance of labile immunomodulatory agents having a short half-life of elimination that otherwise might not have a desirable level of pharmacological activity.
  • the invention relates to certain oligonucleotides.
  • nucleic acid molecules including CpG nucleic acids, GU rich ssRNA and double-stranded RNA.
  • TLR9 Toll-like receptor 9
  • Natural DNA oligonucleotides contain phosphodiester linkages that are rapidly cleaved by nucleases found in the extracellular environment. Yu, D., et al., Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties. Biochem Biophys Res Commun, 2002. 297(1): p. 83-90 (“Yu et al.”); Heeg, K., et al., Structural requirements for uptake and recognition of CpG oligonucleotides. Int J Med Microbiol, 2008. 298(1-2): p. 33-8 (“Heeg et al.”).
  • Such natural oligonucleotides may be considered labile immunomodulatory agents. Accordingly, methods of chemically stabilizing the linkages by replacing the phosphodiester linking group with a phosphorothioate group have been extensively reported in the literature. See U.S. Pat. No. 6,811,975—Phosphorothioate Oligonucleotides Having Modified Internucleoside Linkages.
  • Phosphorothioate CpG containing oligonucleotides have been administered systemically as vaccine adjuvants. Yu et al. However, systemic administration of stabilized CpG oligonucleotides can result in off-target immunostimulatory effects, such as general inflammation, non-specific activation of lymphocytes, and flu-like symptoms.
  • Haas, T., et al. Sequence independent interferon-alpha induction by multimerized phosphodiester DNA depends on spatial regulation of Toll-like receptor-9 activation in plasmacytoid dendritic cells. Immunology, 2009. 126(2): p. 290-8 (“Haas et al.”). Accordingly, such oligonucleotides may be usefully incorporated in the practice of the present invention, as is described in more detail below.
  • composition comprising: synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier; wherein the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship:
  • the immunomodulatory agent preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: IArel(4.5) t %/IArel(7.4) t % ⁇ 1.3, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.4, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.5, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.6, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.7, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.8, IArel(4.5) t %/IArel(7.4) t % ⁇ 1.9, IArel(4.5) t %/IArel(7.4) t % 2, IArel(4.5)
  • the immunomodulatory agent preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: 2 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 2.5 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 3 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 3.5 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 4 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 4.5 ⁇ IArel(4.5) t %/IArel(7.4) t % ⁇ 1.2, 5 ⁇ IArel(4.5) t %/IArel(7.4) t % 1.2, 6 ⁇ IArel(4.5) t
  • this invention relates to compositions and methods comprising synthetic nanocarriers that release immunomodulatory agents at significantly different rates at neutral and acidic pH.
  • immunomodulatory agents to have the most potent effect it is desirable to have the majority of the immunomodulatory agent released inside APCs where they can have a desired effect.
  • immunomodulatory agents are injected in free form, or when they are released from a synthetic nanoparticle outside the APCs, only a small portion of that immunomodulatory agent finds its way to the APCs, while the rest diffuses through the body, where the immune stimulation would be less and may result in deleterious effects.
  • inventive synthetic nanocarriers provided herein are preferentially taken up by APCs.
  • the synthetic nanocarriers Upon being taken up by the APC, the synthetic nanocarriers are presumed to be endocytosed into an endosomal/lysosomal compartment where the pH becomes more acidic, as opposed to the neutral pH outside the cells. Under these conditions, the immunomodulatory agent exhibits a pH sensitive dissociation from the synthetic nanocarrier (e.g., from an immunomodulatory agent coupling moiety) and is released from the synthetic nanocarrier. The immunomodulatory agent is then free to interact with receptors associated with the endosome/lysosome and stimulate a desired immune response.
  • the synthetic nanocarrier e.g., from an immunomodulatory agent coupling moiety
  • the immunomodulatory agents can be coupled to the synthetic nanocarriers by any of a number of methods. Generally, the coupling can be a result of bonding between the immunomodulatory agent and the synthetic nanocarrier. This bonding can result in the immunomodulatory agent being attached to the surface of the synthetic nanocarrier and/or contained within (encapsulated) the synthetic nanocarrier. In some embodiments, however, the immunomodulatory agent is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than bonding to the synthetic nanocarrier.
  • an immunomodulatory agent coupling moiety can be any moiety through which an immunomodulatory agent is bonded to a synthetic nanocarrier.
  • moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunomodulatory agent to the synthetic nanocarrier.
  • molecules include linkers or polymers or a unit thereof.
  • the immunomodulatory agent coupling moiety can comprise a charged polymer to which an immunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically binds.
  • the immunomodulatory agent coupling moiety can comprise a polymer or unit thereof to which the immunomodulatory agent is covalently bonded.
  • the polymer or unit thereof comprises a polyester, polycarbonate, polyamide, or polyether, or unit thereof.
  • the polymer or unit thereof comprises poly(ethylene glycol) (PEG), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof.
  • PEG poly(ethylene glycol)
  • the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable.
  • the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or unit thereof.
  • any of the polymers provided herein can have a weight average molecular weight, as determined by gel permeation chromatography, of about 800 Da to 10,000 Da (e.g., 2,000 Da).
  • the immunomodulatory agent is an adjuvant, such as an imidazoquinoline.
  • Imidazoquinolines include compounds, such as imiquimod and resiquimod (also known as R848).
  • Such adjuvants can be coupled to a polymer as provided above.
  • resiquimod was conjugated to poly-lactic acid (PLA) polymer of ⁇ 2000 Da.
  • PPA poly-lactic acid
  • Table 1 lists the compositions of the particles tested.
  • immunomodulatory agents such as R848, can be coupled to other polymers or units thereof, such as those provided above and elsewhere herein including polylactide-co-glycolide (PLGA) block co-polymer or unit thereof.
  • Immunomodulatory agents, such as R848, can be coupled to such polymers or units thereof by an amide or ester bond. Examples of methods for effecting such coupling are provided elsewhere herein and in the EXAMPLES.
  • composition comprising:
  • synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier; wherein the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship:
  • t1 is 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours
  • t2 is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 hours
  • t1 is 24 hours
  • t2 is 6 hours.
  • the immunomodulatory agent preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: IA(4.5) t1 /IA(4.5) t2 ⁇ 1.5, IA(4.5) t1 /IA(4.5) t2 ⁇ 2, IA(4.5) t1 /IA(4.5) t2 ⁇ 2.5, IA(4.5) t1 /IA(4.5) t2 ⁇ 3, IA(4.5) t1 /IA(4.5) t2 ⁇ 3.5, IA(4.5) t1 /IA(4.5) t2 ⁇ 4, IA(4.5) t1 /IA(4.5) t2 ⁇ 4.5, IA(4.5) t1 /IA(4.5) t2 ⁇ 5, IA(4.5) t1 /IA(4.5) t2 ⁇ 6, IA(4.5) t1 /IA(4.5) t2 ⁇ 7, IA(4.5)
  • the immunomodulatory agent preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 2, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 2.5, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 3, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 3.5, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 4, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 4.5, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 5, 10 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 6, 10 ⁇ IA(
  • Inventive synthetic nanocarriers have also been shown to exhibit the property of augmenting a humoral immune response to a specific antigen. Such augmented humoral immune response has been found to be elevated, in some embodiments, with faster release of immunomodulatory agent.
  • the immunomodulatory agent is a CpG-containing immunostimulatory nucleic acid
  • the CpG-containing immunostimulatory nucleic acid is encapsulated within a synthetic nanocarrier.
  • optimal release of the CpG-containing immunostimulatory nucleic acids from synthetic nanocarriers produced an elevated humoral immune response to nicotine, which was also coupled to the synthetic nanocarriers. In some embodiments, such optimal release was found to better augment an antibody response to an antigen.
  • Optimal release is the dissociation of the immunomodulatory agent from the synthetic nanocarrier that produces the best levels of desired effect(s).
  • the desired effect is an immediate immune response of a desired level (i.e., one that occurs soon after the administration of the synthetic nanocarrier).
  • an immediate immune response is one measured on the order of seconds, minutes, or a few hours.
  • the desired effect is an immune response of a desired level that occurs after a few hours.
  • the desired effect is an immune response of a desired level that is sustained for an extended period of time, such as for 1, 2, 5, 10, 15 or more hours. In other embodiments, the extended period of time is for 1, 2, 5, 10, 15, 20, 25, 30 or more days.
  • the extended period of time is for 1, 2, 5, 10 or more months. In further embodiments, the extended period of time is for 1, 2, 5, 10 or more years.
  • a composition of synthetic nanocarriers that provides optimal release is one wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to one of the above relationships.
  • an immunomodulatory agent preferably a labile immunodmodulatory agent, that dissociates from the synthetic nanocarrier at an intermediate rate satisfies the following relationship: 6 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 5 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 4 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 3 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 2 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 1.2, 6 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 2, 6 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 2.5, 6 ⁇ IA(4.5) t1 /IA(4.5) t2 ⁇ 3, 6 ⁇ IA(4.5) t1 /IA(4.5) t1 /I
  • resiquimod was encapsulated within a synthetic nanocarrier.
  • resiquimod contained in the synthetic nanocarriers augmented humoral immune response against nicotine also coupled to the synthetic nanocarriers.
  • an intermediate release of the resiquimod from the synthetic nanocarriers was optimal, as it resulted in a higher level of antibody induction than fast or slow release of the resiquimod.
  • the synthetic nanocarriers provided herein can also comprise one or more antigens.
  • the antigens can be B cell antigens or T cell antigens or a combination of both.
  • Such antigens can be coupled to the synthetic nanocarriers such that they are present on the surface of the synthetic nanocarriers, encapsulated within the nanocarriers or both, in some embodiments.
  • the immunomodulatory agent augments an immune response to such an antigen.
  • the antigen can also be coupled to the synthetic nanocarriers. In other embodiments, however such as antigen is not coupled to the synthetic nanocarriers. In some of these embodiments, such an antigen can be coadministered to a subject. In still other of these embodiments, such an antigen is not coadministered to the subject.
  • adjuvant means an agent that does not constitute a specific antigen, but boosts the strength and longevity of immune response to an antigen.
  • adjuvants may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium , or Shigella flexneri or specifically with MPL® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIXTTM, emulsions such as MF59TM, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+MPL®), liposomes and liposomal formulations such as AS01, synthe
  • adjuvants comprise agonists for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof.
  • PRR pattern recognition receptors
  • TLRs Toll-Like Receptors
  • adjuvants comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited adjuvants comprise imidazoquinolines; such as resiquimod (also known as R848); adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company); immunostimulatory DNA; or immunostimulatory RNA.
  • synthetic nanocarriers incorporate as adjuvants compounds that are agonists for toll-like receptors (TLRs) 7 & 8 (“TLR 7/8 agonists”).
  • TLR 7/8 agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al., including but not limited to imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines.
  • Preferred adjuvants comprise imiquimod and resiquimod.
  • an adjuvant may be an agonist for the DC surface molecule CD40.
  • a synthetic nanocarrier incorporates an adjuvant that promotes DC maturation (needed for effective priming of naive T cells) and the production of cytokines, such as type I interferons, which in turn stimulate antibody and cytotoxic immune responses against desired antigen.
  • adjuvants also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA or poly I:C (a TLR3 stimulant), and/or those disclosed in F. Heil et al., “Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8” Science 303(5663), 1526-1529 (2004); J.
  • an adjuvant may be a TLR-4 agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or HMGB-1.
  • adjuvants may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos.
  • synthetic nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9, such as immunostimulatory oligonucleotide molecules comprising 5′-CG-3′ motifs, wherein the C is unmethylated, which induce type I interferon secretion, and stimulate T and B cell activation leading to increased antibody production and cytotoxic T cell responses (Krieg et al., CpG motifs in bacterial DNA trigger direct B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thi) immunity. J.
  • TLR Toll-like receptor
  • adjuvants may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals).
  • adjuvants may be activated components of the complement cascade (e.g., CD21, CD35, etc.).
  • adjuvants may be activated components of immune complexes.
  • the adjuvants also include complement receptor agonists, such as a molecule that binds to CD21 or CD35.
  • the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier.
  • adjuvants are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells.
  • the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.
  • administering means providing a drug to a patient in a manner that is pharmacologically useful.
  • APC targeting feature means one or more portions of which the inventive synthetic nanocarriers are comprised that target the synthetic nanocarriers to professional antigen presenting cells (“APCs”), such as but not limited to dendritic cells, SCS macrophages, follicular dendritic cells, and B cells.
  • APC targeting features may comprise immunofeature surface(s) and/or targeting moieties that bind known targets on APCs.
  • APC targeting features may comprise one or more B cell antigens present on a surface of synthetic nanocarriers.
  • APC targeting features may also comprise one or more dimensions of the synthetic nanoparticles that is selected to promote uptake by APCs.
  • targeting moieties for known targets on macrophages comprise any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on macrophages (i.e., subcapsular sinus-Mph markers).
  • entity e.g., protein, lipid, carbohydrate, small molecule, etc.
  • Exemplary SCS-Mph markers include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, MRP-1); CD11a (LFA-1 ⁇ , ⁇ L Integrin chain); CD11b ( ⁇ M Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c ( ⁇ X Integrin, p150, 95, AXb2); CDw12 (p90-120); CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X, SSEA-1,3-FAL); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin ⁇
  • targeting moieties for known targets on dendritic cells comprise any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on DCs (i.e., a DC marker).
  • entity e.g., protein, lipid, carbohydrate, small molecule, etc.
  • DC markers include, but are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b ( ⁇ M Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c ( ⁇ X Integrin, p150, 95, AXb2); CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA-4 ⁇ , ⁇ 4 Integrin); CD49e (VLA-5 ⁇ , ⁇ 5 Integrin); CD58
  • targeting can be accomplished by any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on B cells (i.e., B cell marker).
  • entity e.g., protein, lipid, carbohydrate, small molecule, etc.
  • Exemplary B cell markers include, but are not limited to, CD1c (M241, R7); CD1d (R3); CD2 (E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9 (p24, DRAP-1, MRP-1); CD11a (LFA-1 ⁇ , ⁇ L Integrin chain); CD11b ( ⁇ M Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c ( ⁇ X Integrin, P150, 95, AXb2); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin ⁇ 2, CD11a, b, c ⁇ -subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R, C3dR); CD22 (BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24 (BBA-1
  • B cell targeting can be accomplished by any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on B cells upon activation (i.e., activated B cell marker).
  • entity e.g., protein, lipid, carbohydrate, small molecule, etc.
  • Exemplary activated B cell markers include, but are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80 (B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BLKDD/F12); CD125 (IL-5R ⁇ ); CD126 (IL-6R ⁇ ); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152 (CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253 (TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1);
  • B cell antigen means any antigen that naturally is or could be engineered to be recognized by a B cell, and triggers (naturally or being engineered as known in the art) an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell receptor on a B cell).
  • an antigen that is a T cell antigen is also a B cell antigen.
  • the T cell antigen is not also a B cell antigen.
  • B cell antigens include, but are not limited to proteins, peptides, small molecules, and carbohydrates.
  • the B cell antigen is a non-protein antigen (i.e., not a protein or peptide antigen).
  • the B cell antigen is a carbohydrate associated with an infectious agent. In some embodiments, the B cell antigen is a glycoprotein or glycopeptide associated with an infectious agent.
  • the infectious agent can be a bacterium, virus, fungus, protozoan, parasite or prion.
  • the B cell antigen is a poorly immunogenic antigen. In some embodiments, the B cell antigen is an abused substance or a portion thereof. In some embodiments, the B cell antigen is an addictive substance or a portion thereof. Addictive substances include, but are not limited to, nicotine, a narcotic, a cough suppressant, a tranquilizer, and a sedative.
  • the B cell antigen is a toxin, such as a toxin from a chemical weapon or natural sources, or a pollutant.
  • the B cell antigen may also be a hazardous environmental agent.
  • the B cell antigen is an alloantigen, an allergen, a contact sensitizer, a degenerative disease antigen, a hapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an addictive substance, a xenoantigen, or a metabolic disease enzyme or enzymatic product thereof.
  • Biodegradable polymer means a polymer that degrades over time when introduced into the body of a subject.
  • Biodegradable polymers include but are not limited to, polyesters, polycarbonates, polyketals, or polyamides. Such polymers may comprise poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone.
  • the biodegradable polymer comprises a block-co-polymer of a polyether, such as poly(ethylene glycol), and a polyester, polycarbonate, or polyamide or other biodegradable polymer.
  • the biodegradable polymer comprises a block-co-polymer of poly(ethylene glycol) and poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone.
  • the biodegradable polymer does not comprise a polyether, such as poly(ethylene glycol), or consists solely of the polyether.
  • the biodegradable polymer in embodiments, have a weight average molecular weight ranging from about 800 to about 50,000 Daltons, as determined using gel permeation chromatography.
  • the weight average molecular weight is from about 800 Daltons to about 10,000 Daltons, preferably from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography. In other embodiments, the weight average molecular weight is from 1000 Daltons to 10,000 Daltons, as determined by gel permeation chromatography. In an embodiment, the biodegradable polymer does not comprise polyketal.
  • Coadministered means administering two or more drugs to a subject in a manner that is correlated in time. In embodiments, coadministration may occur through administration of two or more drugs in the same dosage form. In other embodiments, coadministration may encompass administration of two or more drugs in different dosage forms, but within a specified period of time, preferably within 1 month, more preferably within 1 week, still more preferably within 1 day, and even more preferably within 1 hour.
  • “Couple” or “Coupled” or “Couples” means attached to or contained within the synthetic nanocarrier.
  • the coupling is covalent.
  • the covalent coupling is mediated by one or more linkers, polymers or a unit thereof.
  • the coupling is non-covalent.
  • the non-covalent coupling is mediated by charge interactions, affinity interactions, metal coordination, physical adsorption, hostguest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof.
  • the coupling may arise in the context of encapsulation within the synthetic nanocarriers, using conventional techniques. Any of the aforementioned couplings may be arranged to be on a surface or within an inventive synthetic nanocarrier.
  • “Derived” means adapted or modified from the original source.
  • a peptide antigen derived from an infectious strain may have several non-natural amino acid residues substituted for the natural amino acid residues found in the original antigen found in the infectious strain.
  • the adaptations or modifications may be for a variety of reasons, including but not limited to increased specificity, easier antigen processing, or improved safety.
  • Dosage form means a drug in a medium, carrier, vehicle, or device suitable for administration to a subject.
  • Effective amount of an inventive composition is that amount effective for a certain purpose. For example, when the effective amount is for a therapeutic purpose the amount is effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a disease, disorder, and/or condition provided herein.
  • Encapsulate means to enclose within a synthetic nanocarrier, preferably enclose completely within a synthetic nanocarrier. Most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. Encapsulation is distinct from absorbtion, which places most or all of a substance on a surface of a synthetic nanocarrier, and leaves the substance exposed to the local environment external to the synthetic nanocarrier.
  • “Exhibits a pH sensitive dissociation” means that a coupling between two entities, such as the immunomodulatory agent and the synthetic nanocarrier or immunomodulatory agent coupling moiety, is significantly reduced or eliminated by a change in environmental pH.
  • relevant pH sensitive dissociations may satisfy any of the relationships or combinations thereof provided herein.
  • t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 hours.
  • t is 24 hours.
  • t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 hours.
  • t is 24 hours.
  • IA(4.5) t1 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at pH 4.5 for t1 hours taken as an average across a sample of the synthetic nanocarriers.
  • IA(4.5) t2 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at pH 4.5 for t2 hours taken as an average across a sample of the synthetic nanocarriers.
  • t1 is 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours;
  • t2 is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 hours; and t1>t2.
  • t1 is 24 hours
  • t2 is 6 hours.
  • Immunomodulatory agent means an agent that modulates an immune response. “Modulate”, as used herein, refers to inducing, enhancing, stimulating, or directing an immune response. Such agents include adjuvants that stimulate (or boost) an immune response to an antigen but is not an antigen or derived from an antigen.
  • the immunomodulatory agent is on the surface of the synthetic nanocarrier and/or is incorporated within the synthetic nanocarrier. In embodiments, the immunomodulatory agent is coupled to the synthetic nanocarrier via a polymer or unit thereof.
  • all of the immunomodulatory agents of a synthetic nanocarrier are identical to one another.
  • a synthetic nanocarrier comprises a number of different types of immunomodulatory agents.
  • a synthetic nanocarrier comprises multiple individual immunomodulatory agents, all of which are identical to one another.
  • a synthetic nanocarrier comprises exactly one type of immunomodulatory agent.
  • a synthetic nanocarrier comprises exactly two distinct types of immunomodulatory agents.
  • a synthetic nanocarrier comprises greater than two distinct types of immunomodulatory agents.
  • Immunomodulatory agent coupling moiety is any moiety through which an immunomodulatory agent is bonded to a synthetic nanocarrier.
  • Such moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunomodulatory agent to the synthetic nanocarrier.
  • Such molecules include linkers or polymers or a unit thereof.
  • the immunomodulatory agent coupling moiety can comprise a charged polymer to which an immunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically bonds.
  • the immunomodulatory agent coupling moiety can comprise a polymer or unit thereof to which the immunomodulatory agent covalently bonds.
  • the moiety comprises a polyester. In other embodiments, the moiety comprises poly(ethylene glycol), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone. The moiety may also comprise a unit of any of the foregoing polymers, such as a lactide or glycolide.
  • “Labile immunomodulatory agent(s)” means immunomodulatory agent or agents that are unstable under physiological conditions, and degrade to the point where they are no longer pharmacologically active. In embodiments, labile immunomodulatory agents are observed to have systemic half-lives of elimination of less than 24 hours, preferably less than 12 hours, more preferably less than 10 hours, even more preferably less than 8 hours, and still more preferably less than 6 hours. In embodiments, labile immunomodulatory agents comprise imidazoquinolines, adenine derivative, or oligonucleotides that comprise 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
  • the imidazoquinolines comprise imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, imidazoquinoline amines, imiquimod or resiquimod.
  • “Maximum dimension of a synthetic nanocarrier” means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of a synthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would be substantially identical, and would be the size of its diameter. Similarly, for a cubic synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of its height, width or length, while the maximum dimension of a synthetic nanocarrier would be the largest of its height, width or length.
  • a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is greater than 100 nm.
  • a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 5 ⁇ m.
  • a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or greater than 110 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, and more preferably still equal to or greater than 150 nm.
  • a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 3 ⁇ m, more preferably equal to or less than 2 ⁇ m, more preferably equal to or less than 1 ⁇ m, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm.
  • a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm.
  • Measurement of synthetic nanocarrier sizes is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (e.g. using a Brookhaven ZetaPALS instrument).
  • antigens obtained from a source may comprise the original amino acid residue sequence found in that source.
  • antigens obtained from a source may comprise the original molecular structure found in that source.
  • Oligonucleotide means a nucleotide molecule having from 6 to 100 nucleotides, preferably from 8 to 75 nucleotides, more preferably from 10 to 50 nucleotides, still more preferably from 15 to 25 nucleotides, even still more preferably 20 nucleotides.
  • oligonucleotides comprise less than 100 nucleotides, preferably less than 50 nucleotides, more preferably less than 25 nucleotides, and still more preferably less than 10 nucleotides.
  • any cytosine nucleotides (“C”) present in a 5′-CG-3′ sequence of which the oligonucleotide may be comprised are unmethylated, C present in parts of the oligonucleotides other than in a 5′-CG-3′ sequence of which the oligonucleotide may be comprised may be methylated, or may be unmethylated.
  • inventive oligonucleotides comprise a backbone comprising one or more unstabilized internucleotide linkages (meaning internucleotide linkages that are unstable under physiological conditions).
  • Unstabilized internucleotide linkage means a linkage between two nucleotides of which the oligonucleotide is comprised that is not chemically modified to stabilize the backbone, or is chemically modified to destabilize the backbone of the oligonucleotide under physiological conditions.
  • An example of an unstablized internucleotide linkage is a phophodiester internucleotide linkage.
  • the inventive oligonucleotides' backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions.
  • the inventive oligonucleotides' backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • “Pharmaceutically acceptable excipient” means a pharmacologically inactive substance added to an inventive composition to further facilitate administration of the composition.
  • examples, without limitation, of pharmaceutically acceptable excipients include calcium carbonate, calcium phosphate, various diluents, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Release Rate means the rate that an entrapped immunomodulatory agent flows from a composition, such as a synthetic nanocarrier, into a surrounding media in an in vitro release test.
  • the synthetic nanocarrier is prepared for the release testing by placing into the appropriate in vitro release media. This is generally done by exchanging the buffer after centrifugation to pellet the synthetic nanocarrier and reconstitution of the synthetic nanocarriers using a mild condition.
  • the assay is started by placing the sample at 37° C. in an appropriate temperature-controlled apparatus. A sample is removed at various time points.
  • the synthetic nanocarriers are separated from the release media by centrifugation to pellet the synthetic nanocarriers.
  • the release media is assayed for the immunomodulatory agent that has dispersed from the synthetic nanocarriers.
  • the immunomodulatory agent is measured using HPLC to determine the content and quality of the immunomodulatory agent.
  • the pellet containing the remaining entrapped immunomodulatory agent is dissolved in solvents or hydrolyzed by base to free the entrapped immunomodulatory agent from the synthetic nanocarriers.
  • the pellet-containing immunomodulatory agent is then also measured by HPLC to determine the content and quality of the immunomodulatory agent that has not been released at a given time point.
  • the mass balance is closed between immunomodulatory agent that has been released into the release media and what remains in the synthetic nanocarriers. Data are presented as the fraction released or as the net release presented as micrograms released over time.
  • Subject means an animal, including mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; and the like.
  • Synthetic nanocarrier(s) means a discrete object that is not found in nature, and that possesses at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are expressly included as synthetic nanocarriers.
  • Synthetic nanocarriers include polymeric nanoparticles.
  • synthetic nanocarriers can comprise one or more polymeric matrices.
  • the synthetic nanocarriers can also include other nanomaterials and may be, for example, lipid-polymer nanoparticles.
  • a polymeric matrix can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.).
  • the synthetic nanocarrier is not a micelle.
  • a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
  • the various elements of the synthetic nanocarriers can be coupled with the polymeric matrix.
  • the synthetic nanocarriers may comprise one or more lipids.
  • a synthetic nanocarrier may comprise a liposome.
  • a synthetic nanocarrier may comprise a lipid bilayer.
  • a synthetic nanocarrier may comprise a lipid monolayer.
  • a synthetic nanocarrier may comprise a micelle.
  • a synthetic nanocarrier may comprise a non-polymeric core (e.g., metal particle, quantum dot, ceramic particle, bone particle, viral particle, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
  • the synthetic nanocarriers may comprise lipid-based nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles (such as albumin nanoparticles).
  • Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cubic, pyramidal, oblong, cylindrical, toroidal, and the like.
  • Synthetic nanocarriers according to the invention comprise one or more surfaces.
  • Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in U.S. Pat. No.
  • Synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface with hydroxyl groups that activate complement or alternatively comprise a surface that consists essentially of moieties that are not hydroxyl groups that activate complement.
  • synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that substantially activates complement or alternatively comprise a surface that consists essentially of moieties that do not substantially activate complement.
  • synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that activates complement or alternatively comprise a surface that consists essentially of moieties that do not activate complement.
  • synthetic nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
  • synthetic nanocarriers are spheres or spheroids. In some embodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments, synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.
  • a population of synthetic nanocarriers that is relatively uniform in terms of size, shape, and/or composition so that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum dimension or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension. In some embodiments, a population of synthetic nanocarriers may be heterogeneous with respect to size, shape, and/or composition.
  • Synthetic nanocarriers can be solid or hollow and can comprise one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s).
  • synthetic nanocarriers may have a core/shell structure, wherein the core is one layer (e.g., a polymeric core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers may comprise a plurality of different layers.
  • T cell antigen means any antigen that is recognized by and triggers an immune response in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a T cell or an NKT cell via presentation of the antigen or portion thereof bound to a Class I or Class II major histocompatability complex molecule (MHC), or bound to a CD1 complex).
  • an antigen that is a T cell antigen is also a B cell antigen.
  • the T cell antigen is not also a B cell antigen.
  • T cell antigens generally are proteins or peptides.
  • T cell antigens may be an antigen that stimulates a CD8+ T cell response, a CD4+ T cell response, or both. The T cell antigens, therefore, in some embodiments can effectively stimulate both types of responses.
  • the T cell antigen is a T-helper antigen, which is a T cell antigen that can generate an augmented response to an unrelated B cell antigen through stimulation of T cell help.
  • a T-helper antigen may comprise one or more peptides derived from tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE peptide.
  • a T-helper antigen may comprise one or more lipids, or glycolipids, including but not limited to: ⁇ -galactosylceramide ( ⁇ -GalCer), ⁇ -linked glycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols (from Borrelia burgdorferi ), lypophosphoglycan (from Leishmania donovani ), and phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae ).
  • ⁇ -galactosylceramide ⁇ -GalCer
  • ⁇ -linked glycosphingolipids from Sphingomonas spp.
  • galactosyl diacylglycerols from Borrelia burgdorferi
  • lypophosphoglycan from Leishmania donovani
  • PIM4 phosphatidylinositol tetramann
  • CD4+ T-cell antigens may be derivatives of a CD4+ T-cell antigen that is obtained from a source, such as a natural source.
  • CD4+ T-cell antigen sequences such as those peptides that bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity to the antigen obtained from the source.
  • the T cell antigen preferably a T-helper antigen, may be coupled to, or uncoupled from, a synthetic nanocarrier.
  • Unit thereof refers to a monomeric unit of a polymer, the polymer generally being made up of a series of linked monomers.
  • Vaccine means a composition of matter that improves the immune response to a particular pathogen or disease.
  • a vaccine typically contains factors that stimulate a subject's immune system to recognize a specific antigen as foreign and eliminate it from the subject's body.
  • a vaccine also establishes an immunologic ‘memory’ so the antigen will be quickly recognized and responded to if a person is re-challenged.
  • Vaccines can be prophylactic (for example to prevent future infection by any pathogen), or therapeutic (for example a vaccine against a tumor specific antigen for the treatment of cancer).
  • Vaccines according to the invention may comprise one or more of the synthetic nanocarriers or compositions provided herein.
  • the immunomodulatory agent can be coupled to the synthetic nanocarrier in any manner such that the dissociation of the immunomodulatory agent from the synthetic nanocarrier satisfies the dissociation relationships provided herein.
  • Methods for determining whether or not immunomodulatory agents of synthetic nanocarriers satisfy the dissociation relationships provided herein are provided elsewhere above and in the EXAMPLES.
  • Oligonucleotides according to the invention may be encapsulated into synthetic nanocarriers using a variety of methods including but not limited to C. Astete et al., “Synthesis and characterization of PLGA nanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al., “Nanoencapsulation I.
  • the immunomodulatory agent is covalently coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety (e.g., a polymer or unit thereof).
  • an immunomodulatory agent coupling moiety e.g., a polymer or unit thereof.
  • a polymer or unit thereof can be covalently coupled with an immunomodulatory agent in several ways.
  • solvents that may be suitable for use in the invention include, but are not limited to, p-cresol, toluene, xylene, mesitylene, diethyl ether, glycol, petroleum ether, hexane, cyclohexane, pentane, dichloromethane (or methylene chloride), chloroform, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate (EtOAc), triethylamine, acetonitrile, methyl-t-butyl ether (MTBE), N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropan
  • a reaction or any step of the methods provided may be carried out at any suitable temperature. In some cases, a reaction or any step of the methods provided is carried out at about room temperature (e.g., about 25° C., about 20° C., between about 20° C. and about 25° C., or the like).
  • room temperature e.g., about 25° C., about 20° C., between about 20° C. and about 25° C., or the like.
  • the reaction or any step of the methods provided may be carried out at a temperature below or above room temperature, for example, at about ⁇ 20° C., at about ⁇ 10° C., at about 0° C., at about 10° C., at about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C., about 90° C., about 100° C., about 120° C., about 140° C., about 150° C. or greater.
  • the reaction or any step of the methods provided is conducted at temperatures between 0° C. and 120° C.
  • reaction or any step of the methods provided may be carried out at more than one temperature (e.g., reactants added at a first temperature and the reaction mixture agitated at a second wherein the transition from a first temperature to a second temperature may be gradual or rapid).
  • the reaction or any step of the methods provided may be allowed to proceed for any suitable period of time. In some cases, the reaction or any step of the methods provided is allowed to proceed for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 2 days, about 3 days, about 4 days, or more. In some cases, aliquots of the reaction mixture may be removed and analyzed at an intermediate time to determine the progress of the reaction or any step of the methods provided. In some embodiments, a reaction or any step of the methods provided may be carried out under an inert atmosphere in anhydrous conditions (e.g., under an atmosphere of nitrogen or argon, anhydrous solvents, etc.)
  • anhydrous conditions e.g., under an atmosphere of nitrogen or argon, anhydrous solvents, etc.
  • reaction products and/or intermediates may be isolated (e.g., via distillation, column chromatography, extraction, precipitation, etc.) and/or analyzed (e.g., gas liquid chromatography, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, etc.) using commonly known techniques.
  • a synthetic nanocarrier may be analyzed to determine the loading of immunomodulatory agent, for example, using reverse phase HPLC.
  • the polymers may have any suitable molecular weight.
  • the polymers may have a low or high molecular weight.
  • Non-limiting molecular weight values include 100 Da, 200 Da, 300 Da, 500 Da, 750 Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10,000 Da, or greater.
  • the polymers have a weight average molecular weight of about 800 Da to about 10,000 Da.
  • the molecular weight of a polymer may be determined using gel permeation chromatography. Provided below are exemplary reactions that are not intended to be limiting.
  • a polymer e.g., PLA, PLGA
  • a reactive acylating agent such as an acyl halide, acylimidazole, active ester, etc. using an activating reagent commonly used in amide synthesis.
  • the resulting activated polymer or unit thereof e.g., PLA, PLGA
  • an immunomodulatory agent e.g., R848
  • a base e.g., PLA-R848
  • Activating reagents that can be used to convert polymers or units thereof, such as PLA or PLGA, to an activated acylating form include, but are not limited to cyanuric fluoride, N,N-tetramethylfluoroformamidinium hexafluorophosphate (TFFH); Acylimidazoles, such as carbonyl diimidazole (CDI), N,N′-carbonylbis(3-methylimidazolium) triflate (CBMIT); and Active esters, such as N-hydroxylsuccinimide (NHS or HOSu) in the presence of a carbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC), N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) or N,N′-diisopropylcarbodiimide (DIC); N,N′-disucc
  • the activated polymer or unit thereof may be isolated (e.g., via precipitation, extraction, etc.) and/or stored under suitable conditions (e.g., at low temperature, under argon) following activation, or may be used immediately.
  • the activated polymer or unit thereof may be reacted with an immunomodulatory agent under any suitable conditions. In some cases, the reaction is carried out in the presence of a base and/or catalyst.
  • bases/catalysts include diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP).
  • a polymer or unit thereof e.g., PLA, PLGA having any suitable molecular weight
  • an immunomodulatory agent e.g., R848
  • an activating or coupling reagent which converts the polymer or unit thereof (e.g., PLA, PLGA) to a reactive acylating agent in situ, to give the desired conjugate (e.g., PLA-R848, PLGA-R848).
  • Coupling or activating agents include but are not limited to: activating agents used in the presence of an carbodiimide such as EDC or DCC or DIC, such as 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-7-azabenzotriazole (HOAt), 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HO-Dhbt), N-Hydroxysuccinimide (NHS or HOSu), Pentafluorophenol (PFP);
  • Activating agents without carbodiimide: Phosphonium salts such as O-Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-1-yloxytri
  • Immunomodulatory agents can also be coupled to polymers or units thereof that are terminated in a hydroxyl group.
  • polymers or units thereof include polyethylene glycol, polylactide, polylactide-co-glycolide, polycaprolactone, and other like polyesters, or units thereof.
  • the reaction proceeds as follows where an imide of the general structure (IV) will react with the terminal hydroxyl of the aforementioned polymers or units thereof using a catalyst used in lactone ring opening polymerizations.
  • the resulting reaction product (II) links the amide of the agent to the polymer or unit thereof via an ester bond.
  • the compounds of formula (IV) and (II) are as follows:
  • R 1 ⁇ H, OH, SH, NH 2 , or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylamino
  • R 2 ⁇ H, alkyl, or substituted alkyl
  • Y ⁇ N or C R 3 is absent if Y ⁇ N; or is H, alkyl, substituted alkyl, or combined with R 4 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected if Y ⁇ C
  • R 4 is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylamino when not combined with R 3 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected; or is combined with R 3 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected
  • R 5 is a polymer or unit thereof
  • Catalysts include, but are not limited to, phosphazine bases, 1,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD), and N-methyl-1,4,7-triazabicyclodecene (MTDB).
  • DBU 1,8-diazabicycloundec-7-ene
  • TBD 1,4,7-triazabicyclodecene
  • MTDB N-methyl-1,4,7-triazabicyclodecene
  • suitable solvents include methylene chloride, chloroform, and THF.
  • R 5 —OH contains two hydroxyl groups (e.g., a diol, HO—R 5 —OH), each of which are functionalized by reaction with an imide associated with R848.
  • HO—R 5 —OH is a poly-diol such as poly(hexamethyl carbonate) diol or polycaprolactone diol.
  • one of the diol groups may be protected with a protecting group (e.g., t-butyloxycarbonyl), thus the poly-diol would be a compound of formula HO—R 5 —OP, wherein P is a protecting group.
  • the protecting group may be removed and the second diol group may be reacted with any suitable reagent (e.g., PLGA, PLA).
  • a conjugate (e.g., R848-PLA) can be formed via a one-pot ring-opening polymerization of an immunomodulatory agent (e.g., R848) with a polymer or unit thereof (e.g., D/L-lactide) in the presence of a catalyst, for example, as shown in the following scheme:
  • an immunomodulatory agent e.g., R848
  • a polymer or unit thereof e.g., D/L-lactide
  • the immunomodulatory agent and the polymer or unit thereof may be combined into a single reaction mixture comprising a catalyst.
  • the reaction may proceed at a suitable temperature (e.g., at about 150° C.) and the resulting conjugate may be isolated using commonly known techniques.
  • suitable catalysts include DMAP and tin ethylhexanoate.
  • a conjugate can be formed two-step ring opening polymerization of an immunomodulatory agent (e.g., R848) with one or more polymers or units thereof (e.g., D/L-lactide and glycolide) in the presence of a catalyst, for example, as shown in the following scheme:
  • an immunomodulatory agent e.g., R848
  • polymers or units thereof e.g., D/L-lactide and glycolide
  • the polymers or units thereof may be first combined, and in some cases, heated (e.g., to 135° C.) to form a solution.
  • the immunomodulatory agent may be added to a solution comprising the polymers or units thereof, followed by addition of a catalyst (e.g., tin ethylhexanoate).
  • a catalyst e.g., tin ethylhexanoate
  • the resulting conjugate may be isolated using commonly known techniques.
  • suitable catalysts include DMAP and tin ethylhexanoate.
  • the immunomodulatory agent, antigen, and/or targeting moiety can be covalently associated with a polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, the immunomodulatory agent, antigen, and/or targeting moiety can be noncovalently associated with a polymeric matrix. For example, in some embodiments, the immunomodulatory agent, antigen, and/or targeting moiety can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix. Alternatively or additionally, the immunomodulatory agent, antigen, and/or targeting moiety can be associated with a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc.
  • the immunomodulatory agents can also be encapsulated within the nanocarriers.
  • the nanocarriers therefore, can be of any material that is pH sensitive provided that the resulting inventive synthetic nanocarriers satisfy the dissociation relationships provided herein.
  • Such synthetic nanocarriers are well known in the art and include polyketal nanocarriers, pH sensitive liposomes, acid-swelling, cross-linked nanoparticles, such as those of Griset et al., J. Am. Chem. Soc.
  • the pH sensitive synthetic nanocarriers also include those that comprise polymers that dissolve at a pH below 6 or polymers that swell at an acidic pH.
  • the synthetic nanocarriers are of a non-polyketal material. In other embodiment, the synthetic nanocarriers are not micelles.
  • a polymeric matrix comprises one or more polymers.
  • Polymers may be natural or unnatural (synthetic) polymers.
  • Polymers may be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences.
  • polymers in accordance with the present invention are organic polymers.
  • polymers suitable for use in the present invention include, but are not limited to polyethylenes, polycarbonates (e.g., poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic anhydride)), polyhydroxyacids (e.g., poly( ⁇ -hydroxyalkanoate)), polypropylfumerates, polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, polyamines, and polysaccharides (e.g., chitosan).
  • polyethylenes e.g., poly(1,3-dioxan-2one)
  • polyanhydrides
  • polymers in accordance with the present invention include polymers which have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. ⁇ 177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.
  • FDA U.S. Food and Drug Administration
  • polymers can be hydrophilic.
  • polymers may comprise anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationic groups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group, amine group).
  • a synthetic nanocarrier comprising a hydrophilic polymeric matrix generates a hydrophilic environment within the synthetic nanocarrier.
  • polymers can be hydrophobic.
  • a synthetic nanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobic environment within the synthetic nanocarrier. Selection of the hydrophilicity or hydrophobicity of the polymer may have an impact on the nature of materials that are incorporated (e.g., coupled) within the synthetic nanocarrier.
  • polymers may be modified with one or more moieties and/or functional groups.
  • moieties or functional groups can be used in accordance with the present invention.
  • polymers may be modified with PEG, with a carbohydrate, and/or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301).
  • polymers may be modified with a lipid or fatty acid group.
  • a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid.
  • a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
  • polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA.”
  • exemplary polyesters include, for example, polyhydroxyacids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof.
  • polyesters include, for example, polyanhydrides, poly(ortho ester), poly(ortho ester)-PEG copolymers, poly(caprolactone), poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEG copolymers, poly(ethyleneimine), poly(ethylene imine)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[ ⁇ -(4-aminobutyl)-L-glycolic acid], and derivatives thereof.
  • a polymer may be PLGA.
  • PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid.
  • Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid.
  • the degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio.
  • PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
  • polymers may be one or more acrylic polymers.
  • acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers.
  • the acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammoni
  • polymers can be cationic polymers.
  • cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).
  • Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad.
  • polymers can be degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399).
  • polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
  • polymers can be linear or branched polymers. In some embodiments, polymers can be dendrimers. In some embodiments, polymers can be substantially cross-linked to one another. In some embodiments, polymers can be substantially free of cross-links. In some embodiments, polymers can be used in accordance with the present invention without undergoing a cross-linking step. It is further to be understood that inventive compounds and synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, not comprehensive, list of polymers that can be of use in accordance with the present invention.
  • synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc.
  • synthetic nanocarriers may optionally comprise one or more amphiphilic entities.
  • an amphiphilic entity can promote the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity.
  • amphiphilic entities can be associated with the interior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention.
  • amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20
  • amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is an exemplary, not comprehensive, list of substances with surfactant activity. Any amphiphilic entity may be used in the production of synthetic nanocarriers to be used in accordance with the present invention.
  • synthetic nanocarriers may optionally comprise one or more carbohydrates.
  • Carbohydrates may be natural or synthetic.
  • a carbohydrate may be a derivatized natural carbohydrate.
  • a carbohydrate comprises monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid.
  • a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan.
  • the carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.
  • Synthetic nanocarriers may be prepared using a wide variety of methods known in the art.
  • synthetic nanocarriers can be formed by methods as nanoprecipitation, flow focusing fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art.
  • aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat.
  • synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. Conditions used in preparing synthetic nanocarriers may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.). The method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the materials to be coupled to the synthetic nanocarriers and/or the composition of the polymer matrix.
  • Conditions used in preparing synthetic nanocarriers may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.).
  • the method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the materials to be coupled to the synthetic nanocarriers and/or the composition of the polymer matrix.
  • particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve.
  • Coupling can be achieved in a variety of different ways, and can be covalent or non-covalent. Such couplings may be arranged to be on a surface or within an inventive synthetic nanocarrier. Elements of the inventive synthetic nanocarriers (such as moieties of which an immunofeature surface is comprised, targeting moieties, polymeric matrices, and the like) may be directly coupled with one another, e.g., by one or more covalent bonds, or may be coupled by means of one or more linkers.
  • Additional methods of functionalizing synthetic nanocarriers may be adapted from Published US Patent Application 2006/0002852 to Saltzman et al., Published US Patent Application 2009/0028910 to DeSimone et al., or Published International Patent Application WO/2008/127532 A1 to Murthy et al.
  • Linkers may be used to form amide linkages, ester linkages, disulfide linkages, etc.
  • Linkers may contain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).
  • a linker is an aliphatic or heteroaliphatic linker.
  • the linker is a polyalkyl linker.
  • the linker is a polyether linker.
  • the linker is a polyethylene linker.
  • the linker is a polyethylene glycol (PEG) linker.
  • the linker is a cleavable linker.
  • cleavable linkers include protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavable linkers, etc.
  • the linker is not a cleavable linker.
  • a variety of methods can be used to couple a linker or other element of a synthetic nanocarrier with the synthetic nanocarrier.
  • General strategies include passive adsorption (e.g., via electrostatic interactions), multivalent chelation, high affinity non-covalent binding between members of a specific binding pair, covalent bond formation, etc. (Gao et al., 2005, Curr. Op. Biotechnol., 16:63).
  • click chemistry can be used to associate a material with a synthetic nanocarrier.
  • Non-covalent specific binding interactions can be employed.
  • a particle or a biomolecule can be functionalized with biotin with the other being functionalized with streptavidin. These two moieties specifically bind to each other noncovalently and with a high affinity, thereby associating the particle and the biomolecule.
  • Other specific binding pairs could be similarly used.
  • histidine-tagged biomolecules can be associated with particles conjugated to nickel-nitrolotriaceteic acid (Ni-NTA).
  • compositions of the invention can be made in any suitable manner, and the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method may require attention to the properties of the particular moieties being associated.
  • compositions according to the invention comprise inventive synthetic nanocarriers in combination with pharmaceutically acceptable excipients.
  • the compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms.
  • inventive synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
  • inventive synthetic nanocarriers are manufactured under sterile conditions or are terminally sterilized. This can ensure that resulting composition are sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when subjects receiving synthetic nanocarriers have immune defects, are suffering from infection, and/or are susceptible to infection.
  • inventive synthetic nanocarriers may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.
  • compositions may be administered by a variety of routes of administration, including but not limited to subcutaneous, intramuscular, intradermal, oral, parenteral, intranasal, transmucosal, rectal; ophthalmic, transdermal, transcutaneous or by a combination of these routes.
  • compositions and methods described herein can be used to induce, enhance, stimulate, modulate, or direct an immune response.
  • the compositions and methods described herein can be used in the diagnosis, prophylaxis and/or treatment of conditions such as cancers, infectious diseases, metabolic diseases, degenerative diseases, inflammatory diseases, immunological diseases, or other disorders and/or conditions.
  • the compositions and methods described herein can also be used for the prophylaxis or treatment of an addiction, such as an addiction to nicotine or a narcotic.
  • the compositions and methods described herein can also be used for the prophylaxis and/or treatment of a condition resulting from the exposure to a toxin, hazardous substance, environmental toxin, or other harmful agent.
  • PLA dl-polylactide
  • Resomer R202H from Boehringer-Ingelheim, KOH equivalent acid number of 0.21 mmol/g, intrinsic viscosity (iv): 0.21 dl/g) (10 g, 2.1 mmol, 1.0 eq) was dissolved in dichloromethane (DCM) (35 mL).
  • EDC 2.0 g, 10.5 mmol, 5 eq
  • NHS 1.2 g, 10.5 mmol, 5 eq
  • the solution was concentrated to remove most of DCM and the residue was added to a solution of 250 mL of diethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHS ester.
  • the solvents were removed and the polymer was washed twice with ether (2 ⁇ 200 mL) and dried under vacuum to give PLA-NHS activated ester as a white foamy solid ( ⁇ 8 g recovered, H NMR was used to confirm the presence of NHS ester).
  • the PLA-NHS ester was stored under argon in a below ⁇ 10 C freezer before use.
  • the reaction can be performed in DMF, THF, dioxane, or CHCl3 instead of DCM.
  • DCC can be used instead of EDC (resulting DCC-urea is filtered off before precipitation of the PLA-NHS ester from ether).
  • the amount of EDC or DCC and NHS can be in the range of 2-10 eq of the PLA.
  • PLA (R202H, acid number of 0.21 mmol/g) (2.0 g, 0.42 mmol, 1.0 eq) was dissolved in 10 mL of dry acetonitrile. N,N′-disuccinimidyl carbonate (DSC) (215 mg, 1.26 mmol, 3.0 eq) and catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) were added. The resulting mixture was stirred under argon for 1 day. The resulting solution was concentrated to almost dryness.
  • DSC N,N′-disuccinimidyl carbonate
  • DMAP 4-(N,N-dimethylamino)pyridine
  • PLA (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous DCM and 2.5 mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq) and pentafluorophenol (PFP) (580 mg, 3.15 mmol, 5.0 eq) were added. The resulting solution was stirred at room temperature for 6 days and then concentrated to remove DCM. The resulting residue was added to 250 mL of ether to precipitate out the activated PLA polymer which was washed with ether (2 ⁇ 100 mL) and dried under vacuum to give PLA-PFP activated ester as a white foamy solid (4.0 g).
  • PLA-NHS 1.0 g
  • R848 132 mg, 0.42 mmol
  • DIPEA diisopropylethylamine
  • the resulting solution was heated at 50-60 C for 2 days.
  • the solution was cooled to rt and added to 40 mL of de-ionized (DI) water to precipitate out the polymer product.
  • R848-PLA conjugate was then washed with DI water (40 mL) and ether (2 ⁇ 40 mL) and dried at 30 C under vacuum to give R848-PLA conjugate as a white foamy solid (0.8 g, H NMR showed the conjugation of R848 to PLA via the amide bond).
  • the degree of conjugation (loading) of R848 on the polymer was confirmed by HPLC analysis as follows: a weighed amount of polymer was dissolved in THF/MeOH and treated with 15% NaOH. The resulting hydrolyzed polymer products were analyzed for the amount of R848 by HPLC in comparison with a standard curve.
  • PLA-NHS 1.0 g, 0.21 mmol, 1.0 eq
  • R848 132 mg, 0.42 mmol, 2.0 eq
  • DIPEA 0.15 mL, 0.84 mmol, 4.0 eq
  • DMAP 25 mg, 0.21 mmol, 1.0 eq
  • the polymer was then washed with DI water (40 mL) and ether (2 ⁇ 40 mL) and dried at 30 C under vacuum to give PLA-R848 conjugate as a white foamy solid (0.7 g, 20 mg of the polymer was hydrolyzed in solution of 0.2 mL of THF, 0.1 mL of MeOH and 0.1 mL of 15% NaOH.
  • the amount of R848 on the polymer was determined to be about 35 mg/g by reverse phase HPLC analysis (C18 column, mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA in CH3CN, gradient).
  • PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), DCC (260 mg, 1.26 mmol, 3.0 eq), NHS (145 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), DMAP (77 mg, 0.63 mmol, 1.5 eq) and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF. The mixture was heated at 50-55 C for 3 days. The mixture was cooled to rt and diluted with DCM. The DCC-urea was filtered off and the filtrate was concentrated to remove DCM.
  • PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), EDC (242 mg, 1.26 mmol, 3.0 eq), HOAt (171 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF. The mixture was heated at 50-55 C for 2 days. The solution was cooled to rt and added to water (40 mL) to precipitate out the polymer product which was washed with water (40 mL), ether/MeOH (40 mL/2 mL) and ether (40 mL).
  • the orange colored polymer was dissolved in 4 mL of DCM and the resulting solution was added to 40 mL of ether to precipitate out the polymer without much of the orange color.
  • the light colored polymer was washed with ether (40 mL). After drying under vacuum at 30 C, the desired PLA-R848 conjugate was obtained as a light brown foamy solid (1.5 g).
  • PLA (R202H) (1.0 g, 0.21 mmol, 1.0 eq), EDC (161 mg, 0.84 mmol, 4.0 eq), HOBt.H2O (65 mg, 0.42 mmol, 2.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq), and DIPEA (0.150 mL, 0.84 mmol, 4.0 eq) were dissolved in 2 mL of dry DMF. The mixture was heated at 50-55° C. for 2 days. The solution was cooled to room temperature and added to water (40 mL) to precipitate out the polymer product.
  • the orange colored polymer was dissolved in 2 mL of DCM and the resulting solution was added to 40 mL of ether to precipitate out the polymer which was washed with water/acetone (40 mL/2 mL) and ether (40 mL). After drying under vacuum at 30° C., the desired PLA-R848 conjugate was obtained as an off-white foamy solid (1.0 g, loading of R848 on polymer was about 45 mg/g based on HPLC analysis and confirmed by 1 H NMR).
  • PLGA (75% Lactide)-R848 and PLGA (50% lactide)-R848 were prepared.
  • PCADK is synthesized in a 50 mL two-necked flask, connected to a short-path distilling head.
  • 5.5 mg of re-crystallized p-toluenesulfonic acid (0.029 mmol, Aldrich, St. Louis, Mo.)
  • ethyl acetate a 30 mL benzene solution (kept at 100° C.), which contains 1,4-cyclohexanedimethanol (12.98 g, 90.0 mmol, Aldrich).
  • the ethyl acetate is allowed to boil off, and distilled 2,2-dimethoxypropane (10.94 mL, 90.0 mmol, Aldrich) is added to the benzene solution, initiating the polymerization reaction. Additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) are subsequently added to the reaction every hour for 6 hours via a metering funnel to compensate for 2,2-dimethoxypropane and benzene that is distilled off. After 8 hours, the reaction is stopped by addition of 500 ⁇ L of triethylamine. The polymer is isolated by precipitation in cold hexane (stored at ⁇ 20° C.) followed by vacuum filtration.
  • the molecular weight of PCADK is determined by gel permeation chromatography (GPC) (Shimadzu, Kyoto, Japan) equipped with a UV detector. THF is used as the mobile phase at a flow rate of 1 ml/min. Polystyrene standards from Polymer Laboratories (Amherst, Mass.) are used to establish a molecular weight calibration curve. This compound is used to generate the PCADK particles in all subsequent experiments.
  • R848 may be conjugated to the terminal alcohol groups of the PCADK having molecular weight 6000 via imide ring opening, according to the step 2 shown below.
  • Step 2 Conjugation of PCADK to R848
  • step 2 the polymer from step 1 (12 g, 2.0 ⁇ 10 ⁇ 3 moles) is dissolved in methylene chloride 100 mL, and the lactam of R848 (3.3 g, 8.0 ⁇ 10 ⁇ 3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g, 6 ⁇ 10 ⁇ 3 moles) is added in a single portion. After stiffing at room temperature overnight, a clear solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 11.3 grams (81%) of polymer. A portion is hydrolyzed in acid and the R848 content is determined to be 9% by weight.
  • Imide ring opening is used to attach R854 to the terminal alcohol groups of poly-caprolactonediol of molecular weight 2000.
  • the polycaprolactone diol is purchased from Aldrich Chemical Company, Cat. #189421 and has the following structure:
  • the polycaprolactone diol-R854 conjugate has the following structure:
  • the polymer (5 g, 2.5 ⁇ 10 ⁇ 3 moles) is dissolved in methylene chloride 25 mL and the lactam of R854 (2.4 g, 5.0 ⁇ 10 ⁇ 3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4 ⁇ 10 ⁇ 3 moles) is added in a single portion. After stirring at room temperature for 15 minutes, a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.2 grams (70%) of polymer. A portion is hydrolyzed in acid and the R848 content is determined to be 18.5% by weight.
  • Imide ring opening is used to attach R848 to the terminal alcohol groups of poly-(hexamethylene carbonate)diol of molecular weight 2000.
  • the poly(hexamethylene carbonate) diol is purchased from Aldrich Chemical Company, Cat # 461164, and has the following structure:
  • the poly(hexamethylene carbonate) diol-R848 conjugate has the following structure:
  • the polymer (5 g, 2.5 ⁇ 10 ⁇ 3 moles) is dissolved in methylene chloride 25 mL and the lactam of R848 (2.06 g, 5.0 ⁇ 10 ⁇ 3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4 ⁇ 10 ⁇ 3 moles) is added in a single portion. After stirring at room temperature overnight a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.9 grams (84%) of polymer. NMR is used to determine the R848 content which is determined to be 21%.
  • the solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give 3.59 grams of polylactic acid-R-848 conjugate.
  • a portion of the polymer was hydrolyzed in base and examined by HPLC for R-848 content. By comparison to a standard curve of R-848 concentration vs. HPLC response, it was determined that the polymer contained 4.51 mg of R-848 per gram of polymer.
  • the molecular weight of the polymer was determined by GPC to be about 19,000.
  • the imidazoquinoline (R-848), D/L lactide, and associated glassware were all dried under vacuum at 50° C. for 8 hours prior to use.
  • To a round bottom flask equipped with a stir bar and condenser was added the R-848 (33 mg, 1.05 ⁇ 10 ⁇ 4 moles), and dry toluene (5 mL). This was heated to reflux to dissolve all of the R-848.
  • the solution was stirred under nitrogen and cooled to room temperature to provide a suspension of finely divided R-848.
  • a solution of lithium diisopropyl amide 2.0 M in THF, 50 ⁇ L, 1.0 ⁇ 10 ⁇ 4 moles
  • the polymer was dissolved in methylene chloride (10 mL) and the solution was dripped into stirred hexane (200 mL). The precipitated polymer was isolated by decantation and was dried under vacuum to give 1.47 grams of the polylactic acid—R-848 conjugate as a white solid. A portion of the polymer was hydrolyzed in base and examined by HPLC for R-848 content. By comparison to a standard curve of R-848 concentration vs. HPLC response, it was determined that the polymer contained 10.96 mg of R-848 per gram of polymer.
  • PLA D/L-polylactide
  • DCM dichloromethane
  • EDC 2.0 g, 10.5 mmol, 5 eq
  • NHS 1.2 g, 10.5 mmol, 5 eq
  • the resulting solution is stirred at room temperature for 3 days.
  • the solution is concentrated to remove most of DCM and the residue is added to a solution of 250 mL of diethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHS ester.
  • PLA-NHS activated ester is stored under argon in a below ⁇ 10° C. freezer before use.
  • the reaction can be performed in DMF, THF, dioxane, or CHCl3 instead of DCM.
  • DCC can be used instead of EDC (resulting DCC-urea is filtered off before precipitation of the PLA-NHS ester from ether).
  • the amount of EDC or DCC and NHS can be in the range of 2-10 eq of the PLA.
  • low MW PLGA with 50% to 75% glycolide is converted to the corresponding PLGA-NHS activated ester and is stored under argon in a below ⁇ 10° C. freezer before use.
  • the polymer was dried under vacuum at 30° C. to give an off-white puffy solid (5.0 g). Polymeric structure was confirmed by 1 H NMR in CDCl 3 . A small sample of the polymer was treated with 2 N NaOH aq in THF/MeOH to determine the loading of R848 on the polymer by reverse phase HPLC. The loading of R848 is 3 mg per gram of polymer (0.3% loading-27.5% of theory).
  • t-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid (I) is prepared by ring opening polymerization of glycine N-carboxyanhydride (Aldrich cat #369772) using 6-aminohexanoic acid benzyl ester (Aldrich cat #S33465) by the method of Aliferis et al. ( Biomacromolecules, 5, 1653, (2004)). Protection of the end amino group as the t-BOC carbamate followed by hydrogenation over palladium on carbon to remove the benzyl ester completes the synthesis of BOC protected polyglycine carboxylic acid (I).
  • the polymer is isolated by filtration and the polymer is then washed with 2-propanol (4 ⁇ 25 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 3 days.
  • the polymer is isolated as an off white solid in a yield of 5.1 g (88%).
  • the R848 loading can be determined by NMR is 10.1%.
  • the t-BOC protecting group is removed using trifluoroacetic acid and the resulting polymer is grafted to PLA with carboxyl end groups by conventional methods.
  • Step 1 A t-BOC protected polyglycine/R848 conjugate (5 g) is dissolved in trifluoroacetic acid (25 mL) and this solution is warmed at 50° C. for one hour. After cooling, the trifluoroacetic acid is removed under vacuum and the residue is triturated in ethyl acetate (25 mL). The polymer is isolated by filtration and is washed well with 2-propanol. After drying under vacuum there is obtained 4.5 grams of polymer as an off white solid.
  • Step 2 A mixture of PLGA (Lakeshores Polymers, MW ⁇ 5000, 7525DLG1A, acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous DMF (100 mL) is stirred at RT under argon for 50 minutes.
  • the polymer from above (1.4 g, 7 mmol) dissolved in dry DMF (20 mL) is added, followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture is stirred at RT for 6 h and then at 50-55° C. overnight (16 h).
  • DIPEA diisopropylethylamine
  • the poly(hexamethylene carbonate) diol is purchased from Aldrich Chemical Company, Cat # 461164.
  • the polymer (5 g, 2.5 ⁇ 10 ⁇ 3 moles) is dissolved in methylene chloride 25 mL and the lactam of 2-pentyl-8-hydroxy-9-benzyladenine (2.05 g, 5.0 ⁇ 10 ⁇ 3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4 ⁇ 10 ⁇ 3 moles) is added in a single portion. After stirring at room temperature overnight a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL), and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.5 grams (78%) of polymer. NMR is used to determine the benzyladenine content which is 18%.
  • a 3-nicotine-PEG-PLA polymer was synthesized as follows:
  • the cotinine/PEG polymer (0.20 gm, 5.7 ⁇ 10 ⁇ 5 moles) was dissolved in dry tetrahydrofuran (10 mL) under nitrogen and the solution was stirred as a solution of lithium aluminum hydride in tetrahydrofuran (1.43 mL of 2.0M, 2.85 ⁇ 10 ⁇ 3 moles) was added. The addition of the lithium aluminum hydride caused the polymer to precipitate as a gelatinous mass. The reaction was heated to 80° C. under a slow stream of nitrogen and the tetrahydrofuran was allowed to evaporate. The residue was then heated at 80° C. for 2 hours. After cooling, water (0.5 mL) was cautiously added.
  • the flask was placed in an oil bath set at 120° C., and once the lactide had dissolved, tin ethylhexanoate (5.5 mg, 1.36 ⁇ 10 ⁇ 5 moles) was added. The reaction was allowed to proceed at 120° C. for 16 hours. After cooling to room temperature, water (15 mL) was added and stirring was continued for 30 minutes. Methylene chloride (200 mL) was added, and after agitation in a separatory funnel, the phases were allowed to settle. The methylene chloride layer was isolated and dried over anhydrous magnesium sulfate. After filtration to remove the drying agent, the filtrates were evaporated under vacuum to give the polymer as a colorless foam.
  • Resiquimod (aka R848) was synthesized according to the synthesis provided in Example 99 of U.S. Pat. No. 5,389,640 to Gerster et al.
  • R848 was conjugated to PLA by a method provided above, and the PLA structure was confirmed by NMR.
  • PLA-PEG-nicotine conjugate was prepared according to Example 31.
  • PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 North Normandy Drive, Orlando, Va. 23805).
  • Ovalbumin peptide 323-339 was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part #406-4565).
  • Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL), solution #3 (0.25 to 0.5 mL) and solution #4 (0.1 mL) were combined in a small vial and the mixture was sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • solution #5 2.0 mL
  • sonication at 35% amplitude for 40 seconds using the Branson Digital Sonifier 250 forms the second emulsion.
  • This was added to a beaker containing phosphate buffer solution (30 mL) and this mixture was stirred at room temperature for 2 hours to form the nanoparticles.
  • nanoparticle dispersion a portion of the nanoparticle dispersion (7.4 mL) was transferred to a centrifuge tube and spun at 5,300 g for one hour, supernatant was removed, and the pellet was re-suspended in 7.4 mL of phosphate buffered saline. The centrifuge procedure was repeated and the pellet was re-suspended in 2.2 mL of phosphate buffered saline for a final nanoparticle dispersion of about 10 mg/mL.
  • Ovalbumin peptide 323-339 a 17 amino acid peptide known to be a T cell epitope of Ovalbumin protein, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.)
  • Resiquimod (aka R848) was synthesized according to a method provided in U.S. Pat. No. 6,608,201.
  • PLA-R848, resiquimod was conjugated to PLA with a molecular weight of approximately 2,500 Da according to a method provided above.
  • PLGA-R848, resiquimod was conjugated to PLGA with a molecular weight of approximately 4,100 Da according to a method provided above.
  • PS-1826 DNA oligonucleotide with fully phosphorothioated backbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodium counter-ion was purchased from Oligos Etc (9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.)
  • PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodium counter-ion was purchased from Oligos Etc. (9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.) ⁇
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 ⁇ L 2A.)
  • PLA with an inherent viscosity of 0.71 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 ⁇ L 7A.)
  • PLA with an inherent viscosity of 0.19 dL/g was purchased from Boehringer Ingelheim Chemicals, Inc. (Petersburg, Va. Product Code R202H.)
  • PLA-PEG-nicotine with a molecular weight of approximately 18,500 to 22,000 Da was prepared according to a method provided above.
  • PLA-PEG-R848 with a molecular weight of approximately 15,000 Da was synthesized was prepared according to a method provided above.
  • Solution 1A Ovalbumin peptide 323-339 @ 35 mg/mL in dilute hydrochloric acid aqueous solution.
  • the solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 1B Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloric acid aqueous solution.
  • the solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 2A 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride.
  • the solution was prepared by first preparing two separate solutions at room temperature: 0.21-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride.
  • the final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 2B 0.71-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride.
  • the solution was prepared by first preparing two separate solutions at room temperature: 0.71-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride.
  • the final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 2C 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride.
  • the solution was prepared by first preparing two separate solutions at room temperature: 0.19-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride.
  • the final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 3A Oligonucleotide (either PS-1826 or P0-1826) @ 200 mg/ml in purified water.
  • the solution was prepared by dissolving oligonucleotide in purified water at room temperature.
  • Solution 4A Same as Solution #2A.
  • Solution 4B Same as Solution #2B.
  • Solution 5A Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
  • W1/O2 was prepared by combining solution 1 and solution 2 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • W3/O4 was prepared by combining solution 3 and solution 4 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • a third emulsion with two inner emulsion ([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.5 ml of each primary emulsion (W1/O2 and W3/O4) and solution 5 and sonicating at 30% amplitude for 40 to 60 seconds using the Branson Digital Sonifier 250.
  • the third emulsion was added to a beaker containing 70 mM phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow for the methylene chloride to evaporate and for the nanocarriers to form.
  • a portion of the nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube and spinning at 13,823 g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. The washing procedure was repeated and the pellet was re-suspended in phosphate buffered saline for a final nanocarrier dispersion of about 10 mg/mL.
  • oligonucleotide and peptide in the nanocarrier were determined by HPLC analysis.
  • Solution 1A Ovalbumin peptide 323-339 @ 69 mg/mL in de-ionized water. The solution was prepared by slowly adding ovalbumin peptide to the water while mixing at room temperature.
  • Solution 1B Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloric acid aqueous solution.
  • the solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 1C Oligonucleotide (PS-1826) @ 50 mg/ml in purified water. The solution was prepared by dissolving oligonucleotide in purified water at room temperature.
  • Solution 1D Ovalbumin peptide 323-339 @ 17.5 mg/mL in dilute hydrochloric acid aqueous solution.
  • the solution was prepared by dissolving ovalbumin peptide @ 70 mg/ml in 0.13N hydrochloric acid solution at room temperature and then diluting the solution with 3 parts purified water per one part of starting solution.
  • Solution 2A R848 @ 10 mg/ml and 0.19-IV PLA @ 100 mg/mL in pure methylene chloride prepared at room temperature.
  • Solution 2B PLA-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 2C PLGA-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 2D PLA-PEG-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 3A PLA-PEG-nicotine @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 4A 0.19-IV PLA @ 100 mg/mL in pure methylene chloride prepared at room temperature.
  • Solution 5A Polyvinyl alcohol @ 50 mg/mL in de-ionized water.
  • Solution 5B Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
  • the water in oil (W/O) primary emulsion was prepared by combining solution 1 and solution 2, solution 3, and solution 4 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
  • the water/oil/water (W/O/W) double emulsion was prepared by adding solution 5 to the primary emulsion and sonicating at 30% to 35% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • the double emulsion was added to a beaker containing phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow for the methylene chloride to evaporate and for the nanocarriers to form.
  • a portion of the nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube and spinning at 5,000 to 9,500 RPM for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. The washing procedure was repeated and the pellet was re-suspended in phosphate buffered saline for a final nanocarrier dispersion of about 10 mg/mL.
  • CpG immunological agent
  • Agilent 1100 reverse phase HPLC on Agilent 1100 system at 260 nm equipped with Waters XBridge C-18 (2.5 micron particle, 50 ⁇ 4.6 mm ID (part No. 186003090), column temp. 600 C) using mobile phase A of 2% acetonitrile in 100 mM TEA-acetic acid buffer, pH about 8.0 and mobile B as 90% acetonitrile, 10% water (column equilibrated at 5% B, increased to 55% B in 8.5 min, then ramped to 90% B to 12 minutes. Strength of B was rapidly decreased to 5% in one minute and equilibrated until stop time, 16 minutes. The flow rate was 1 mL/min until end of the method, 16 minutes).
  • Nicotine analog was measured using reverse phase HPLC on Agilent 1100 system at 254 nm equipped with Waters X-Bridge C-18 (5 micron particle, 100 ⁇ 4.6 mm ID, column temp at 400 C) using Mobile Phase A (MPA) of 95% water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90% acetonitrile/10% water/0.09% TFA (gradient: column was equilibrated at 5% B increased to 45% B in 14 minutes. Then ramped up to 95% B from 14 to 20 minutes. Mobile B strength was quickly decreased back to 5% and requilibrated until the end of the method. The flow rate of the method was maintained at 0.5 ml/min with total run time of 25 minutes.
  • the NC suspension was centrifuged @14000 rpm for about 15-30 minutes depending on particle size.
  • the collected pellets were treated with 200 uL of conc. NH 4 OH (8 M) for 2 h with agitation until the solution turns clear.
  • a 200 uL of 1% TFA was added to neutralize the mixture solution, which brought the total volume of the pellet solution to 200 uL.
  • An aliquot of 50 uL of the solution was diluted with MPA(or water) to 200 uL and analyzed on HPLC as above to determine the amount present in the pellets.
  • NC suspension from the manufacture (about 10 mg/mL suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes depending on particle size.
  • the collected pellets were re-suspended with 500 uL of water and sonicated for 30 minutes to fully disperse the particles.
  • the NC was then heated at 600° C. for 10 minutes. Additional 200 uL of 1 N NaOH was added to the mixture, heated for another 5 minutes where the mixture becomes clear.
  • the hydrolyzed NC solution was centrifuged briefly at 14000 rpm. A final 2 ⁇ dilution of the clear solution using water was then made and assayed on the reverse HPLC described above.
  • Encapsulated T Cell Antigens e.g., Ova Peptide, or Human Peptide, TT2pDT5t
  • NC suspension from the manufacture (about 10 mg/mL suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes.
  • 100 uL of acetonitrile was added to the pellets to dissolve the polymer components of the NC.
  • the mixture was vortexed and sonicated for 1 to 5 minutes.
  • 100 uL 0.2% TFA was added to the mixture to extract the peptides and sonicated for another 5 minutes to ensure the break down of the aggregates.
  • the mixture was centrifuged at 14000 rpm for 15 minutes to separate any insoluble materials (e.g., polymers).
  • a 50 uL aliquot of the supernatant diluted with 150 uL of MPA (or water) was taken and assayed on the reverse phase HPLC as described above.
  • the total amount of R848 and ova peptide present in the nanoparticles was as shown in Table 1.
  • An aqueous suspension of the tested synthetic nanocarriers was then diluted to a final stock volume of 4.4 mL with PBS.
  • a 200 ⁇ L aliquot was immediately removed from each of the NP sample and centrifuged @ 14000 rpm in a microcentrifuge tubes using a Microcentrifuge (Model: Galaxy 16). 100 ⁇ L of supernatant was removed and diluted to 200 ⁇ L in HPLC Mobile Phase A (MPA) and assayed for the amount of R848 and ova peptide released on the reverse phase HPLC.
  • MPA HPLC Mobile Phase A
  • T0 sample a 200 ⁇ L aliquot was removed from each of the samples and centrifuged @ 6000 rpm for 20 minutes and the supernatant was removed. The residue nanoparticles was resuspended in 200 uL of citrate buffer and centrifuged @ 14000 rpm for 15 minutes. 100 uL of the supernatant was removed and diluted to 200 uL with MPA and assayed for R848 and peptide as above.
  • the remaining pellets (conjugated R848 samples only) from each sample was treated with 200 uL of conc. NH4OH (8 M) for 3 h with mixing. After the mixture was settled, 200 uL of 1% TFA was added to bring total volume of the pellet to 400 uL. An aliquot of 50 uL of the solution was diluted with MPA to 200 uL and analyzed on HPLC as above to determine the amount of R848 and ova peptide that remained in the pellet after in vitro release to close the mass balance. For unconjugated samples, the sample was diluted with TFA in acetonitrile and assayed as above for R848 and peptide.
  • HPLC grade water EMD—#WX0008-1.
  • antigen e.g., ova peptide, T cell antigen
  • immunostimulatory agents e.g., R848, CpG
  • PBS phosphate buffered saline solution
  • the release of R848 from the nanocarrier composed of conjugated R848 and the ova peptide was achieved by exchanging desired amount of the aqueous suspension of the tested synthetic nanocarriers obtained from the manufacture (e.g., about 10 mg/mL in PBS) into the same volume of the appropriate release media (Citrate buffer 100 mM) via centrifugation and re-suspension.
  • desired amount of the aqueous suspension of the tested synthetic nanocarriers obtained from the manufacture e.g., about 10 mg/mL in PBS
  • the appropriate release media e.g., about 10 mg/mL in PBS
  • a 150 ⁇ L aliquot was immediately removed from NC suspension prior placing the NC suspension to 37° C. thermal chamber and centrifuged @ 14000 rpm in microcentrifuge tubes using a microcentrifuge (Model: Galaxy 16). 100 ⁇ L of the supernatant was removed and diluted to 200 ⁇ L with HPLC Mobile Phase A (MPA) or water and assayed for the amount of R848 and ova peptide released on the reverse phase HPLC.
  • MPA HPLC Mobile Phase A
  • the remaining pellets from each time point were treated with 100 uL of NH 4 OH (8 M) for 2 h (or more) with agitation until solution turn clear.
  • a 100 uL of 1% TFA was added to neutralize the mixture, which brought the total volume of the pellet solution to 200 uL.
  • the release of CpG was determined similar to the measurement of R848 and ova peptide in terms of sample preparation and monitored time points. However, the amount of the CpG in the release media was assayed by the reverse phase HPLC method described above.
  • NC-Nic was a composition of nanocarriers exhibiting nicotine on the outer surface and, for all groups of mice except for Group 1, carrying CpG-1826 (thioated) adjuvant, which was released from the nanocarriers at different rates.
  • the nanocarriers were prepared according to a method provided above. Serum anti-nicotine antibodies were then measured on days 26 and 40. EC 50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 4 .
  • the Group 1 mice were administered NC-Nic w/o CpG-1826 containing Ova peptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic.
  • the Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.2% CpG-1826; release rate at 24 hours: 4.2 ⁇ g CpG per mg of NC.
  • the Group 3 mice were administered NC-Nic containing polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours: 15 ⁇ g CpG per mg of NC. Release was determined at a pH of 4.5.
  • results shown in FIG. 4 demonstrate that entrapment of adjuvant into nanocarriers is beneficial for the immune response against NC-associated antigen, and, furthermore, that the higher release rate of entrapped CpG adjuvant from within the nanocarriers (NC) at 24 hours produced an immune response, which was elevated compared to one induced by NC with a slower release rate of CpG adjuvant (a TLR9 agonist).
  • NC-Nic was a composition of nanocarriers exhibiting nicotine on the outer surface and carrying one of two forms of CpG-1826 adjuvant.
  • the nanocarriers were prepared according to a method provided above. EC 50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 5 .
  • the Group 1 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 6.2% CpG-1826 (thioated); release rate at 24 hours: 16.6 ⁇ g CpG per mg of NC.
  • the Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.2% CpG-1826 (thioated); release rate at 24 hours: 13.2 ⁇ g CpG per mg of NC.
  • the Group 3 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.9% CpG-1826 (phosphodiester or PO, non-thioated); release rate at 24 hours: 19.6 ⁇ g CpG per mg of NC.
  • the Group 4 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 8.5% CpG-1826 (PO, non-thioated); release rate at 24 hours: 9.3 ⁇ g CpG per mg of NC. Release was determined at a pH of 4.5.
  • mice Groups of five mice were immunized three times (subcutaneously, hind limbs) at 2-week intervals (days 0, 14 and 28) with 100 ⁇ g of NC-Nic and serum anti-nicotine antibodies were then measured on days 26, 40 and 54.
  • the nanocarriers were prepared according to a method provided above. EC 50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 6 .
  • the Group 1 mice were administered NC-Nic containing ova peptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, but without adjuvant.
  • the Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 1.0% R848; of which 92% is released at 2 hours and more than 96% is released at 6 hours.
  • the Group 3 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and 25% were PLA-PEG-Nic, and 1.3% R848, of which 29.4% is released at 6 hours and 67.8% is released at 24 hours.
  • mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and 25% were PLA-PEG-Nic, and 1.4% of R848, of which 20.4% is released at 6 hours and 41.5% is released at 24 hours.
  • the Group 5 mice were administered NC-Nic containing ova peptide, polymers, 25% of which were PLA-PEG-R848, 50% PLA, and 25% were PLA-PEG-Nic, and 0.7% of R848; of which less than 1% is released at 24 hours. Release was determined at a pH of 4.5.
  • mice Groups of five mice were immunized three times (subcutaneously, hind limbs) at 2-week intervals (days 0, 14 and 28) with 100 ⁇ g of NC-Nic (nanocarrier exhibiting nicotine on the outer surface) with entrapped PO-CpG or not containing entrapped PO-CpG admixed with free PO-CpG.
  • the synthetic nanocarriers were prepared according to methods provided above. Serum anti-nicotine antibodies were then measured in both groups on days 26 and 40. EC 50 for anti-nicotine antibodies as determined in standard ELISA against polylysine-nicotine are shown in FIG. 7 .
  • mice were immunized with a NC-Nic with 1826 PO-CpG and MHC-II helper peptide from ovalbumin (Ov-II) encapsulated (6.6% PO-CpG; 2.3% Ov-II).
  • the group 2 mice were immunized with a NC-Nic with 0.7% of entrapped Ov-II admixed with 20 ⁇ g of free 1826 PO-CpG.

Abstract

This invention relates to compositions, and related methods, of synthetic nanocarriers that target sites of action in cells, such as antigen presenting cells (APCs), and comprise immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner. Also disclosed are compositions and methods relating to synthetic nanocarriers that encapsulate labile immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner.

Description

    RELATED APPLICATIONS
  • This application claims the benefit under 35 U.S.C. §119 of U.S. provisional applications 61/217,129, 61/217,117, 61/217,124, and 61/217,116, each filed May 27, 2009, the contents of each of which are incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • This invention relates to compositions, and related methods, of synthetic nanocarriers that target sites of action in cells, such as antigen presenting cells (APCs), and comprise immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner. The invention additionally relates to protection of labile immunomodulatory agents by means of their encapsulation in synthetic nanocarriers.
    • BACKGROUND
  • Immunomodulatory agents are used to produce immune responses in subjects. Stimulation of the immune system, which includes stimulation of either or both innate immunity and adaptive immunity, is a complex phenomenon that can result in either protective or adverse physiologic outcomes for the host. In recent years there has been increased interest in the mechanisms underlying innate immunity, which is believed to initiate and support adaptive immunity. This interest has been fueled in part by the recent discovery of a family of highly conserved pattern recognition receptor proteins known as Toll-like receptors (TLRs) believed to be involved in innate immunity as receptors for pathogen-associated molecular patterns (PAMPs).
  • Compositions and methods useful for modulating innate immunity are therefore of great interest, as they may affect therapeutic approaches to conditions involving inflammation, allergy, asthma, infection, cancer, and immunodeficiency, etc.
  • It is at times advantageous to couple such agents to delivery vehicles. However, information regarding how the release of such agents, especially labile immunomodulatory agents, from delivery vehicles can be controlled and what kind of release provides for optimal in vivo effects is lacking.
  • There is a need for new delivery vehicles for delivering immunomodulatory agents that allow for optimal release as well as related methods.
    • SUMMARY OF THE INVENTION
  • Aspects of the invention relate to compositions comprising synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier, wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship: IArel(4.5)24%/IArel(7.4)24%≧1.2, wherein IArel(4.5)24% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers, and wherein IArel(7.4)24% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers.
  • In some embodiments, the immunomodulatory agent is coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety. In certain embodiments, the immunomodulatory agent is encapsulated within the synthetic nanocarrier. In some embodiments, the immunomodulatory agent comprises a labile immunomodulatory agent such as an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages. In certain embodiments, the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod.
  • In some embodiments, the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications. In some embodiments, the immunomodulatory agent is an adjuvant. In certain embodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist such as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.
  • In some embodiments, the TLR agonist is an immunostimulatory nucleic acid such as an immunostimulatory DNA or immunostimulatory RNA. In certain embodiments, the immunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid that comprises one or more stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the adjuvant comprises a universal T-cell antigen.
  • In some embodiments, the synthetic nanocarriers further comprise a B cell antigen and/or a T cell antigen. In certain embodiments, the synthetic nanocarriers further comprise an antigen presenting cell (APC) targeting feature. In some embodiments, the synthetic nanocarriers comprise one or more biodegradable polymers. In some embodiments, the immunomodulatory agent is coupled to the one or more biodegradable polymers via the immunomodulatory agent coupling moiety. In certain embodiments, the biodegradable polymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).
  • In some embodiments, the biodegradable polymers have a weight average molecular weight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography. In certain embodiments, the immunomodulatory agent coupling moiety comprises an amide bond. In some embodiments, the immunomodulatory agent coupling moiety comprises an ester bond.
  • In some embodiments, the synthetic nanocarriers comprise lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles, nanoparticles that comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidal nanoparticles.
  • Aspects of the invention relate to compositions comprising synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier, wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship: IA(4.5)24/IA(4.5)6≧1.2, wherein IA(4.5)24 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours taken as an average across a sample of the synthetic nanocarriers, and wherein IA(4.5)6 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 6 hours taken as an average across a sample of the synthetic nanocarriers.
  • In some embodiments, the immunomodulatory agent comprises a labile immunomodulatory agent encapsulated within the synthetic nanocarrier. In certain embodiments, the labile immunomodulatory agent comprises an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages. In certain embodiments, the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod. In some embodiments, the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • Further aspects of the invention relate to compositions comprising synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier, wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship: 6≦IA(4.5)24/IA(4.5)6≧1.2, wherein IA(4.5)24 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours taken as an average across a sample of the synthetic nanocarriers, and wherein IA(4.5)6 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 6 hours taken as an average across a sample of the synthetic nanocarriers.
  • In some embodiments, the immunomodulatory agent comprises a labile immunomodulatory agent encapsulated within the synthetic nanocarrier. In some embodiments, the labile immunomodulatory agent comprises an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages. In certain embodiments, the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, a imidazoquinoline amine, imiquimod, or resiquimod.
  • In some embodiments, the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In some embodiments, the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications. In certain embodiments, the immunomodulatory agent is coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety. In some embodiments, the immunomodulatory agent is encapsulated within the synthetic nanocarrier.
  • In some embodiments, the immunomodulatory agent is an adjuvant. In certain embodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist such as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist. In certain embodiments, the TLR agonist is an immunostimulatory nucleic acid such as an immunostimulatory DNA or immunostimulatory RNA.
  • In some embodiments, the immunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid that comprises one or more stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In certain embodiments, the adjuvant comprises a universal T-cell antigen. In some embodiments, the synthetic nanocarriers further comprise a B cell antigen and/or a T cell antigen.
  • In some embodiments, the synthetic nanocarriers further comprise an antigen presenting cell (APC) targeting feature. In certain embodiments, the synthetic nanocarriers comprise one or more biodegradable polymers. In some embodiments, the immunomodulatory agent is coupled to the one or more biodegradable polymers via the immunomodulatory agent coupling moiety. In certain embodiments, the biodegradable polymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).
  • In some embodiments, the biodegradable polymers have a weight average molecular weight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography. In certain embodiments, the immunomodulatory agent coupling moiety comprises an amide bond. In some embodiments, the immunomodulatory agent coupling moiety comprises an ester bond.
  • In some embodiments, the synthetic nanocarriers comprise lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles, nanoparticles that comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidal nanoparticles. In certain embodiments, compositions associated with the invention further comprise a pharmaceutically acceptable excipient.
  • Further aspects of the invention relate to compositions comprising a vaccine comprising any of the compositions associated with the invention.
  • Further aspects of the invention involve methods comprising administering any of the compositions associated with the invention to a subject. In some embodiments, the composition is in an amount effective to induce or enhance an immune response. In some embodiments, the subject has cancer, an infectious disease, a non-autoimmune metabolic disease, a degenerative disease, or an addiction.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 demonstrates the release of resiquimod (R848) from synthetic nanocarrier formulations at pH 7.4, 37° C.
  • FIG. 2 demonstrates the release of R848 from synthetic nanocarrier formulations at pH 4.5, 37° C.
  • FIG. 3 demonstrates the release of R848 from synthetic nanocarrier formulations at pH 7.4 and pH 4.5 at 24 hours.
  • FIG. 4 shows the level of antibody induction by synthetic nanocarriers with a CpG-containing immunostimulatory nucleic acid (Groups 2 and 3) as compared to the level of antibody induction by synthetic nanocarriers without the CpG-containing immunostimulatory nucleic acid (Group 1).
  • FIG. 5 shows the level of antibody induction by synthetic nanocarriers that release a phosphodiester, non-thioated CpG-containing immunostimulatory nucleic acid or a thioated CpG-containing immunostimulatory nucleic acid.
  • FIG. 6 shows the level of antibody induction by synthetic nanocarriers that release R848 at different rates.
  • FIG. 7 shows the level of antibody induction by synthetic nanocarriers carrying entrapped phosphodiester (PO) CpG, designated as NC-Nic/PO-CpG.
  • FIG. 8 shows the release of entrapped PO-CpG from nanocarriers at a pH of 4.5 versus pH 7.5. The data demonstrates that a labile immunomodulatory agent, such as PO-CpG, is protected by encapsulation within a synthetic nanocarrier. Such a labile agent can be released at a desired site of action with a pH of 4.5 (e.g., in the endosome/lysosome) with low levels of release occurring at a pH of 7.4 (e.g., generally the pH outside of the endosome/lysosome).
    •  DETAILED DESCRIPTION
  • Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting of the use of alternative terminology to describe the present invention.
  • All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.
  • As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a polymer” includes a mixture of two or more such molecules, reference to “a solvent” includes a mixture of two or more such solvents, reference to “an adhesive” includes mixtures of two or more such materials, and the like.
    • INTRODUCTION
  • This invention is useful in that it provides a way to release immunomodulatory agents more directly at the sites of action in cells of interest, in particular antigen presenting cells, which would result in beneficial immune response and/or reduce off-target effects and toxicity, as the majority of the release of the immunomodulatory agents would be at a site of action in the cells of interest. This is of particular interest for the delivery of adjuvants. The controlled release properties offer for the first time a controlled way of delivering immunomodulatory agents to the immune cells of interest and allow for a more precise intervention on the immune system, including the ability to release immunomodulatory agents over an extended period. All of this leads to a very tunable system to get the optimum release of immunomodulatory agent such that it will release primarily at a site of action in the desired cells.
  • The inventors have further recognized that coupling labile immunomodulatory agents within the inventive synthetic nanocarriers through encapsulating the labile immunomodulatory agents within the inventive synthetic nanocarriers, and providing a controlled way of delivering labile immunomodulatory agents to immune cells of interest, preferably over an extended period, results in targeted delivery of the labile immunomodulatory agents while minimizing off-target effects of the immunomodulatory agents, especially off-target effects associated with systemic administration of the immunomodulatory agents. Additionally, this approach can enhance the performance of labile immunomodulatory agents having a short half-life of elimination that otherwise might not have a desirable level of pharmacological activity.
  • In one embodiment, the invention relates to certain oligonucleotides. Recently, there have been a number of reports describing the immunostimulatory effect of certain types of nucleic acid molecules, including CpG nucleic acids, GU rich ssRNA and double-stranded RNA. Of note, it was recently reported that Toll-like receptor 9 (TLR9) recognizes bacterial DNA and oligonucleotides containing a CpG motif wherein the cytosine is unmethylated. Hemmi H et al. (2000) Nature 408:740-5; Bauer S. et al. (2001) Proc Natl Acad Sci USA 98:9237-42. The effects of CpG containing oligonucleotides on immune modulation have been described extensively in U.S. patents such as U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116; and 6,218,371, and published international patent applications, such as WO98/37919, WO98/40100, WO98/52581, and WO99/56755. The entire immunostimulatory nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5′-CG-3′ must be unmethylated.
  • Natural DNA oligonucleotides contain phosphodiester linkages that are rapidly cleaved by nucleases found in the extracellular environment. Yu, D., et al., Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties. Biochem Biophys Res Commun, 2002. 297(1): p. 83-90 (“Yu et al.”); Heeg, K., et al., Structural requirements for uptake and recognition of CpG oligonucleotides. Int J Med Microbiol, 2008. 298(1-2): p. 33-8 (“Heeg et al.”). Such natural oligonucleotides may be considered labile immunomodulatory agents. Accordingly, methods of chemically stabilizing the linkages by replacing the phosphodiester linking group with a phosphorothioate group have been extensively reported in the literature. See U.S. Pat. No. 6,811,975—Phosphorothioate Oligonucleotides Having Modified Internucleoside Linkages.
  • Phosphorothioate CpG containing oligonucleotides have been administered systemically as vaccine adjuvants. Yu et al. However, systemic administration of stabilized CpG oligonucleotides can result in off-target immunostimulatory effects, such as general inflammation, non-specific activation of lymphocytes, and flu-like symptoms. Haas, T., et al., Sequence independent interferon-alpha induction by multimerized phosphodiester DNA depends on spatial regulation of Toll-like receptor-9 activation in plasmacytoid dendritic cells. Immunology, 2009. 126(2): p. 290-8 (“Haas et al.”). Accordingly, such oligonucleotides may be usefully incorporated in the practice of the present invention, as is described in more detail below.
  • The inventors have unexpectedly and surprisingly discovered that the problems and limitations noted above can be overcome by practicing the invention disclosed herein. In particular, the inventors have unexpectedly discovered that it is possible to provide, together with related methods, a composition comprising: synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier; wherein the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship:

  • IArel(4.5)t%/IArel(7.4)t%≧1.2;
  • wherein IArel(4.5)t% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers; and wherein IArel(7.4)t% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers; and wherein t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 hours.
  • In some embodiments, the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: IArel(4.5)t%/IArel(7.4)t%≧1.3, IArel(4.5)t%/IArel(7.4)t%≧1.4, IArel(4.5)t%/IArel(7.4)t%≧1.5, IArel(4.5)t%/IArel(7.4)t%≧1.6, IArel(4.5)t%/IArel(7.4)t%≧1.7, IArel(4.5)t%/IArel(7.4)t%≧1.8, IArel(4.5)t%/IArel(7.4)t%≧1.9, IArel(4.5)t%/IArel(7.4)t% 2, IArel(4.5)t%/IArel(7.4)t%≧2.2, IArel(4.5)t%/IArel(7.4)t%≧2.5, IArel(4.5)t%/IArel(7.4)t%≧2.7, IArel(4.5)t%/IArel(7.4)t%≧3, IArel(4.5)t%/IArel(7.4)t%≧3.5, IArel(4.5)t%/IArel(7.4)t%≧4, IArel(4.5)t%/IArel(7.4)t%≧4.5, IArel(4.5)t%/IArel(7.4)t% 5, IArel(4.5)t%/IArel(7.4)t%≧5.5, IArel(4.5)t%/IArel(7.4)t%≧6, IArel(4.5)t%/IArel(7.4)t%≧6.5, IArel(4.5)t%/IArel(7.4)t%≧7, IArel(4.5)t%/IArel(7.4)t%≧7.5, IArel(4.5)t%/IArel(7.4)t%≧8, IArel(4.5)t%/IArel(7.4)t%≧8.5, IArel(4.5)t%/IArel(7.4)t% 9, IArel(4.5)t%/IArel(7.4)t%≧9.5, IArel(4.5)t%/IArel(7.4)t%≧10, IArel(4.5)t%/IArel(7.4)t%≧10.5, or IArel(4.5)t%/IArel(7.4)t%≧11, wherein IArel(4.5)t%, IArel(7.4)t%, and t are as defined above.
  • In other embodiments, the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: 2≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 2.5≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 3≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 3.5≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 4≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 4.5≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 5≦IArel(4.5)t%/IArel(7.4)t% 1.2, 6≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 7≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 8≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 9≦IArel(4.5)t%/IArel(7.4)t%≧1.2, 10≦IArel(4.5)t%/IArel(7.4)t% 1.2, 10≦IArel(4.5)t%/IArel(7.4)t%≧2, 10≦IArel(4.5)t%/IArel(7.4)t%≧2.5, 10≦IArel(4.5)t%/IArel(7.4)t%≧3, 10≦IArel(4.5)t%/IArel(7.4)t%≧3.5, 10≦IArel(4.5)t%/IArel(7.4)t%≧4, 10≦IArel(4.5)t%/IArel(7.4)t%≧4.5, 10≦IArel(4.5)t%/IArel(7.4)t%≧5, 10≦IArel(4.5)t%/IArel(7.4)t%≧6, 10≦IArel(4.5)t%/IArel(7.4)t%≧7, 10≦IArel(4.5)t%/IArel(7.4)t%≧8, 10≦IArel(4.5)t%/IArel(7.4)t%≧9, 3≦IArel(4.5)t%/IArel(7.4)t%≧2, 4≦IArel(4.5)t%/IArel(7.4)t%≧3, 5≦IArel(4.5)t%/IArel(7.4)t%≧4, 6≦IArel(4.5)t%/IArel(7.4)t%≧5, 7≦IArel(4.5)t%/IArel(7.4)t%≧6, 8≦IArel(4.5)t%/IArel(7.4)t%≧7, or 9≦IArel(4.5)t%/IArel(7.4)t%≧8, wherein IArel(4.5)t%, IArel(7.4)t%, and t are as defined above. In some embodiments, t is 24 hours.
  • Accordingly, this invention relates to compositions and methods comprising synthetic nanocarriers that release immunomodulatory agents at significantly different rates at neutral and acidic pH. In delivering immunomodulatory agents, to have the most potent effect it is desirable to have the majority of the immunomodulatory agent released inside APCs where they can have a desired effect. When immunomodulatory agents are injected in free form, or when they are released from a synthetic nanoparticle outside the APCs, only a small portion of that immunomodulatory agent finds its way to the APCs, while the rest diffuses through the body, where the immune stimulation would be less and may result in deleterious effects. The inventive synthetic nanocarriers provided herein are preferentially taken up by APCs. Upon being taken up by the APC, the synthetic nanocarriers are presumed to be endocytosed into an endosomal/lysosomal compartment where the pH becomes more acidic, as opposed to the neutral pH outside the cells. Under these conditions, the immunomodulatory agent exhibits a pH sensitive dissociation from the synthetic nanocarrier (e.g., from an immunomodulatory agent coupling moiety) and is released from the synthetic nanocarrier. The immunomodulatory agent is then free to interact with receptors associated with the endosome/lysosome and stimulate a desired immune response. The property of the inventive synthetic nanocarriers of having lower release of immunomodulatory agents at or about neutral pH, or in embodiments at or about physiological pH (i.e., pH=7.4), but increased release at or about a pH of 4.5 is desirable for it targets the immunomodulatory agents to the endosomal/lysosomal compartment of APCs to which the synthetic nanocarriers target.
  • The immunomodulatory agents can be coupled to the synthetic nanocarriers by any of a number of methods. Generally, the coupling can be a result of bonding between the immunomodulatory agent and the synthetic nanocarrier. This bonding can result in the immunomodulatory agent being attached to the surface of the synthetic nanocarrier and/or contained within (encapsulated) the synthetic nanocarrier. In some embodiments, however, the immunomodulatory agent is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than bonding to the synthetic nanocarrier.
  • When coupling occurs as a result of bonding between the immunomodulatory agent and synthetic nanocarrier, the coupling occurs via an immunomodulatory agent coupling moiety. An immunomodulatory agent coupling moiety can be any moiety through which an immunomodulatory agent is bonded to a synthetic nanocarrier. Such moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunomodulatory agent to the synthetic nanocarrier. Such molecules include linkers or polymers or a unit thereof. For example, the immunomodulatory agent coupling moiety can comprise a charged polymer to which an immunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically binds. As another example, the immunomodulatory agent coupling moiety can comprise a polymer or unit thereof to which the immunomodulatory agent is covalently bonded.
  • In some embodiments, the polymer or unit thereof comprises a polyester, polycarbonate, polyamide, or polyether, or unit thereof. In other embodiments, the polymer or unit thereof comprises poly(ethylene glycol) (PEG), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof. In some embodiments, it is preferred that the polymer is biodegradable. Therefore, in these embodiments, it is preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or unit thereof, the polymer comprises a block-co-polymer of a polyether and a biodegradable polymer such that the polymer is biodegradable. In other embodiments, the polymer does not solely comprise a polyether or unit thereof, such as poly(ethylene glycol) or unit thereof. The immunomodulatory agent coupling moiety as provided herein, therefore, can comprise one of the aforementioned polymers or a unit thereof (e.g., a lactide or glycolide).
  • In some embodiments, for use as part of a synthetic nanocarrier, the polymer of the compounds or conjugates provided herein is insoluble in water at pH=7.4 and at 25° C., is biodegradable, or both. In other embodiments, the polymer is insoluble in water at pH=7.4 and at 25° C. but soluble at pH=4.5 and at 25° C. In still other embodiments, the polymer is insoluble in water at pH=7.4 and at 25° C. but soluble at pH=4.5 and at 25° C. and biodegradable. In other embodiments, any of the polymers provided herein can have a weight average molecular weight, as determined by gel permeation chromatography, of about 800 Da to 10,000 Da (e.g., 2,000 Da).
  • In one embodiment, the immunomodulatory agent is an adjuvant, such as an imidazoquinoline. Imidazoquinolines include compounds, such as imiquimod and resiquimod (also known as R848). Such adjuvants can be coupled to a polymer as provided above. As an example, resiquimod was conjugated to poly-lactic acid (PLA) polymer of ˜2000 Da. In in vitro release studies, such an embodiment demonstrated an increase in R848 release of 3- to 6-fold when the pH was dropped from 7.4 to 4.5. Table 1 lists the compositions of the particles tested. These included two formulations that encapsulated R848, 2 formulations with the PLA coupled covalently to R848 through the R848 amine, and four formulations with PLA coupled covalently to R848 (via a ring opening method). In all formulations, the release of R848 was significantly increased at the lower pH. The encapsulated release rate is much faster than the conjugated release rates, and there are also differences in release rates between the conjugation methods.
  • TABLE 1
    Formulation Targets With A Covalent R848
    Ova PLA- PLA-R848
    R848 peptide PEG- conjugate PLA (15-20K,
    Formulation load* load NIC type** BI R202H) Chemistry
    1 E1.5% 1.1-2.2% 25% 75%
    2 E1.5%++ 1.1-2.2% 25% 75%
    3 C75% 0.15-0.31% 25% Method 1 Amine
    4 C75% 0.15-0.31% 25% Method 1 Amine
    5 C75% 0.15-0.31% 25% Method 5 ROP-hi MW
    6 C75% 0.15-0.31% 25% Method 5 ROP-lo MW
    7 C50% 0.15-0.31% 25% Method 5 25% ROP-lo MW
    8 C25% 0.15-0.31% 25% Method 5 50% ROP-lo MW
    *C = covalent R848; E = encapsulation of R848
  • Although the above example was with PLA, immunomodulatory agents, such as R848, can be coupled to other polymers or units thereof, such as those provided above and elsewhere herein including polylactide-co-glycolide (PLGA) block co-polymer or unit thereof. Immunomodulatory agents, such as R848, can be coupled to such polymers or units thereof by an amide or ester bond. Examples of methods for effecting such coupling are provided elsewhere herein and in the EXAMPLES.
  • The inventors have also unexpectedly discovered that it is possible to provide, together with related methods, a composition comprising:
  • synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier; wherein the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship:

  • IA(4.5)t1/IA(4.5)t2≧1.2;
  • wherein IA(4.5)t1 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t1 hours taken as an average across a sample of the synthetic nanocarriers; and wherein IA(4.5)t2 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t2 hours taken as an average across a sample of the synthetic nanocarriers; and wherein t1 is 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours; t2 is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 hours; and t1>t2. In some embodiments, t1 is 24 hours, and t2 is 6 hours.
  • In some embodiments, the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: IA(4.5)t1/IA(4.5)t2≧1.5, IA(4.5)t1/IA(4.5)t2≧2, IA(4.5)t1/IA(4.5)t2≧2.5, IA(4.5)t1/IA(4.5)t2≧3, IA(4.5)t1/IA(4.5)t2≧3.5, IA(4.5)t1/IA(4.5)t2≧4, IA(4.5)t1/IA(4.5)t2≧4.5, IA(4.5)t1/IA(4.5)t2≧5, IA(4.5)t1/IA(4.5)t2≧6, IA(4.5)t1/IA(4.5)t2≧7, IA(4.5)t1/IA(4.5)t2≧8, IA(4.5)t1/IA(4.5)t2≧9, or IA(4.5)t1/IA(4.5)t2≧10; wherein IA(4.5)t1, IA(4.5)t2, t1, and t2 are as defined above. In some embodiments, t1 is 24 hours, and t2 is 6 hours.
  • In other embodiments, the immunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates from the synthetic nanocarrier according to the following relationship: 10≦IA(4.5)t1/IA(4.5)t2≧1.2, 10≦IA(4.5)t1/IA(4.5)t2≧2, 10≦IA(4.5)t1/IA(4.5)t2≧2.5, 10≦IA(4.5)t1/IA(4.5)t2≧3, 10≦IA(4.5)t1/IA(4.5)t2≧3.5, 10≦IA(4.5)t1/IA(4.5)t2≧4, 10≦IA(4.5)t1/IA(4.5)t2≧4.5, 10≦IA(4.5)t1/IA(4.5)t2≧5, 10≦IA(4.5)t1/IA(4.5)t2≧6, 10≦IA(4.5)t1/IA(4.5)t2≧7, 10≦IA(4.5)t1/IA(4.5)t2≧8, 10≦IA(4.5)t1/IA(4.5)t2≧9, 9≦IA(4.5)t1/IA(4.5)t2≧1.2, 8≦IA(4.5)t1/IA(4.5)t2≧1.2, 7≦IA(4.5)t1/IA(4.5)t2≧1.2, 6≦IA(4.5)t1/IA(4.5)t2≧1.2, 5≦IA(4.5)t1/IA(4.5)t2≧1.2, 4.5≦IA(4.5)t1/IA(4.5)t2≧1.2, 4≦IA(4.5)t1/IA(4.5)t2≧1.2, 3.5≦IA(4.5)t1/IA(4.5)t2≧1.2, 3≦IA(4.5)t1/IA(4.5)t2≧1.2, 2.5≦IA(4.5)t1/IA(4.5)t2≧1.2, 2≦IA(4.5)t1/IA(4.5)t2≧1.2, 1.5≦IA(4.5)t1/IA(4.5)t2≧1.2, 3≦IA(4.5)t1/IA(4.5)t2≧2, 4≦IA(4.5)t1/IA(4.5)t2≧3, 5≦IA(4.5)t1/IA(4.5)t2≧4, 6≦IA(4.5)t1/IA(4.5)t2≧5, 7≦IA(4.5)t1/IA(4.5)t2≧6, 8≦IA(4.5)t1/IA(4.5)t2≧7, or 9≦IA(4.5)t1/IA(4.5)t2≧8; wherein IA(4.5)t1, IA(4.5)t2, t1, and t2 are as defined above. In some embodiments, t1 is 24 hours, and t2 is 6 hours.
  • Inventive synthetic nanocarriers have also been shown to exhibit the property of augmenting a humoral immune response to a specific antigen. Such augmented humoral immune response has been found to be elevated, in some embodiments, with faster release of immunomodulatory agent. In one embodiment, the immunomodulatory agent is a CpG-containing immunostimulatory nucleic acid, and the CpG-containing immunostimulatory nucleic acid is encapsulated within a synthetic nanocarrier. In in vitro studies, described further below in the EXAMPLES, it was found that optimal release of the CpG-containing immunostimulatory nucleic acids from synthetic nanocarriers produced an elevated humoral immune response to nicotine, which was also coupled to the synthetic nanocarriers. In some embodiments, such optimal release was found to better augment an antibody response to an antigen.
  • Optimal release is the dissociation of the immunomodulatory agent from the synthetic nanocarrier that produces the best levels of desired effect(s). In some embodiments, the desired effect is an immediate immune response of a desired level (i.e., one that occurs soon after the administration of the synthetic nanocarrier). Generally, an immediate immune response is one measured on the order of seconds, minutes, or a few hours. In other embodiments, the desired effect is an immune response of a desired level that occurs after a few hours. In still other embodiments, the desired effect is an immune response of a desired level that is sustained for an extended period of time, such as for 1, 2, 5, 10, 15 or more hours. In other embodiments, the extended period of time is for 1, 2, 5, 10, 15, 20, 25, 30 or more days. In further embodiments, the extended period of time is for 1, 2, 5, 10 or more months. In further embodiments, the extended period of time is for 1, 2, 5, 10 or more years. In some embodiments, a composition of synthetic nanocarriers that provides optimal release is one wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to one of the above relationships.
  • In embodiments, an immunomodulatory agent, preferably a labile immunodmodulatory agent, that dissociates from the synthetic nanocarrier at an intermediate rate satisfies the following relationship: 6≦IA(4.5)t1/IA(4.5)t2≧1.2, 5≦IA(4.5)t1/IA(4.5)t2≧1.2, 4≦IA(4.5)t1/IA(4.5)t2≧1.2, 3≦IA(4.5)t1/IA(4.5)t2≧1.2, 2≦IA(4.5)t1/IA(4.5)t2≧1.2, 6≦IA(4.5)t1/IA(4.5)t2≧2, 6≦IA(4.5)t1/IA(4.5)t2≧2.5, 6≦IA(4.5)t1/IA(4.5)t2≧3, 6≦IA(4.5)t1/IA(4.5)t2≧3.5, 6≦IA(4.5)t1/IA(4.5)t2≧4, 6≦IA(4.5)t1/IA(4.5)t2≧5, 4≦IA(4.5)t1/IA(4.5)t2≧1.5, 3.5≦IA(4.5)t1/IA(4.5)t2≧1.5, 3≦IA(4.5)t1/IA(4.5)t2≧1.5, 2.5≦IA(4.5)t1/IA(4.5)t2≧1.5, 5≦IA(4.5)t1/IA(4.5)t2≧2, 4≦IA(4.5)t1/IA(4.5)t2≧2, or 3≦IA(4.5)t1/IA(4.5)t2≧2; wherein IA(4.5)t1, IA(4.5)2, t1, and t2 are as defined above. In some embodiments, t1 is 24 hours, and t2 is 6 hours.
  • As another example, resiquimod was encapsulated within a synthetic nanocarrier. In in vitro studies, described further below in the EXAMPLES, it was found that resiquimod contained in the synthetic nanocarriers augmented humoral immune response against nicotine also coupled to the synthetic nanocarriers. It was also found that an intermediate release of the resiquimod from the synthetic nanocarriers was optimal, as it resulted in a higher level of antibody induction than fast or slow release of the resiquimod.
  • Accordingly, the synthetic nanocarriers provided herein can also comprise one or more antigens. The antigens can be B cell antigens or T cell antigens or a combination of both. Such antigens can be coupled to the synthetic nanocarriers such that they are present on the surface of the synthetic nanocarriers, encapsulated within the nanocarriers or both, in some embodiments. In embodiments, the immunomodulatory agent augments an immune response to such an antigen. As mentioned above, the antigen can also be coupled to the synthetic nanocarriers. In other embodiments, however such as antigen is not coupled to the synthetic nanocarriers. In some of these embodiments, such an antigen can be coadministered to a subject. In still other of these embodiments, such an antigen is not coadministered to the subject.
    • DEFINITIONS
  • “Adjuvant” means an agent that does not constitute a specific antigen, but boosts the strength and longevity of immune response to an antigen. Such adjuvants may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL® (AS04), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIXT™, emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+MPL®), liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic® block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments. In embodiments, adjuvants comprise agonists for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof. In other embodiments, adjuvants comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited adjuvants comprise imidazoquinolines; such as resiquimod (also known as R848); adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company); immunostimulatory DNA; or immunostimulatory RNA. In specific embodiments, synthetic nanocarriers incorporate as adjuvants compounds that are agonists for toll-like receptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8 agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al., including but not limited to imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. Preferred adjuvants comprise imiquimod and resiquimod. In specific embodiments, an adjuvant may be an agonist for the DC surface molecule CD40. In certain embodiments, a synthetic nanocarrier incorporates an adjuvant that promotes DC maturation (needed for effective priming of naive T cells) and the production of cytokines, such as type I interferons, which in turn stimulate antibody and cytotoxic immune responses against desired antigen. In embodiments, adjuvants also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA or poly I:C (a TLR3 stimulant), and/or those disclosed in F. Heil et al., “Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8” Science 303(5663), 1526-1529 (2004); J. Vollmer et al., “Immune modulation by chemically modified ribonucleosides and oligoribonucleotides” WO 2008033432 A2; A. Forsbach et al., “Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway” WO 2007062107 A2; E. Uhlmann et al., “Modified oligoribonucleotide analogs with enhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., “Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections” WO 2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods” WO 2003086280 A2. In some embodiments, an adjuvant may be a TLR-4 agonist, such as bacterial lipopolysacccharide (LPS), VSV-G, and/or HMGB-1. In some embodiments, adjuvants may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725. In specific embodiments, synthetic nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9, such as immunostimulatory oligonucleotide molecules comprising 5′-CG-3′ motifs, wherein the C is unmethylated, which induce type I interferon secretion, and stimulate T and B cell activation leading to increased antibody production and cytotoxic T cell responses (Krieg et al., CpG motifs in bacterial DNA trigger direct B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thi) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al. CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J. Immunol. 1998. 160:870-876; Lipford et al., Bacterial DNA as immune cell activator. Trends Microbiol. 1998. 6:496-500. In some embodiments, adjuvants may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, adjuvants may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, adjuvants may be activated components of immune complexes. The adjuvants also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some embodiments, adjuvants are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.
  • “Administering” or “administration” means providing a drug to a patient in a manner that is pharmacologically useful.
  • “APC targeting feature” means one or more portions of which the inventive synthetic nanocarriers are comprised that target the synthetic nanocarriers to professional antigen presenting cells (“APCs”), such as but not limited to dendritic cells, SCS macrophages, follicular dendritic cells, and B cells. In embodiments, APC targeting features may comprise immunofeature surface(s) and/or targeting moieties that bind known targets on APCs. In embodiments, APC targeting features may comprise one or more B cell antigens present on a surface of synthetic nanocarriers. In embodiments, APC targeting features may also comprise one or more dimensions of the synthetic nanoparticles that is selected to promote uptake by APCs.
  • In embodiments, targeting moieties for known targets on macrophages (“Mphs”) comprise any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on macrophages (i.e., subcapsular sinus-Mph markers). Exemplary SCS-Mph markers include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, MRP-1); CD11a (LFA-1α, α L Integrin chain); CD11b (αM Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CDw12 (p90-120); CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X, SSEA-1,3-FAL); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a,b,c β-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein); CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV, PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin, Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49a (VLA-1α, α1 Integrin); CD49b (VLA-2α, gpla, α2 Integrin); CD49c (VLA-3α, α3 Integrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α, α6 Integrin, gplc); CD50 (ICAM-3); CD51 (Integrin α, VNR-α, Vitronectin-Rα); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL, P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP Ma, β3 Integrin); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1, gp55, NGA, LAMP-3, ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); CD65s (Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD74 (Ii, invariant chain); CD75 (sialo-masked Lactosamine); CD75S (α2,6 sialylated Lactosamine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 (4F9, C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor, FcaR); CD91 (α2M-R, LRP); CDw92 (p70); CDw93 (GR11); CD95 (APO-1, FAS, TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2); CD99R (CD99 Mab restricted); CD100 (SEMA4D); CD101 (IGSF2, P126, V7); CD102 (ICAM-2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); CD112 (HveB, PRR2, PVRL2, Nectin2); CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms, CSF-1R, M-CSFR); CD116 (GMCSFRα); CDw119 (IFNγR, IFNγRA); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); CD123 (IL-3Rα); CD124 (IL-4Rα); CD127 (p90, IL-7R, IL-7Rα); CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb, CSCR2, (Tentatively renamed as CD182)); CD130 (gp130); CD131 (Common β subunit); CD132 (Common γ chain, IL-2Rγ); CDw136 (MSP-R, RON, p158-ron); CDw137 (4-1BB, ILA); CD139; CD141 (Thrombomodulin, Fetomodulin); CD147 (Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260, DEP-1); CD155 (PVR); CD156a (CD156, ADAMS, MS2); CD156b (TACE, ADAM17, cSVP); CDw156C (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD165 (AD2, gp37); CD168 (RHAMM, 1HABP, HMMR); CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1, (Formerly known as CD128a)); CD182 (CXCR2, (Formerly known as CD128b)); CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195 (CCRS); CDw197 (CCR7 (was CDw197)); CDw198 (CCR8); CD204 (MSR); CD205 (DEC-25); CD206 (MMR); CD207 (Langerin); CDw210 (CK); CD213a (CK); CDw217 (CK); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD226 (DNAM-1, PTA1); CD230 (Prion Protein (PrP)); CD232 (VESP-R); CD244 (2B4, P38, NAIL); CD245 (p220/240); CD256 (APRIL, TALL2, TNF (ligand) superfamily, member 13); CD257 (BLYS, TALL1, TNF (ligand) superfamily, member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNBF-R superfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD277 (BT3.1, B7 family: Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1, TOLL-like receptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4, TOLL-like receptor 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase, β3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e (CMRF-35L1); CD302 (DCL1); CD305 (LAIR1); CD312 (EMR2); CD315 (CD9P1); CD317 (BST2); CD321 (JAM1); CD322 (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 (gp 110, Macrosialin); and/or mannose receptor; wherein the names listed in parentheses represent alternative names.
  • In embodiments, targeting moieties for known targets on dendritic cells (“DCs”) comprise any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on DCs (i.e., a DC marker). Exemplary DC markers include, but are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b (αM Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA-4α, α4 Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (LFA-3); CD64 (FcγRI); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′ nucloticlase); CD74 (Ii, invariant chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15); CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97 (BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFRα); CD120a (TMFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD123 (IL-3Rα); CD139; CD148 (HPTP-η, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP); CD157 (Mo5, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, 1HABP, HMMR); CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE); CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CD206 (MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 (DCSIGN); CDw218a (IL18Rα); CDw218b (IL8Rβ); CD227 (MUC1, PUM, PEM, EMA); CD230 (Prion Protein (PrP)); CD252 (OX40L, TNF (ligand) superfamily, member 4); CD258 (LIGHT, TNF (ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily, member 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276 (B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-like receptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase β3 submit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68 (gp110, Macrosialin); class II MHC; BDCA-1; Siglec-H; wherein the names listed in parentheses represent alternative names.
  • In embodiments, targeting can be accomplished by any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on B cells (i.e., B cell marker). Exemplary B cell markers include, but are not limited to, CD1c (M241, R7); CD1d (R3); CD2 (E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9 (p24, DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD11b (αM Integrin chain, CR3, Mol, C3niR, Mac-1); CD11c (αX Integrin, P150, 95, AXb2); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a, b, c β-subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R, C3dR); CD22 (BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24 (BBA-1, HSA); CD25 (Tac antigen, IL-2Rα, p55); CD26 (DPP IV ectoeneyme, ADA binding protein); CD27 (T14, S152); CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD35 (CR1, C3b/C4b receptor); CD37 (gp52-40); CD38 (ADPribosyl cyclase, T10); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD44 (ECMRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophilin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gpla, α2 Integrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 Integrin); CD50 (ICAM-3); CD52 (CAMPATH-1, HES); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′-nuciotidase); CD74 (Ii, invariant chain); CD75 (sialo-masked Lactosamine); CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk antigen, BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, B29); CD80; CD81 (TAPA-1); CD82 (4F9, C33, IA4, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j (ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD102 (ICAM-2); CD108 (SEMA7A, JMH blood group antigen); CDw119 (IFNγR, IFNγRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); CD124 (IL-4Rα); CD130 (gp130); CD132 (Common γ chain, IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN, M6, OX47); CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD166 (ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD167a (DDR1, trkE, cak); CD171 (L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197 (CCR7 (was CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CDw210 (CK); CD213a (CK); CDw217 (CK); CDw218a (IL18Rα); CDw218b (IL18R(3); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD225 (Leu13); CD226 (DNAM-1, PTA1); CD227 (MUC1, PUM, PEM, EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232 (VESP-R); CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD267 (TACI, TNF-R superfamily, member 13B); CD268 (BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA, TNF-R superfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3.1.B7 family: Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD307 (IRTA2); CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321 (JAM1); CD322 (JAM2); CDw327 (Siglec6, CD33L); CD68 (gp 100, Macrosialin); CXCR5; VLA-4; class II MHC; surface IgM; surface IgD; APRL; and/or BAFF-R; wherein the names listed in parentheses represent alternative names. Examples of markers include those provided elsewhere herein.
  • In some embodiments, B cell targeting can be accomplished by any targeting moiety that specifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that is prominently expressed and/or present on B cells upon activation (i.e., activated B cell marker). Exemplary activated B cell markers include, but are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80 (B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BLKDD/F12); CD125 (IL-5Rα); CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152 (CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253 (TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1); CD289 (TLR9, TOLL-like receptor 9); and CD312 (EMR2); wherein the names listed in parentheses represent alternative names. Examples of markers include those provided elsewhere herein.
  • “B cell antigen” means any antigen that naturally is or could be engineered to be recognized by a B cell, and triggers (naturally or being engineered as known in the art) an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell receptor on a B cell). In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is not also a B cell antigen. B cell antigens include, but are not limited to proteins, peptides, small molecules, and carbohydrates. In some embodiments, the B cell antigen is a non-protein antigen (i.e., not a protein or peptide antigen). In some embodiments, the B cell antigen is a carbohydrate associated with an infectious agent. In some embodiments, the B cell antigen is a glycoprotein or glycopeptide associated with an infectious agent. The infectious agent can be a bacterium, virus, fungus, protozoan, parasite or prion. In some embodiments, the B cell antigen is a poorly immunogenic antigen. In some embodiments, the B cell antigen is an abused substance or a portion thereof. In some embodiments, the B cell antigen is an addictive substance or a portion thereof. Addictive substances include, but are not limited to, nicotine, a narcotic, a cough suppressant, a tranquilizer, and a sedative. In some embodiments, the B cell antigen is a toxin, such as a toxin from a chemical weapon or natural sources, or a pollutant. The B cell antigen may also be a hazardous environmental agent. In other embodiments, the B cell antigen is an alloantigen, an allergen, a contact sensitizer, a degenerative disease antigen, a hapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an addictive substance, a xenoantigen, or a metabolic disease enzyme or enzymatic product thereof.
  • “Biodegradable polymer” means a polymer that degrades over time when introduced into the body of a subject. Biodegradable polymers, include but are not limited to, polyesters, polycarbonates, polyketals, or polyamides. Such polymers may comprise poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone. In some embodiments, the biodegradable polymer comprises a block-co-polymer of a polyether, such as poly(ethylene glycol), and a polyester, polycarbonate, or polyamide or other biodegradable polymer. In embodiments, the biodegradable polymer comprises a block-co-polymer of poly(ethylene glycol) and poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone. In some embodiments, however, the biodegradable polymer does not comprise a polyether, such as poly(ethylene glycol), or consists solely of the polyether. Generally, for use as part of a synthetic nanocarrier the biodegradable polymer is insoluble in water at pH=7.4 and at 25° C. The biodegradable polymer, in embodiments, have a weight average molecular weight ranging from about 800 to about 50,000 Daltons, as determined using gel permeation chromatography. In some embodiments, the weight average molecular weight is from about 800 Daltons to about 10,000 Daltons, preferably from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography. In other embodiments, the weight average molecular weight is from 1000 Daltons to 10,000 Daltons, as determined by gel permeation chromatography. In an embodiment, the biodegradable polymer does not comprise polyketal.
  • “Coadministered” means administering two or more drugs to a subject in a manner that is correlated in time. In embodiments, coadministration may occur through administration of two or more drugs in the same dosage form. In other embodiments, coadministration may encompass administration of two or more drugs in different dosage forms, but within a specified period of time, preferably within 1 month, more preferably within 1 week, still more preferably within 1 day, and even more preferably within 1 hour.
  • “Couple” or “Coupled” or “Couples” (and the like) means attached to or contained within the synthetic nanocarrier. In some embodiments, the coupling is covalent. In some embodiments, the covalent coupling is mediated by one or more linkers, polymers or a unit thereof. In some embodiments, the coupling is non-covalent. In some embodiments, the non-covalent coupling is mediated by charge interactions, affinity interactions, metal coordination, physical adsorption, hostguest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. In embodiments, the coupling may arise in the context of encapsulation within the synthetic nanocarriers, using conventional techniques. Any of the aforementioned couplings may be arranged to be on a surface or within an inventive synthetic nanocarrier.
  • “Derived” means adapted or modified from the original source. For example, as a non-limiting example, a peptide antigen derived from an infectious strain may have several non-natural amino acid residues substituted for the natural amino acid residues found in the original antigen found in the infectious strain. The adaptations or modifications may be for a variety of reasons, including but not limited to increased specificity, easier antigen processing, or improved safety.
  • “Dosage form” means a drug in a medium, carrier, vehicle, or device suitable for administration to a subject.
  • “Effective amount” of an inventive composition is that amount effective for a certain purpose. For example, when the effective amount is for a therapeutic purpose the amount is effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a disease, disorder, and/or condition provided herein.
  • “Encapsulate” means to enclose within a synthetic nanocarrier, preferably enclose completely within a synthetic nanocarrier. Most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. Encapsulation is distinct from absorbtion, which places most or all of a substance on a surface of a synthetic nanocarrier, and leaves the substance exposed to the local environment external to the synthetic nanocarrier.
  • “Exhibits a pH sensitive dissociation” means that a coupling between two entities, such as the immunomodulatory agent and the synthetic nanocarrier or immunomodulatory agent coupling moiety, is significantly reduced or eliminated by a change in environmental pH. In embodiments, relevant pH sensitive dissociations may satisfy any of the relationships or combinations thereof provided herein.
  • “IArel(4.5)t%” is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for t hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers. In embodiments, t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 hours. In preferred embodiments, t is 24 hours.
  • “IArel(7.4)t%” is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for t hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers. In embodiments, t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 hours. In preferred embodiments, t is 24 hours.
  • “IA(4.5)t1” is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at pH 4.5 for t1 hours taken as an average across a sample of the synthetic nanocarriers. “IA(4.5)t2” is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at pH 4.5 for t2 hours taken as an average across a sample of the synthetic nanocarriers. t1 is 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours; t2 is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 hours; and t1>t2. In preferred embodiments, t1 is 24 hours, and t2 is 6 hours.
  • “Immunomodulatory agent” means an agent that modulates an immune response. “Modulate”, as used herein, refers to inducing, enhancing, stimulating, or directing an immune response. Such agents include adjuvants that stimulate (or boost) an immune response to an antigen but is not an antigen or derived from an antigen. In some embodiments, the immunomodulatory agent is on the surface of the synthetic nanocarrier and/or is incorporated within the synthetic nanocarrier. In embodiments, the immunomodulatory agent is coupled to the synthetic nanocarrier via a polymer or unit thereof.
  • In some embodiments, all of the immunomodulatory agents of a synthetic nanocarrier are identical to one another. In some embodiments, a synthetic nanocarrier comprises a number of different types of immunomodulatory agents. In some embodiments, a synthetic nanocarrier comprises multiple individual immunomodulatory agents, all of which are identical to one another. In some embodiments, a synthetic nanocarrier comprises exactly one type of immunomodulatory agent. In some embodiments, a synthetic nanocarrier comprises exactly two distinct types of immunomodulatory agents. In some embodiments, a synthetic nanocarrier comprises greater than two distinct types of immunomodulatory agents.
  • “Immunomodulatory agent coupling moiety” is any moiety through which an immunomodulatory agent is bonded to a synthetic nanocarrier. Such moieties include covalent bonds, such as an amide bond or ester bond, as well as separate molecules that bond (covalently or non-covalently) the immunomodulatory agent to the synthetic nanocarrier. Such molecules include linkers or polymers or a unit thereof. For example, the immunomodulatory agent coupling moiety can comprise a charged polymer to which an immunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically bonds. As another example, the immunomodulatory agent coupling moiety can comprise a polymer or unit thereof to which the immunomodulatory agent covalently bonds. In some embodiments, the moiety comprises a polyester. In other embodiments, the moiety comprises poly(ethylene glycol), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or a polycaprolactone. The moiety may also comprise a unit of any of the foregoing polymers, such as a lactide or glycolide.
  • “Labile immunomodulatory agent(s)” means immunomodulatory agent or agents that are unstable under physiological conditions, and degrade to the point where they are no longer pharmacologically active. In embodiments, labile immunomodulatory agents are observed to have systemic half-lives of elimination of less than 24 hours, preferably less than 12 hours, more preferably less than 10 hours, even more preferably less than 8 hours, and still more preferably less than 6 hours. In embodiments, labile immunomodulatory agents comprise imidazoquinolines, adenine derivative, or oligonucleotides that comprise 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages. In embodiments, the imidazoquinolines comprise imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, imidazoquinoline amines, imiquimod or resiquimod.
  • “Maximum dimension of a synthetic nanocarrier” means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of a synthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would be substantially identical, and would be the size of its diameter. Similarly, for a cubic synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of its height, width or length, while the maximum dimension of a synthetic nanocarrier would be the largest of its height, width or length. In an embodiment, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 100 nm. In an embodiment, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 μm. Preferably, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 110 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, and more preferably still equal to or greater than 150 nm. Preferably, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 3 μm, more preferably equal to or less than 2 μm, more preferably equal to or less than 1 μm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In preferred embodiments, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm. Measurement of synthetic nanocarrier sizes is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (e.g. using a Brookhaven ZetaPALS instrument).
  • “Obtained” means taken without adaptation or modification from the original source. For example, in embodiments, antigens obtained from a source may comprise the original amino acid residue sequence found in that source. In other embodiments, for example, antigens obtained from a source may comprise the original molecular structure found in that source.
  • “Oligonucleotide” means a nucleotide molecule having from 6 to 100 nucleotides, preferably from 8 to 75 nucleotides, more preferably from 10 to 50 nucleotides, still more preferably from 15 to 25 nucleotides, even still more preferably 20 nucleotides. In an embodiment according to the invention, oligonucleotides comprise less than 100 nucleotides, preferably less than 50 nucleotides, more preferably less than 25 nucleotides, and still more preferably less than 10 nucleotides. Any cytosine nucleotides (“C”) present in a 5′-CG-3′ sequence of which the oligonucleotide may be comprised are unmethylated, C present in parts of the oligonucleotides other than in a 5′-CG-3′ sequence of which the oligonucleotide may be comprised may be methylated, or may be unmethylated. In embodiments, inventive oligonucleotides comprise a backbone comprising one or more unstabilized internucleotide linkages (meaning internucleotide linkages that are unstable under physiological conditions). “Unstabilized internucleotide linkage” means a linkage between two nucleotides of which the oligonucleotide is comprised that is not chemically modified to stabilize the backbone, or is chemically modified to destabilize the backbone of the oligonucleotide under physiological conditions. An example of an unstablized internucleotide linkage is a phophodiester internucleotide linkage. In embodiments, the inventive oligonucleotides' backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions. In embodiments, the inventive oligonucleotides' backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
  • “Pharmaceutically acceptable excipient” means a pharmacologically inactive substance added to an inventive composition to further facilitate administration of the composition. Examples, without limitation, of pharmaceutically acceptable excipients include calcium carbonate, calcium phosphate, various diluents, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • “Release Rate” means the rate that an entrapped immunomodulatory agent flows from a composition, such as a synthetic nanocarrier, into a surrounding media in an in vitro release test. First, the synthetic nanocarrier is prepared for the release testing by placing into the appropriate in vitro release media. This is generally done by exchanging the buffer after centrifugation to pellet the synthetic nanocarrier and reconstitution of the synthetic nanocarriers using a mild condition. The assay is started by placing the sample at 37° C. in an appropriate temperature-controlled apparatus. A sample is removed at various time points.
  • The synthetic nanocarriers are separated from the release media by centrifugation to pellet the synthetic nanocarriers. The release media is assayed for the immunomodulatory agent that has dispersed from the synthetic nanocarriers. The immunomodulatory agent is measured using HPLC to determine the content and quality of the immunomodulatory agent. The pellet containing the remaining entrapped immunomodulatory agent is dissolved in solvents or hydrolyzed by base to free the entrapped immunomodulatory agent from the synthetic nanocarriers. The pellet-containing immunomodulatory agent is then also measured by HPLC to determine the content and quality of the immunomodulatory agent that has not been released at a given time point.
  • The mass balance is closed between immunomodulatory agent that has been released into the release media and what remains in the synthetic nanocarriers. Data are presented as the fraction released or as the net release presented as micrograms released over time.
  • “Subject” means an animal, including mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; and the like.
  • “Synthetic nanocarrier(s)” means a discrete object that is not found in nature, and that possesses at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are expressly included as synthetic nanocarriers.
  • Synthetic nanocarriers include polymeric nanoparticles. In some embodiments, synthetic nanocarriers can comprise one or more polymeric matrices. The synthetic nanocarriers, however, can also include other nanomaterials and may be, for example, lipid-polymer nanoparticles. In some embodiments, a polymeric matrix can be surrounded by a coating layer (e.g., liposome, lipid monolayer, micelle, etc.). In some embodiments, the synthetic nanocarrier is not a micelle. In some embodiments, a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some embodiments, the various elements of the synthetic nanocarriers can be coupled with the polymeric matrix.
  • The synthetic nanocarriers may comprise one or more lipids. In some embodiments, a synthetic nanocarrier may comprise a liposome. In some embodiments, a synthetic nanocarrier may comprise a lipid bilayer. In some embodiments, a synthetic nanocarrier may comprise a lipid monolayer. In some embodiments, a synthetic nanocarrier may comprise a micelle. In some embodiments, a synthetic nanocarrier may comprise a non-polymeric core (e.g., metal particle, quantum dot, ceramic particle, bone particle, viral particle, proteins, nucleic acids, carbohydrates, etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).
  • The synthetic nanocarriers may comprise lipid-based nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles (such as albumin nanoparticles). Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cubic, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers according to the invention comprise one or more surfaces. Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric nanoparticles of Published U.S. Patent Application 20060002852 to Saltzman et al., (3) the lithographically constructed nanoparticles of Published U.S. Patent Application 20090028910 to DeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian et al., or (5) the nanoparticles disclosed in Published U.S. Patent Application 2008/0145441 to Penades et al.
  • Synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface with hydroxyl groups that activate complement or alternatively comprise a surface that consists essentially of moieties that are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that substantially activates complement or alternatively comprise a surface that consists essentially of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that activates complement or alternatively comprise a surface that consists essentially of moieties that do not activate complement. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.
  • In some embodiments, synthetic nanocarriers are spheres or spheroids. In some embodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments, synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers are ovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, or pyramids.
  • It is often desirable to use a population of synthetic nanocarriers that is relatively uniform in terms of size, shape, and/or composition so that each synthetic nanocarrier has similar properties. For example, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum dimension or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension. In some embodiments, a population of synthetic nanocarriers may be heterogeneous with respect to size, shape, and/or composition.
  • Synthetic nanocarriers can be solid or hollow and can comprise one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). To give but one example, synthetic nanocarriers may have a core/shell structure, wherein the core is one layer (e.g., a polymeric core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers may comprise a plurality of different layers.
  • “T cell antigen” means any antigen that is recognized by and triggers an immune response in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a T cell or an NKT cell via presentation of the antigen or portion thereof bound to a Class I or Class II major histocompatability complex molecule (MHC), or bound to a CD1 complex). In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is not also a B cell antigen. T cell antigens generally are proteins or peptides. T cell antigens may be an antigen that stimulates a CD8+ T cell response, a CD4+ T cell response, or both. The T cell antigens, therefore, in some embodiments can effectively stimulate both types of responses.
  • In some embodiments the T cell antigen is a T-helper antigen, which is a T cell antigen that can generate an augmented response to an unrelated B cell antigen through stimulation of T cell help. In embodiments, a T-helper antigen may comprise one or more peptides derived from tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE peptide. In other embodiments, a T-helper antigen may comprise one or more lipids, or glycolipids, including but not limited to: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan (from Leishmania donovani), and phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae). For additional lipids and/or glycolipids useful as T-helper antigens, see V. Cerundolo et al., “Harnessing invariant NKT cells in vaccination strategies.” Nature Rev Immun, 9:28-38 (2009). In embodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cell antigen that is obtained from a source, such as a natural source. In such embodiments, CD4+ T-cell antigen sequences, such as those peptides that bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity to the antigen obtained from the source. In embodiments, the T cell antigen, preferably a T-helper antigen, may be coupled to, or uncoupled from, a synthetic nanocarrier.
  • “Unit thereof” refers to a monomeric unit of a polymer, the polymer generally being made up of a series of linked monomers.
  • “Vaccine” means a composition of matter that improves the immune response to a particular pathogen or disease. A vaccine typically contains factors that stimulate a subject's immune system to recognize a specific antigen as foreign and eliminate it from the subject's body. A vaccine also establishes an immunologic ‘memory’ so the antigen will be quickly recognized and responded to if a person is re-challenged. Vaccines can be prophylactic (for example to prevent future infection by any pathogen), or therapeutic (for example a vaccine against a tumor specific antigen for the treatment of cancer). Vaccines according to the invention may comprise one or more of the synthetic nanocarriers or compositions provided herein.
    • Methods of Making the Inventive Compounds, Conjugates, or Synthetic Nanocarriers
  • The immunomodulatory agent can be coupled to the synthetic nanocarrier in any manner such that the dissociation of the immunomodulatory agent from the synthetic nanocarrier satisfies the dissociation relationships provided herein. Methods for determining whether or not immunomodulatory agents of synthetic nanocarriers satisfy the dissociation relationships provided herein are provided elsewhere above and in the EXAMPLES.
  • Oligonucleotides according to the invention may be encapsulated into synthetic nanocarriers using a variety of methods including but not limited to C. Astete et al., “Synthesis and characterization of PLGA nanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al., “Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles” Nanomedicine 2:8-21 (2006). Other methods suitable for encapsulating oligonucleotides into synthetic nanocarriers may be used, including without limitation methods disclosed in U.S. Pat. No. 6,632,671 to Unger Oct. 14, 2003.
  • In some embodiments, the immunomodulatory agent is covalently coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety (e.g., a polymer or unit thereof). In general, a polymer or unit thereof can be covalently coupled with an immunomodulatory agent in several ways.
  • The following methods or any step of the methods provided are exemplary and may be carried out under any suitable conditions. In some cases, the reaction or any step of the methods provided may be carried out in the presence of a solvent or a mixture of solvents. Non-limiting examples of solvents that may be suitable for use in the invention include, but are not limited to, p-cresol, toluene, xylene, mesitylene, diethyl ether, glycol, petroleum ether, hexane, cyclohexane, pentane, dichloromethane (or methylene chloride), chloroform, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate (EtOAc), triethylamine, acetonitrile, methyl-t-butyl ether (MTBE), N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropanol (IPA), mixtures thereof, or the like. In some cases, the solvent is selected from the group consisting of ethyl acetate, methylene chloride, THF, DMF, NMP, DMAC, DMSO, and toluene, or a mixture thereof.
  • A reaction or any step of the methods provided may be carried out at any suitable temperature. In some cases, a reaction or any step of the methods provided is carried out at about room temperature (e.g., about 25° C., about 20° C., between about 20° C. and about 25° C., or the like). In some cases, however, the reaction or any step of the methods provided may be carried out at a temperature below or above room temperature, for example, at about −20° C., at about −10° C., at about 0° C., at about 10° C., at about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C., about 90° C., about 100° C., about 120° C., about 140° C., about 150° C. or greater. In particular embodiments, the reaction or any step of the methods provided is conducted at temperatures between 0° C. and 120° C. In some embodiments, the reaction or any step of the methods provided may be carried out at more than one temperature (e.g., reactants added at a first temperature and the reaction mixture agitated at a second wherein the transition from a first temperature to a second temperature may be gradual or rapid).
  • The reaction or any step of the methods provided may be allowed to proceed for any suitable period of time. In some cases, the reaction or any step of the methods provided is allowed to proceed for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 2 days, about 3 days, about 4 days, or more. In some cases, aliquots of the reaction mixture may be removed and analyzed at an intermediate time to determine the progress of the reaction or any step of the methods provided. In some embodiments, a reaction or any step of the methods provided may be carried out under an inert atmosphere in anhydrous conditions (e.g., under an atmosphere of nitrogen or argon, anhydrous solvents, etc.)
  • The reaction products and/or intermediates may be isolated (e.g., via distillation, column chromatography, extraction, precipitation, etc.) and/or analyzed (e.g., gas liquid chromatography, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, etc.) using commonly known techniques. In some cases, a synthetic nanocarrier may be analyzed to determine the loading of immunomodulatory agent, for example, using reverse phase HPLC.
  • The polymers may have any suitable molecular weight. For example, the polymers may have a low or high molecular weight. Non-limiting molecular weight values include 100 Da, 200 Da, 300 Da, 500 Da, 750 Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10,000 Da, or greater. In some embodiments, the polymers have a weight average molecular weight of about 800 Da to about 10,000 Da. The molecular weight of a polymer may be determined using gel permeation chromatography. Provided below are exemplary reactions that are not intended to be limiting.
    • Method 1
  • A polymer (e.g., PLA, PLGA) or unit thereof with at least one acid end groups is converted to a reactive acylating agent such as an acyl halide, acylimidazole, active ester, etc. using an activating reagent commonly used in amide synthesis.
  • In this two-step method, the resulting activated polymer or unit thereof (e.g., PLA, PLGA) is isolated and then reacted with an immunomodulatory agent (e.g., R848) in the presence of a base to give the desired conjugate (e.g., PLA-R848), for example, as shown in the following scheme:
  • Figure US20110020388A1-20110127-C00001
  • Activating reagents that can be used to convert polymers or units thereof, such as PLA or PLGA, to an activated acylating form include, but are not limited to cyanuric fluoride, N,N-tetramethylfluoroformamidinium hexafluorophosphate (TFFH); Acylimidazoles, such as carbonyl diimidazole (CDI), N,N′-carbonylbis(3-methylimidazolium) triflate (CBMIT); and Active esters, such as N-hydroxylsuccinimide (NHS or HOSu) in the presence of a carbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC), N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) or N,N′-diisopropylcarbodiimide (DIC); N,N′-disuccinimidyl carbonate (DSC); pentafluorophenol in the presence of DCC or EDC or DIC; pentafluorophenyl trifluoroacetate.
  • The activated polymer or unit thereof may be isolated (e.g., via precipitation, extraction, etc.) and/or stored under suitable conditions (e.g., at low temperature, under argon) following activation, or may be used immediately. The activated polymer or unit thereof may be reacted with an immunomodulatory agent under any suitable conditions. In some cases, the reaction is carried out in the presence of a base and/or catalyst. Non-limiting examples of bases/catalysts include diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP).
    • Method 2
  • A polymer or unit thereof (e.g., PLA, PLGA having any suitable molecular weight) with an acid end group reacts with an immunomodulatory agent (e.g., R848) in the presence of an activating or coupling reagent, which converts the polymer or unit thereof (e.g., PLA, PLGA) to a reactive acylating agent in situ, to give the desired conjugate (e.g., PLA-R848, PLGA-R848).
  • Figure US20110020388A1-20110127-C00002
  • Coupling or activating agents include but are not limited to: activating agents used in the presence of an carbodiimide such as EDC or DCC or DIC, such as 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-7-azabenzotriazole (HOAt), 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HO-Dhbt), N-Hydroxysuccinimide (NHS or HOSu), Pentafluorophenol (PFP); Activating agents without carbodiimide: Phosphonium salts, such as O-Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP); uronium salts such as O-Benzotriazol-1-yloxytris-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and hexafluorophosphate (HBTU), O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TPTU); Halouronium and halophosphonium salts such as bis(tetramethylene)fluoroformamidinium hexafluorophosphate (BTFFH), bromotris(dimethylamino)phosphonium hexafluoro-phosphate (BroP), bromotripyrrolidino phosphonium hexafluorophosphate (PyBroP) and chlorotripyrrolidino phosphonium hexafluorophosphate (PyClop); Benzotriazine derivatives such as O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TDBTU) and 3-(diethyloxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT). Non-limiting examples of suitable solvents include DMF, DCM, toluene, ethyl acetate, etc., as described herein.
    • Method 3
  • Immunomodulatory agents, such as R848, can also be coupled to polymers or units thereof that are terminated in a hydroxyl group. Such polymers or units thereof include polyethylene glycol, polylactide, polylactide-co-glycolide, polycaprolactone, and other like polyesters, or units thereof. In general, the reaction proceeds as follows where an imide of the general structure (IV) will react with the terminal hydroxyl of the aforementioned polymers or units thereof using a catalyst used in lactone ring opening polymerizations. The resulting reaction product (II) links the amide of the agent to the polymer or unit thereof via an ester bond. The compounds of formula (IV) and (II) are as follows:
  • Figure US20110020388A1-20110127-C00003
  • wherein R1═H, OH, SH, NH2, or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylamino; R2═H, alkyl, or substituted alkyl; Y═N or C; R3 is absent if Y═N; or is H, alkyl, substituted alkyl, or combined with R4 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected if Y═C; R4 is H, or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylamino when not combined with R3 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected; or is combined with R3 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which they are connected; R5 is a polymer or unit thereof; X is C, N, O, or S; R6 and R7 are each independently H or substituted; and R9, R10, R11, and R12 are each independently H, a halogen, OH, thio, NH2, or substituted or unsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or arylamino.
  • Catalysts include, but are not limited to, phosphazine bases, 1,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD), and N-methyl-1,4,7-triazabicyclodecene (MTDB). Other catalysts are known in the art and provided, for example, in Kamber et al., Organocatalytic Ring-Opening Polymerization, Chem. Rev. 2007, 107, 58-13-5840. Non-limiting examples of suitable solvents include methylene chloride, chloroform, and THF.
  • A specific example of a reaction completed by such a method is shown here:
  • Figure US20110020388A1-20110127-C00004
  • wherein R5—OH contains two hydroxyl groups (e.g., a diol, HO—R5—OH), each of which are functionalized by reaction with an imide associated with R848. In some cases, HO—R5—OH is a poly-diol such as poly(hexamethyl carbonate) diol or polycaprolactone diol.
  • In embodiments where a poly-diol is employed, one of the diol groups may be protected with a protecting group (e.g., t-butyloxycarbonyl), thus the poly-diol would be a compound of formula HO—R5—OP, wherein P is a protecting group. Following reaction with an immunomodulatory agent to form a immunomodulatory agent-R5—OP conjugate, the protecting group may be removed and the second diol group may be reacted with any suitable reagent (e.g., PLGA, PLA).
    • Method 4
  • A conjugate (e.g., R848-PLA) can be formed via a one-pot ring-opening polymerization of an immunomodulatory agent (e.g., R848) with a polymer or unit thereof (e.g., D/L-lactide) in the presence of a catalyst, for example, as shown in the following scheme:
  • Figure US20110020388A1-20110127-C00005
  • In a one-step procedure, the immunomodulatory agent and the polymer or unit thereof may be combined into a single reaction mixture comprising a catalyst. The reaction may proceed at a suitable temperature (e.g., at about 150° C.) and the resulting conjugate may be isolated using commonly known techniques. Non-limiting examples of suitable catalysts include DMAP and tin ethylhexanoate.
    • Method 5
  • A conjugate can be formed two-step ring opening polymerization of an immunomodulatory agent (e.g., R848) with one or more polymers or units thereof (e.g., D/L-lactide and glycolide) in the presence of a catalyst, for example, as shown in the following scheme:
  • Figure US20110020388A1-20110127-C00006
  • The polymers or units thereof may be first combined, and in some cases, heated (e.g., to 135° C.) to form a solution. The immunomodulatory agent may be added to a solution comprising the polymers or units thereof, followed by addition of a catalyst (e.g., tin ethylhexanoate). The resulting conjugate may be isolated using commonly known techniques. Non-limiting examples of suitable catalysts include DMAP and tin ethylhexanoate.
  • In some embodiments, the immunomodulatory agent, antigen, and/or targeting moiety can be covalently associated with a polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, the immunomodulatory agent, antigen, and/or targeting moiety can be noncovalently associated with a polymeric matrix. For example, in some embodiments, the immunomodulatory agent, antigen, and/or targeting moiety can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric matrix. Alternatively or additionally, the immunomodulatory agent, antigen, and/or targeting moiety can be associated with a polymeric matrix by hydrophobic interactions, charge interactions, van der Waals forces, etc.
  • The immunomodulatory agents can also be encapsulated within the nanocarriers. The nanocarriers, therefore, can be of any material that is pH sensitive provided that the resulting inventive synthetic nanocarriers satisfy the dissociation relationships provided herein. Such synthetic nanocarriers are well known in the art and include polyketal nanocarriers, pH sensitive liposomes, acid-swelling, cross-linked nanoparticles, such as those of Griset et al., J. Am. Chem. Soc. 2009, 131, 2469-2471, which in their initial state are hydrophobic, but upon cellular internalization transform to a hydrophilic structure (a hydrogel particle), and polymeric nanoparticles, such as those of Griset, Dissertation entitled: Delivery of Paclitaxel via pH-Responsive Polymeric Nanoparticles for Prevention of Lung Cancer and Mesothelioma Recurrence, Ohio State University, 2003. The pH sensitive synthetic nanocarriers also include those that comprise polymers that dissolve at a pH below 6 or polymers that swell at an acidic pH. In some embodiments, the synthetic nanocarriers are of a non-polyketal material. In other embodiment, the synthetic nanocarriers are not micelles.
  • A wide variety of polymers and methods for forming polymeric matrices therefrom are known conventially. In general, a polymeric matrix comprises one or more polymers. Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences. Typically, polymers in accordance with the present invention are organic polymers.
  • Examples of polymers suitable for use in the present invention include, but are not limited to polyethylenes, polycarbonates (e.g., poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic anhydride)), polyhydroxyacids (e.g., poly(β-hydroxyalkanoate)), polypropylfumerates, polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, polyamines, and polysaccharides (e.g., chitosan).
  • In some embodiments, polymers in accordance with the present invention include polymers which have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, including but not limited to polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.
  • In some embodiments, polymers can be hydrophilic. For example, polymers may comprise anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationic groups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group, amine group). In some embodiments, a synthetic nanocarrier comprising a hydrophilic polymeric matrix generates a hydrophilic environment within the synthetic nanocarrier. In some embodiments, polymers can be hydrophobic. In some embodiments, a synthetic nanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobic environment within the synthetic nanocarrier. Selection of the hydrophilicity or hydrophobicity of the polymer may have an impact on the nature of materials that are incorporated (e.g., coupled) within the synthetic nanocarrier.
  • In some embodiments, polymers may be modified with one or more moieties and/or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, polymers may be modified with PEG, with a carbohydrate, and/or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301).
  • In some embodiments, polymers may be modified with a lipid or fatty acid group. In some embodiments, a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
  • In some embodiments, polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA.” In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEG copolymers and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof. In some embodiments, polyesters include, for example, polyanhydrides, poly(ortho ester), poly(ortho ester)-PEG copolymers, poly(caprolactone), poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEG copolymers, poly(ethyleneimine), poly(ethylene imine)-PEG copolymers, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.
  • In some embodiments, a polymer may be PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid:glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
  • In some embodiments, polymers may be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
  • In some embodiments, polymers can be cationic polymers. In general, cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids (e.g., DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively-charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.
  • In some embodiments, polymers can be degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633).
  • The properties of these and other polymers and methods for preparing them are well known in the art (see, for example, U.S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, a variety of methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
  • In some embodiments, polymers can be linear or branched polymers. In some embodiments, polymers can be dendrimers. In some embodiments, polymers can be substantially cross-linked to one another. In some embodiments, polymers can be substantially free of cross-links. In some embodiments, polymers can be used in accordance with the present invention without undergoing a cross-linking step. It is further to be understood that inventive compounds and synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, not comprehensive, list of polymers that can be of use in accordance with the present invention.
  • In some embodiments, synthetic nanocarriers may comprise metal particles, quantum dots, ceramic particles, etc.
  • In some embodiments, synthetic nanocarriers may optionally comprise one or more amphiphilic entities. In some embodiments, an amphiphilic entity can promote the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic entities can be associated with the interior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention. Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60); polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85 (Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethylene glycol)-400-monostearate; phospholipids; synthetic and/or natural detergents having high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof. An amphiphilic entity component may be a mixture of different amphiphilic entities. Those skilled in the art will recognize that this is an exemplary, not comprehensive, list of substances with surfactant activity. Any amphiphilic entity may be used in the production of synthetic nanocarriers to be used in accordance with the present invention.
  • In some embodiments, synthetic nanocarriers may optionally comprise one or more carbohydrates. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate comprises monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In certain embodiments, the carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.
  • Synthetic nanocarriers may be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods as nanoprecipitation, flow focusing fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods have been described in the literature (see, e.g., Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, δ: 275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755, and also U.S. Pat. Nos. 5,578,325 and 6,007,845).
  • In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. Conditions used in preparing synthetic nanocarriers may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.). The method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the materials to be coupled to the synthetic nanocarriers and/or the composition of the polymer matrix.
  • If particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve.
  • Coupling can be achieved in a variety of different ways, and can be covalent or non-covalent. Such couplings may be arranged to be on a surface or within an inventive synthetic nanocarrier. Elements of the inventive synthetic nanocarriers (such as moieties of which an immunofeature surface is comprised, targeting moieties, polymeric matrices, and the like) may be directly coupled with one another, e.g., by one or more covalent bonds, or may be coupled by means of one or more linkers. Additional methods of functionalizing synthetic nanocarriers may be adapted from Published US Patent Application 2006/0002852 to Saltzman et al., Published US Patent Application 2009/0028910 to DeSimone et al., or Published International Patent Application WO/2008/127532 A1 to Murthy et al.
  • Any suitable linker can be used in accordance with the present invention. Linkers may be used to form amide linkages, ester linkages, disulfide linkages, etc. Linkers may contain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.). In some embodiments, a linker is an aliphatic or heteroaliphatic linker. In some embodiments, the linker is a polyalkyl linker. In certain embodiments, the linker is a polyether linker. In certain embodiments, the linker is a polyethylene linker. In certain specific embodiments, the linker is a polyethylene glycol (PEG) linker.
  • In some embodiments, the linker is a cleavable linker. To give but a few examples, cleavable linkers include protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavable linkers, etc. In some embodiments, the linker is not a cleavable linker.
  • A variety of methods can be used to couple a linker or other element of a synthetic nanocarrier with the synthetic nanocarrier. General strategies include passive adsorption (e.g., via electrostatic interactions), multivalent chelation, high affinity non-covalent binding between members of a specific binding pair, covalent bond formation, etc. (Gao et al., 2005, Curr. Op. Biotechnol., 16:63). In some embodiments, click chemistry can be used to associate a material with a synthetic nanocarrier.
  • Non-covalent specific binding interactions can be employed. For example, either a particle or a biomolecule can be functionalized with biotin with the other being functionalized with streptavidin. These two moieties specifically bind to each other noncovalently and with a high affinity, thereby associating the particle and the biomolecule. Other specific binding pairs could be similarly used. Alternately, histidine-tagged biomolecules can be associated with particles conjugated to nickel-nitrolotriaceteic acid (Ni-NTA).
  • For additional general information on coupling, see the journal Bioconjugate Chemistry, published by the American Chemical Society, Columbus Ohio, PO Box 3337, Columbus, Ohio, 43210; “Cross-Linking,” Pierce Chemical Technical Library, available at the Pierce web site and originally published in the 1994-95 Pierce Catalog, and references cited therein; Wong S S, Chemistry of Protein Conjugation and Cross-linking, CRC Press Publishers, Boca Raton, 1991; and Hermanson, G. T., Bioconjugate Techniques, Academic Press, Inc., San Diego, 1996.
  • It is to be understood that the compositions of the invention can be made in any suitable manner, and the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method may require attention to the properties of the particular moieties being associated.
    • Pharmaceutical Compositions and Methods of Use
  • Compositions according to the invention comprise inventive synthetic nanocarriers in combination with pharmaceutically acceptable excipients. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. In an embodiment, inventive synthetic nanocarriers are suspended in sterile saline solution for injection together with a preservative.
  • In some embodiments, inventive synthetic nanocarriers are manufactured under sterile conditions or are terminally sterilized. This can ensure that resulting composition are sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when subjects receiving synthetic nanocarriers have immune defects, are suffering from infection, and/or are susceptible to infection. In some embodiments, inventive synthetic nanocarriers may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.
  • The inventive compositions may be administered by a variety of routes of administration, including but not limited to subcutaneous, intramuscular, intradermal, oral, parenteral, intranasal, transmucosal, rectal; ophthalmic, transdermal, transcutaneous or by a combination of these routes.
  • The compositions and methods described herein can be used to induce, enhance, stimulate, modulate, or direct an immune response. The compositions and methods described herein can be used in the diagnosis, prophylaxis and/or treatment of conditions such as cancers, infectious diseases, metabolic diseases, degenerative diseases, inflammatory diseases, immunological diseases, or other disorders and/or conditions. The compositions and methods described herein can also be used for the prophylaxis or treatment of an addiction, such as an addiction to nicotine or a narcotic. The compositions and methods described herein can also be used for the prophylaxis and/or treatment of a condition resulting from the exposure to a toxin, hazardous substance, environmental toxin, or other harmful agent.
    • EXAMPLES Example 1 Preparation of Activated Polymer
  • PLA (dl-polylactide) (Resomer R202H from Boehringer-Ingelheim, KOH equivalent acid number of 0.21 mmol/g, intrinsic viscosity (iv): 0.21 dl/g) (10 g, 2.1 mmol, 1.0 eq) was dissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2 g, 10.5 mmol, 5 eq) were added. The solids were dissolved with the aid of sonication. The resulting solution was stirred at room temperature for 6 days. The solution was concentrated to remove most of DCM and the residue was added to a solution of 250 mL of diethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHS ester. The solvents were removed and the polymer was washed twice with ether (2×200 mL) and dried under vacuum to give PLA-NHS activated ester as a white foamy solid (˜8 g recovered, H NMR was used to confirm the presence of NHS ester). The PLA-NHS ester was stored under argon in a below −10 C freezer before use.
  • Alternatively, the reaction can be performed in DMF, THF, dioxane, or CHCl3 instead of DCM. DCC can be used instead of EDC (resulting DCC-urea is filtered off before precipitation of the PLA-NHS ester from ether). The amount of EDC or DCC and NHS can be in the range of 2-10 eq of the PLA.
  • In the same manner, PLA with iv of 0.33 dl/g and acid number of 0.11 mmol/g or PLGA (Resomer RG653H, 65% lactide-35% glycolide, iv: 0.39 dl/g and acid number 0.08 mmol/g) or PLGA (Resomer RG752H, 75% lactide-25% glycolide, iv: 0.19 dl/g and acid number of 0.22 mmol/g) is converted to the corresponding PLA-NHS or PLGA-NHS activated ester and stored under argon in a below −10 C freezer before use.
    • Example 2 Preparation of Activated Polymer
  • PLA (R202H, acid number of 0.21 mmol/g) (2.0 g, 0.42 mmol, 1.0 eq) was dissolved in 10 mL of dry acetonitrile. N,N′-disuccinimidyl carbonate (DSC) (215 mg, 1.26 mmol, 3.0 eq) and catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) were added. The resulting mixture was stirred under argon for 1 day. The resulting solution was concentrated to almost dryness. The residue was then added to 40 mL of ether to precipitate out the polymer which was washed twice with ether (2×30 mL) and dried under vacuum to give PLA-NHS activated ester (1H NMR showed the amount of NHS ester at about 80%).
    • Example 3 Preparation of Activated Polymer
  • PLA (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous DCM and 2.5 mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq) and pentafluorophenol (PFP) (580 mg, 3.15 mmol, 5.0 eq) were added. The resulting solution was stirred at room temperature for 6 days and then concentrated to remove DCM. The resulting residue was added to 250 mL of ether to precipitate out the activated PLA polymer which was washed with ether (2×100 mL) and dried under vacuum to give PLA-PFP activated ester as a white foamy solid (4.0 g).
    • Example 4 Conjugation of Immunomodulatory Agent
  • PLA-NHS (1.0 g), R848 (132 mg, 0.42 mmol) and diisopropylethylamine (DIPEA) (0.073 mL, 0.42 mmol) were dissolved in 2 mL of dry DMF under argon. The resulting solution was heated at 50-60 C for 2 days. The solution was cooled to rt and added to 40 mL of de-ionized (DI) water to precipitate out the polymer product. The polymer was then washed with DI water (40 mL) and ether (2×40 mL) and dried at 30 C under vacuum to give R848-PLA conjugate as a white foamy solid (0.8 g, H NMR showed the conjugation of R848 to PLA via the amide bond). The degree of conjugation (loading) of R848 on the polymer was confirmed by HPLC analysis as follows: a weighed amount of polymer was dissolved in THF/MeOH and treated with 15% NaOH. The resulting hydrolyzed polymer products were analyzed for the amount of R848 by HPLC in comparison with a standard curve.
    • Example 5 Conjugation of Immunomodulatory Agent
  • PLA-NHS (1.0 g, 0.21 mmol, 1.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq), DIPEA (0.15 mL, 0.84 mmol, 4.0 eq) and DMAP (25 mg, 0.21 mmol, 1.0 eq) were dissolved in 2 mL of dry DMF under argon. The resulting solution was heated at 50-60 C for 2 days. The solution was cooled to rt and added to 40 mL of de-ionized (DI) water to precipitate out the polymer product. The polymer was then washed with DI water (40 mL) and ether (2×40 mL) and dried at 30 C under vacuum to give PLA-R848 conjugate as a white foamy solid (0.7 g, 20 mg of the polymer was hydrolyzed in solution of 0.2 mL of THF, 0.1 mL of MeOH and 0.1 mL of 15% NaOH. The amount of R848 on the polymer was determined to be about 35 mg/g by reverse phase HPLC analysis (C18 column, mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA in CH3CN, gradient).
    • Example 6 Conjugation of Immunomodulatory Agent
  • PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), DCC (260 mg, 1.26 mmol, 3.0 eq), NHS (145 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), DMAP (77 mg, 0.63 mmol, 1.5 eq) and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF. The mixture was heated at 50-55 C for 3 days. The mixture was cooled to rt and diluted with DCM. The DCC-urea was filtered off and the filtrate was concentrated to remove DCM. The resulting residue in DMF was added to water (40 mL) to precipitate out the polymer product which was washed with water (40 mL), ether/DCM (40 mL/4 mL) and ether (40 mL). After drying under vacuum at 30 C, the desired PLA-R848 conjugate was obtained as a white foamy solid (1.5 g).
    • Example 7 Conjugation of Immunomodulatory Agent
  • PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), EDC (242 mg, 1.26 mmol, 3.0 eq), HOAt (171 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF. The mixture was heated at 50-55 C for 2 days. The solution was cooled to rt and added to water (40 mL) to precipitate out the polymer product which was washed with water (40 mL), ether/MeOH (40 mL/2 mL) and ether (40 mL). The orange colored polymer was dissolved in 4 mL of DCM and the resulting solution was added to 40 mL of ether to precipitate out the polymer without much of the orange color. The light colored polymer was washed with ether (40 mL). After drying under vacuum at 30 C, the desired PLA-R848 conjugate was obtained as a light brown foamy solid (1.5 g).
    • Example 8 Conjugation of Immunomodulatory Agent
  • PLA (R202H) (1.0 g, 0.21 mmol, 1.0 eq), EDC (161 mg, 0.84 mmol, 4.0 eq), HOBt.H2O (65 mg, 0.42 mmol, 2.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq), and DIPEA (0.150 mL, 0.84 mmol, 4.0 eq) were dissolved in 2 mL of dry DMF. The mixture was heated at 50-55° C. for 2 days. The solution was cooled to room temperature and added to water (40 mL) to precipitate out the polymer product. The orange colored polymer was dissolved in 2 mL of DCM and the resulting solution was added to 40 mL of ether to precipitate out the polymer which was washed with water/acetone (40 mL/2 mL) and ether (40 mL). After drying under vacuum at 30° C., the desired PLA-R848 conjugate was obtained as an off-white foamy solid (1.0 g, loading of R848 on polymer was about 45 mg/g based on HPLC analysis and confirmed by 1H NMR). In the same manner, PLGA (75% Lactide)-R848 and PLGA (50% lactide)-R848 were prepared.
    • Example 9 Conjugation of Immunomodulatory Agent
  • Figure US20110020388A1-20110127-C00007
  • To a round bottom flask equipped with a stir bar and condenser was added the imidazoquinoline, resiquimod (R-848, 218 mg, 6.93×10−4 moles), D/L lactide (1.0 g, 6.93×10−3 moles) and anhydrous sodium sulfate (800 mg). The flask and contents were dried under vacuum at 55° C. for 8 hours. After cooling, the flask was then flushed with argon and toluene (50 mL) was added. The reaction was stirred in an oil bath set at 120° C. until all of the lactide had dissolved and then tin ethylhexanoate (19 mg, 15 μL) was added via pipette. Heating was continued under argon for 16 hours. After cooling, the reaction was diluted with ether (200 mL) and the solution was washed with water (200 mL). The solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give 880 mg. of crude polylactic acid-R-848 conjugate. The crude polymer was chromatographed on silica using 10% methanol in methylene chloride as eluent. The fractions containing the conjugate were pooled and evaporated to give the purified conjugate. This was dried under high vacuum to provide the conjugate as a solid foam in a yield of 702 mg (57.6%). By integrating the NMR signals for the aromatic protons of the quinoline and comparing this to the integrated intensity of the lactic acid CH proton it was determined that the molecular weight of the conjugate was approximately 2 KD. GPC showed that the conjugate contained less than 5% of free R848.
    • Example 10 Preparation of Low MW PLA-R848 Conjugate
  • Figure US20110020388A1-20110127-C00008
  • A solution of PLA-CO2H (average MW: 950, DPI:1.32; 5.0 g, 5.26 mmol) and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at room temperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol) was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture was stirred at room temperature for 6 h and then at 50-55° C. for 15 h. After cooling, the mixture was diluted with EtOAc (150 mL) and washed with 1% citric acid solution (2×40 mL), water (40 mL) and brine solution (40 mL). The solution was dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then added and the polymer conjugate precipitated out of solution. The polymer was then washed with MTBE (50 mL) and dried under vacuum at room temperature for 2 days as a white foam (5.3 g, average MW by GPC is 1200, PDI: 1.29; R848 loading is 20% by HPLC).
    • Example 11 Preparation of Low MW PLA-R848 Conjugate
  • Figure US20110020388A1-20110127-C00009
  • A solution of PLA-CO2H (average MW: 1800, DPI:1.44; 9.5 g, 5.26 mmol) and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at room temperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol) was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture was stirred at room temperature for 6 h and then at 50-55° C. for 15 h. After cooling, the mixture was diluted with EtOAc (150 mL) and washed with 1% citric acid solution (2×40 mL), water (40 mL) and brine solution (40 mL). The solution was dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then added and the polymer conjugate precipitated out of solution. The polymer was then washed with MTBE (50 mL) and dried under vacuum at room temperature for 2 days as a white foam (9.5 g, average MW by GPC is 1900, PDI: 1.53; R848 loading is 17% by HPLC).
    • Example 12 Conjugation of R848 to PCADK Via Imide Ring Opening
  • The following example describes the synthesis of a polyketal, PCADK, according to a method provided in Pulendran et al, WO 2008/127532, as illustrated in step 1 below.
  • PCADK is synthesized in a 50 mL two-necked flask, connected to a short-path distilling head. First, 5.5 mg of re-crystallized p-toluenesulfonic acid (0.029 mmol, Aldrich, St. Louis, Mo.), is dissolved in 6.82 mL of ethyl acetate, and added to a 30 mL benzene solution (kept at 100° C.), which contains 1,4-cyclohexanedimethanol (12.98 g, 90.0 mmol, Aldrich). The ethyl acetate is allowed to boil off, and distilled 2,2-dimethoxypropane (10.94 mL, 90.0 mmol, Aldrich) is added to the benzene solution, initiating the polymerization reaction. Additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) are subsequently added to the reaction every hour for 6 hours via a metering funnel to compensate for 2,2-dimethoxypropane and benzene that is distilled off. After 8 hours, the reaction is stopped by addition of 500 μL of triethylamine. The polymer is isolated by precipitation in cold hexane (stored at −20° C.) followed by vacuum filtration. The molecular weight of PCADK is determined by gel permeation chromatography (GPC) (Shimadzu, Kyoto, Japan) equipped with a UV detector. THF is used as the mobile phase at a flow rate of 1 ml/min. Polystyrene standards from Polymer Laboratories (Amherst, Mass.) are used to establish a molecular weight calibration curve. This compound is used to generate the PCADK particles in all subsequent experiments.
  • R848 may be conjugated to the terminal alcohol groups of the PCADK having molecular weight 6000 via imide ring opening, according to the step 2 shown below.
    • Step 1: Preparation of PCADK
  • Figure US20110020388A1-20110127-C00010
    • Step 2: Conjugation of PCADK to R848
  • Figure US20110020388A1-20110127-C00011
  • In step 2, the polymer from step 1 (12 g, 2.0×10−3 moles) is dissolved in methylene chloride 100 mL, and the lactam of R848 (3.3 g, 8.0×10−3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g, 6×10−3 moles) is added in a single portion. After stiffing at room temperature overnight, a clear solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 11.3 grams (81%) of polymer. A portion is hydrolyzed in acid and the R848 content is determined to be 9% by weight.
    • Example 13 Conjugation of R848 to Poly-Caprolactonediol Via Imide Ring Opening
  • Imide ring opening is used to attach R854 to the terminal alcohol groups of poly-caprolactonediol of molecular weight 2000. The polycaprolactone diol is purchased from Aldrich Chemical Company, Cat. #189421 and has the following structure:
  • Figure US20110020388A1-20110127-C00012
  • The polycaprolactone diol-R854 conjugate has the following structure:
  • Figure US20110020388A1-20110127-C00013
  • The polymer (5 g, 2.5×10−3 moles) is dissolved in methylene chloride 25 mL and the lactam of R854 (2.4 g, 5.0×10−3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10−3 moles) is added in a single portion. After stirring at room temperature for 15 minutes, a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.2 grams (70%) of polymer. A portion is hydrolyzed in acid and the R848 content is determined to be 18.5% by weight.
    • Example 14 Conjugation of R848 to Poly-(Hexamethylene Carbonate)Diol Via Imide Ring Opening
  • Imide ring opening is used to attach R848 to the terminal alcohol groups of poly-(hexamethylene carbonate)diol of molecular weight 2000. The poly(hexamethylene carbonate) diol is purchased from Aldrich Chemical Company, Cat # 461164, and has the following structure:

  • HO—[CH2(CH2)4CH2OCO2 ]nCH2(CH2)4CH2—OH.
  • The poly(hexamethylene carbonate) diol-R848 conjugate has the following structure:
  • Figure US20110020388A1-20110127-C00014
  • The polymer (5 g, 2.5×10−3 moles) is dissolved in methylene chloride 25 mL and the lactam of R848 (2.06 g, 5.0×10−3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10−3 moles) is added in a single portion. After stirring at room temperature overnight a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL) and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.9 grams (84%) of polymer. NMR is used to determine the R848 content which is determined to be 21%.
    • Example 15 Polylactic Acid Conjugates of an Imidazoquinoline Using a Tin Ethylhexanoate Catalyst
  • Figure US20110020388A1-20110127-C00015
  • To a two necked round bottom flask equipped with a stir bar and condenser was added the imidazoquinoline resiquimod (R-848, 100 mg, 3.18×10−4 moles), D/L lactide (5.6 g, 3.89×10−2 moles) and anhydrous sodium sulfate (4.0 g). The flask and contents were dried under vacuum at 50° C. for 8 hours. The flask was then flushed with argon and toluene (100 mL) was added. The reaction was stirred in an oil bath set at 120° C. until all of the lactide had dissolved and then tin ethylhexanoate (75 mg, 60 μL) was added via pipette. Heating was continued under argon for 16 hours. After cooling, water (20 mL) was added and stiffing was continued for 30 minutes. The reaction was diluted with additional toluene (200 mL) and was then washed with water (200 mL). The toluene solution was then washed in turn with 10% sodium chloride solution containing 5% conc. Hydrochloric acid (200 mL) followed by saturated sodium bicarbonate (200 mL). TLC (silica, 10% methanol in methylene chloride) showed that the solution contained no free R-848. The solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give 3.59 grams of polylactic acid-R-848 conjugate. A portion of the polymer was hydrolyzed in base and examined by HPLC for R-848 content. By comparison to a standard curve of R-848 concentration vs. HPLC response, it was determined that the polymer contained 4.51 mg of R-848 per gram of polymer. The molecular weight of the polymer was determined by GPC to be about 19,000.
    • Example 16 Low Molecular Weight Polylactic Acid Conjugates of an Imidazoquinoline
  • Figure US20110020388A1-20110127-C00016
  • To a round bottom flask equipped with a stir bar and condenser was added the imidazoquinoline, resiquimod (R-848, 218 mg, 6.93×10−4 moles), D/L lactide (1.0 g, 6.93×10−3 moles) and anhydrous sodium sulfate (800 mg). The flask and contents were dried under vacuum at 55° C. for 8 hours. After cooling, the flask was then flushed with argon and toluene (50 mL) was added. The reaction was stirred in an oil bath set at 120° C. until all of the lactide had dissolved and then tin ethylhexanoate (19 mg, 15 μL) was added via pipette. Heating was continued under argon for 16 hours. After cooling, the reaction was diluted with ether (200 mL) and the solution was washed with water (200 mL). The solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give 880 mg. of crude polylactic acid-R-848 conjugate. The crude polymer was chromatographed on silica using 10% methanol in methylene chloride as eluent. The fractions containing the conjugate were pooled and evaporated to give the purified conjugate. This was dried under high vacuum to provide the conjugate as a solid foam in a yield of 702 mg (57.6%). By integrating the NMR signals for the aromatic protons of the quinoline and comparing this to the integrated intensity of the lactic acid CH proton it was determined that the molecular weight of the conjugate was approximately 2 KD. GPC showed that the conjugate contained less than 5% of free R848.
    • Example 17 Low Molecular Weight Polylactic Acid Co-Glycolic Acid Conjugates of an Imidazoquinoline
  • Figure US20110020388A1-20110127-C00017
  • To a round bottom flask equipped with a stir bar and condenser was added the imidazoquinoline, resiquimod (R-848, 436 mg, 1.39×10−3 moles), glycolide (402 mg, 3.46×10−3 moles), D/L lactide (2.0 g, 1.39×10−2 moles) and anhydrous sodium sulfate (1.6 g). The flask and contents were dried under vacuum at 55° C. for 8 hours. After cooling, the flask was then flushed with argon and toluene (60 mL) was added. The reaction was stirred in an oil bath set at 120° C. until all of the R848, glycolide and lactide had dissolved and then tin ethylhexanoate (50 mg, 39 μL) was added via pipette. Heating was continued under argon for 16 hours. After cooling, the reaction was diluted with ethyl acetate (200 mL) and the solution was washed with water (200 mL). The solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give crude PLGA-R-848 conjugate. The crude polymer was chromatographed on silica using 10% methanol in methylene chloride as eluent. The fractions containing the conjugate were pooled and evaporated to give the purified conjugate. This was dried under high vacuum to provide the conjugate as a solid foam in a yield of 1.55 g (54.6%). By integrating the NMR signals for the aromatic protons of the quinoline and comparing this to the integrated intensity of the lactic acid CH proton it was determined that the molecular weight of the conjugate was approximately 2 KD. GPC showed that the conjugate contained no detectable free R848.
    • Example 18 Polylactic Acid Conjugates of an Imidazoquinoline Using A Lithium Diisopropylamide Catalysis
  • The imidazoquinoline (R-848), D/L lactide, and associated glassware were all dried under vacuum at 50° C. for 8 hours prior to use. To a round bottom flask equipped with a stir bar and condenser was added the R-848 (33 mg, 1.05×10−4 moles), and dry toluene (5 mL). This was heated to reflux to dissolve all of the R-848. The solution was stirred under nitrogen and cooled to room temperature to provide a suspension of finely divided R-848. To this suspension was added a solution of lithium diisopropyl amide (2.0 M in THF, 50 μL, 1.0×10−4 moles) after which stirring was continued at room temperature for 5 minutes. The pale yellow solution that had formed was added via syringe to a hot (120° C.) solution of D/L lactide (1.87 g, 1.3×10−2 moles) under nitrogen. The heat was removed and the pale yellow solution was stirred at room temperature for one hour. The solution was diluted with methylene chloride (200 mL) and this was then washed with 1% hydrochloric acid (2×50 mL) followed by saturated sodium bicarbonate solution (50 mL). The solution was dried over magnesium sulfate, filtered and evaporated under vacuum to give the polylactic acid-R-848 conjugate. TLC (silica, 10% methanol in methylene chloride) showed that the solution contained no free R-848. The polymer was dissolved in methylene chloride (10 mL) and the solution was dripped into stirred hexane (200 mL). The precipitated polymer was isolated by decantation and was dried under vacuum to give 1.47 grams of the polylactic acid—R-848 conjugate as a white solid. A portion of the polymer was hydrolyzed in base and examined by HPLC for R-848 content. By comparison to a standard curve of R-848 concentration vs. HPLC response, it was determined that the polymer contained 10.96 mg of R-848 per gram of polymer.
    • Example 19 Attachment of Immunomodulatory Agent to Low MW PLA
  • PLA (D/L-polylactide) with MW of 5000 (10.5 g, 2.1 mmol, 1.0 eq) is dissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2 g, 10.5 mmol, 5 eq) are added. The resulting solution is stirred at room temperature for 3 days. The solution is concentrated to remove most of DCM and the residue is added to a solution of 250 mL of diethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHS ester. The solvents are removed and the polymer is washed twice with ether (2×200 mL) and dried under vacuum to give PLA-NHS activated ester as a white foamy solid (˜8 g recovered, H NMR can be used to confirm the presence of NHS ester). The PLA-NHS ester is stored under argon in a below −10° C. freezer before use.
  • Alternatively, the reaction can be performed in DMF, THF, dioxane, or CHCl3 instead of DCM. DCC can be used instead of EDC (resulting DCC-urea is filtered off before precipitation of the PLA-NHS ester from ether). The amount of EDC or DCC and NHS can be in the range of 2-10 eq of the PLA.
    • Example 20 Attachment of Immunomodulatory Agent to Low MW PLGA
  • In the same manner as provided above for polymer activation, low MW PLGA with 50% to 75% glycolide is converted to the corresponding PLGA-NHS activated ester and is stored under argon in a below −10° C. freezer before use.
    • Example 21 One-Pot Ring-Opening Polymerization of R848 with D/L-Lactide in the Presence of a Catalyst
  • Figure US20110020388A1-20110127-C00018
  • A mixture of R848 (0.2 mmol, 63 mg), D/L-lactide (40 mmol, 5.8 g), and 4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol) in 2 mL of anhydrous toluene was heated slowly to 150° C. (oil bath temperature) and maintained at this temperature for 18 h (after 3 hr, no R848 was left). The mixture was cooled to ambient temperature and the resulting mixture was quenched with water (50 mL) to precipitate out the resulting polymer, R848-PLA. The polymer was then washed sequentially with 45 mL each of MeOH, iPrOH, and ethyl ether. The polymer was dried under vacuum at 30° C. to give an off-white puffy solid (5.0 g). Polymeric structure was confirmed by 1H NMR in CDCl3. A small sample of the polymer was treated with 2 N NaOH aq in THF/MeOH to determine the loading of R848 on the polymer by reverse phase HPLC. The loading of R848 is 3 mg per gram of polymer (0.3% loading-27.5% of theory).
    • Example 22 Two Step Ring Opening Polymerization of R848 with D/L-Lactide and Glycolide
  • Figure US20110020388A1-20110127-C00019
  • A mixture of D/L-lactide (10.8 g, 0.075 moles) and glycolide (2.9 g, 0.025 moles) was heated to 135° C. under argon. Once all of the materials had melted and a clear solution had resulted, R848 (1.08 g, 3.43×10−3 moles) was added. This solution was stirred at 135° C. under a slow stream of argon for one hour. Tin ethylhexanoate (150 μL) was added and heating was continued for 4 hours. After cooling, the solid pale brown mass was dissolved in methylene chloride (250 mL) and the solution was washed with 5% tartaric acid solution (2×200 mL). The methylene chloride solution was dried over magnesium sulfate, filtered, and then concentrated under vacuum. The residue was dissolved in methylene chloride (20 mL) and 2-propanol (250 mL) was added with stirring. The polymer that separated was isolated by decantation of the 2-propanol and was dried under high vacuum. NMR showed that the polymer was 71.4% lactide and 28.6% glycolide with a molecular weight of 4000. The loading of R848 was close to theoretical by NMR.
    • Example 23 Preparation of PLGA-R848 Conjugate
  • Figure US20110020388A1-20110127-C00020
  • A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc (160 mL) was stirred at room temperature under argon for 50 minutes. Compound R848 (2.2 g, 7 mmol) was added, followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture was stirred at room temperature for 6 h and then at 50-55° C. overnight (about 16 h). After cooling, the mixture was diluted with EtOAc (200 mL) and washed with saturated NH4Cl solution (2×40 mL), water (40 mL) and brine solution (40 mL). The solution was dried over Na2SO4 (20 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) was then added and the polymer conjugate precipitated out of solution. The polymer was then washed with IPA (4×50 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 3 days as a white powder (10.26 g, MW by GPC is 5200, R848 loading is 12% by HPLC).
    • Example 24 Preparation of PLGA-854A Conjugate
  • Figure US20110020388A1-20110127-C00021
  • A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (20 mL) was stirred at room temperature under argon for 45 minutes. Compound 845A (0.29 g, 0.7 mmol) was added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture was stirred at room temperature for 6 h and then at 50-55° C. overnight (about 15 h). After cooling, the mixture was diluted with EtOAc (100 mL) and washed with saturated NH4Cl solution (2×20 mL), water (20 mL) and brine solution (20 mL). The solution was dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (40 mL) was then added and the polymer conjugate precipitated out of solution. The polymer was then washed with IPA (4×25 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 2 days as a white powder (1.21 g, MW by GPC is 4900, 854A loading is 14% by HPLC).
    • Example 25 Preparation of PLGA-BBHA Conjugate
  • Figure US20110020388A1-20110127-C00022
  • A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) was stirred at room temperature under argon for 30 minutes. Compound BBHA (0.22 g, 0.7 mmol) in 2 mL of dry DMSO was added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture was stirred at room temperature for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) were added and the mixture was heated at 50-55° C. for 4 h. After cooling, the mixture was diluted with EtOAc (100 mL) and washed with saturated NH4Cl solution 20 mL), water (2×20 mL) and brine solution (20 mL). The solution was dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) was then added and the brownish polymer conjugate precipitated out of solution. The polymer was then washed with IPA (2×20 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 2 days as a brownish powder (1.1 g).
    • Example 26 Conjugation of R848 to Polyglycine, a Polyamide
  • Figure US20110020388A1-20110127-C00023
  • The t-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid (I) is prepared by ring opening polymerization of glycine N-carboxyanhydride (Aldrich cat #369772) using 6-aminohexanoic acid benzyl ester (Aldrich cat #S33465) by the method of Aliferis et al. (Biomacromolecules, 5, 1653, (2004)). Protection of the end amino group as the t-BOC carbamate followed by hydrogenation over palladium on carbon to remove the benzyl ester completes the synthesis of BOC protected polyglycine carboxylic acid (I).
  • A mixture of BOC-protected polyglycine carboxylic acid (5 gm, MW=2000, 2.5×10−3 moles) and HBTU (3.79 gm, 1.0×10−2 moles) in anhydrous DMF (100 mL) is stirred at room temperature under argon for 50 minutes. Then R848 (1.6 gm, 5.0×10−3 moles) is added, followed by diisopropylethylamine (4 mL, 2.2×10−2 moles). The mixture is stirred at RT for 6 h and then at 50-55° C. overnight (16 h). After cooling, the DMF is evaporated under vacuum and the residue is triturated in EtOAc (100 mL). The polymer is isolated by filtration and the polymer is then washed with 2-propanol (4×25 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 3 days. The polymer is isolated as an off white solid in a yield of 5.1 g (88%). The R848 loading can be determined by NMR is 10.1%.
  • The t-BOC protecting group is removed using trifluoroacetic acid and the resulting polymer is grafted to PLA with carboxyl end groups by conventional methods.
    • Example 27 Preparation of a PLGA Conjugate of the Polyglycine/R848 Polymer
  • Step 1: A t-BOC protected polyglycine/R848 conjugate (5 g) is dissolved in trifluoroacetic acid (25 mL) and this solution is warmed at 50° C. for one hour. After cooling, the trifluoroacetic acid is removed under vacuum and the residue is triturated in ethyl acetate (25 mL). The polymer is isolated by filtration and is washed well with 2-propanol. After drying under vacuum there is obtained 4.5 grams of polymer as an off white solid.
  • Step 2: A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous DMF (100 mL) is stirred at RT under argon for 50 minutes. The polymer from above (1.4 g, 7 mmol) dissolved in dry DMF (20 mL) is added, followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture is stirred at RT for 6 h and then at 50-55° C. overnight (16 h). After cooling, the DMF is evaporated under vacuum, and the residue is dissolved in methylene chloride (50 mL). The polymer is precipitated by the addition of 2-propanol (200 mL). The polymer is isolated by decantation and is washed with 2-propanol (4×50 mL) to remove residual reagents and then dried under vacuum at 35-40 C overnight. There is obtained 9.8 g (86%) of the block copolymer.
    • Example 28 Preparation of PLGA-2-Butoxy-8-Hydroxy-9-Benzyl Adenine Conjugate
  • Figure US20110020388A1-20110127-C00024
  • A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (I) (0.22 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture is stirred at room temperature for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at 50-55° C. for 4 h. After cooling, the mixture is diluted with EtOAc (100 mL) and washed with saturated NH4Cl solution 20 mL), water (2×20 mL) and brine solution (20 mL). The solution is dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) is then added and the brownish polymer conjugate precipitates out of solution. The polymer is then washed with IPA (2×20 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 2 days as a brownish powder (1.0 g).
    • Example 29 Preparation of PLGA-2,9-Dibenzyl-8-Hydroxyadenine Conjugate
  • Figure US20110020388A1-20110127-C00025
  • A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number 0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (II) (0.24 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture is stirred at RT for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at 50-55° C. for 4 h. After cooling, the mixture is diluted with EtOAc (100 mL) and washed with saturated NH4Cl solution 20 mL), water (2×20 mL) and brine solution (20 mL). The solution is dried over Na2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) is then added and the brownish polymer conjugate precipitated out of solution. The polymer is then washed with IPA (2×20 mL) to remove residual reagents and dried under vacuum at 35-40° C. for 2 days as a brownish powder (1.2 g).
    • Example 30 Imide Ring Opening Used to Attach 2-Pentyl-8-Hydroxy-9-Benzyladenine to the Terminal Alcohol Groups of Poly-Hexamethylene Carbonate) Diol of Molecular Weight 2000
  • The poly(hexamethylene carbonate) diol is purchased from Aldrich Chemical Company, Cat # 461164.
  • Poly(hexamethylene carbonate) diol:

  • HO—[CH2(CH2)4CH2OCO2 ]nCH2(CH2)4CH2—OH
  • Poly(hexamethylene carbonate) diol-8-oxoadenine conjugate:
  • Figure US20110020388A1-20110127-C00026
  • The polymer (5 g, 2.5×10−3 moles) is dissolved in methylene chloride 25 mL and the lactam of 2-pentyl-8-hydroxy-9-benzyladenine (2.05 g, 5.0×10−3 moles) is added. This slurry is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10−3 moles) is added in a single portion. After stirring at room temperature overnight a clear pale yellow solution forms. The solution is diluted with methylene chloride (100 mL), and the solution is washed with 5% citric acid. This solution is dried over sodium sulfate after which it is filtered and evaporated under vacuum. After drying under high vacuum there is obtained 5.5 grams (78%) of polymer. NMR is used to determine the benzyladenine content which is 18%.
    • Example 31 Nicotine-PEG-PLA Conjugates
  • A 3-nicotine-PEG-PLA polymer was synthesized as follows:
  • First, monoamino poly(ethylene glycol) from JenKem® with a molecular weight of 3.5 KD (0.20 gm, 5.7×10−5 moles) and an excess of 4-carboxycotinine (0.126 gm, 5.7×10−4 moles) were dissolved in dimethylformamide (5.0 mL). The solution was stirred and dicyclohexylcarbodiimide (0.124 gm, 6.0×10−4 moles) was added. This solution was stirred overnight at room temperature. Water (0.10 mL) was added and stiffing was continued for an additional 15 minutes. The precipitate of dicyclohexylurea was removed by filtration and the filtrates were evaporated under vacuum. The residue was dissolved in methylene chloride (4.0 mL) and this solution was added to diethyl ether (100 mL). The solution was cooled in the refrigerator for 2 hours and the precipitated polymer was isolated by filtration. After washing with diethyl ether, the solid white polymer was dried under high vacuum. The yield was 0.188 gm. This polymer was used without further purification for the next step.
  • The cotinine/PEG polymer (0.20 gm, 5.7×10−5 moles) was dissolved in dry tetrahydrofuran (10 mL) under nitrogen and the solution was stirred as a solution of lithium aluminum hydride in tetrahydrofuran (1.43 mL of 2.0M, 2.85×10−3 moles) was added. The addition of the lithium aluminum hydride caused the polymer to precipitate as a gelatinous mass. The reaction was heated to 80° C. under a slow stream of nitrogen and the tetrahydrofuran was allowed to evaporate. The residue was then heated at 80° C. for 2 hours. After cooling, water (0.5 mL) was cautiously added. Once the hydrogen evolution had stopped, 10% methanol in methylene chloride (50 mL) was added and the reaction mixture was stirred until the polymer had dissolved. This mixture was filtered through Celite® brand diatomaceous earth (available from EMD Inc. as Celite® 545, part #CX0574-3) and the filtrates were evaporated to dryness under vacuum. The residue was dissolved in methylene chloride (4.0 mL) and this solution was slowly added to diethyl ether (100 mL). The polymer separated as a white flocculent solid and was isolated by centrifugation. After washing with diethyl ether, the solid was dried under vacuum. The yield was 0.129 gm.
  • Next, a 100 mL round bottom flask, equipped with a stir bar and reflux condenser was charged with the PEG/nicotine polymer (0.081 gm, 2.2×10−5 moles), D/L lactide (0.410 gm, 2.85×10−3 moles) and anhydrous sodium sulfate (0.380 gm). This was dried under vacuum at 55° C. for 8 hours. The flask was cooled and flushed with argon and then dry toluene (10 mL) was added. The flask was placed in an oil bath set at 120° C., and once the lactide had dissolved, tin ethylhexanoate (5.5 mg, 1.36×10−5 moles) was added. The reaction was allowed to proceed at 120° C. for 16 hours. After cooling to room temperature, water (15 mL) was added and stirring was continued for 30 minutes. Methylene chloride (200 mL) was added, and after agitation in a separatory funnel, the phases were allowed to settle. The methylene chloride layer was isolated and dried over anhydrous magnesium sulfate. After filtration to remove the drying agent, the filtrates were evaporated under vacuum to give the polymer as a colorless foam. The polymer was dissolved in tetrahydrofuran (10 mL) and this solution was slowly added to water (150 mL) with stiffing. The precipitated polymer was isolated by centrifugation and the solid was dissolved in methylene chloride (10 mL). The methylene chloride was removed under vacuum and the residue was dried under vacuum. 3-nicotine-PEG-PLA polymer yield was 0.38 gm.
    • Example 32 Synthetic Nanocarrier Formulation
  • For encapsulated adjuvant formulations, Resiquimod (aka R848) was synthesized according to the synthesis provided in Example 99 of U.S. Pat. No. 5,389,640 to Gerster et al.
  • R848 was conjugated to PLA by a method provided above, and the PLA structure was confirmed by NMR.
  • PLA-PEG-nicotine conjugate was prepared according to Example 31.
  • PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 North Normandy Drive, Petersburg, Va. 23805). The polyvinyl alcohol (Mw=11 KD-31 KD, 85-89% hydrolyzed) was purchased from VWR scientific. Ovalbumin peptide 323-339 was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part #406-4565).
  • The above materials were used to prepare the following solutions:
      • 1. Resiquimod (R848) @ 10 mg/mL and PLA @ 100 mg/mL in methylene chloride or PLA-R848 conjugate @ 100 mg/mL in methylene chloride
      • 2. PLA-PEG-nicotine in methylene chloride @ 100 mg/mL
      • 3. PLA in methylene chloride @ 100 mg/mL
      • 4. Ovalbumin peptide 323-339 in water @ 10 or 69 mg/mL
      • 5. Polyvinyl alcohol in water @50 mg/mL.
  • Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL), solution #3 (0.25 to 0.5 mL) and solution #4 (0.1 mL) were combined in a small vial and the mixture was sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. To this emulsion was added solution #5 (2.0 mL) and sonication at 35% amplitude for 40 seconds using the Branson Digital Sonifier 250 forms the second emulsion. This was added to a beaker containing phosphate buffer solution (30 mL) and this mixture was stirred at room temperature for 2 hours to form the nanoparticles.
  • To wash the particles a portion of the nanoparticle dispersion (7.4 mL) was transferred to a centrifuge tube and spun at 5,300 g for one hour, supernatant was removed, and the pellet was re-suspended in 7.4 mL of phosphate buffered saline. The centrifuge procedure was repeated and the pellet was re-suspended in 2.2 mL of phosphate buffered saline for a final nanoparticle dispersion of about 10 mg/mL.
    • Example 33 Double Emulsion with Multiple Primary Emulsions Materials
  • Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T cell epitope of Ovalbumin protein, was purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505.)
  • Resiquimod (aka R848) was synthesized according to a method provided in U.S. Pat. No. 6,608,201.
  • PLA-R848, resiquimod, was conjugated to PLA with a molecular weight of approximately 2,500 Da according to a method provided above.
  • PLGA-R848, resiquimod, was conjugated to PLGA with a molecular weight of approximately 4,100 Da according to a method provided above.
  • PS-1826 DNA oligonucleotide with fully phosphorothioated backbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodium counter-ion was purchased from Oligos Etc (9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.)
  • PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodium counter-ion was purchased from Oligos Etc. (9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.)\
  • PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 □L 2A.)
  • PLA with an inherent viscosity of 0.71 dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. Product Code 100 □L 7A.)
  • PLA with an inherent viscosity of 0.19 dL/g was purchased from Boehringer Ingelheim Chemicals, Inc. (Petersburg, Va. Product Code R202H.)
  • PLA-PEG-nicotine with a molecular weight of approximately 18,500 to 22,000 Da was prepared according to a method provided above.
  • PLA-PEG-R848 with a molecular weight of approximately 15,000 Da was synthesized was prepared according to a method provided above.
  • Polyvinyl alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J. T. Baker (Part Number U232-08).
  • Batches were produced using a double emulsion process with multiple primary emulsions. The table below references the solution suffix (e.g., B in Solution #1 column indicates Solution #1B was used) and volume of solution used.
  • Solution Solution Solution Solution Solution
    Sample #
    1 #2 #3 #4 #5
    Number (Volume) (Volume) (Volume) (Volume) (Volume)
    1 B (0.1 ml) C (1.0 ml) A (0.1 ml) C (1.0 ml) A (2.0 ml)
    2 A (0.2 ml) A (1.0 ml) A (0.1 ml) A (1.0 ml) A (3.0 ml)
    3 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A (3.0 ml)
    4 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A (3.0 ml)
  • Solution 1A: Ovalbumin peptide 323-339 @ 35 mg/mL in dilute hydrochloric acid aqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloric acid aqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 2A: 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride. The solution was prepared by first preparing two separate solutions at room temperature: 0.21-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride. The final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 2B: 0.71-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride. The solution was prepared by first preparing two separate solutions at room temperature: 0.71-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride. The final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 2C, 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride. The solution was prepared by first preparing two separate solutions at room temperature: 0.19-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride. The final solution was prepared by adding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.
  • Solution 3A: Oligonucleotide (either PS-1826 or P0-1826) @ 200 mg/ml in purified water. The solution was prepared by dissolving oligonucleotide in purified water at room temperature.
  • Solution 4A: Same as Solution #2A.
  • Solution 4B: Same as Solution #2B.
  • Solution 4C: Same as Solution #2C.
  • Solution 5A: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
  • Two separate primary water in oil emulsions were prepared. W1/O2 was prepared by combining solution 1 and solution 2 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. W3/O4 was prepared by combining solution 3 and solution 4 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. A third emulsion with two inner emulsion ([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.5 ml of each primary emulsion (W1/O2 and W3/O4) and solution 5 and sonicating at 30% amplitude for 40 to 60 seconds using the Branson Digital Sonifier 250.
  • The third emulsion was added to a beaker containing 70 mM phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow for the methylene chloride to evaporate and for the nanocarriers to form. A portion of the nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube and spinning at 13,823 g for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. The washing procedure was repeated and the pellet was re-suspended in phosphate buffered saline for a final nanocarrier dispersion of about 10 mg/mL.
  • The amounts of oligonucleotide and peptide in the nanocarrier were determined by HPLC analysis.
    • Example 34 Standard Double Emulsion Materials
  • As provided in Example 33 above.
  • Batches were produced using a standard double emulsion process. The table below references the solution suffix (e.g., B in Solution #1 column indicates Solution #1B was used) and volume of solution used.
  • Solution #1 Solution #2 Solution #3 Solution #4 Solution #5
    Sample Number (Volume) (Volume) (Volume) (Volume) (Volume)
    1 A (0.1 ml) A (0.75 ml) A (0.25 ml) None A (2.0 ml)
    2 A (0.1 ml) None A (0.25 ml) A (0.75 ml) A (2.0 ml)
    3 A (0.1 ml) B (0.75 ml) A (0.25 ml) None A (2.0 ml)
    4 B (0.1 ml) C (0.75 ml) A (0.25 ml) None B (2.0 ml)
    5 B (0.1 ml) D (0.25 ml) A (0.25 ml) A (0.50 ml) B (2.0 ml)
    6 C (0.2 ml) None A (0.25 ml) A (0.75 ml) B (2.0 ml)
    7 D (0.1 ml) None A (0.25 ml) A (0.75 ml) B (2.0 ml)
  • Solution 1A: Ovalbumin peptide 323-339 @ 69 mg/mL in de-ionized water. The solution was prepared by slowly adding ovalbumin peptide to the water while mixing at room temperature.
  • Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloric acid aqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13N hydrochloric acid solution at room temperature.
  • Solution 1C: Oligonucleotide (PS-1826) @ 50 mg/ml in purified water. The solution was prepared by dissolving oligonucleotide in purified water at room temperature.
  • Solution 1D: Ovalbumin peptide 323-339 @ 17.5 mg/mL in dilute hydrochloric acid aqueous solution. The solution was prepared by dissolving ovalbumin peptide @ 70 mg/ml in 0.13N hydrochloric acid solution at room temperature and then diluting the solution with 3 parts purified water per one part of starting solution.
  • Solution 2A: R848 @ 10 mg/ml and 0.19-IV PLA @ 100 mg/mL in pure methylene chloride prepared at room temperature.
  • Solution 2B: PLA-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 2C: PLGA-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 2D: PLA-PEG-R848 @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 3A: PLA-PEG-nicotine @ 100 mg/ml in pure methylene chloride prepared at room temperature.
  • Solution 4A: 0.19-IV PLA @ 100 mg/mL in pure methylene chloride prepared at room temperature.
  • Solution 5A: Polyvinyl alcohol @ 50 mg/mL in de-ionized water.
  • Solution 5B: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
  • The water in oil (W/O) primary emulsion was prepared by combining solution 1 and solution 2, solution 3, and solution 4 in a small pressure tube and sonicating at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250. The water/oil/water (W/O/W) double emulsion was prepared by adding solution 5 to the primary emulsion and sonicating at 30% to 35% amplitude for 40 seconds using the Branson Digital Sonifier 250.
  • The double emulsion was added to a beaker containing phosphate buffer solution (30 mL) and stirred at room temperature for 2 hours to allow for the methylene chloride to evaporate and for the nanocarriers to form. A portion of the nanocarriers were washed by transferring the nanocarrier suspension to a centrifuge tube and spinning at 5,000 to 9,500 RPM for one hour, removing the supernatant, and re-suspending the pellet in phosphate buffered saline. The washing procedure was repeated and the pellet was re-suspended in phosphate buffered saline for a final nanocarrier dispersion of about 10 mg/mL.
    • Example 35 Determination of Amount of Agents
  • Method for R848 and Peptides (e.g., Ova Peptide, Human Peptide, TT2pDT5t)
  • The amount of R848 (immunostimulatory agent) and ova peptide (T cell antigen) was measured using reverse phase HPLC on an Agilent 1100 system at appropriate wavelengths (λ=254 nm for R848 and 215 nm for ova peptide) equipped with an Agilent Zorbax SB-C18 column (3.5 μm. 75×4.6 mm. Column Temp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA) of 95% water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90% acetonitrile/10% water/0.09% TFA (Gradient: B=5 to 45% in 7 minutes; ramp to 95% B to 9 min; decrease back to 5% B to 9.5 min and kept equilibrating to end. Total run time was 13 minute with flow rate of 1 mL/min).
    • Method for CpG
  • The amount of CpG (immunostimulatory agent) was measured using reverse phase HPLC on Agilent 1100 system at 260 nm equipped with Waters XBridge C-18 (2.5 micron particle, 50×4.6 mm ID (part No. 186003090), column temp. 600 C) using mobile phase A of 2% acetonitrile in 100 mM TEA-acetic acid buffer, pH about 8.0 and mobile B as 90% acetonitrile, 10% water (column equilibrated at 5% B, increased to 55% B in 8.5 min, then ramped to 90% B to 12 minutes. Strength of B was rapidly decreased to 5% in one minute and equilibrated until stop time, 16 minutes. The flow rate was 1 mL/min until end of the method, 16 minutes).
    • Method for Nicotine Analog
  • Nicotine analog was measured using reverse phase HPLC on Agilent 1100 system at 254 nm equipped with Waters X-Bridge C-18 (5 micron particle, 100×4.6 mm ID, column temp at 400 C) using Mobile Phase A (MPA) of 95% water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90% acetonitrile/10% water/0.09% TFA (gradient: column was equilibrated at 5% B increased to 45% B in 14 minutes. Then ramped up to 95% B from 14 to 20 minutes. Mobile B strength was quickly decreased back to 5% and requilibrated until the end of the method. The flow rate of the method was maintained at 0.5 ml/min with total run time of 25 minutes. The NC suspension was centrifuged @14000 rpm for about 15-30 minutes depending on particle size. The collected pellets were treated with 200 uL of conc. NH4OH (8 M) for 2 h with agitation until the solution turns clear. A 200 uL of 1% TFA was added to neutralize the mixture solution, which brought the total volume of the pellet solution to 200 uL. An aliquot of 50 uL of the solution was diluted with MPA(or water) to 200 uL and analyzed on HPLC as above to determine the amount present in the pellets.
    • Encapsulated Free R848 in Nanocarrier
  • 0.5 mL of the NC suspension was centrifuged @14000 Orpm for about 15 minutes. The collected pellet was dissolved with 0.3 mL of acetonitrile and centrifuged briefly @ 14000 rpm to remove any residual insolubles. The clear solution was further diluted with 4 times equivalent volume of MPA and assayed on reverse phase HPLC described above.
  • Encapsulated CpG in Nanocarrier
  • 330 uL of NC suspension from the manufacture (about 10 mg/mL suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes depending on particle size. The collected pellets were re-suspended with 500 uL of water and sonicated for 30 minutes to fully disperse the particles. The NC was then heated at 600° C. for 10 minutes. Additional 200 uL of 1 N NaOH was added to the mixture, heated for another 5 minutes where the mixture becomes clear. The hydrolyzed NC solution was centrifuged briefly at 14000 rpm. A final 2× dilution of the clear solution using water was then made and assayed on the reverse HPLC described above.
  • Encapsulated T Cell Antigens (e.g., Ova Peptide, or Human Peptide, TT2pDT5t)
  • 330 uL of NC suspension from the manufacture (about 10 mg/mL suspension in PBS) was spun down at 14000 rpm for 15 to 30 minutes. 100 uL of acetonitrile was added to the pellets to dissolve the polymer components of the NC. The mixture was vortexed and sonicated for 1 to 5 minutes. 100 uL 0.2% TFA was added to the mixture to extract the peptides and sonicated for another 5 minutes to ensure the break down of the aggregates. The mixture was centrifuged at 14000 rpm for 15 minutes to separate any insoluble materials (e.g., polymers). A 50 uL aliquot of the supernatant diluted with 150 uL of MPA (or water) was taken and assayed on the reverse phase HPLC as described above.
    • Amount of Conjugated Nicotine Analog (B Cell Antigen) in Nanocarriers
  • 1.5 mL of NC suspension was spun down @ 14000 rpm for about 15 minutes, the pellets were hydrolyzed using 150 uL of concentrated NH4OH (8M) for about 2-3 h until the solution turns clear. A 150 uL of 2% TFA(aq) solution was added to the pellet mixture to neutralize the solution. A 100 uL aliquot of the mixture was diluted with 200 uL of water and assayed on reverse phase HPLC described above and quantified based on the standard curve established using the precursor (PEG-nicotine) of the PLA-PEG-nicotine used in the manufacture.
    • Example 36 Release Rate Testing
  • The release of T-cell antigen, ova peptide and adjuvant, R848 from the synthetic nanocarrier (nanoparticles) in PBS (100 mM, pH=7.4) and Citrate buffer (100 mM, pH=4.5) at 37° C. were performed as follows:
  • Analytical Method: The amount of R848 and ova peptide released is measured using reverse phase HPLC on a Agilent 1100 system at λ=215 nm equipped with an Agilent Zorbax SB-C18 column (3.5 μm. 75×4.6 mm. Column Temp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA) of 98% water/2% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90% acetonitrile/10% water/0.09% TFA with Gradient: B=5 to 45% in 7 minutes; ramp to 95% B to 9 min; re-EQ to end. 13 minute run time. Flow=1 mL/min.
  • The total amount of R848 and ova peptide present in the nanoparticles was as shown in Table 1. An aqueous suspension of the tested synthetic nanocarriers was then diluted to a final stock volume of 4.4 mL with PBS.
  • (A) In Vitro Release Rate Measurement in PBS (pH=7.4):
  • For T0 sample, a 200 μL aliquot was immediately removed from each of the NP sample and centrifuged @ 14000 rpm in a microcentrifuge tubes using a Microcentrifuge (Model: Galaxy 16). 100 μL of supernatant was removed and diluted to 200 μL in HPLC Mobile Phase A (MPA) and assayed for the amount of R848 and ova peptide released on the reverse phase HPLC.
  • For time point measurements: 9×200 μL of each of the samples were added to microcentrifuge tubes (3×200 for unconjugated) and 300 μL of 37 C PBS was added to each above aliquot and the samples were placed immediately in 37° C. oven. At the following time points: 24 hr, 48 hr, 96 hr and 144 hr (for conjugated R848) or 2 h, 16 h and 24 h (for unconjugated (encapsulated) R848), the samples were centrifuged and assayed for the amount of R848 and ova peptide released as above for T0 sample.
  • (B) In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5):
  • For T0 sample, a 200 μL aliquot was removed from each of the samples and centrifuged @ 6000 rpm for 20 minutes and the supernatant was removed. The residue nanoparticles was resuspended in 200 uL of citrate buffer and centrifuged @ 14000 rpm for 15 minutes. 100 uL of the supernatant was removed and diluted to 200 uL with MPA and assayed for R848 and peptide as above.
  • For time point measurements: 9×200 uL of each of the samples were added to microcentrifuge tubes (3×200 for unconjugated) and centrifuged for 20 minutes @ 6000 rpm and the supernatants were removed. The residue NPs were then resuspended in 500 uL of citrate buffer and placed in 37° C. oven. At the following time points: 24 hr, 48 hr, 96 hr and 144 hr (for conjugated R848) or 2 h, 16 h and 24 h (for unconjugated (encapsulated) R848), the samples were centrifuged and assayed for the amount of R848 and ova peptide released as above for T0 sample.
  • In order to complete the mass balance from above measurements in PBS and Citrate buffer, the remaining pellets (conjugated R848 samples only) from each sample was treated with 200 uL of conc. NH4OH (8 M) for 3 h with mixing. After the mixture was settled, 200 uL of 1% TFA was added to bring total volume of the pellet to 400 uL. An aliquot of 50 uL of the solution was diluted with MPA to 200 uL and analyzed on HPLC as above to determine the amount of R848 and ova peptide that remained in the pellet after in vitro release to close the mass balance. For unconjugated samples, the sample was diluted with TFA in acetonitrile and assayed as above for R848 and peptide.
  • The results are summarized in FIGS. 1-3.
    • Materials and Method—
  • HPLC—Agilent 1100. λ=215 nm. Column Temp=40° C.
  • Column—Agilent Zorbax SB-C18, 3.5 μm. 75×4.6 mm. (part no. 866953-902)
  • C18 guard column
  • Mobile Phase A (MPA)—98% water/2% acetonitrile/0.1% TFA
  • Mobile Phase B (MPB)—90% acetonitrile/10% water/0.09% TFA
      • Gradient: B=5 to 45% in 7 minutes; ramp to 95% B to 9 min; re-EQ to end. 13 minute run time. Flow=1 mL/min.
  • PBS—100 mM, pH=7.4.
  • Citrate Buffer—100 mM, pH=4.5.
  • Oven—
  • Microcentrifuge—Galaxy 16
  • Microcentrifuge tubes
  • Sonicator
  • Pipets—20, 200, 1000 μL adjustable
  • HPLC grade water—EMD—#WX0008-1.
  • NH4OH—˜8M. Mallinkcrodt.
  • TFA, 0.2%. Prep Apr. 27, 2009.
  • TFA, 1%. Prep May 13, 2009.
  • Thermometer
    • SAMPLES—“6-1” and “6-2” have entrapped R848. All of the rest have conjugated R848. The estimated values are based on the loading results from the “62” series.
  • TABLE 2
    Estimated R848 and Ova peptide in synthetic nanocarriers:
    Estimated R848 in Estimated Ova in
    Sample ID NPs (μg/mL) NPs (μg/mL)
    1 54 146
    2 166 184
    3 119 32
    4 114 34
    5 465 37
    6 315 34
    7 116 40
  • Sample volumes were slightly below what was planned. To ensure enough material is available for all time points, the following volumes of PBS were added to the samples to bring them all to 4.4 mL.
  • TABLE 3
    Sample Volume PBS added
    Sample ID Volume (mL) (mL)
    1 4.35 0.05
    2 4.23 0.17
    3 4.21 0.19
    4 4.20 0.20
    5 4.21 0.19
    6 4.19 0.21
    7 4.20 0.20
    • Procedure—
      • 1) T=0 Sample Prep
        • a. PBS
          • i. Remove a 200 μL aliquot from each of the samples. Microcentrifuge @ 14000 rpm. Remove supernatant.
          • ii. Dilute supernatant 100 μL>200 μL in MPA. (DF=2).
          • iii. Assay for peptide and R848.
        • b. Citrate
          • i. Remove a 200 μL aliquot from each of the samples. Microcentrifuge @ 6000 rpm for 20 minutes. Remove supernatant.
          • ii. Add 200 μL of citrate buffer and thoroughly resuspend.
          • iii. Microcentrifuge @ 14000 rpm for 15 minutes. Remove supernatant.
          • iv. Dilute supernatant 100 μL>200 μL in MPA. (DF=2)
          • v. Assay for peptide and R848.
      • 2) PBS IVR
        • a. Add 9×200 μL of each of the samples to microcentrifuge tubes. (3×200 for unconjugated)
        • b. To each aliquot add 300 μL of 37 C PBS.
        • c. Immediately place samples in 37 C oven.
      • 3) Citrate IVR
        • a. Add 9×200 μL of each of the samples to microcentrifuge tubes. (3×200 for unconjugated)
        • b. Centrifuge for 20 minutes @ 6000 rpm.
        • c. Remove the supernatants.
        • d. To each tube, add 500 μL of citrate buffer and resuspend thoroughly.
        • e. Place samples in 37 C oven
      • 4) For lots 1-4 and 8, remove the samples (see step 6) at the following time points:
        • a. Conjugated
          • i. 24 hr
          • ii. 48 hr (2 days)
          • iii. 96 hr (4 days)
          • iv. 144 hr (6 days)
          • v. Further time points TBD based on the above data.
        • b. Non conjugated
          • i. 2 hr
          • ii. 16 hr
          • iii. 24 hr
      • 5) For lots 6 and 7, remove samples at the following time points:
        • a. PBS
          • i. 24 hr
          • ii. 48 hr (2 days)
          • iii. 96 hr (4 days)
          • iv. 144 hr (6 days)
          • v. Further time points TBD based on the above data.
        • b. Citrate
          • i. 2 hr
          • ii. 16 hr
          • iii. 24 hr
          • iv. 48 hr (2 days)
          • v. 72 hr (3 days)
          • vi. 96 hr (4 days)
          • vii. 120 hr (5 days)
          • viii. Further time points TBD based on the above data.
      • 6) Sample as follows:
        • a. Microcentrifuge @ 14000 rpm for 15 minutes.
        • b. Remove supernatant.
        • c. Dilute 100 μL to 200 μL in MPA. (DF=2)
      • 7) Assay for peptide and R848. This will provide the amount released at each time point.
  • To Complete Mass Balance, Perform the Following:
      • 8) To the remaining pellets (conjugated only) add 200 uL NH4OH.
      • 9) Vortex briefly and sonicate to disperse.
      • 10) Add stir bar. Allow to sit until clear (at least 3 hours).
      • 11) Add 200 uL of 1% TFA (total pellet volume=400 μL).
      • 12) Dilute 50 μL to 200 μL in MPA. Analyze by HPLC to determine peptide and R848 remaining in the pellet. (DF=4).
      • 13) For unconjugated lots, assay for peptide and R848 with typical AcN/TFA method.
    Example 37 Release Rate Testing
  • The release of antigen (e.g., ova peptide, T cell antigen) and immunostimulatory agents (e.g., R848, CpG) from synthetic nanocarriers in phosphate buffered saline solution (PBS) (100 mM, pH=7.4) and citrate buffer (100 mM, pH=4.5) at 37° C. was determined as follows:
  • The release of R848 from the nanocarrier composed of conjugated R848 and the ova peptide was achieved by exchanging desired amount of the aqueous suspension of the tested synthetic nanocarriers obtained from the manufacture (e.g., about 10 mg/mL in PBS) into the same volume of the appropriate release media (Citrate buffer 100 mM) via centrifugation and re-suspension.
  • In Vitro Release Rate Measurement in PBS (pH=7.4)
  • 1 mL of the PBS suspension NC was centrifuged @ 14000 rpm in microcentrifuge tubes generally from 15-30 minutes depending on particle size. The collected supernatant was then diluted with equal volume of the mobile phase A (MPA) or water and assayed on reverse phase HPLC for the amount of the R848 release during the storage. The remaining pellet was re-suspended to homogeneous suspension in 1 mL of PBS and placed to 37° C. thermal chamber with constant gentle agitation
  • For T0 sample, a 150 μL aliquot was immediately removed from NC suspension prior placing the NC suspension to 37° C. thermal chamber and centrifuged @ 14000 rpm in microcentrifuge tubes using a microcentrifuge (Model: Galaxy 16). 100 μL of the supernatant was removed and diluted to 200 μL with HPLC Mobile Phase A (MPA) or water and assayed for the amount of R848 and ova peptide released on the reverse phase HPLC.
  • For time point measurements, 150 μL aliquot was removed from the 37° C. NC sample suspension, and the samples were centrifuged and assayed for the amount of R848 and ova peptide released in the same manner as for T0 sample. The R848 and ova peptide released was tested at 6 h, 24 h for routine monitoring with additional 2 h, 48 h, 96 h and 144 h for complete release profile establishment.
  • In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5)
  • A 100 mM sodium citrate buffer (pH=4.5) was applied to exchange the original NC storage solution (e.g., PBS) instead of the PBS buffer, pH=7.4. In order to complete the mass balance from above measurements in PBS and Citrate buffer, the remaining pellets from each time point were treated with 100 uL of NH4OH (8 M) for 2 h (or more) with agitation until solution turn clear. A 100 uL of 1% TFA was added to neutralize the mixture, which brought the total volume of the pellet solution to 200 uL. An aliquot of 50 uL of the mixture was diluted with MPA (or water) to 200 uL and analyzed on HPLC as above to determine the amount of unreleased R848 remaining in the pellets after in vitro release to close the mass balance. For unconjugated samples, the sample was diluted with TFA in acetonitrile and assayed as above for R848.
  • The release of CpG was determined similar to the measurement of R848 and ova peptide in terms of sample preparation and monitored time points. However, the amount of the CpG in the release media was assayed by the reverse phase HPLC method described above.
    • Example 38 Immunization with NC-Nic Carrying CpG Adjuvant
  • Groups of five mice were immunized three times (subcutaneously, hind limbs) at 2-week intervals ( days 0, 14 and 28) with 100 μg of NC-Nic. NC-Nic was a composition of nanocarriers exhibiting nicotine on the outer surface and, for all groups of mice except for Group 1, carrying CpG-1826 (thioated) adjuvant, which was released from the nanocarriers at different rates. The nanocarriers were prepared according to a method provided above. Serum anti-nicotine antibodies were then measured on days 26 and 40. EC50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 4.
  • The Group 1 mice were administered NC-Nic w/o CpG-1826 containing Ova peptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic. The Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.2% CpG-1826; release rate at 24 hours: 4.2 μg CpG per mg of NC. The Group 3 mice were administered NC-Nic containing polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours: 15 μg CpG per mg of NC. Release was determined at a pH of 4.5.
  • The results shown in FIG. 4 demonstrate that entrapment of adjuvant into nanocarriers is beneficial for the immune response against NC-associated antigen, and, furthermore, that the higher release rate of entrapped CpG adjuvant from within the nanocarriers (NC) at 24 hours produced an immune response, which was elevated compared to one induced by NC with a slower release rate of CpG adjuvant (a TLR9 agonist).
    • Example 39 Immunization with NC-Nic Carrying Two Forms of CpG Adjuvant
  • Groups of five mice were immunized two times (subcutaneously, hind limbs) at 4-week intervals (days 0, and 28) with 100 μg of NC-Nic and serum anti-nicotine antibodies were then measured on days 12, 24 and 40. NC-Nic was a composition of nanocarriers exhibiting nicotine on the outer surface and carrying one of two forms of CpG-1826 adjuvant. The nanocarriers were prepared according to a method provided above. EC50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 5.
  • The Group 1 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 6.2% CpG-1826 (thioated); release rate at 24 hours: 16.6 μg CpG per mg of NC. The Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.2% CpG-1826 (thioated); release rate at 24 hours: 13.2 μg CpG per mg of NC. The Group 3 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.9% CpG-1826 (phosphodiester or PO, non-thioated); release rate at 24 hours: 19.6 μg CpG per mg of NC. The Group 4 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 8.5% CpG-1826 (PO, non-thioated); release rate at 24 hours: 9.3 μg CpG per mg of NC. Release was determined at a pH of 4.5.
  • The results shown in FIG. 5 demonstrate that the rate of release of entrapped adjuvant (CpG, TLR9 agonist) from nanocarriers influenced production of an antibody to NC-bound antigen (nicotine) with the nanocarrier exhibiting higher release rate at 24 hours induced stronger humoral immune response (group 1>group 2 and group 3>group 4). This was true irrespective of CpG form used (more stable, thioated or less stable non-thioated).
    • Example 40 Immunization with NC-Nic Carrying R848
  • Groups of five mice were immunized three times (subcutaneously, hind limbs) at 2-week intervals ( days 0, 14 and 28) with 100 μg of NC-Nic and serum anti-nicotine antibodies were then measured on days 26, 40 and 54. The nanocarriers were prepared according to a method provided above. EC50 for anti-nicotine antibodies as measured in standard ELISA against polylysine-nicotine are shown in FIG. 6.
  • The Group 1 mice were administered NC-Nic containing ova peptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, but without adjuvant. The Group 2 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 1.0% R848; of which 92% is released at 2 hours and more than 96% is released at 6 hours. The Group 3 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and 25% were PLA-PEG-Nic, and 1.3% R848, of which 29.4% is released at 6 hours and 67.8% is released at 24 hours. The Group 4 mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and 25% were PLA-PEG-Nic, and 1.4% of R848, of which 20.4% is released at 6 hours and 41.5% is released at 24 hours. The Group 5 mice were administered NC-Nic containing ova peptide, polymers, 25% of which were PLA-PEG-R848, 50% PLA, and 25% were PLA-PEG-Nic, and 0.7% of R848; of which less than 1% is released at 24 hours. Release was determined at a pH of 4.5.
  • The results shown in FIG. 6 demonstrate that R848 adjuvant (a TLR 7/8 agonist) contained in the NC augments humoral immune response against NC-associated antigen (groups 2-5>>group 1). Furthermore, neither fast (group 2), nor slow (group 5) release of R848 was elevated an immune response to the same level as NC releasing R848 at intermediate rate (group 3≈group 4>group 2≈group 5).
    • Example 41 Immunization with NC-Nic Carrying Entrapped PO CpG
  • Groups of five mice were immunized three times (subcutaneously, hind limbs) at 2-week intervals ( days 0, 14 and 28) with 100 μg of NC-Nic (nanocarrier exhibiting nicotine on the outer surface) with entrapped PO-CpG or not containing entrapped PO-CpG admixed with free PO-CpG. The synthetic nanocarriers were prepared according to methods provided above. Serum anti-nicotine antibodies were then measured in both groups on days 26 and 40. EC50 for anti-nicotine antibodies as determined in standard ELISA against polylysine-nicotine are shown in FIG. 7.
  • The group 1 mice were immunized with a NC-Nic with 1826 PO-CpG and MHC-II helper peptide from ovalbumin (Ov-II) encapsulated (6.6% PO-CpG; 2.3% Ov-II). The group 2 mice were immunized with a NC-Nic with 0.7% of entrapped Ov-II admixed with 20 μg of free 1826 PO-CpG.
  • This experiment demonstrates that the entrapment of PO-CpG within the nanocarrier (NC) generates a humoral immune response, which was superior to one induced when a ˜3-fold higher dose of free PO-CpG is admixed to NC without entrapped PO-CpG (antibody titer in group 1>antibody titer in group 2).

Claims (31)

1. A composition comprising:
synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier;
wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship:

IArel(4.5)24%/IArel(7.4)24%≧1.2;
wherein IArel(4.5)24% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers; and
wherein IArel(7.4)24% is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours divided by the sum of the weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours, expressed as weight percent, and taken as an average across a sample of the synthetic nanocarriers.
2. The composition of claim 1, wherein the immunomodulatory agent is coupled to the synthetic nanocarrier via an immunomodulatory agent coupling moiety.
3. The composition of claim 1, wherein the immunomodulatory agent is encapsulated within the synthetic nanocarrier.
4. The composition of claim 3, wherein the immunomodulatory agent comprises a labile immunomodulatory agent.
5. The composition of claim 4, wherein the labile immunomodulatory agent comprises an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotide comprises a backbone comprising one or more unstabilized internucleotide linkages.
6. The composition of claim 5, wherein the imidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod.
7. The composition of claim 5, wherein the oligonucleotide's backbone comprises no stabilizing chemical modifications that function to stabilize the backbone under physiological conditions.
8. The composition of claim 7, wherein the oligonucleotide's backbone comprises a backbone that is not modified to incorporate phosphorothioate stabilizing chemical modifications.
9. The composition of claim 1, wherein the immunomodulatory agent is an adjuvant.
10. The composition of claim 9, wherein the adjuvant comprises a Toll-like receptor (TLR) agonist.
11. The composition of claim 10, wherein the TLR agonist is a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.
12. The composition of claim 10, wherein the TLR agonist is an immunostimulatory nucleic acid.
13. The composition of claim 12, wherein the immunostimulatory nucleic acid is an immunostimulatory DNA or immunostimulatory RNA.
14. The composition of claim 12, wherein the immunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid that comprises one or more stabilizing chemical modifications that function to stabilize the backbone under physiological conditions.
15. The composition of claim 9, wherein the adjuvant comprises a universal T-cell antigen.
16. The composition of claim 1, wherein the synthetic nanocarriers further comprise a B cell antigen and/or a T cell antigen.
17. The composition of claim 1, wherein the synthetic nanocarriers further comprise an antigen presenting cell (APC) targeting feature.
18. The composition of claim 1, wherein the synthetic nanocarriers comprise one or more biodegradable polymers.
19. The composition of claim 18, wherein the immunomodulatory agent is coupled to the one or more biodegradable polymers via the immunomodulatory agent coupling moiety.
20. The composition of claim 18, wherein the biodegradable polymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).
21. The composition of claim 18, wherein the biodegradable polymers have a weight average molecular weight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeation chromatography.
22. The composition of claim 2, wherein the immunomodulatory agent coupling moiety comprises an amide bond.
23. The composition of claim 2, wherein the immunomodulatory agent coupling moiety comprises an ester bond.
24. The composition of claim 1, wherein the synthetic nanocarriers comprise lipid-based nanoparticles, polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles, peptide or protein-based particles, nanoparticles that comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidal nanoparticles.
25. A composition comprising:
synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier;
wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship:

IA(4.5)24/IA(4.5)6≧1.2;
wherein IA(4.5)24 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours taken as an average across a sample of the synthetic nanocarriers; and
wherein IA(4.5)6 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 6 hours taken as an average across a sample of the synthetic nanocarriers.
26-30. (canceled)
31. A composition comprising:
synthetic nanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier;
wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according to the following relationship:

6≦IA(4.5)24/IA(4.5)6≧1.2;
wherein IA(4.5)24 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours taken as an average across a sample of the synthetic nanocarriers; and
wherein IA(4.5)6 is defined as a weight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for 6 hours taken as an average across a sample of the synthetic nanocarriers.
32-55. (canceled)
56. A composition comprising a vaccine comprising the composition claim 1.
57. A method comprising:
administering the composition of claim 1 to a subject.
58-59. (canceled)
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Publication number Priority date Publication date Assignee Title
US20110110965A1 (en) * 2009-08-26 2011-05-12 Selecta Biosciences, Inc. Compositions that induce t cell help
US20110223201A1 (en) * 2009-04-21 2011-09-15 Selecta Biosciences, Inc. Immunonanotherapeutics Providing a Th1-Biased Response
WO2012149411A1 (en) * 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Controlled release of immunosuppressants from synthetic nanocarriers
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US9066978B2 (en) 2010-05-26 2015-06-30 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9152988B2 (en) 2000-12-05 2015-10-06 Open Invention Network Method and device utilizing polymorphic data in E-commerce
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
US9603944B2 (en) 2013-09-27 2017-03-28 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
WO2017070626A2 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Respiratory virus vaccines
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WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
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US11426451B2 (en) 2017-03-11 2022-08-30 Selecta Biosciences, Inc. Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant
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US11472856B2 (en) 2016-06-13 2022-10-18 Torque Therapeutics, Inc. Methods and compositions for promoting immune cell function
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
US11524033B2 (en) 2017-09-05 2022-12-13 Torque Therapeutics, Inc. Therapeutic protein compositions and methods of making and using the same
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
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US20030129251A1 (en) * 2000-03-10 2003-07-10 Gary Van Nest Biodegradable immunomodulatory formulations and methods for use thereof
JP4817599B2 (en) * 2003-12-25 2011-11-16 独立行政法人科学技術振興機構 Immune activity enhancer and method for enhancing immune activity using the same
EP2323628B1 (en) 2008-08-13 2022-04-13 California Institute of Technology Carrier nanoparticles and related compositions, methods and systems
US11446374B2 (en) 2008-12-09 2022-09-20 Novavax, Inc. Modified RSV F proteins and methods of their use
RU2477753C2 (en) * 2008-12-09 2013-03-20 Коули Фармасьютикал Груп, Инк. Immunostimulating oligonucleotides
JP5813513B2 (en) 2008-12-09 2015-11-17 ノババックス，インコーポレイテッド Modified RSVF protein and method of using the same
KR101908445B1 (en) 2010-11-12 2018-10-17 코어 파마슈티칼스 디벨롭먼트 컴퍼니 인크. Modified immune-modulating particles
EP2648759A4 (en) * 2010-12-10 2016-06-15 California Inst Of Techn Targeting kidney mesangium with nanoparticles of defined diameter
EP2495567A1 (en) * 2011-03-04 2012-09-05 Erasmus University Medical Center Rotterdam Methods and means for monitoring disruption of tissue homeostasis in the total body
CA2830948A1 (en) * 2011-03-25 2012-10-04 Selecta Biosciences, Inc. Osmotic mediated release synthetic nanocarriers
MX346094B (en) 2011-04-18 2017-03-07 Univ Georgia Vaccine delivery method.
CA3211102A1 (en) 2012-06-21 2013-12-27 Northwestern University Peptide conjugated particles
US9468681B2 (en) 2013-03-01 2016-10-18 California Institute Of Technology Targeted nanoparticles
BR112015022347A2 (en) 2013-03-11 2017-07-18 Cristal Delivery B V vaccination composition, method for producing a vaccination composition and use of composition
KR20230025948A (en) 2013-03-13 2023-02-23 코어 파마슈티칼스 디벨롭먼트 컴퍼니 인크. Immune-modifying particles for the treatment of inflammation
CA2910579C (en) * 2013-05-03 2023-09-26 Selecta Biosciences, Inc. Dosing combinations for reducing undesired humoral immune responses
BR112016003084A2 (en) 2013-08-13 2017-09-12 Univ Northwestern peptide conjugated particles
DE102013015112B4 (en) 2013-09-13 2016-07-14 Holger Frey Cleavable polyethylene glycol (PEG) macromolecules for inclusion of (glyco) proteins / antigens / allergens in degradable polyethylene glycol (PEG) nanoparticles and process for their preparation
EP3094314B1 (en) 2014-01-16 2021-06-23 MUSC Foundation For Research Development Targeted nanocarriers for the administration of immunosuppressive agents
US9919058B2 (en) 2014-07-15 2018-03-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polyketal particles including a CpG oligodeoxynucleotide for the treatment of lung cancer
WO2016019126A1 (en) * 2014-07-30 2016-02-04 The Research Foundation For The State University Of New York System and method for delivering genetic material or protein to cells
EP3795609B1 (en) 2015-07-01 2024-04-03 California Institute of Technology Cationic mucic acid polymer-based delivery systems
CN114796474A (en) 2015-09-03 2022-07-29 诺瓦瓦克斯股份有限公司 Vaccine compositions with improved stability and immunogenicity
EP3347047A1 (en) * 2015-09-09 2018-07-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Expression vector delivery system and use thereof for inducing an immune response
EP3377047A1 (en) 2015-11-20 2018-09-26 Cristal Delivery B.V. Nanoparticles with active targeting
JP2019511483A (en) * 2016-03-02 2019-04-25 ザ ボード オブ リージェンツ オブ ザ ユニバーシティー オブ テキサス システム STING activated nanovaccine for immunotherapy
US11433123B2 (en) 2016-07-21 2022-09-06 Case Western Reserve University Sustained release cowpea mosaic virus or virus-like particle therapeutic constructs for the treatment of cancer
AU2018207283B2 (en) 2017-01-10 2024-02-15 Nektar Therapeutics Multi-arm polymer conjugates of TLR agonist compounds and related immunotherapeutic treatment methods
IL305911A (en) 2018-03-19 2023-11-01 Novavax Inc Multivalent influenza nanoparticle vaccines
CN111971296A (en) * 2018-05-30 2020-11-20 韩国亿诺生物有限公司 Pharmaceutical composition for preventing or treating cancer comprising CD300E inhibitor as active ingredient
WO2020033791A1 (en) 2018-08-09 2020-02-13 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
CA3125541A1 (en) * 2019-01-04 2020-07-09 Ascendis Pharma Oncology Division A/S Minimization of systemic inflammation
AU2020204970A1 (en) * 2019-01-04 2021-06-24 Ascendis Pharma Oncology Division A/S Conjugates of pattern recognition receptor agonists
CN111249453B (en) * 2020-02-26 2021-11-19 浙江大学 Nano vaccine and preparation method thereof
WO2021231702A1 (en) * 2020-05-15 2021-11-18 Rutgers, The State University Of New Jersey Compositions and methods for treating wounds
US20230310504A1 (en) * 2020-07-01 2023-10-05 Cello Therapeutics, Inc. Platelet membrane coated nanoparticles and uses thereof
WO2022044025A1 (en) * 2020-08-31 2022-03-03 Rav Bariach (08) Industries Ltd. Mechanical muti-point lock with an electro-mechanical unit for remote operation
CN112494460B (en) * 2020-12-10 2022-03-11 浙江大飞龙动物保健品股份有限公司 Tilmicosin powder and preparation method thereof
EP4277654A1 (en) 2021-01-18 2023-11-22 Conserv Bioscience Limited Coronavirus immunogenic compositions, methods and uses thereof
US20230086755A1 (en) * 2021-09-17 2023-03-23 Illumina Cambridge Limited Reagent compositions, methods, cartridges, and systems
CN117224699B (en) * 2023-09-05 2024-03-19 贵州大学 Nanocomposite and preparation method and application thereof

Citations (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4009257A (en) * 1970-02-27 1977-02-22 Burroughs Wellcome, Inc. Preparation of immunosuppressive materials
US4021364A (en) * 1972-12-04 1977-05-03 Prof. Dr. Peter Speiser Microcapsules in the nanometric range and a method for their production
US4929624A (en) * 1989-03-23 1990-05-29 Minnesota Mining And Manufacturing Company Olefinic 1H-imidazo(4,5-c)quinolin-4-amines
US4994281A (en) * 1986-11-12 1991-02-19 Sanraku Incorporated Polylactic acid microspheres and process for producing the same
US5114703A (en) * 1989-05-30 1992-05-19 Alliance Pharmaceutical Corp. Percutaneous lymphography using particulate fluorocarbon emulsions
US5213812A (en) * 1990-08-01 1993-05-25 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Preparation process of sustained release compositions and the compositions thus obtained
US5500161A (en) * 1993-09-21 1996-03-19 Massachusetts Institute Of Technology And Virus Research Institute Method for making hydrophobic polymeric microparticles
US5514378A (en) * 1993-02-01 1996-05-07 Massachusetts Institute Of Technology Biocompatible polymer membranes and methods of preparation of three dimensional membrane structures
US5620708A (en) * 1993-02-22 1997-04-15 Alza Corporation Compositions and methods for the oral delivery of active agents
US5733572A (en) * 1989-12-22 1998-03-31 Imarx Pharmaceutical Corp. Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles
US5733925A (en) * 1993-01-28 1998-03-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US5869103A (en) * 1994-06-18 1999-02-09 Danbiosyst Uk Limited Polymer microparticles for drug delivery
US5871747A (en) * 1992-09-11 1999-02-16 Institut Pasteur Antigen-carrying microparticles and their use in the indication of humoral or cellular responses
US6060082A (en) * 1997-04-18 2000-05-09 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
US6197229B1 (en) * 1997-12-12 2001-03-06 Massachusetts Institute Of Technology Method for high supercoiled DNA content microspheres
US6225460B1 (en) * 1993-09-17 2001-05-01 Gilead Sciences, Inc. Nucleotide analogs
US6365187B2 (en) * 1992-04-24 2002-04-02 Brown University Research Foundation Bioadhesive microspheres and their use as drug delivery and imaging systems
US20020055477A1 (en) * 2000-03-10 2002-05-09 Nest Gary Van Immunomodulatory formulations and methods for use thereof
US20030009029A1 (en) * 1998-06-18 2003-01-09 Merck Patent Gmbh Catalytic titanium (IV) oxide, mediated geminal symmetric dialkylation of carboxamides
US20030035804A1 (en) * 1996-09-16 2003-02-20 Beth Israel Deaconess Medical Center Drug complex for treatment of metastatic prostate cancer
US20040022840A1 (en) * 2002-04-12 2004-02-05 Nagy Jon O. Nanoparticle vaccines
US20040038406A1 (en) * 2002-04-08 2004-02-26 Genesegues, Inc. Nanoparticle delivery systems and methods of use thereof
US20040059094A1 (en) * 2002-07-18 2004-03-25 Bachmann Martin F. Hapten-carrier conjugates and uses thereof
US20040067503A1 (en) * 2002-04-22 2004-04-08 Weihong Tan Functionalized nanoparticles and methods of use
US6723429B2 (en) * 1998-08-28 2004-04-20 Celanese Ventures Gmbh Method for preparing smooth-surface spherical microparticles completely or partially made of at least one water-insoluble linear polysaccharide and microparticles produced according to this method
US20040092470A1 (en) * 2002-06-18 2004-05-13 Leonard Sherry A. Dry powder oligonucleotide formualtion, preparation and its uses
US20050020525A1 (en) * 2002-02-20 2005-01-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US6849270B2 (en) * 1999-04-23 2005-02-01 Alza Corporation Releasable linkage and compositions containing same
US20050032733A1 (en) * 2001-05-18 2005-02-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US20050037075A1 (en) * 2003-06-06 2005-02-17 Farokhzad Omid C. Targeted delivery of controlled release polymer systems
US20050042298A1 (en) * 2003-08-20 2005-02-24 Pardridge William M. Immunonanoparticles
US20050048063A1 (en) * 2002-08-28 2005-03-03 Erkki Ruoslahti Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
US20050074812A1 (en) * 1998-03-13 2005-04-07 The Burnham Institute Molecules that home to various selected organs or tissues
US6881421B1 (en) * 1998-02-27 2005-04-19 Bioalliance Pharma S.A. Nanoparticles comprising at least one polymer and at least one compound able to complex one or more active ingredients
US20050107322A1 (en) * 2003-04-30 2005-05-19 O'hagan Derek Compositions for inducing immune responses
US20050113697A1 (en) * 1999-08-13 2005-05-26 Point Biomedical Corporation Method of imaging lymphatic system using nanocapsule compositions
US20060002852A1 (en) * 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
US20060002995A1 (en) * 2002-12-13 2006-01-05 Ian Harwigsson Pharmaceutical porous particles
US6989435B2 (en) * 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US20060017339A1 (en) * 2004-06-03 2006-01-26 Lalit Chordia Brushless canned motor
US7008411B1 (en) * 2002-09-30 2006-03-07 Advanced Cardiovascular Systems, Inc. Method and apparatus for treating vulnerable plaque
US20060051317A1 (en) * 2004-09-07 2006-03-09 Batrakova Elena V Amphiphilic polymer-protein conjugates and methods of use thereof
US20060111271A1 (en) * 2004-11-24 2006-05-25 Cerny Erich H Active and passive immunization against pharmacologically active hapten molecules using a synthetic carrier compound composed of similar elements
US20070014807A1 (en) * 2003-09-03 2007-01-18 Maida Anthony E Iii Multiplex vaccine
US20070014804A1 (en) * 2003-02-17 2007-01-18 Peter Burkhard Peptidic nanoparticles as drug delivery and antigen display systems
US20070087986A1 (en) * 2004-01-26 2007-04-19 Brett Premack Compositions and methods for enhancing immunity by chemoattractant adjuvants
US20070116768A1 (en) * 2003-12-09 2007-05-24 Michael Chorny Sustained release preparations composed of biocompatible complex microparticles
US20080014281A1 (en) * 2006-06-16 2008-01-17 Florida Atlantic University Chitin Micro-Particles As An Adjuvant
US20080031899A1 (en) * 2006-02-21 2008-02-07 Reddy Sai T Nanoparticles for immunotherapy
US20080044484A1 (en) * 2004-07-08 2008-02-21 The Regents Of The University Of California Use of polymeric nanoparticles for vaccine delivery
US20080081074A1 (en) * 2006-05-15 2008-04-03 Massachusetts Institute Of Technology Polymers for functional particles
US7375234B2 (en) * 2002-05-30 2008-05-20 The Scripps Research Institute Copper-catalysed ligation of azides and acetylenes
US20080160089A1 (en) * 2003-10-14 2008-07-03 Medivas, Llc Vaccine delivery compositions and methods of use
US20090004118A1 (en) * 2004-10-07 2009-01-01 Shuming Nie Multifunctional Nanoparticle Conjugates And Their Use
US20090028910A1 (en) * 2003-12-19 2009-01-29 University Of North Carolina At Chapel Hill Methods for Fabrication Isolated Micro-and Nano-Structures Using Soft or Imprint Lithography
US20090053293A1 (en) * 2005-09-09 2009-02-26 Beijing Diacrid Medical Technology Co., Ltd. Nano Anticancer Micelles of Vinca Alkaloids Entrapped in Polyethylene Glycolylated Phospholipids
US7501134B2 (en) * 2002-02-20 2009-03-10 Novartis Vaccines And Diagnostics, Inc. Microparticles with adsorbed polypeptide-containing molecules
US20090074828A1 (en) * 2007-04-04 2009-03-19 Massachusetts Institute Of Technology Poly(amino acid) targeting moieties
US7517520B2 (en) * 2003-03-26 2009-04-14 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US20090104268A1 (en) * 2004-10-01 2009-04-23 Midatech Limited Nanoparticles Comprising Antigens and Adjuvants, and Immunogenic Structures
US20100022680A1 (en) * 2006-06-23 2010-01-28 Massachusetts Institute Of Technology Microfluidic Synthesis of Organic Nanoparticles
US20100055189A1 (en) * 2008-08-29 2010-03-04 Hubbell Jeffrey A Nanoparticles for immunotherapy
US20100062968A1 (en) * 2005-05-10 2010-03-11 Bali Pulendran Novel strategies for delivery of active agents using micelles and particles
US20100068285A1 (en) * 2008-06-16 2010-03-18 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20100069426A1 (en) * 2008-06-16 2010-03-18 Zale Stephen E Therapeutic polymeric nanoparticles with mTor inhibitors and methods of making and using same
US20100075995A1 (en) * 2008-08-11 2010-03-25 Smithkline Beecham Corporation Compounds
US20100092425A1 (en) * 2008-10-12 2010-04-15 Von Andrian Ulrich Nicotine Immunonanotherapeutics
US20100129439A1 (en) * 2008-10-12 2010-05-27 Frank Alexis Adjuvant Incorporation in Immunonanotherapeutics
US20100129392A1 (en) * 2008-10-12 2010-05-27 Jinjun Shi Targeting of Antigen Presenting Cells with Immunonanotherapeutics
US20110008435A1 (en) * 1998-11-02 2011-01-13 Elan Corporation Plc Nanoparticulate and Controlled Release Compositions Comprising Aryl-Heterocyclic Compounds
US20110027217A1 (en) * 2009-05-27 2011-02-03 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US20110052697A1 (en) * 2006-05-17 2011-03-03 Gwangju Institute Of Science & Technology Aptamer-Directed Drug Delivery
US20110110965A1 (en) * 2009-08-26 2011-05-12 Selecta Biosciences, Inc. Compositions that induce t cell help
US20120027806A1 (en) * 2010-05-26 2012-02-02 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US20120058154A1 (en) * 2010-08-20 2012-03-08 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising peptides obtained or derived from human influenza a virus m2e
US20120070493A1 (en) * 2010-08-23 2012-03-22 Selecta Biosciences, Inc. Targeted multi-epitope dosage forms for induction of an immune response to antigens
US20130028857A1 (en) * 2011-07-29 2013-01-31 Selecta Biosciences, Inc. Synthetic nanocarriers comprising polymers comprising multiple immunomodulatory agents
US20130058977A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Compositions and methods related to induced tolerogenic dendritic cells externally loaded with mhc class i-restricted epitopes
US8652487B2 (en) * 2011-04-29 2014-02-18 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory B cells

Family Cites Families (181)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR7461M (en) 1968-06-19 1970-01-05
DK143689C (en) 1975-03-20 1982-03-15 J Kreuter PROCEDURE FOR THE PREPARATION OF AN ADVERTISED VACCINE
US4756907A (en) 1978-10-17 1988-07-12 Stolle Research & Development Corp. Active/passive immunization of the internal female reproductive organs
US4946929A (en) 1983-03-22 1990-08-07 Massachusetts Institute Of Technology Bioerodible articles useful as implants and prostheses having predictable degradation rates
US6309669B1 (en) 1984-03-16 2001-10-30 The United States Of America As Represented By The Secretary Of The Army Therapeutic treatment and prevention of infections with a bioactive materials encapsulated within a biodegradable-biocompatible polymeric matrix
US4638045A (en) 1985-02-19 1987-01-20 Massachusetts Institute Of Technology Non-peptide polyamino acid bioerodible polymers
US4631211A (en) 1985-03-25 1986-12-23 Scripps Clinic & Research Foundation Means for sequential solid phase organic synthesis and methods using the same
US4806621A (en) 1986-01-21 1989-02-21 Massachusetts Institute Of Technology Biocompatible, bioerodible, hydrophobic, implantable polyimino carbonate article
US5736372A (en) 1986-11-20 1998-04-07 Massachusetts Institute Of Technology Biodegradable synthetic polymeric fibrous matrix containing chondrocyte for in vivo production of a cartilaginous structure
CA1340581C (en) 1986-11-20 1999-06-08 Joseph P. Vacanti Chimeric neomorphogenesis of organs by controlled cellular implantation using artificial matrices
US5804178A (en) 1986-11-20 1998-09-08 Massachusetts Institute Of Technology Implantation of cell-matrix structure adjacent mesentery, omentum or peritoneum tissue
FR2608988B1 (en) 1986-12-31 1991-01-11 Centre Nat Rech Scient PROCESS FOR THE PREPARATION OF COLLOIDAL DISPERSIBLE SYSTEMS OF A SUBSTANCE, IN THE FORM OF NANOPARTICLES
US5912017A (en) 1987-05-01 1999-06-15 Massachusetts Institute Of Technology Multiwall polymeric microspheres
US5229490A (en) 1987-05-06 1993-07-20 The Rockefeller University Multiple antigen peptide system
US5019379A (en) 1987-07-31 1991-05-28 Massachusetts Institute Of Technology Unsaturated polyanhydrides
US6130082A (en) 1988-05-05 2000-10-10 American Cyanamid Company Recombinant flagellin vaccines
US5010167A (en) 1989-03-31 1991-04-23 Massachusetts Institute Of Technology Poly(amide-and imide-co-anhydride) for biological application
US5389640A (en) 1991-03-01 1995-02-14 Minnesota Mining And Manufacturing Company 1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines
DE69221568T2 (en) 1991-04-02 1998-03-19 Biotech Australia Pty Ltd ORAL RELEASE SYSTEMS OF MICROPARTICLES
EP0654973A4 (en) 1992-07-21 1995-08-09 Gen Hospital Corp System of drug delivery to the lymphatic tissues.
GB9216082D0 (en) 1992-07-28 1992-09-09 Univ Nottingham Lymphatic delivery composition
US6608201B2 (en) 1992-08-28 2003-08-19 3M Innovative Properties Company Process for preparing 1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines
WO1994007469A1 (en) 1992-09-25 1994-04-14 Dynagen, Inc. An immunobooster for delayed release of immunogen
US5399665A (en) 1992-11-05 1995-03-21 Massachusetts Institute Of Technology Biodegradable polymers for cell transplantation
EP0678034B1 (en) 1993-01-11 1999-05-26 Dana Farber Cancer Institute Inducing cytotoxic t lymphocyte responses
US5512600A (en) 1993-01-15 1996-04-30 Massachusetts Institute Of Technology Preparation of bonded fiber structures for cell implantation
ATE156706T1 (en) 1993-03-17 1997-08-15 Silica Gel Gmbh SUPERPARAMAGNETIC PARTICLES, METHOD FOR THE PRODUCTION AND USE OF THE SAME
WO1995003035A1 (en) 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Polymerized liposomes with enhanced stability for oral delivery
US5543158A (en) 1993-07-23 1996-08-06 Massachusetts Institute Of Technology Biodegradable injectable nanoparticles
US5565215A (en) 1993-07-23 1996-10-15 Massachusettes Institute Of Technology Biodegradable injectable particles for imaging
CA2184242C (en) 1994-02-28 2000-05-02 Jorg Kreuter Drug targeting system, method for preparing same and its use
PT772619E (en) 1994-07-15 2006-10-31 Univ Iowa Res Found OLIGONUCLEOTIDOS IMUNOMODULADORES
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6007845A (en) 1994-07-22 1999-12-28 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US5716404A (en) 1994-12-16 1998-02-10 Massachusetts Institute Of Technology Breast tissue engineering
JPH10511957A (en) 1995-01-05 1998-11-17 ザ ボード オブ リージェンツ オブ ザ ユニヴァーシティ オブ ミシガン Surface-modified nanoparticles and methods for their production and use
US6123727A (en) 1995-05-01 2000-09-26 Massachusetts Institute Of Technology Tissue engineered tendons and ligaments
WO1997004747A1 (en) 1995-07-27 1997-02-13 Dunn James M Drug delivery systems for macromolecular drugs
US5916597A (en) 1995-08-31 1999-06-29 Alkermes Controlled Therapeutics, Inc. Composition and method using solid-phase particles for sustained in vivo release of a biologically active agent
US6095148A (en) 1995-11-03 2000-08-01 Children's Medical Center Corporation Neuronal stimulation using electrically conducting polymers
US5902599A (en) 1996-02-20 1999-05-11 Massachusetts Institute Of Technology Biodegradable polymer networks for use in orthopedic and dental applications
US5874064A (en) 1996-05-24 1999-02-23 Massachusetts Institute Of Technology Aerodynamically light particles for pulmonary drug delivery
US5922695A (en) 1996-07-26 1999-07-13 Gilead Sciences, Inc. Antiviral phosphonomethyoxy nucleotide analogs having increased oral bioavarilability
JP4391592B2 (en) 1996-10-25 2009-12-24 スリーエム カンパニー Immune response modifying compounds for the treatment of TH2-mediated and related diseases
US6042820A (en) 1996-12-20 2000-03-28 Connaught Laboratories Limited Biodegradable copolymer containing α-hydroxy acid and α-amino acid units
JP2001513776A (en) 1997-02-28 2001-09-04 ユニバーシティ オブ アイオワ リサーチ ファウンデーション Use of nucleic acids containing unmethylated CpG dinucleotides in the treatment of LPS-related disorders
DE69841122D1 (en) 1997-03-10 2009-10-15 Coley Pharm Gmbh Use of non-methylated CpG dinucleotide in combination with aluminum as adjuvants
US6211159B1 (en) 1997-04-11 2001-04-03 University Of Toronto Flagellin gene, FlaC of campylobacter
ATE370740T1 (en) 1997-05-20 2007-09-15 Ottawa Health Research Inst METHOD FOR PRODUCING NUCLEIC ACID CONSTRUCTS
US5837752A (en) 1997-07-17 1998-11-17 Massachusetts Institute Of Technology Semi-interpenetrating polymer networks
DE19745950A1 (en) 1997-10-17 1999-04-22 Dds Drug Delivery Service Ges Drug carrier particle for site specific drug delivery, especially to CNS
DE69817393T2 (en) * 1997-11-28 2004-06-17 Sumitomo Pharmaceuticals Co., Ltd. NEW HETEROCYCLIC CONNECTIONS
US6254890B1 (en) 1997-12-12 2001-07-03 Massachusetts Institute Of Technology Sub-100nm biodegradable polymer spheres capable of transporting and releasing nucleic acids
US6686446B2 (en) 1998-03-19 2004-02-03 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6632922B1 (en) 1998-03-19 2003-10-14 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
US6506577B1 (en) 1998-03-19 2003-01-14 The Regents Of The University Of California Synthesis and crosslinking of catechol containing copolypeptides
AU760549B2 (en) 1998-04-03 2003-05-15 University Of Iowa Research Foundation, The Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
JP2002513763A (en) 1998-05-06 2002-05-14 ユニバーシティ オブ アイオワ リサーチ ファウンデーション Methods for preventing and treating parasitic infections and related diseases using CPG oligonucleotides
US6242589B1 (en) 1998-07-14 2001-06-05 Isis Pharmaceuticals, Inc. Phosphorothioate oligonucleotides having modified internucleoside linkages
PT1100468E (en) 1998-07-29 2006-07-31 Chiron Corp MICROPARTICLES WITH ADSORVENT SURFACES, PROCESSES OF MANUFACTURE AND USE OF THE SAME
US6306640B1 (en) 1998-10-05 2001-10-23 Genzyme Corporation Melanoma antigenic peptides
US7521068B2 (en) 1998-11-12 2009-04-21 Elan Pharma International Ltd. Dry powder aerosols of nanoparticulate drugs
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US6444192B1 (en) 1999-02-05 2002-09-03 The Regents Of The University Of California Diagnostic imaging of lymph structures
US7390780B2 (en) 1999-04-23 2008-06-24 Alza Corporation Gene delivery mediated by liposome-DNA complex with cleavable peg surface modification
US7238368B2 (en) 1999-04-23 2007-07-03 Alza Corporation Releasable linkage and compositions containing same
US6800296B1 (en) 1999-05-19 2004-10-05 Massachusetts Institute Of Technology Modification of surfaces using biological recognition events
US6815170B1 (en) 1999-06-30 2004-11-09 John Wayne Cancer Institute Methods for lymph node identification
CA2391534A1 (en) 1999-11-15 2001-05-25 Drug Innovation & Design, Inc. Selective cellular targeting: multifunctional delivery vehicles
WO2001051665A2 (en) 2000-01-13 2001-07-19 Nanosphere Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
CN1406140A (en) 2000-02-28 2003-03-26 吉倪塞思公司 Nano capsule encapsulation system and method
US20030129251A1 (en) 2000-03-10 2003-07-10 Gary Van Nest Biodegradable immunomodulatory formulations and methods for use thereof
US7192725B2 (en) 2000-05-19 2007-03-20 University Of Toronto Flagellin gene, flaC of Campylobacter
AU2001297913A1 (en) 2000-10-13 2002-12-23 Ligocyte Pharmaceuticals, Inc. Polyvalent nanoparticles
GB0025414D0 (en) 2000-10-16 2000-11-29 Consejo Superior Investigacion Nanoparticles
CA2426692C (en) 2000-10-19 2011-01-25 Eidgenossische Technische Hochschule Zurich Block copolymers for multifunctional self-assembled systems
US7592008B2 (en) 2000-11-20 2009-09-22 The Board Of Trustees Of The University Of Illinois, A Body Corporate And Politic Of The State Of Illinois Membrane scaffold proteins
US7097837B2 (en) 2001-02-19 2006-08-29 Pharmexa A/S Synthetic vaccine agents
US20030175950A1 (en) 2001-05-29 2003-09-18 Mcswiggen James A. RNA interference mediated inhibition of HIV gene expression using short interfering RNA
WO2004030608A2 (en) 2001-06-05 2004-04-15 The Regents Of The University Of Michigan Nanoemulsion vaccines
EP1417236A4 (en) 2001-07-10 2010-03-17 Corixa Corp Compositions and methods for delivery of proteins and adjuvants encapsulated in microspheres
US20030133988A1 (en) 2001-08-07 2003-07-17 Fearon Karen L. Immunomodulatory compositions, formulations, and methods for use thereof
US6818732B2 (en) 2001-08-30 2004-11-16 The Regents Of The University Of California Transition metal initiators for controlled poly (beta-peptide) synthesis from beta-lactam monomers
US8088388B2 (en) 2002-02-14 2012-01-03 United Biomedical, Inc. Stabilized synthetic immunogen delivery system
US20030232013A1 (en) 2002-02-22 2003-12-18 Gary Sieckman Therapeutic and diagnostic targeting of cancers cells with tumor homing peptides
US7635463B2 (en) * 2002-02-27 2009-12-22 Pharmain Corporation Compositions for delivery of therapeutics and other materials
EP1499187B1 (en) 2002-04-04 2015-06-17 Zoetis Belgium S.A. Immunostimulatory g,u-containing oligoribonucleotides
US20080233181A1 (en) 2002-04-12 2008-09-25 Nagy Jon O Nanoparticle adjuvants for sub-unit vaccines
US20040142887A1 (en) 2002-07-10 2004-07-22 Chengji Cui Antigen-polymer compositions
US20060189554A1 (en) 2002-09-24 2006-08-24 Russell Mumper Nanoparticle-Based vaccine delivery system containing adjuvant
NO20024755D0 (en) 2002-10-03 2002-10-03 Amersham Health As Method
US20040156846A1 (en) 2003-02-06 2004-08-12 Triton Biosystems, Inc. Therapy via targeted delivery of nanoscale particles using L6 antibodies
US20040191215A1 (en) 2003-03-25 2004-09-30 Michael Froix Compositions for induction of a therapeutic response
WO2004084871A1 (en) 2003-03-26 2004-10-07 Ltt Bio-Pharma Co., Ltd. Intravenous nanoparticles for targenting drug delivery and sustained drug release
US20060182793A1 (en) 2003-07-22 2006-08-17 Cytos Biotechnology Ag Cpg-packaged liposomes
US7943179B2 (en) 2003-09-23 2011-05-17 Massachusetts Institute Of Technology pH triggerable polymeric particles
JP2007514519A (en) 2003-10-20 2007-06-07 ウィリアム・マーシュ・ライス・ユニバーシティ Method for producing microcapsules comprising polymer and charged nanoparticles
WO2005065418A2 (en) 2003-12-31 2005-07-21 Board Of Regents, The University Of Texas System Compositions and methods of use of targeting peptides for diagnosis and therapy
AU2005230938A1 (en) 2004-02-19 2005-10-20 Coley Pharmaceutical Gmbh Immunostimulatory viral RNA oligonucleotides
JP2007532572A (en) * 2004-04-09 2007-11-15 スリーエム イノベイティブ プロパティズ カンパニー Methods, compositions and preparations for delivering immune response modifiers
ES2246695B1 (en) 2004-04-29 2007-05-01 Instituto Cientifico Y Tecnologico De Navarra, S.A. STIMULATING COMPOSITION OF THE IMMUNE RESPONSE THAT INCLUDES NANOPARTICLES BASED ON A COPYLIMER OF METHYL VINYL ETER AND MALEIC ANHYDRIDE.
ES2389080T3 (en) 2004-06-11 2012-10-23 Riken Drug presenting a regulatory cell ligand contained in the liposome
US7713550B2 (en) 2004-06-15 2010-05-11 Andrx Corporation Controlled release sodium valproate formulation
CN1692943A (en) 2004-09-17 2005-11-09 四川大学 Preparation and application of CpG DNA molecule anti-infection and immunity prepn
GB0421296D0 (en) 2004-09-24 2004-10-27 Angiogene Pharm Ltd Bioreductively-activated prodrugs
US9492400B2 (en) 2004-11-04 2016-11-15 Massachusetts Institute Of Technology Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals
JP2008519052A (en) 2004-11-05 2008-06-05 ザ ジェネラル ホスピタル コーポレイション Optimum behavior of human migratory cells by drugs
WO2007013893A2 (en) 2004-11-15 2007-02-01 Novartis Vaccines And Diagnostics Inc. Immunogenic compositions containing anthrax antigen, biodegradable polymer microparticles, and polynucleotide-containing immunological adjuvant
ATE544774T1 (en) 2004-12-14 2012-02-15 Alnylam Pharmaceuticals Inc RNAI MODULATION OF MLL-AF4 AND USES THEREOF
US20060257359A1 (en) 2005-02-28 2006-11-16 Cedric Francois Modifying macrophage phenotype for treatment of disease
JP2008534508A (en) 2005-03-22 2008-08-28 メドスター ヘルス インコーポレイテッド Delivery system and method for diagnosing and treating cardiovascular disease
US20080305161A1 (en) 2005-04-13 2008-12-11 Pfizer Inc Injectable depot formulations and methods for providing sustained release of nanoparticle compositions
US20060241076A1 (en) 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
WO2006117217A2 (en) 2005-05-04 2006-11-09 Noxxon Pharma Ag Novel use of spiegelmers
WO2006122127A1 (en) * 2005-05-10 2006-11-16 Naomi Balaban Compositions for administering rnaiii-inhibiting peptides
US9290617B2 (en) 2005-07-06 2016-03-22 Molly S. Shoichet Method of biomolecule immobilization on polymers using click-type chemistry
ES2556974T3 (en) 2005-08-12 2016-01-21 Jiang Liu Devices for lymphatic system orientation
BRPI0618857B1 (en) 2005-11-25 2022-07-19 Zoetis Belgium S.A ISOLATED RNA OLIGONUCLEOTIDE, AND METHOD TO NEGATIVELY REGULATE IMMUNOSUPPRESSANT CD4+ REGULATORY CELLS
WO2008051245A2 (en) 2005-12-02 2008-05-02 Novartis Ag Nanoparticles for use in immunogenic compositions
AU2006325225B2 (en) 2005-12-14 2013-07-04 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
WO2007070682A2 (en) 2005-12-15 2007-06-21 Massachusetts Institute Of Technology System for screening particles
US7842312B2 (en) 2005-12-29 2010-11-30 Cordis Corporation Polymeric compositions comprising therapeutic agents in crystalline phases, and methods of forming the same
EP1986685A4 (en) * 2006-01-31 2009-02-25 Medivas Llc Vaccine delivery compositions and methods of use
EP1991204A2 (en) 2006-02-24 2008-11-19 Novartis AG Microparticles containing biodegradable polymer and cationic polysaccharide for use in immunogenic compositions
ES2776100T3 (en) 2006-03-31 2020-07-29 Massachusetts Inst Technology System for targeted delivery of therapeutic agents
US20100247653A1 (en) 2006-04-11 2010-09-30 Hans Lautenschlager Nanoparticles containing nicotine and/or cotinine, dispersions, and use thereof
US8541559B2 (en) 2006-06-12 2013-09-24 Cytos Biotechnology Ag Process for producing aggregated oligonucleotides
US20100144845A1 (en) 2006-08-04 2010-06-10 Massachusetts Institute Of Technology Oligonucleotide systems for targeted intracellular delivery
US20080171059A1 (en) 2006-08-07 2008-07-17 Shanshan Wu Howland Methods and compositions for increased priming of t-cells through cross-presentation of exogenous antigens
CA2657829A1 (en) 2006-08-11 2008-05-29 Panacea Biotec Limited Particles for delivery of active ingredients, process of making and compositions thereof
WO2008033432A2 (en) 2006-09-12 2008-03-20 Coley Pharmaceutical Group, Inc. Immune modulation by chemically modified ribonucleosides and oligoribonucleotides
JP2010505883A (en) 2006-10-12 2010-02-25 ザ ユニバーシティー オブ クイーンズランド Compositions and methods for modulating immune responses
WO2008147456A2 (en) 2006-11-20 2008-12-04 Massachusetts Institute Of Technology Drug delivery systems using fc fragments
WO2008073856A2 (en) 2006-12-08 2008-06-19 Massachusetts Institute Of Technology Delivery of nanoparticles and/or agents to cells
WO2008071774A1 (en) 2006-12-14 2008-06-19 Cytos Biotechnology Ag Purification process for coat protein of rna bacteriophages
US20080149123A1 (en) * 2006-12-22 2008-06-26 Mckay William D Particulate material dispensing hairbrush with combination bristles
EP2510946B1 (en) * 2007-02-07 2015-08-05 The Regents of The University of California Conjugates of synthetic tlr agonists and uses therefor
EP2134830A2 (en) 2007-02-09 2009-12-23 Massachusetts Institute of Technology Oscillating cell culture bioreactor
WO2008115641A2 (en) 2007-02-15 2008-09-25 Yale University Modular nanoparticles for adaptable vaccines
WO2008118861A2 (en) 2007-03-23 2008-10-02 The University Of North Carolina At Chapel Hill Discrete size and shape specific organic nanoparticles designed to elicit an immune response
EP2142216B1 (en) 2007-03-30 2015-04-22 Particle Sciences, Inc. Particle formulations and uses thereof
WO2008124634A1 (en) 2007-04-04 2008-10-16 Massachusetts Institute Of Technology Polymer-encapsulated reverse micelles
CA2683063A1 (en) 2007-04-09 2008-10-16 Chimeros, Inc. Self-assembling nanoparticle drug delivery system
WO2008127532A1 (en) 2007-04-12 2008-10-23 Emory University Novel strategies for delivery of active agents using micelles and particles
US8394914B2 (en) 2007-08-24 2013-03-12 Board Of Trustees Of Michigan State University Functional polyglycolide nanoparticles derived from unimolecular micelles
WO2009038685A1 (en) 2007-09-18 2009-03-26 The Scripps Research Institute Ligands for copper-catalyzed azide-alkyne cycloaddition reactions
WO2009051837A2 (en) 2007-10-12 2009-04-23 Massachusetts Institute Of Technology Vaccine nanotechnology
WO2009052837A2 (en) * 2007-10-24 2009-04-30 Tallinn University Of Technology Maldi ms-based high-throughput screening method for substances inhibiting aggregation of alzheimer's amyloid beta peptides
WO2009076158A1 (en) * 2007-12-07 2009-06-18 Novartis Ag Compositions for inducing immune responses
JP6088123B2 (en) 2008-02-01 2017-03-01 アルファ−オー・ペプチドズ・アーゲーＡｌｐｈａ−Ｏ Ｐｅｐｔｉｄｅｓ Ａｇ Self-assembling peptide nanoparticles useful as vaccines
WO2009106999A2 (en) 2008-02-28 2009-09-03 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Hollow nanoparticles and uses thereof
CN102014874A (en) 2008-03-04 2011-04-13 流体科技公司 Immunomodulator particles and methods of treating
US20090297621A1 (en) 2008-06-03 2009-12-03 Abbott Cardiovascular Systems Inc. Microparticles For The Treatment Of Disease
US20110104293A1 (en) * 2008-07-01 2011-05-05 Bali Pulendran Synergistic induction of humoral and cellular immunity by combinatorial activation of toll-like receptors
WO2010017330A1 (en) 2008-08-06 2010-02-11 Novartis Ag Microparticles for use in immunogenic compositions
ES2433371T3 (en) 2008-08-11 2013-12-10 Glaxosmithkline Llc Purine derivatives for use in the treatment of allergic, inflammatory and infectious diseases
WO2010018132A1 (en) 2008-08-11 2010-02-18 Smithkline Beecham Corporation Compounds
BRPI0917458A2 (en) 2008-08-11 2015-12-01 Glaxosmithkline Llc compound, method of treating disease and conditions, pharmaceutical composition, method for treating or preventing disease, and use of a compound
US8889635B2 (en) 2008-09-30 2014-11-18 The Regents Of The University Of Michigan Dendrimer conjugates
US8277812B2 (en) 2008-10-12 2012-10-02 Massachusetts Institute Of Technology Immunonanotherapeutics that provide IgG humoral response without T-cell antigen
CA2756996C (en) 2009-04-01 2018-12-11 University Of Miami Vaccine compositions and methods of use thereof
EA201101530A1 (en) 2009-04-21 2012-03-30 Селекта Байосайенсиз, Инк. IMMUNONANOTHERAPY, PROVIDING TH1-DISPERSED RESPONSE
CN102414327B (en) 2009-05-01 2015-04-08 奇亚根盖瑟斯堡股份有限公司 A non-target amplification method for detection of RNA splice-forms in a sample
WO2011005850A1 (en) 2009-07-07 2011-01-13 The Research Foundation Of State University Of New York Lipidic compositions for induction of immune tolerance
EP2515942B1 (en) 2009-12-15 2020-02-12 Pfizer Inc. Therapeutic polymeric nanoparticle compositions with high glass transition temperature or high molecular weight copolymers
WO2011085231A2 (en) 2010-01-08 2011-07-14 Selecta Biosciences, Inc. Synthetic virus-like particles conjugated to human papillomavirus capsid peptides for use as vaccines
US20110229556A1 (en) 2010-03-19 2011-09-22 Massachusetts Institute Of Technology Lipid-coated polymer particles for immune stimulation
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
US20110272836A1 (en) 2010-04-12 2011-11-10 Selecta Biosciences, Inc. Eccentric vessels
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
US20120171229A1 (en) 2010-12-30 2012-07-05 Selecta Biosciences, Inc. Synthetic nanocarriers with reactive groups that release biologically active agents
CA2830948A1 (en) 2011-03-25 2012-10-04 Selecta Biosciences, Inc. Osmotic mediated release synthetic nanocarriers
WO2014179772A2 (en) 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce or prevent anaphylaxis in response to a non-allergenic antigen
CN112933218A (en) 2013-06-04 2021-06-11 西莱克塔生物科技公司 Repeated administration of non-immunosuppressive antigen-specific immunotherapeutics
US20150359865A1 (en) 2014-06-17 2015-12-17 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for t-cell-mediated autoimmune disease
US20160220501A1 (en) 2015-02-03 2016-08-04 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
WO2015200728A1 (en) 2014-06-25 2015-12-30 Selecta Biosciences, Inc. Methods and compositions for treatment with synthetic nanocarriers and immune checkpoint inhibitors
WO2016037165A1 (en) 2014-09-07 2016-03-10 Selecta Biosciences, Inc. Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses
WO2016073798A1 (en) 2014-11-05 2016-05-12 Selecta Biosciences, Inc. Methods and compositions related to the use of low hlb surfactants in the production of synthetic nanocarriers comprising a rapalog

Patent Citations (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4009257A (en) * 1970-02-27 1977-02-22 Burroughs Wellcome, Inc. Preparation of immunosuppressive materials
US4021364A (en) * 1972-12-04 1977-05-03 Prof. Dr. Peter Speiser Microcapsules in the nanometric range and a method for their production
US4994281A (en) * 1986-11-12 1991-02-19 Sanraku Incorporated Polylactic acid microspheres and process for producing the same
US4929624A (en) * 1989-03-23 1990-05-29 Minnesota Mining And Manufacturing Company Olefinic 1H-imidazo(4,5-c)quinolin-4-amines
US5114703A (en) * 1989-05-30 1992-05-19 Alliance Pharmaceutical Corp. Percutaneous lymphography using particulate fluorocarbon emulsions
US5733572A (en) * 1989-12-22 1998-03-31 Imarx Pharmaceutical Corp. Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles
US5213812A (en) * 1990-08-01 1993-05-25 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Preparation process of sustained release compositions and the compositions thus obtained
US6365187B2 (en) * 1992-04-24 2002-04-02 Brown University Research Foundation Bioadhesive microspheres and their use as drug delivery and imaging systems
US5871747A (en) * 1992-09-11 1999-02-16 Institut Pasteur Antigen-carrying microparticles and their use in the indication of humoral or cellular responses
US5733925A (en) * 1993-01-28 1998-03-31 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
US5514378A (en) * 1993-02-01 1996-05-07 Massachusetts Institute Of Technology Biocompatible polymer membranes and methods of preparation of three dimensional membrane structures
US5620708A (en) * 1993-02-22 1997-04-15 Alza Corporation Compositions and methods for the oral delivery of active agents
US6225460B1 (en) * 1993-09-17 2001-05-01 Gilead Sciences, Inc. Nucleotide analogs
US5500161A (en) * 1993-09-21 1996-03-19 Massachusetts Institute Of Technology And Virus Research Institute Method for making hydrophobic polymeric microparticles
US5869103A (en) * 1994-06-18 1999-02-09 Danbiosyst Uk Limited Polymer microparticles for drug delivery
US20030035804A1 (en) * 1996-09-16 2003-02-20 Beth Israel Deaconess Medical Center Drug complex for treatment of metastatic prostate cancer
US6387397B1 (en) * 1997-04-18 2002-05-14 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
US6060082A (en) * 1997-04-18 2000-05-09 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
US6989435B2 (en) * 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US20060073114A1 (en) * 1997-09-11 2006-04-06 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US6197229B1 (en) * 1997-12-12 2001-03-06 Massachusetts Institute Of Technology Method for high supercoiled DNA content microspheres
US6881421B1 (en) * 1998-02-27 2005-04-19 Bioalliance Pharma S.A. Nanoparticles comprising at least one polymer and at least one compound able to complex one or more active ingredients
US20050074812A1 (en) * 1998-03-13 2005-04-07 The Burnham Institute Molecules that home to various selected organs or tissues
US20030009029A1 (en) * 1998-06-18 2003-01-09 Merck Patent Gmbh Catalytic titanium (IV) oxide, mediated geminal symmetric dialkylation of carboxamides
US6723429B2 (en) * 1998-08-28 2004-04-20 Celanese Ventures Gmbh Method for preparing smooth-surface spherical microparticles completely or partially made of at least one water-insoluble linear polysaccharide and microparticles produced according to this method
US20110008435A1 (en) * 1998-11-02 2011-01-13 Elan Corporation Plc Nanoparticulate and Controlled Release Compositions Comprising Aryl-Heterocyclic Compounds
US6849270B2 (en) * 1999-04-23 2005-02-01 Alza Corporation Releasable linkage and compositions containing same
US20050113697A1 (en) * 1999-08-13 2005-05-26 Point Biomedical Corporation Method of imaging lymphatic system using nanocapsule compositions
US20020055477A1 (en) * 2000-03-10 2002-05-09 Nest Gary Van Immunomodulatory formulations and methods for use thereof
US20050032733A1 (en) * 2001-05-18 2005-02-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
US20050020525A1 (en) * 2002-02-20 2005-01-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7501134B2 (en) * 2002-02-20 2009-03-10 Novartis Vaccines And Diagnostics, Inc. Microparticles with adsorbed polypeptide-containing molecules
US20040038406A1 (en) * 2002-04-08 2004-02-26 Genesegues, Inc. Nanoparticle delivery systems and methods of use thereof
US20070098713A1 (en) * 2002-04-08 2007-05-03 Genesegues, Inc. Nanoparticle delivery systems and methods of use thereof
US20040022840A1 (en) * 2002-04-12 2004-02-05 Nagy Jon O. Nanoparticle vaccines
US20040067503A1 (en) * 2002-04-22 2004-04-08 Weihong Tan Functionalized nanoparticles and methods of use
US7375234B2 (en) * 2002-05-30 2008-05-20 The Scripps Research Institute Copper-catalysed ligation of azides and acetylenes
US20040092470A1 (en) * 2002-06-18 2004-05-13 Leonard Sherry A. Dry powder oligonucleotide formualtion, preparation and its uses
US20040059094A1 (en) * 2002-07-18 2004-03-25 Bachmann Martin F. Hapten-carrier conjugates and uses thereof
US20050048063A1 (en) * 2002-08-28 2005-03-03 Erkki Ruoslahti Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
US7008411B1 (en) * 2002-09-30 2006-03-07 Advanced Cardiovascular Systems, Inc. Method and apparatus for treating vulnerable plaque
US20060002995A1 (en) * 2002-12-13 2006-01-05 Ian Harwigsson Pharmaceutical porous particles
US20070014804A1 (en) * 2003-02-17 2007-01-18 Peter Burkhard Peptidic nanoparticles as drug delivery and antigen display systems
US7517520B2 (en) * 2003-03-26 2009-04-14 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US20050107322A1 (en) * 2003-04-30 2005-05-19 O'hagan Derek Compositions for inducing immune responses
US20050037075A1 (en) * 2003-06-06 2005-02-17 Farokhzad Omid C. Targeted delivery of controlled release polymer systems
US20050042298A1 (en) * 2003-08-20 2005-02-24 Pardridge William M. Immunonanoparticles
US20070014807A1 (en) * 2003-09-03 2007-01-18 Maida Anthony E Iii Multiplex vaccine
US20080160089A1 (en) * 2003-10-14 2008-07-03 Medivas, Llc Vaccine delivery compositions and methods of use
US20070116768A1 (en) * 2003-12-09 2007-05-24 Michael Chorny Sustained release preparations composed of biocompatible complex microparticles
US20090028910A1 (en) * 2003-12-19 2009-01-29 University Of North Carolina At Chapel Hill Methods for Fabrication Isolated Micro-and Nano-Structures Using Soft or Imprint Lithography
US20070087986A1 (en) * 2004-01-26 2007-04-19 Brett Premack Compositions and methods for enhancing immunity by chemoattractant adjuvants
US20060017339A1 (en) * 2004-06-03 2006-01-26 Lalit Chordia Brushless canned motor
US20080014144A1 (en) * 2004-07-01 2008-01-17 Yale University Methods of Treatment with Drug Loaded Polymeric Materials
US20060002852A1 (en) * 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
US20080044484A1 (en) * 2004-07-08 2008-02-21 The Regents Of The University Of California Use of polymeric nanoparticles for vaccine delivery
US20060051317A1 (en) * 2004-09-07 2006-03-09 Batrakova Elena V Amphiphilic polymer-protein conjugates and methods of use thereof
US20090104268A1 (en) * 2004-10-01 2009-04-23 Midatech Limited Nanoparticles Comprising Antigens and Adjuvants, and Immunogenic Structures
US20090004118A1 (en) * 2004-10-07 2009-01-01 Shuming Nie Multifunctional Nanoparticle Conjugates And Their Use
US20060111271A1 (en) * 2004-11-24 2006-05-25 Cerny Erich H Active and passive immunization against pharmacologically active hapten molecules using a synthetic carrier compound composed of similar elements
US20100062968A1 (en) * 2005-05-10 2010-03-11 Bali Pulendran Novel strategies for delivery of active agents using micelles and particles
US20090053293A1 (en) * 2005-09-09 2009-02-26 Beijing Diacrid Medical Technology Co., Ltd. Nano Anticancer Micelles of Vinca Alkaloids Entrapped in Polyethylene Glycolylated Phospholipids
US20080031899A1 (en) * 2006-02-21 2008-02-07 Reddy Sai T Nanoparticles for immunotherapy
US20080081074A1 (en) * 2006-05-15 2008-04-03 Massachusetts Institute Of Technology Polymers for functional particles
US8367113B2 (en) * 2006-05-15 2013-02-05 Massachusetts Institute Of Technology Polymers for functional particles
US20110052697A1 (en) * 2006-05-17 2011-03-03 Gwangju Institute Of Science & Technology Aptamer-Directed Drug Delivery
US20080014281A1 (en) * 2006-06-16 2008-01-17 Florida Atlantic University Chitin Micro-Particles As An Adjuvant
US20100022680A1 (en) * 2006-06-23 2010-01-28 Massachusetts Institute Of Technology Microfluidic Synthesis of Organic Nanoparticles
US20090074828A1 (en) * 2007-04-04 2009-03-19 Massachusetts Institute Of Technology Poly(amino acid) targeting moieties
US20100068285A1 (en) * 2008-06-16 2010-03-18 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20100069426A1 (en) * 2008-06-16 2010-03-18 Zale Stephen E Therapeutic polymeric nanoparticles with mTor inhibitors and methods of making and using same
US20100075995A1 (en) * 2008-08-11 2010-03-25 Smithkline Beecham Corporation Compounds
US20100055189A1 (en) * 2008-08-29 2010-03-04 Hubbell Jeffrey A Nanoparticles for immunotherapy
US20100129392A1 (en) * 2008-10-12 2010-05-27 Jinjun Shi Targeting of Antigen Presenting Cells with Immunonanotherapeutics
US20100129439A1 (en) * 2008-10-12 2010-05-27 Frank Alexis Adjuvant Incorporation in Immunonanotherapeutics
US20100092425A1 (en) * 2008-10-12 2010-04-15 Von Andrian Ulrich Nicotine Immunonanotherapeutics
US20110027217A1 (en) * 2009-05-27 2011-02-03 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US20140030344A1 (en) * 2009-05-27 2014-01-30 Selecta Biosciences, Inc. Targeted synthetic nanocarriers with ph sensitive release of immunostimulatory agents
US8629151B2 (en) * 2009-05-27 2014-01-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US20110110965A1 (en) * 2009-08-26 2011-05-12 Selecta Biosciences, Inc. Compositions that induce t cell help
US20120027806A1 (en) * 2010-05-26 2012-02-02 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US20120064110A1 (en) * 2010-08-20 2012-03-15 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising peptides obtained or derived from human influenza a virus hemagglutinin
US20120058153A1 (en) * 2010-08-20 2012-03-08 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising proteins obtained or derived from human influenza a virus hemagglutinin
US20120058154A1 (en) * 2010-08-20 2012-03-08 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising peptides obtained or derived from human influenza a virus m2e
US20120070493A1 (en) * 2010-08-23 2012-03-22 Selecta Biosciences, Inc. Targeted multi-epitope dosage forms for induction of an immune response to antigens
US8652487B2 (en) * 2011-04-29 2014-02-18 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory B cells
US20130028857A1 (en) * 2011-07-29 2013-01-31 Selecta Biosciences, Inc. Synthetic nanocarriers comprising polymers comprising multiple immunomodulatory agents
US20130028941A1 (en) * 2011-07-29 2013-01-31 Selecta Biosciences, Inc. Synthetic nanocarriers that generate humoral and cytotoxic t lymphocyte (ctl) immune responses
US20130039954A1 (en) * 2011-07-29 2013-02-14 Selecta Biosciences, Inc. Control of antibody responses to synthetic nanocarriers
US20130058901A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Transplantable graft-specific induced tolerogenic dendritic cells and methods of use
US20130058976A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Allergen-specific induced tolerogenic dendritic cells for allergy therapy
US20130058894A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Therapeutic protein-specific induced tolerogenic dendritic cells and methods of use
US20130058970A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Induced tolerogenic dendritic cells to reduce systemic inflammatory cytokines
US20130059009A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Compositions and methods for producing antigen-specific induced tolerogenic dendritic cells with synthetic nanocarriers
US20130058963A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Induced tolerogenic dendritic cells for generating cd8+ regulatory t cells
US20130058974A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Antigen-specific induced tolerogenic dendritic cells to reduce cytotoxic t lymphocyte responses
US20130058975A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Antigen-specific induced tolerogenic dendritic cells to reduce antibody responses
US20130058902A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Dendritic cell subsets for generating induced tolerogenic dendritic cells and related compositions and methods
US20130058978A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Induced tolerogenic dendritic cells for inducing regulatory b cells
US20130058977A1 (en) * 2011-09-06 2013-03-07 Selecta Biosciences, Inc. Compositions and methods related to induced tolerogenic dendritic cells externally loaded with mhc class i-restricted epitopes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Diwan et al, J. Cont. Rel. 85:247-262, 2002 *

Cited By (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9152988B2 (en) 2000-12-05 2015-10-06 Open Invention Network Method and device utilizing polymorphic data in E-commerce
US20110223201A1 (en) * 2009-04-21 2011-09-15 Selecta Biosciences, Inc. Immunonanotherapeutics Providing a Th1-Biased Response
US9006254B2 (en) 2009-05-27 2015-04-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US9884112B2 (en) 2009-05-27 2018-02-06 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US8629151B2 (en) 2009-05-27 2014-01-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US20110110965A1 (en) * 2009-08-26 2011-05-12 Selecta Biosciences, Inc. Compositions that induce t cell help
US9764031B2 (en) 2010-05-26 2017-09-19 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9066978B2 (en) 2010-05-26 2015-06-30 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
US9987354B2 (en) 2011-04-29 2018-06-05 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells
CN107837402A (en) * 2011-04-29 2018-03-27 西莱克塔生物科技公司 The control release immunodepressant from synthesis nano-carrier
KR20140027360A (en) * 2011-04-29 2014-03-06 셀렉타 바이오사이언시즈, 인크. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
CN114306638A (en) * 2011-04-29 2022-04-12 西莱克塔生物科技公司 Controlled release of immunosuppressants from synthetic nanocarriers
US11235057B2 (en) 2011-04-29 2022-02-01 Selecta Biosciences, Inc. Methods for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses
KR102344744B1 (en) 2011-04-29 2021-12-30 셀렉타 바이오사이언시즈, 인크. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US8652487B2 (en) 2011-04-29 2014-02-18 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory B cells
US11717569B2 (en) 2011-04-29 2023-08-08 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers
KR20200055145A (en) * 2011-04-29 2020-05-20 셀렉타 바이오사이언시즈, 인크. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
KR102112002B1 (en) 2011-04-29 2020-05-18 셀렉타 바이오사이언시즈, 인크. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
CN103517707A (en) * 2011-04-29 2014-01-15 西莱克塔生物科技公司 Controlled release of immunosuppressants from synthetic nanocarriers
US11779641B2 (en) 2011-04-29 2023-10-10 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for allergy therapy
US10441651B2 (en) 2011-04-29 2019-10-15 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells
US10420835B2 (en) 2011-04-29 2019-09-24 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells
US20150320856A1 (en) * 2011-04-29 2015-11-12 Selecta Biosciences, Inc. Controlled release of immunosuppressants from synthetic nanocarriers
WO2012149411A1 (en) * 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Controlled release of immunosuppressants from synthetic nanocarriers
US9265815B2 (en) 2011-04-29 2016-02-23 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers
US9289476B2 (en) 2011-04-29 2016-03-22 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for allergy therapy
US9289477B2 (en) 2011-04-29 2016-03-22 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce cytotoxic T lymphocyte responses
US9295718B2 (en) 2011-04-29 2016-03-29 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
CN105999295A (en) * 2011-04-29 2016-10-12 西莱克塔生物科技公司 Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US20190076522A1 (en) * 2011-04-29 2019-03-14 Selecta Biosciences, Inc. Methods for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses
US10039822B2 (en) * 2011-04-29 2018-08-07 Selecta Biosciences, Inc. Method for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses
US10004802B2 (en) 2011-04-29 2018-06-26 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells
EA027103B1 (en) * 2011-04-29 2017-06-30 Селекта Байосайенсиз, Инк. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US9993548B2 (en) 2011-04-29 2018-06-12 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory B cells
WO2012149255A2 (en) * 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
CN107320734A (en) * 2011-04-29 2017-11-07 西莱克塔生物科技公司 The control release immunodepressant from synthesis nano-carrier
WO2012149255A3 (en) * 2011-04-29 2013-01-03 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US10933129B2 (en) 2011-07-29 2021-03-02 Selecta Biosciences, Inc. Methods for administering synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte responses
CN103702687A (en) * 2011-07-29 2014-04-02 西莱克塔生物科技公司 Synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte (CTL) immune responses
WO2013019669A3 (en) * 2011-07-29 2013-07-04 Selecta Biosciences, Inc. Synthetic nanocarriers that generate humoral and cytotoxic t lymphocyte (ctl) immune responses
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2014152540A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US10335395B2 (en) 2013-05-03 2019-07-02 Selecta Biosciences, Inc. Methods of administering immunosuppressants having a specified pharmacodynamic effective life and therapeutic macromolecules for the induction of immune tolerance
US10668053B2 (en) 2013-05-03 2020-06-02 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce or prevent anaphylaxis in response to a non-allergenic antigen
US10357483B2 (en) 2013-05-03 2019-07-23 Selecta Biosciences, Inc. Methods comprising dosing combinations for reducing undesired humoral immune responses
US10357482B2 (en) 2013-05-03 2019-07-23 Selecta Biosciences, Inc. Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity
US11298342B2 (en) 2013-05-03 2022-04-12 Selecta Biosciences, Inc. Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity
US10434088B2 (en) 2013-05-03 2019-10-08 Selecta Biosciences, Inc. Methods related to administering immunosuppressants and therapeutic macromolecules at a reduced pharmacodynamically effective dose
WO2014197616A1 (en) * 2013-06-04 2014-12-11 Selecta Biosciences, Inc. Repeated administration of non-immunosupressive antigen specific immunotherapeutics
WO2015006747A2 (en) 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use.
EP3971287A1 (en) 2013-07-11 2022-03-23 ModernaTX, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
US10226510B2 (en) 2013-09-27 2019-03-12 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
US10588942B2 (en) 2013-09-27 2020-03-17 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
US11529392B2 (en) 2013-09-27 2022-12-20 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
US10357544B2 (en) 2013-09-27 2019-07-23 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
US9603944B2 (en) 2013-09-27 2017-03-28 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
EP3594348A1 (en) 2013-11-22 2020-01-15 Mina Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
EP3985118A1 (en) 2013-11-22 2022-04-20 MiNA Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
US11633422B2 (en) 2014-09-07 2023-04-25 Selecta Biosciences, Inc. Methods and compositions for attenuating anti-viral transfer vector immune responses
US10071114B2 (en) 2014-09-07 2018-09-11 Selecta Biosciences, Inc. Methods and compositions for attenuating gene expression modulating anti-viral transfer vector immune responses
US10046064B2 (en) 2014-09-07 2018-08-14 Selecta Biosciences, Inc. Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses
US11034752B2 (en) 2015-08-12 2021-06-15 Massachusetts Institute Of Technology Cell surface coupling of nanoparticles
US11261226B2 (en) 2015-08-12 2022-03-01 Massachusetts Institute Of Technology (Mitn1) Cell surface coupling of nanoparticles
EP4349405A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4349404A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4011451A1 (en) 2015-10-22 2022-06-15 ModernaTX, Inc. Metapneumovirus mrna vaccines
WO2017070626A2 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Respiratory virus vaccines
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
US11472856B2 (en) 2016-06-13 2022-10-18 Torque Therapeutics, Inc. Methods and compositions for promoting immune cell function
US11426451B2 (en) 2017-03-11 2022-08-30 Selecta Biosciences, Inc. Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant
US11524033B2 (en) 2017-09-05 2022-12-13 Torque Therapeutics, Inc. Therapeutic protein compositions and methods of making and using the same
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4233880A2 (en) 2017-09-08 2023-08-30 MiNA Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4219715A2 (en) 2017-09-08 2023-08-02 MiNA Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
EP4242307A2 (en) 2018-04-12 2023-09-13 MiNA Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
US11697827B2 (en) 2018-05-16 2023-07-11 Synthego Corporation Systems and methods for gene modification
US11345932B2 (en) 2018-05-16 2022-05-31 Synthego Corporation Methods and systems for guide RNA design and use
US11802296B2 (en) 2018-05-16 2023-10-31 Synthego Corporation Methods and systems for guide RNA design and use
US11884918B2 (en) 2019-01-25 2024-01-30 Synthego Corporation Systems and methods for modulating CRISPR activity
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use

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