Targeted synthetic nanocarriers with ph sensitive release of immunomodulatory agents

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TARGETED SYNTHETIC NANOCARRIERS WITH PH SENSITIVE RELEASE OFIMMUNOMODULATORY AGENTS

FIELD OF THE INVENTION

This invention relates to compositions, and related methods, of synthetic nanocarriers thattarget sites of action in cells, such as antigen presenting cells (APCs), and compriseimmunomodulatory 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 theirencapsulation in synthetic nanocarriers.

BACKGROUND

Immunomodulatory agents are used to produce immune responses in subjects. Stimulation ofthe immune system, which includes stimulation of either or both innate immunity and adaptiveimmunity, is a complex phenomenon that can result in either protective or adverse physiologicoutcomes for the host. In recent years there has been increased interest in the mechanisms underlyinginnate immunity, which is believed to initiate and support adaptive immunity. This interest has beenfueled in part by the recent discovery of a family of highly conserved pattern recognition receptorproteins known as Toll-like receptors (TLRs) believed to be involved in innate immunity as receptorsfor pathogen-associated molecular patterns (PAMPs).

Compositions and methods useful for modulating innate immunity are therefore of greatinterest, 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, informationregarding how the release of such agents, especially labile immunomodulatory agents, from deliveryvehicles can be controlled and what kind of release provides for optimal in vivo effects is lacking.WO 2009/051837 dislcoses PLGA nanoparticles comprising imiquimod as immunostimulatoryagents, the nanoparticles being vacines. US 2008/160089 discloses nanoparticles composed ofpolyester amide polymer comprising an immunostimulatory agent (TLR agonist) that can bemiquimod of CpG nucleic acid. WO 2004/098509 discloses vaccinal comprisiong an antigen, apolylactic acid of PLGA polymer and an immunomodulatory agent. Diwan M. et al, (2002) J.Controlled Release 85, 247-262 discloses PLGA nanspheres encapsulating an immunomodulatoryagent (CpG adjuvant) and antigen, trhe nanospheres can be used for immunization. WO 2008/115319discloses immunomodulatory agents (adenine derivatives) which can be covalently linked topolymeric (PLGA) nanoparticles.

There is a need for new delivery vehicles for delivering immunomodulatory agents that allowfor optimal release as well as related methods. 02128201\158-01 -2- PCT/US2010/001560 WO 2010/138193

SUMMARY OF THE INVENTION

Aspects of the invention relate to compositions comprising synthetic nanocarriers thatcomprise an immunomodulatory agent coupled to the synthetic nanocarrier, wherein theimmunomodulatory agent dissociates from the synthetic nanocarrier according to thefollowing relationship: IArel(4.5)24 % / IArel(7.4)24 % > 1.2, wherein IArel(4.5)24 % isdefined as a weight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH = 4.5 for 24 hours divided by the sumof the weight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH = 4.5 for 24 hours plus a weight ofimmunomodulatory agent retained in the synthetic nanocarrier upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH = 4.5 for 24 hours, expressed asweight percent, and taken as an average across a sample of the synthetic nanocarriers, andwherein IArel(7.4)24 % is defined as a weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4 for 24hours divided by the sum of the weight of immunomodulatory agent released upon exposureof the synthetic nanocanrier to an in vitro aqueous environment at a pH = 7.4 for 24 hoursplus a weight of immunomodulatory agent retained in the synthetic nanocarrier uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4 for 24hours, expressed as weight percent, and taken as an average across a sample of the syntheticnanocarriers.

In some embodiments, the immunomodulatory agent is coupled to the syntheticnanocarrier via an immunomodulatory agent coupling moiety. In certain embodiments, theimmunomodulatory agent is encapsulated within the synthetic nanocarrier. In someembodiments, the immunomodulatory agent comprises a labile immunomodulatory agentsuch as an imidazoquinoline, an adenine derivative, or an oligonucleotide that comprises 5’ -CG - 3’, wherein C is unmethylated and wherein the oligonucleotide comprises a backbonecomprising one or more unstabilized intemucleotide linkages. In certain embodiments, theimidazoquinoline comprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, orresiquimod.

In some embodiments, the oligonucleotide’s backbone comprises no stabilizingchemical modifications that function to stabilize the backbone under physiological -3- WO 2010/138193 PCT/US2010/001560 conditions. In some embodiments, the oligonucleotide’s backbone comprises a backbone thatis not modified to incorporate phosphorothioate stabilizing chemical modifications. In someembodiments, the immunomodulatory agent is an adjuvant. In certain embodiments, theadjuvant comprises a Τόΐΐ-like receptor (TLR) agonist such as a TLR 3 agonist, TLR Ίagonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.

In some embodiments, the TLR agonist is an immunostimulatory nucleic acid such asan immunostimulatory DNA or immunostimulatory RNA. In certain embodiments, theimmunostimulatory nucleic acid is a CpG-containing immunostimulatory nucleic acid thatcomprises one or more stabilizing chemical modifications that function to stabilize the / backbone under physiological conditions. In some embodiments, the adjuvant comprises auniversal T-cell antigen.

In some embodiments, the synthetic nanocarriers further comprise a B cell antigenand/or a T cell antigen. In certain embodiments, the synthetic nanocarriers further comprisean antigen presenting cell (APC) targeting feature. In some embodiments, the syntheticnanocarriers comprise one or more biodegradable polymers. In some embodiments, theimmunomodulatory agent is coupled to the one or more biodegradable polymers via theimmunomodulatory agent coupling moiety. In certain embodiments, the biodegradablepolymer comprises poly(Iactide), poly(glycolide), or poly(Iactide-co-glycolide).

In some embodiments, the biodegradable polymers have a weight average molecularweight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeationchromatography. In certain embodiments, the immunomodulatory agent coupling moietycomprises an amide bond. In some embodiments, the immunomodulatory agent couplingmoiety 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, nanoparticlesthat comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles,pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidalnanoparticles.

Aspects of the invention relate to compositions comprising synthetic nanocarriers thatcomprise an immunomodulatory agent coupled to the synthetic nanocarrier, wherein theimmunomodulatory agent dissociates from the synthetic nanocarrier according to thefollowing relationship: IA(4.5)24 / ΙΑ(4.5)ό > 1.2, wherein LA(4.5)24 is defined as a weight ofimmunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitro -4- PCT/US2010/001560 WO 2010/138193 aqueous environment at a pH = 4.5 for 24 hours taken as an average across a sample of thesynthetic nanocarriers, and wherein IA(4.5)g is defined as a weight of immunomodulatoryagent released upon exposure of the synthetic nanocarrier to an in vitro aqueous environmentat 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 labileimmunomodulatory agent encapsulated within the synthetic nanocarrier. In certainembodiments, the labile immunomodulatory agent comprises an imidazoquinoline, anadenine derivative, or an oligonucleotide that comprises 5’ - CG - 3’, wherein C isunmethylated and wherein the oligonucleotide comprises a backbone comprising one or moreunstabilized intemucleotide linkages. In certain embodiments, the imidazoquinolinecomprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fusedcycloalkylimidazopyridine amine, an imidazoquinoline amine, imiquimod, or resiquimod. Insome embodiments, the oligonucleotide’s backbone comprises no stabilizing chemicalmodifications that function to stabilize the backbone under physiological conditions. In someembodiments, the oligonucleotide’s backbone comprises a backbone that is not modified toincorporate phosphorothioate stabilizing chemical modifications.

Further aspects of the invention relate to compositions comprising syntheticnanocarriers that comprise an immunomodulatory agent coupled to the synthetic nanocarrier,wherein the immunomodulatory agent dissociates from the synthetic nanocarrier according tothe following relationship: 6 < IA(4.5)24 / IA(4.5)e > 1.2, wherein IA(4.5)24 is defined as aweight of immunomodulatory agent released upon exposure of the synthetic nanocarrier to anin vitro aqueous environment at a pH = 4.5 for 24 hours taken as an average across a sampleof the synthetic nanocarriers, and wherein IA(4.5)s is defined as a weight ofimmunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH = 4.5 for 6 hours taken as an average across a sample of thesynthetic nanocarriers.

In some embodiments, the immunomodulatory ageijt Comprises a labileimmunomodulatory agent encapsulated within the synthetic nanocarrier. In someembodiments, the labile immunomodulatory agent comprises an imidazoquinoline, anadenine derivative, or an oligonucleotide that comprises 5’ - CG - 3’, wherein C isunmethylated and wherein the oligonucleotide comprises a backbone comprising one or moreunstabilized intemucleotide linkages. In certain embodiments, the imidazoquinolinecomprises an imidazoquinoline amine, an imidazopyridine amine, a 6,7-fusedcycloalkylimidazopyridine amine, a imidazoquinoline amine, imiquimod, or resiquimod. -5- PCT/US2010/001560 WO 2010/138193

In some embodiments, the oligonucleotide’s backbone comprises no stabilizingchemical modifications that function to stabilize the backbone under physiologicalconditions. In some embodiments, the oligonucleotide’s backbone comprises a backbone thatis not modified to incorporate phosphorothioate stabilizing chemical modifications. Incertain embodiments, the immunomodulatory agent is coupled to the synthetic nanocarriervia an immunomodulatory agent coupling moiety. In some embodiments, theimmunomodulatory agent is encapsulated within the synthetic nanocarrier.

In some embodiments, the immunomodulatory agent is an adjuvant. In certainembodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist such as a TLR 3agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist, or a TLR 9 agonist. In certainembodiments, the TLR agonist is an immunostimulatory nucleic acid such as animmunostimulatory DNA or immunostimulatory RNA.

In some embodiments, the immunostimulatory nucleic acid is a CpG-containingimmunostimulatory nucleic acid that comprises one or more stabilizing chemicalmodifications that function to stabilize the backbone under physiological conditions. Incertain embodiments, the adjuvant comprises a universal T-cell antigen. In someembodiments, the synthetic nanocarriers further comprise a B cell antigen and/or a T cellantigen.

In some embodiments, the synthetic nanocarriers further comprise an antigenpresenting cell (APC) targeting feature. In certain embodiments, the synthetic nanocarrierscomprise one or more biodegradable polymers. In some embodiments, theimmunomodulatory agent is coupled to the one or more biodegradable polymers via theimmunomodulatory agent coupling moiety. In certain embodiments, the biodegradablepolymer comprises poly(lactide), poly(glycolide), or poly(lactide-co-glycolide).

In some embodiments, the biodegradable polymers have a weight average molecularweight ranging from 800 Daltons to 10,000 Daltons, as determined using gel permeationchromatography. In certain embodiments, the immunomodulatory agent coupling moietycomprises an amide bond. In some embodiments, the immunomodulatory agent couplingmoiety 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, nanoparticlesthat comprise a combination of nanomaterials, spheroidal nanoparticles, cubic nanoparticles, -6- PCT/US2010/001560 WO 2010/138193 pyramidal nanoparticles, oblong nanoparticles, cylindrical nanoparticles, or toroidalnanoparticles. In certain embodiments, compositions associated with the invention furthercomprise a pharmaceutically acceptable excipient.

Further aspects of the invention relate to compositions comprising a vaccinecomprising any of the compositions associated with the invention.

Further aspects of the invention involve methods comprising administering any of thecompositions associated with the invention to a subject. In some embodiments, thecomposition is in an amount effective to induce or enhance an immune response. In someembodiments, the subject has cancer, an infectious disease, a non-autoimmune metabolicdisease, a degenerative disease, or an addiction.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 demonstrates the release of resiquimod (R848) from synthetic nanocarrierformulations at pH 7.4, 37°C.

Fig· 2 demonstrates the release of R848 from synthetic nanocarrier formulations at pH4.5, 37°C.

Fig. 3 demonstrates the release of R848 from synthetic nanocarrier formulations at pH7.4 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 ofantibody induction by synthetic nanocarriers without the CpG-containing immunostimulatorynucleic acid (Group 1).

Fig. 5 shows the level of antibody induction by synthetic nanocarriers that release aphosphodiester, non-thioated CpG-containing immunostimulatory nucleic acid or a thioatedCpG-containing immunostimulatory nucleic acid.

Fig. 6 shows the level of antibody induction by synthetic nanocarriers that releaseR848 at different rates.

Fig. 7 shows the level of antibody induction by synthetic nanocarriers carryingentrapped phosphodiester (PO) CpG, designated as NC-Nic/PO-CpG.

Fig. 8 shows the release of entrapped ΡΟ-CpG from nanocarriers at a pH of 4.5 versuspH 7.5. The data demonstrates that a labile imunomodulatory agent, such as ΡΟ-CpG, isprotected by encapsulation within a synthetic nanocarrier. Such a labile agent can be releasedat a desired site of action with a pH of 4.5 (e.g., in the endosome/lysosome) with low levelsof release occurring at a pH of 7.4 (e.g., generally the pH outside of the endosome/lysosome). -7- 216548/2

DETAILED DESCRIPTION

Before describing the present invention in detail, it is to be understood that thisinvention is not limited to particularly exemplified materials or process parameters as suchmay, of course, vary. It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, and is not intended to belimiting of the use of alternative terminology to describe the present invention.

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 "asolvent" includes a mixture of two or more such solvents, reference to "an adhesive" includesmixtures of two or more such materials, and the like.

INTRODUCTION

This invention is useful in that it provides a way to release immunomodulatory agentsmore 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 andtoxicity, as the majority of the release of the immunomodulatory agents would be at a site ofaction in the cells of interest. This is of particular interest for the delivery of adjuvants. Thecontrolled release properties offer for the first time a controlled way of deliveringimmunomodulatory agents to the immune cells of interest and allow for a more preciseintervention on the immune system, including the ability to release immunomodulatoryagents over an extended period. All of this leads to a very tunable system to get the optimumrelease of immunomodulatory agent such that it will release primarily at a site of action in thedesired cells.

The inventors have further recognized that coupling labile immunomodulatory agentswithin the inventive synthetic nanocarriers through encapsulating the labileimmunomodulatory agents within the inventive synthetic nanocarriers, and providing acontrolled way of delivering labile immunomodulatory agents to immune cells of interest,preferably over an extended period, results in targeted delivery of the labileimmunomodulatory agents while minimizing off-target effects of the immunomodulatoryagents, especially off-target effects associated with systemic administration of the 02128201400-01 -8- PCT/US2010/001560 WO 2010/138193 immunomodulatory agents. Additonally, this approach can enhance the performance oflabile immunomodulatory agents having a short half-life of elimination that otherwise mightnot have a desirable level of pharmacological activity.

In one embodiment, the invention relates to certain oligonucleotides. Recently, therehave been a number of reports describing the immunostimulatory effect of certain types ofnucleic acid molecules, including CpG nucleic acids, GU rich ssRNA and double-strandedRNA. Of note, it was recently reported that Toll-like receptor 9 (TLR9) recognizes bacterialDNA 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 USA98:9237-42. The effects of CpG containing oligonucleotides on immune modulation havebeen 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 asW098/37919, W098/40100, W098/52581, and W099/56755. The entire immunostimulatorynucleic 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 rapidlycleaved by nucleases found in the extracellular environment. Yu, D., et al., Potent CpGoligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatoryproperties. Biochem Biophys Res Commun, 2002. 297(1): p. 83-90 (“Yu et al.”); Heeg, K., etal., Structural requirements for uptake and recognition of CpG oligonucleotides. Int J MedMicrobiol, 2008. 298(1-2): p. 33-8 (“Heeg et al.”). Such natural oligonucleotides may beconsidered labile immunomodulatory agents. Accordingly, methods of chemically stabilizingthe linkages by replacing the phosphodiester linking group with a phosphorothioate grouphave been extensively reported in the literature. See US Patent 6811975 - PhosphorothioateOligonucleotides Having Modified Intemucleoside Linkages.

Phosphorothioate CpG containing oligonucleotides have been administeredsystemically as vaccine adjuvants. Yu et al. However, systemic administration of stabilizedCpG oligonucleotides can result in off-target immunostimulatory effects, such as generalinflammation, non-specific activation of lymphocytes, and flu-like symptoms. Haas, T., etal., Sequence independent interferon-alpha induction by multimerized phosphodiester DNAdepends on spatial regulation of Toll-like receptor-9 activation in plasmacytoid dendriticcells. Immunology, 2009. 126(2): p. 290-8 (“Haas et al.”). Accordingly, sucholigonucleotides may be usefully incorporated in the practice of the present invention, as isdescribed in more detail below. -9- PCT/US2010/001560 WO 2010/138193

The inventors have unexpectedly and surprisingly discovered that the problems andlimitations noted above can be overcome by practicing the invention disclosed herein. Inparticular, the inventors have unexpectedly discovered that it is possible to provide, togetherwith related methods, a composition comprising: synthetic nanocarriers that comprise animmunomodulatory agent coupled to the synthetic nanocarrier; wherein theimmunomodulatory agent, preferably a labile immunodmodulatory agent, dissociates fromthe 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 releasedupon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5for t hours divided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5 for thours plus a weight of immunomodulatory agent retained in the synthetic nanocarrier uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5 for thours, expressed as weight percent, and taken as an average across a sample of the syntheticnanocarriers; and wherein IArel(7.4)t % is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitro aqueous environment at apH = 7.4 for t hours divided by the sum of the weight of immunomodulatory agent releasedupon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4for t hours plus a weight of immunomodulatory agent retained in the synthetic nanocarrierupon exposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4for t hours, expressed as weight percent, and taken as an average across a sample of thesynthetic nanocarriers; and wherein t is 2,4, 6, 8, 10,12,14,16,18,20,22, 24,26,28, or 30hours.

In some embodiments, the immunomodulatory agent, preferably a labileimmunodmodulatory agent, dissociates from the synthetic nanocarrier according to thefollowing 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 % / IAreI(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, -10- PCT/US2010/001560 WO 2010/138193 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, orIArel(4.5)t % I IArel(7.4)t % > 11, wherein IArel(4.5)t %, IArel(7.4)t %, and t are as definedabove.

In other embodiments, the immunomodulatory agent, preferably a labileimmunodmodulatory agent, dissociates from the synthetic nanocarrier according to thefollowing 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 %I 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, tis 24 hours.

Accordingly, this invention relates to compositions and methods comprising syntheticnanocarriers that release immunomodulatory agents at significantly different rates at neutraland acidic pH. In delivering immunomodulatory agents, to have the most potent effect it isdesirable to have the majority of the immunomodulatory agent released inside APCs wherethey can have a desired effect. When immunomodulatory agents are injected in free form, orwhen they are released from a synthetic nanoparticle outside the APCs, only a small portionof that immunomodulatory agent finds its way to the APCs, while the rest diffuses throughthe 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. -11- PCT/US2010/001560 WO 2010/138193

Upon being taken up by the APC, the synthetic nanocarriers are presumed to be endocytosedinto an endosomal/lysosomal compartment where the pH becomes more acidic, as opposed tothe neutral pH outside the cells. Under these conditions, the immunomodulatory agentexhibits a pH sensitive dissociation from the synthetic nanocarrier (e.g., from animmunomodulatory agent coupling moiety) and is released from the synthetic nanocarrier.The immunomodulatory agent is then free to interact with receptors associated with theendosome/lysosome and stimulate a desired immune response. The property of the inventivesynthetic nanocarriers of having lower release of immunomodulatory agents at or aboutneutral pH, or in embodiments at or about physiological pH (i.e., pH = 7.4), but increasedrelease at or about a pH of 4.5 is desirable for it targets the immunomodulatory agents to theendosomal/lysosomal compartment of APCs to which the synthetic nanocarriers target.

The immunomodulatory agents can be coupled to the synthetic nanocarriers by any ofa number of methods. Generally, the coupling can be a result of bonding between theimmunomodulatory agent and the synthetic nanocarrier. This bonding can result in theimmunomodulatory agent being attached to the surface of the synthetic nanocarrier and/orcontained within (encapsulated) the synthetic nanocarrier. In some embodiments, however,the immunomodulatory agent is encapsulated by the synthetic nanocarrier as a result of thestructure of the synthetic nanocarrier rather than bonding to the synthetic nanocarrier.

When coupling occurs as a result of bonding between the immunomodulatory agentand synthetic nanocarrier, the coupling occurs via an immunomodulatory agent couplingmoiety. An immunomodulatory agent coupling moiety can be any moiety through which animmunomodulatory agent is bonded to a synthetic nanocarrier. Such moieties includecovalent 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, theimmunomodulatory agent coupling moiety can comprise a charged polymer to which animmunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically binds.As another example, the immunomodulatory agent coupling moiety can comprise a polymeror 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 polymeror unit thereof comprises polyethylene glycol) (PEG), poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or a polycaprolactone, or unit thereof. In some embodiments, itis preferred that the polymer is biodegradable. Therefore, in these embodiments, it is -12- PCT/US2010/001560 WO 2010/138193 preferred that if the polymer comprises a polyether, such as poly(ethylene glycol) or unitthereof, the polymer comprises a block-co-polymer of a polyether and a biodegradablepolymer such that the polymer is biodegradable. In other embodiments, the polymer does notsolely comprise a polyether or unit thereof, such as poly(ethylene glycol) or unit thereof. Theimmunomodulatory agent coupling moiety as provided herein, therefore, can comprise one ofthe 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 thecompounds or conjugates provided herein is insoluble in water at pH = 7.4 and at 25°C, isbiodegradable, or both. In other embodiments, the polymer is insoluble in water at pH = 7.4and at 25°C but soluble at pH = 4.5 and at 25°C. In still other embodiments, the polymer isinsoluble in water at pH = 7.4 and at 25°C but soluble at pH = 4.5 and at 25°C andbiodegradable. In other embodiments, any of the polymers provided herein can have aweight average molecular weight, as determined by gel permeation chromatography, of about800 Da to 10,000 Da (e.g., 2,000 Da).

In one embodiment, the immunomodulatory agent is an adjuvant, such as animidazoquinoline. Imidazoquinolines include compounds, such as imiquimod andresiquimod (also known as R848). Such adjuvants can be coupled to a polymer as providedabove. 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 R848release of 3- to 6-fold when the pH was dropped from 7.4 to 4.5. Table 1 lists thecompositions of the particles tested. These included two formulations that encapsulatedR848,2 formulations with the PLA coupled covalently to R848 through the R848 amine, andfour formulations with PLA coupled covalently to R848 (via a ring opening method). In allformulations, the release of R848 was significantly increased at the lower pH. Theencapsulated release rate is much faster than the conjugated release rates, and there are alsodifferences in release rates between the conjugation methods.

Table 1. Formulation Targets With A Covalent R848

Formulation R848load* Ovapeptideload PLA-PEG-NIC PLA-R848conjugatetype** PLA (15-20K, BI R202H) Chemistry 1 El.5% 1.1-2.2% 25% 75% 2 E1.5%++ 1.1-2.2% 25% 75% 3 C75% 0.15- 25% Method 1 Amine -13- PCT/US2010/001560 WO 2010/138193

0.31% 4 €75% 0.15- 0.31% 25% Method 1 Amine 5 €75% 0.15- 0.31% 25% Method 5 ROP-hiMW 6 €75% 0.15- 0.31% 25% Method 5 ROP-loMW 7 €50% 0.15- 0.31% 25% Method 5 25% ROP-loMW 8 €25% 0.15- 0.31% 25% Method 5 50% ROP-loMW *C=covalent R848; E=encapsulation of R848

Although the above example was with PLA, immunomodulatory agents, such asR848, can be coupled to other polymers or units thereof, such as those provided above andelsewhere herein including polylactide-co-glycolide (PLGA) block co-polymer or unitthereof. Immunomodulatory agents, such as R848, can be coupled to such polymers or unitsthereof by an amide or ester bond. Examples of methods for effecting such coupling areprovided 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 thesynthetic nanocarrier; wherein the immunomodulatory agent, preferably a labileimmunodmodulatory agent, dissociates from the synthetic nanocarrier according to thefollowing relationship: IA(4.5)ti/IA(4.5)t2> 1.2; wherein IA(4.5)ti is defined as a weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5 for tlhours taken as an average across a sample of the synthetic nanocarriers; and wherein IA(4.5)t2is defined as a weight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH = 4.5 for t2 hours taken as an averageacross a sample of the synthetic nanocarriers; and wherein tl 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; andtl > t2. In some embodiments, tl is 24 hours, and t2 is 6 hours.

In some embodiments, the immunomodulatory agent, preferably a labileimmunodmodulatory agent, dissociates from the synthetic nanocarrier according to the -14- PCT/US2010/001560 WO 2010/138193 following relationship: IA(4.5)U / ΙΑ(4.5)<2 > 1.5, IA(4.5)tI / ΙΑ(4.5)β > 2, IA(4.5)ti / IA(4.5)C> 2.5, IA(4.5)U / IA(4.5)t2 > 3, IA(4.5)ti / IA(4.5)t2 > 3.5, IA(4.5)ti / IA(4.5)a > 4, IA(4.5)tl /IA(4.5)t2 > 4.5, IA(4.5)ti / IA(4.5)t2 > 5, IA(4.5)tI / IA(4.5)t2 > 6, IA(4.5)tI / ΙΑ(4.5)β > 7,IA(4.5)ti / ΙΑ(4.5)β > 8, IA(4.5)tl / ΙΑ(4.5)α > 9, or IA(4.5)ti / ΙΑ(4.5)β > 10; whereinIA(4.5)ti, IA(4.5)t2, tl, and t2 are as defined above. In some embodiments, tl is 24 hours, andt2 is 6 hours.

In other embodiments, the immunomodulatory agent, preferably a labileimmunodmodulatory agent, dissociates from the synthetic nanocarrier according to thefollowing relationship: 10 < IA(4.5)ti / ΙΑ(4.5)β > 1.2,10 < IA(4.5)ti / ΙΑ(4.5)β 2,10 <IA(4.5)ti / IA(4.5)t2 2.5,10 < IA(4.5)ti / IA(4.5)q > 3,10 < IA(4.5)U / IA(4.5)t2 > 3.5,10 <IA(4.5)ti / IA(4.5)t2 > 4,10 < IA(4.5)tl / ΙΑ(4.5)α > 4.5,10 < IA(4.5)U / ΙΑ(4.5)α > 5, 10 <IA(4.5)ti / IA(4.5)t2 > 6,10 < IA(4.5)d / IA(4.5)t2 > 7,10 < IA(4.5)ti / IA(4.5)q > 8,10 <IA(4.5)ti / IA(4.5)t2 > 9, 9 < IA(4.5)tJ / ΙΑ(4.5)α > 1.2, 8 < IA(4.5)tl / IA(4.5)t2 > 1.2, 7 <IA(4.5)ti / IA(4.5)t2 > 1.2, 6 < IA(4.5)d / ΙΑ(4.5)β > 1.2, 5 < IA(4.5)n / ΙΑ(4.5)β > 1.2,4.5 <IA(4.5)ti / IA(4.5)t2 > 1.2, 4 < IA(4.5)d / IA(4.5)G > 1.2, 3.5 < IA(4.5)U / ΙΑ(4.5)β > 1.2,3 <IA(4.5)ti / IA(4.5)t2 > 1.2,2.5 < IA(4.5)ti / ΙΑ(4.5)α > 1.2,2 < IA(4.5)ti / IA(4.5)t2 > 1.2,1.5 <IA(4.5)ti / ΙΑ(4.5)α £ 1.2, 3 < IA(4.5)d / IA(4.5)Q > 2,4 < IA(4.5)U / IA(4.5)t2 > 3, 5 <IA(4.5)ti / IA(4.5)t2 > 4,6 < IA(4.5)U / ΙΑ(4.5)α > 5,7 < IA(4.5)ti / ΙΑ(4.5)α > 6, 8 < IA(4.5)tiI IA(4.5)t2 > 7, or 9 < IA(4.5)U / ΙΑ(4.5)α 8; wherein IA(4.5)ti, ΙΑ(4.5)β, tl, and t2 are asdefined above. In some embodiments, tl is 24 hours, and t2 is 6 hours.

Inventive synthetic nanocarriers have also been shown to exhibit the property ofaugmenting a humoral immune response to a specific antigen. Such augmented humoralimmune response has been found to be elevated, in some embodiments, with faster release ofimmunomodulatory agent.. In one embodiment, the immunomodulatory agent is a CpG-containing immunostimulatory nucleic acid, and the CpG-containing immunostimulatorynucleic acid is encapsulated within a synthetic nanocarrier. In in vitro studies, describedfurther below in the EXAMPLES, it was found that optimal release of the CpG-containingimmunostimulatory nucleic acids from synthetic nanocarriers produced an elevated humoralimmune response to nicotine, which was also coupled to the synthetic nanocarriers. In someembodiments, such optimal release was found to better augment an antibody response to anantigen.

Optimal release is the dissociation of the immunomodulatory agent from the syntheticnanocarrier that produces the best levels of desired effect(s). In some embodiments, the -15- PCT/US2010/001560 WO 2010/138193 desired effect is an immediate immune response of a desired level (i.e., one that occurs soonafter the administration of the synthetic nanocarrier). Generally, an immediate immuneresponse is one measured on the order of seconds, minutes, or a few hours. In otherembodiments, the desired effect is an immune response of a desired level that occurs after afew hours. In still other embodiments, the desired effect is an immune response of a desiredlevel that is sustained for an extended period of time, such as for 1,2, 5,10,15 or morehours. In other embodiments, the extended period of time is for 1,2, 5,10,15,20,25,30 ormore days. In further embodiments, the extended period of time is for 1, 2, 5, 10 or moremonths. 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 releaseis one wheren the immunomodulatory agent dissociates from the synthetic nanocarrieraccording to one of the above relationships.

In embodiments, an immunomodulatory agent, preferably a labileimmunodmodulatory agent, that dissociates from the synthetic nanocarrier at an intermediaterate satisfies the following relationship: 6 < IA(4.5)ti / IA(4.5)t2 1.2, 5 < IA(4.5)n / IA(4.5)G> 1.2,4 < IA(4.5)U / IA(4.5)t2 > 1.2, 3 < IA(4.5)ti / ΙΑ(4.5)α > 1.2,2 < IA(4.5)U / IA(4.5)G >1.2,6 < IA(4.5)u / IA(4.5)t2 > 2,6 < IA(4.5)tI / IA(4.5)G > 2.5, 6 < IA(4.5)U / ΙΑ(4.5)α >3,6< IA(4.5)U / IA(4.5)G > 3.5, 6 < IA(4.5)U / IA(4.5)t2 > 4, 6 < IA(4.5)ti / IA(4.5)C > 5, 4 <IA(4.5)U / IA(4.5)t2 > 1.5, 3.5 < IA(4.5)ti / IA(4.5)t2 > 1.5, 3 < IA(4.5)ti / IA(4.5)G > 1.5,2.5 <IA(4.5)ti / IA(4.5)t2 > 1.5, 5 < IA(4.5)U / ΙΑ(4.5)β > 2, 4 < IA(4.5)U / ΙΑ(4.5)<2 > 2, or 3 <IA(4.5)tj / IA(4.5)g 2; wherein IA(4.5)ti, ΙΑ(4.5)α, tl, and t2 are as defined above. In someembodiments, tl is 24 hours, and t2 is 6 hours.

As another example, resiquimod was encapsulated within a synthetic nanocarrier. Inin vitro studies, described further below in the EXAMPLES, it was found that resiquimodcontained in the synthetic nanocarriers augmented humoral immune response against nicotinealso coupled to the synthetic nanocarriers. It was also found that an intermediate release ofthe resiquimod from the synthetic nanocarriers was optimal, as it resulted in a higher level ofantibody induction than fast or slow release of the resiquimod.

Accordingly, the synthetic nanocarriers provided herein can also comprise one ormore antigens. The antigens can be B cell antigens or T cell antigens or a combination ofboth. Such antigens can be coupled to the synthetic nanocarriers such that they are present onthe surface of the synthetic nanocarriers, encapsulated within the nanocarriers or both, insome embodiments. In embodiments, the immunomodulatory agent augments an immune -16- PCT/US2010/001560 WO 2010/138193 response to such an antigen. As mentioned above, the antigen can also be coupled to thesynthetic nanocarriers. In other embodiments, however such as antigen is not coupled to thesynthetic nanocarriers. In some of these embodiments, such an antigen can becoadministered to a subject. In still other of these embodiments, such an antigen is notcoadministered to the subject.

DEFINITIONS “Adjuvant” means an agent that does not constitute a specific antigen, but boosts thestrength and longevity of immune response to an antigen. Such adjuvants may include, butare 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 withmonphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonellaminnesota, Salmonella typhimurium, or Shigella fiexneri or specifically with MPL® (AS04),MPL A of above-mentioned bacteria separately, saponins, such as QS-21,Quil-A, ISCOMs,ISCOMATRIX™, emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02(QS21+squalene+ MPL®), liposomes and liposomal formulations such as AS01, synthesizedor specifically prepared microparticles and microcarriers such as bacteria-derived outermembrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosanparticles, depot-forming agents, such as Pluronic® block co-polymers, specifically modifiedor 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 notlimited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3,4, 5, 7, 8, 9 and/orcombinations thereof. In other embodiments, adjuvants comprise agonists for Toll-LikeReceptors 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 knownas R848); adenine derivatives, such as those disclosed in US patent 6,329,381 (SumitomoPharmaceutical Company); immunostimulatory DNA; or immunostimulatory RNA. Inspecific embodiments, synthetic nanocarriers incorporate as adjuvants compounds that areagonists for toll-like receptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8agonist compounds disclosed in US Patent 6,696,076 to Tomai et al., including but notlimited to imidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. Preferredadjuvants comprise imiquimod and resiquimod. In specific embodiments, an adjuvant may -17- PCT/US2010/001560 WO 2010/138193 be an agonist for the DC surface molecule CD40. In certain embodiments, a syntheticnanocarrier incorporates an adjuvant that promotes DC maturation (needed for effectivepriming of naive T cells) and the production of cytokines, such as type I interferons, which inturn stimulate antibody and cytotoxic immune responses against desired antigen. Inembodiments, adjuvants also may comprise immunostimulatory RNA molecules, such as butnot 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 chemicallymodified ribonucleosides and oligoribonucleotides” WO 2008033432 A2; A. Forsbach et al.,“Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targetingthe Toll-like receptor 8 pathway” WO 2007062107 A2; E. Uhlmann et al., “Modifiedoligoribonucleotide analogs with enhanced immunostimulatory activity” U.S. Pat. Appl.Publ. US 2006241076; G. Lipford et al., “Immunostimulatory viral RNA oligonucleotidesand use for treating cancer and infections” WO 2005097993 A2; G. Lipford et al.,“Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screeningmethods” 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 someembodiments, adjuvants may comprise TLR-5 agonists, such as flagellin, or portions orderivatives thereof, including but not limited to those disclosed in US Patents 6,130,082,6,585,980, and 7,192,725. In specific embodiments, synthetic nanocarriers incorporate aligand for Toll-like receptor (TLR)-9, such as immunostimulatory oligonucleotide moleculescomprising 5’ - CG - 3’ motifs, wherein the C is unmethylated, which induce type Iinterferon secretion, and stimulate T and B cell activation leading to increased antibodyproduction and cytotoxic T cell responses (Krieg et al., CpG motifs in bacterial DNA triggerdirect B cell activation. Nature. 1995. 374:546-549; Chu et al. CpG oligodeoxynucleotidesact as adjuvants that switch on T helper 1 (Thl) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al. CpG-containing synthetic oligonucleotides promote B and cytotoxic Tcell 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 potentenhancer of specific immunity in mice immunized with recombinant hepatitis B surfaceantigen. J. Immunol. 1998. 160:870-876; Lipford et al., Bacterial DNA as immune cellactivator. Trends Microbiol. 1998. 6:496-500. In some embodiments, adjuvants may beproinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some -18- WO 2010/138193 PCT/US2010/001560 embodiments, adjuvants may be activated components of the complement cascade (e.g.,CD21, CD35, etc.). In some embodiments, adjuvants may be activated components ofimmune complexes. The adjuvants also include complement receptor agonists, such as amolecule that binds to CD21 or CD35. In some embodiments, the complement receptoragonist induces endogenous complement opsonization of the synthetic nanocarrier. In someembodiments, adjuvants are cytokines, which are small proteins or biological factors (in therange of 5 kD - 20 kD) that are released by cells and have specific effects on cell-cellinteraction, communication and behavior of other cells. In some embodiments, the cytokinereceptor agonist is a small molecule, antibody, fusion protein, or aptamer. "Administering” or "administration" means providing a drug to a patient in a mannerthat is pharmacologically useful. “APC targeting feature” means one or more portions of which the inventive syntheticnanocarriers are comprised that target the synthetic nanocarriers to professional antigenpresenting cells (“APCs”), such as but not limited to dendritic cells, SCS macrophages,follicular dendritic cells, and B cells. In embodiments, APC targeting features may compriseimmunofeature surface(s) and/or targeting moieties that bind known targets on APCs. Inembodiments, APC targeting features may comprise one or more B cell antigens present on asurface of synthetic nanocarriers. In embodiments, APC targeting features may alsocomprise one or more dimensions of the synthetic nanoparticles that is selected to promoteuptake 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 onmacrophages (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); CD1 la (LFA-la, a L Integrin chain); CD1 lb (aM Integrin chain, CR3, Mol, C3niR, Mac-1); CD1 lc (aXIntegrin, pl50,95, AXb2); CDwl2 (p90-120); CD13 (APN, gpl50, 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); CD 16b (FcgRIIIb);CDwl7 (Lactosylceramide, LacCer); CD 18 (Integrin β2, CD1 la,b,c β-subunit); CD26 (DPPIV ectoeneyme, ADA binding protein); CD29 (Platelet GPIIa, β-l integrin, GP); CD31(PECAM-1, Endocam); CD32 (FCyRII); 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, -19- PCT/US2010/001560 WO 2010/138193

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-Ια, al Integrin); CD49b (VLA-2a, gpla, a2 Integrin); CD49c (VLA-3a, a3 Integrin);CD49e (VLA-5a, aS Integrin); CD49f (VLA-6a, a6 Integrin, gplc); CD50 (ICAM-3); CDS 1(Integrin a, VNR-a, Vitronectin-Ra); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54(ICAM-1); CD55 (DAF); CD58 (LFA-3); CD59 (lFSAg, H19, Protectin, MACIF, MIRL, P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP Illa, β3 Integrin); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1, gp55, NGA,LAMP-3, ME491); CD64 (FcyRI); CD65 (Ceramide, VIM-2); CD65s (Sialylated-CD65,VIM2); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD74 (li, invariant chain); CD75 (sialo-maskedLactosamine); CD75S (a2,6 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 (a2M-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); GDI 12 (HveB, PRR2, PVRL2, Nectin2); GDI 14 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms, CSF-1R, M-CSFR); CD116 (GMCSFRa); CDwl 19 (IFNyR,IFNyRA); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFRp80); CDI21b (Type 2IL-1R); CD122 (IL2RP); CD123 (IL-3Ra); CD124 (IL-4Ra); CD127 (p90, IL-7R, IL-7Ra);CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb, CSCR2,(Tentatively renamed as CD182)); CD 130 (gpl30); CD131 (Common β subunit); CD132(Common γ chain, IL-2Ry); CDwl36 (MSP-R, RON, pl58-ron); CDwl37 (4-1BB, ILA); CD 139; CD141 (Thrombomodulin, Fetomodulin); CD 147 (Basigin, EMMPRIN, M6,0X47);CD148 (ΗΡΤΡ-η, p260, DEP-1); CD155 (PVR); CD156a (CD156, ADAM8, MS2); CD156b(TACE, ADAM17, cSVP); CDwl56C (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD165 (AD2, gp37); CD168 (RHAMM, IHABP, HMMR);CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-la, MyD-1); CD172b (SIIU^); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1, (Formerlyknown as CD128a)); CD182 (CXCR2, (Formerly known as CD128b)); CD184 (CXCR4,NPY3R); CD191 (CCR1); CD192 (CCR2); CD195 (CCR5); CDwl97 (CCR7 (wasCDwl97)); CDwl98 (CCR8); CD204 (MSR); CD205 (DEC-25); CD206 (MMR); CD207 -20- PCT/US2010/001560 WO 2010/138193 (Langerin); CDw210 (CK); CD213a (CK); CDw217 (CK); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD226 (DNAM-1, PTA1); CD230 (PrionProtein (PrP)); CD232 (VESP-R); CD244 (2B4, P38, NAIL); CD245 (p220/240); CD256(APRIL, TALL2, INF (ligand) superfamily, member 13); CD257 (BLYS, TALL1, INF(ligand) superfamily, member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member 10a);CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNBF-Rsuperfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member lOd); 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 (JAMI); CD322 (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 (gp110, Macrosialin); and/or mannose receptor; wherein the names listed in parenthesesrepresent 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, CD la (R4, T6, HTA-1); CDlb (Rl); CDlc (M241, R7); CDld (R3); CDle (R2); CD1 lb (aM Integrin chain, CR3,Mol, C3niR, Mac-1); CD1 lc (aX Integrin, pl50, 95, AXb2); CDwll7 (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-4a,a4 Integrin); CD49e (VLA-5a, a5 Integrin); CD58 (LFA-3); CD64 (FcyRI); 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-CSFRa);CD120a (TMFRI, p55); CD120b (TNFRII, p75, INFR p80); CD123 (IL-3Ra); CD139;CD148 (ΗΡΤΡ-η, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP);CD157 (Mo5, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR);CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE); CD172 (SIRP-la, MyD-1); CD172b (SIRPfi); CD180 (RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); WO 2010/138193 PCT/US2010/001560 CD193 (CCR3); CD196 (CCR6); CD197 (CCR7 (ws CDwl97)); CDwl97 (CCR7, EBI1,BLR2); CD200 (0X2); CD205 (DEC-205); CD206 (MMR); CD207 (Langerin); CD208(DC-LAMP); CD209 (DCSIGN); CDw218a (IL18Ra); CDw218b (IL8RP); 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 1 la); 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-likereceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298 (ATP1B3, Na KATPase 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 (gpl 10, Macrosialin); classII MHC; BDCA-1; Siglec-H; wherein the names listed in parentheses represent alternativenames.

In embodiments, targeting can be accomplished by any targeting moiety thatspecifically binds to any entity (e.g., protein, lipid, carbohydrate, small molecule, etc.) that isprominently expressed and/or present on B cells (i.e., B cell marker). Exemplary B cellmarkers include, but are not limited to, CDlc (M241, R7); CD Id (R3); CD2 (E-rosette R,Til, LFA-2); CD5 (Tl, Tp67, Leu-1, Ly-1); CD6 (T12); CD9 (p24,DRAP-1, MRP-1);CD1 la (LFA-Ια, aL Integrin chain); CD1 lb (aM Integrin chain, CR3, Mol, C3niR, Mac-1);CD1 lc (aX Integrin, P150, 95, AXb2); CDwl7 (Lactosylceramide, LacCer); CD18 (Integrinβ2, CD1 la, b, c β-subunit); CD19 (B4); CD20 (Bl, 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-2Ra, p55); CD26 (DPP IV ectoeneyme, ADA bindingprotein); CD27 (T14, S152); CD29 (Platelet GPIIa, β-l integrin, GP); CD31 (PECAM-1,Endocam); CD32 (FCyRII); 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-2a, gpla, a2 Integrin); CD49c (VLA-3a, a3 Integrin); CD49d (VLA-4a, a4 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 -22- WO 2010/138193 PCT/US2010/001560 chain); CD75 (sialo-masked Lactosamine); CD75S (a2, 6 sialytated Lactosamine); CD77 (Pkantigen, BLA, CTH/Gb3); CD79a (Iga, MB1); CD79b (IgP, 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 groupantigen); CDwl 19 (IFNyR, IFNyRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFRp80); CD121b (Type 2 IL-1R); CD122 (IL2Rp); CD124 (IL-4Ra); CD130 (gpl30); CD132(Common y chain, IL-2Ry); CDwl37 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPR1N,M6, 0X47); 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(LICMA, NILE); CD175s (Sialyl-Τη (S-Τη)); CD180 (RP105, Bgp95, Ly64); CD184(CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197 (CCR7 (wasCDwl97)); CDwl97 (CCR7, EBI1, BLR2); CD200 (0X2); CD205 (DEC-205); CDw210(CK); CD213a (CK); CDw217 (CK); CDw218a (IL18Rct); CDw218b (IL18RP); CD220(Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD225 (Leul3);CD226 (DNAM-1, PTA1); CD227 (MUC1, PUM, PEM, EMA); CD229 (Ly9); CD230(Prion Protein (Pip)); CD232 (VESP-R); CD245 (p220/240); CD247 (CD3 Zeta Chain);CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-Rsuperfamily, member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264(TRAIL-R4, TNF-R superfamily, member lOd); CD265 (TRANCE-R, TNF-R superfamily,member 1 la); CD267 (TACI, TNF-R superfamily, member 13B); CD268 (BAFFR, TNF-Rsuperfamily, 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); CD3O5(LAIR1); CD307 (IRTA2); CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319(CRACC, SLAMF7); CD321 (JAMI); 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 moietythat 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 cellmarker). Exemplary activated B cell markers include, but are not limited to, CD la (R4, T6,HTA-1); CDlb (Rl); CD 15s (Sialyl Lewis X); CD15u (3’ sulpho Lewis X); CD15su (6 -23- WO 2010/138193 PCI7US2010/001560 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-5Ra); CD126 (IL-6Ra); CD138 (Syndecan-1, Heparan sulfateproteoglycan); CD 152 (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 parenthesesrepresent alternative names. Examples of markers include those provided elsewhere herein. “B cell antigen” means any antigen that naturally is or could be engineered to berecognized by a B cell, and triggers (naturally or being engineered as known in the art) animmune response in a B cell (e.g., an antigen that is specifically recognized by a B cellreceptor on a B cell). In some embodiments, an antigen that is a T cell antigen is also a B cellantigen. In other embodiments, the T cell antigen is not also a B cell antigen. B cell antigensinclude, but are not limited to proteins, peptides, small molecules, and carbohydrates. Insome embodiments, the B cell antigen is a non-protein antigen (i.e., not a protein or peptideantigen). In some embodiments, the B cell antigen is a carbohydrate associated with aninfectious agent. In some embodiments, the B cell antigen is a glycoprotein or glycopeptideassociated 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 poorlyimmunogenic antigen. In some embodiments, the B cell antigen is an abused substance or aportion thereof. In some embodiments, the B cell antigen is an addictive substance or aportion thereof. Addictive substances include, but are not limited to, nicotine, a narcotic, acough suppressant, a tranquilizer, and a sedative. In some embodiments, the B cell antigen isa toxin, such as a toxin from a chemical weapon or natural sources, or a pollutant. The B cellantigen may also be a hazardous environmental agent. In other embodiments, the B cellantigen is an alloantigen, an allergen, a contact sensitizer, a degenerative disease antigen, ahapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an addictivesubstance, a xenoantigen, or a metabolic disease enzyme or enzymatic product thereof. “Biodegradable polymer” means a polymer that degrades over time when introducedinto 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(Iactic-co-glycolic acid), or polycaprolactone. In someembodiments, the biodegradable polymer comprises a block-co-polymer of a polyether, suchas polyethylene glycol), and a polyester, polycarbonate, or polyamide or other biodegradablepolymer. In embodiments, the biodegradable polymer comprises a block-co-polymer of -24- WO 2010/138193 PCT/US2010/001560 polyethylene glycol) and poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or polycaprolactone. In some embodiments, however, the biodegradable polymer does notcomprise a polyether, such as polyethylene glycol), or consists solely of the polyether.Generally, for use as part of a synthetic nanocarrier the biodegradable polymer is insoluble inwater at pH = 7.4 and at 25°C. The biodegradable polymer, in embodiments, have a weightaverage molecular weight ranging from about 800 to about 50,000 Daltons, as determinedusing gel permeation chromatography. In some embodiments, the weight average molecularweight is from about 800 Daltons to about 10,000 Daltons, preferably from 800 Daltons to10,000 Daltons, as determined using gel permeation chromatography. In other embodiments,the weight average molecular weight is from 1000 Daltons to 10,000 Daltons, as determinedby gel permeation chromatography. In an embodiment, the biodegradable polymer does notcomprise polyketal. “Coadministered” means-administering two or more drugs to a subject in a mannerthat is correlated in time. In embodiments, coadministration may occur throughadministration of two or more drugs in the same dosage form. In other embodiments,coadministration may encompass administration of two or more drugs in different dosageforms, but within a specified period of time, preferably within 1 month, more preferablywithin 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 containedwithin the synthetic nanocarrier. In some embodiments, the coupling is covalent. In someembodiments, the covalent coupling is mediated by one or more linkers, polymers or a unitthereof. In some embodiments, the coupling is non-covalent. In some embodiments, thenon-covalent coupling is mediated by charge interactions, affinity interactions, metalcoordination, physical adsorption, hostguest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waals interactions, magneticinteractions, electrostatic interactions, dipole-dipole interactions, and/or combinationsthereof. In embodiments, the coupling may arise in the context of encapsulation within thesynthetic nanocarriers, using conventional techniques. Any of the aforementioned couplingsmay 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 anon-limiting example, a peptide antigen derived from an infectious strain may have severalnon-natural amino acid residues substituted for the natural amino acid residues found in theoriginal antigen found in the infectious strain. The adaptations or modifications may be for a -25- WO 2010/138193 PCT/US2010/001560 variety of reasons, including but not limited to increased specificity, easier antigenprocessing, or improved safety. "Dosage form" means a drug in a medium, carrier, vehicle, or device suitable foradministration to a subject. “Effective amount” of an inventive composition is that amount effective for a certainpurpose. For example, when the effective amount is for a therapeutic purpose the amount iseffective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibitingprogression of, reducing severity of, and/or reducing incidence of one or more symptoms orfeatures of a disease, disorder, and/or condition provided herein. “Encapsulate” means to enclose within a synthetic nanocarrier, preferably enclosecompletely within a synthetic nanocarrier. Most or all of a substance that is encapsulated isnot exposed to the local environment external to the synthetic nanocarrier. Encapsulation isdistinct from absorbtion, which places most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environment external to thesynthetic nanocarrier. “Exhibits a pH sensitive dissociation” means that a coupling between two entities,such as the immunomodulatory agent and the synthetic nanocarrier or immunomodulatoryagent coupling moiety, is significantly reduced or eliminated by a change in environmentalpH. In embodiments, relevant pH sensitive dissociations may satisfy any of the relationshipsor combinations thereof provided herein. “IArel(4.5)t %” is defined as a weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5 for thours divided by the sum of the weight of immunomodulatory agent released upon exposureof the synthetic nanocarrier to an in vitro aqueous environment at a pH = 4.5 for t hours plusa weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure ofthe 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 syntheticnanocarriers. In embodiments, t is 2, 4, 6, 8, 10,12, 14, 16,18, 20, 22, 24, 26, 28, or 30hours. In preferred embodiments, t is 24 hours. “IArel(7.4)t %” is defined as a weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4 for thours divided by the sum of the weight of immunomodulatory agent released upon exposureof the synthetic nanocarrier to an in vitro aqueous environment at a pH = 7.4 for t hours plusa weight of immunomodulatory agent retained in the synthetic nanocarrier upon exposure of -26- WO 2010/138193 PCT/US2010/001560 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 syntheticnanocarriers. In embodiments, t is 2,4, 6, 8, 10, 12, 14, 16,18,20, 22, 24, 26, 28, or 30hours. In preferred embodiments, t is 24 hours. “I A(4.5)u” is defined as a weight of immunomodulatory agent released upon exposureof the synthetic nanocarrier to an in vitro aqueous environment at pH 4.5 for tl hours taken asan average across a sample of the synthetic nanocarriers. “IA(4.5)t2” is defined as a weight ofimmunomodulatory agent released upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at pH 4.5 for t2 hours taken as an average across a sample of thesynthetic nanocarriers, tl 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 tl > t2. In preferred embodiments,tl 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 animmune response. Such agents include adjuvants that stimulate (or boost) an immuneresponse to an antigen but is not an antigen or derived from an antigen. In someembodiments, the immunomodulatory agent is on the surface of the synthetic nanocarrierand/or is incorporated within the synthetic nanocarrier. In embodiments, theimmunomodulatory agent is coupled to the synthetic nanocarrier via a polymer or unitthereof.

In some embodiments, all of the immunomodulatory agents of a synthetic nanocarrierare identical to one another. In some embodiments, a synthetic nanocarrier comprises anumber of different types of immunomodulatory agents. In some embodiments, a syntheticnanocarrier comprises multiple individual immunomodulatory agents, all of which areidentical to one another. In some embodiments, a synthetic nanocarrier comprises exactlyone type of immunomodulatory agent. In some embodiments, a synthetic nanocarriercomprises exactly two distinct types of immunomodulatory agents. In some embodiments, asynthetic nanocarrier comprises greater than two distinct types of immunomodulatory agents. “Immunomodulatory agent coupling moiety” is any moiety through which animmunomodulatory agent is bonded to a synthetic nanocarrier. Such moieties includecovalent 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, theimmunomodulatory agent coupling moiety can comprise a charged polymer to which an -27- WO 2010/138193 PCT/US2010/001560 immunomodulatory agent (e.g., an immunostimulatory nucleic acid) electrostatically bonds.As another example, the immunomodulatory agent coupling moiety can comprise a polymeror unit thereof to which the immunomodulatory agent covalently bonds. In someembodiments, the moiety comprises a polyester. In other embodiments, the moietycomprises polyethylene glycol), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), or a polycaprolactone. The moiety may also comprise a unit of any of the foregoingpolymers, such as a lactide or glycolide. “Labile immunomodulatory agent(s)” means immunomodulatory agent or agents thatare unstable under physiological conditions, and degrade to the point where they are nolonger pharmacologically active. In embodiments, labile immunomodulatory agents areobserved to have systemic half-lives of elimination of less than 24 hours, preferably less than12 hours, more preferably less than 10 hours, even more preferably less than 8 hours, and stillmore preferably less than 6 hours. In embodiments, labile immunomodulatory agentscomprise imidazoquinolines, adenine derivative, or oligonucleotides that comprise 5’ - CG -3’, wherein C is unmethylated and wherein the oligonucleotide comprises a backbonecomprising one or more unstabilized intemucleotide linkages. In embodiments, theimidazoquinolines comprise imidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, imidazoquinoline amines, imiquimod or resiquimod. “Maximum dimension of a synthetic nanocarrier” means the largest dimension of ananocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of asynthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measuredalong any axis of the synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would besubstantially identical, and would be the size of its diameter. Similarly, for a cubic syntheticnanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of itsheight, width or length, while the maximum dimension of a synthetic nanocarrier would bethe largest of its height, width or length. In an embodiment, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in the sample, is greater than 100nm. In an embodiment, a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample, based on the total numberof synthetic nanocarriers in the sample, is equal to or less than 5 pm. Preferably, a minimumdimension of at least 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the -28- WO 2010/138193 PCT/US2010/001560 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 greaterthan 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 totalnumber of synthetic nanocarriers in the sample is equal to or less than 3 pm, more preferablyequal to or less than 2 pm, more preferably equal to or less than 1 pm, more preferably equalto or less than 800 nm, more preferably equal to or less than 600 nm, and more preferablystill equal to or less than 500 nm. In preferred embodiments, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in the sample, is equal to orgreater than 1 OOnm, more preferably equal to or greater than 120 nm, more preferably equalto or greater than 130 nm, more preferably equal to or greater than 140 nm, and morepreferably still equal to or greater than 150 nm. Measurement of synthetic nanocarrier sizesis obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media andusing 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 originalamino 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 thatsource. “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 morepreferably from 15 to 25 nucleotides, even still more preferably 20 nucleotides. In anembodiment according to the invention, oligonucleotides comprise less than 100 nucleotides,preferably less than 50 nucleotides, more preferably less than 25 nucleotides, and still morepreferably 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 inparts of the oligonucleotides other than in a 5’ - CG - 3’ sequence of which theoligonucleotide may be comprised may be methylated, or may be unmethylated. Inembodiments, inventive oligonucleotides comprise a backbone comprising one or moreunstabilized intemucleotide linkages (meaning intemucleotide linkages that are unstableunder physiological conditions). “Unstabilized intemucleotide linkage” means a linkagebetween two nucleotides of which the oligonucleotide is comprised that is not chemically -29- WO 2010/138193 PCT/US2010/001560 modified to stabilize the backbone, or is chemically modified to destabilize the backbone ofthe oligonucleotide under physiological conditions. An example of an unstablizedintemucleotide linkage is a phophodiester intemucleotide linkage. In embodiments, theinventive oligonucleotides’ backbone comprises no stabilizing chemical modifications thatfunction to stabilize the backbone under physiological conditions. In embodiments, theinventive oligonucleotides’ backbone comprises a backbone that is not modified toincorporate phosphorothioate stabilizing chemical modifications. “Pharmaceutically acceptable excipient” means a pharmacologically inactivesubstance added to an inventive composition to further facilitate administration of thecomposition. Examples, without limitation, of pharmaceutically acceptable excipientsinclude calcium carbonate, calcium phosphate, various diluents, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. “Release Rate” means the rate that an entrapped immunomodulatory agent flows froma composition, such as a synthetic nanocarrier, into a surrounding media in an in vitro releasetest. First, the synthetic nanocarrier is prepared for the release testing by placing into theappropriate in vitro release media. This is generally done by exchanging the buffer aftercentrifugation to pellet the synthetic nanocarrier and reconstitution of the syntheticnanocarriers using a mild condition. The assay is started by placing the sample at 37°C in anappropriate temperature-controlled apparatus. A sample is removed at various time points.

The synthetic nanocarriers are separated from the release media by centrifugation topellet the synthetic nanocarriers. The release media is assayed for the immunomodulatoryagent that has dispersed from the synthetic nanocarriers. The immunomodulatory agent ismeasured using HPLC to determine the content and quality of the immunomodulatory agent.The pellet containing the remaining entrapped immunomodulatory agent is dissolved insolvents or hydrolyzed by base to free the entrapped immunomodulatory agent from thesynthetic nanocarriers. The pellet-containing immunomodulatoiy agent is then also measuredby HPLC to determine the content and quality of the immunomodulatory agent that has notbeen released at a given time point.

The mass balance is closed between immunomodulatory agent that has been releasedinto the release media and what remains in the synthetic nanocarriers. Data are presented asthe 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, horsesand pigs; laboratory animals such as mice, rats and guinea pigs; fish; and the like. WO 2010/138193 -30- PCT/US2010/001560 “Synthetic nanocarrier(s)” means a discrete object that is not found in nature, and thatpossesses at least one dimension that is less than or equal to 5 microns in size. Albuminnanoparticles are expressly included as synthetic nanocarriers.

Synthetic nanocarriers include polymeric nanoparticles. In some embodiments,synthetic nanocarriers can comprise one or more polymeric matrices. The syntheticnanocarriers, however, can also include other nanomaterials and may be, for example, lipid-polymer nanoparticles. In some embodiments, a polymeric matrix can be surrounded by acoating layer (e.g., liposome, lipid monolayer, micelle, etc.). In some embodiments, thesynthetic nanocarrier is not a micelle. In some embodiments, a synthetic nanocarrier maycomprise 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 syntheticnanocarriers can be coupled with the polymeric matrix.

The synthetic nanocarriers may comprise one or more lipids. In some embodiments, asynthetic nanocarrier may comprise a liposome. In some embodiments, a syntheticnanocarrier may comprise a lipid bilayer. In some embodiments, a synthetic nanocarrier maycomprise a lipid monolayer. In some embodiments, a synthetic nanocarrier may comprise amicelle. 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, lipidmonolayer, etc.).

The synthetic nanocarriers may comprise lipid-based nanoparticles, metallicnanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-likeparticles, peptide or protein-based particles (such as albumin nanoparticles). Syntheticnanocarriers may be a variety of different shapes, including but not limited to spheroidal,cubic, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers accordingto the invention comprise one or more surfaces. Exemplary synthetic nanocarriers that can beadapted for use in the practice of the present invention comprise: (1) the biodegradablenanoparticles disclosed in U.S. Patent 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published U.S. Patent Application 20060002852 to Saltzman et al., (3) thelithographically constructed nanoparticles of Published U.S. Patent Application 20090028910to DeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian et al., or (5) thenanoparticles disclosed in Published U.S. Patent Application 2008/0145441 to Penades et al.

Synthetic nanocarriers according to the invention that have a minimum dimension ofequal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a -31- WO 2010/138193 PCT/US2010/001560 surface with hydroxyl groups that activate complement or alternatively comprise a surfacethat consists essentially of moieties that are not hydroxyl groups that activate complement. Ina preferred embodiment, synthetic nanocarriers according to the invention that have aminimum dimension of equal to or less than about 100 nm, preferably equal to or less than100 nm, do not comprise a surface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do not substantially activatecomplement. In a more preferred embodiment, synthetic nanocarriers according to theinvention that have a minimum dimension of equal to or less than about 100 nm, preferablyequal to or less than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moieties that do not activatecomplement. In embodiments, synthetic nanocarriers may possess an aspect ratio greaterthan 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 someembodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments,synthetic nanocarriers are cubes or cubic. In some embodiments, synthetic nanocarriers areovals or ellipses. In some embodiments, synthetic nanocarriers are cylinders, cones, orpyramids.

It is often desirable to use a population of synthetic nanocarriers that is relativelyuniform in terms of size, shape, and/or composition so that each synthetic nanocarrier hassimilar properties. For example, at least 80%, at least 90%, or at least 95% of the syntheticnanocarriers 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, apopulation of synthetic nanocarriers may be heterogeneous with respect to size, shape, and/orcomposition.

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 tothe other Iayer(s). To give but one example, synthetic nanocarriers may have a core/shellstructure, 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 ofdifferent layers. “T cell antigen” means any antigen that is recognized by and triggers an immuneresponse in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a Tcell or an NKT cell via presentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to a CD1 complex). -32- WO 2010/138193 PCT/US2010/001560

In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. T cell antigens generally areproteins or peptides. T cell antigens may be an antigen that stimulates a CD8+ T cellresponse, a CD4+ T cell response, or both. The T cell antigens, therefore, in someembodiments can effectively stimulate both types of responses.

In some embodiments the T cell antigen is a T-helper antigen, which is a T cellantigen that can generate an augmented response to an unrelated B cell antigen throughstimulation of T cell help. In embodiments, a T-helper antigen may comprise one or morepeptides derived from tetanus toxoid, Epstein-Barr virus, influenza virus, respiratorysyncytial virus, measles virus, mumps virus, rubella virus, cytomegalovirus, adenovirus,diphtheria toxoid, or a PADRE peptide. In other embodiments, a T-helper antigen maycomprise one or more lipids, or glycolipids, including but not limited to: a-galactosylceramide (a-GalCer), α-linked glycosphingolipids (from Sphingomonas spp.),galactosyl diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan (from Leishmaniadonovani), 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 antigenthat is obtained from a source, such as a natural source. In such embodiments, CD4+ T-cellantigen 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 cellantigen, preferably a T-helper antigen, may be coupled to, or uncoupled from, a syntheticnanocarrier. “Unit thereof’ refers to a monomeric unit of a polymer, the polymer generally beingmade up of a series of linked monomers. “Vaccine” means a composition of matter that improves the immune response to aparticular pathogen or disease. A vaccine typically contains factors that stimulate a subject'simmune system to recognize a specific antigen as foreign and eliminate it from the subject’sbody. A vaccine also establishes an immunologic ‘memory’ so the antigen will be quicklyrecognized and responded to if a person is re-challenged. Vaccines can be prophylactic (forexample to prevent future infection by any pathogen), or therapeutic (for example a vaccineagainst a tumor specific antigen for the treatment of cancer). Vaccines according to the -33- WO 2010/138193 PCT/US2010/001560 invention may comprise one or more of the synthetic nanocarriers or compositions providedherein.

METHODS OF MAKING THE INVENTIVE COMPOUNDS, CONJUGATES, ORSYNTHETIC NANOCARRIERS

The immunomodulatory agent can be coupled to the synthetic nanocarrier in anymanner such that the dissociation of the immunomodulatory agent from the syntheticnanocarrier satisfies the dissociation relationships provided herein. Methods for determiningwhether or not immunomodulatory agents of synthetic nanocarriers satisfy the dissociationrelationships provided herein are provided elsewhere above and in the EXAMPLES.

Oligonucleotides according to the invention may be encapsulated into syntheticnanocarriers 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 DrugDelivery” 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 nanocarriersmay be used, including without limitation methods disclosed in United States Patent6,632,671 to Unger October 14, 2003.

In some embodiments, the immunomodulatory agent is covalently coupled to thesynthetic nanocarrier via an immunomodulatory agent coupling moiety (e.g., a polymer orunit thereof). In general, a polymer or unit thereof can be covalently coupled with animmunomodulatory agent in several ways.

The following methods or any step of the methods provided are exemplary and maybe carried out under any suitable conditions. In some cases, the reaction or any step of themethods 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, butare 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-/-butyl ether (MTBE), N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropanol (IPA), mixturesthereof, or the like. In some cases, the solvent is selected from the group consisting of ethyl -34- WO 2010/138193 PCT/US2010/001560 acetate, methylene chloride, THF, DMF, NMP, DMAC, DMSO, and toluene, or a mixturethereof. A reaction or any step of the methods provided may be carried out at any suitabletemperature. In some cases, a reaction or any step of the methods provided is carried out atabout 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 providedmay 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 themethods provided is conducted at temperatures between 0 °C and 120 °C.' In someembodiments, the reaction or any step of the methods provided may be carried out at morethan one temperature (e.g., reactants added at a first temperature and the reaction mixtureagitated at a second wherein the transition from a first temperature to a second temperaturemay be gradual or rapid).

The reaction or any step of the methods provided may be allowed to proceed for anysuitable period of time. In some cases, the reaction or any step of the methods provided isallowed to proceed for about 10 minutes, about 20 minutes, about 30 minutes, about 40minutes, about 50. minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about12 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 anintermediate time to determine the progress of the reaction or any step of the methodsprovided. In some embodiments, a reaction or any step of the methods provided may becarried out under an inert atmosphere in anhydrous conditions (e.g., under an atmosphere ofnitrogen 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 liquidchromatography, high performance liquid chromatography, nuclear magnetic resonancespectroscopy, etc.) using commonly known techniques. In some cases, a syntheticnanocarrier may be analyzed to determine the loading of immunomodulatory agent, forexample, using reverse phase HPLC.

The polymers may have any suitable molecular weight. For example, the polymersmay have a low or high molecular weight. Non-limiting molecular weight values include 100Da, 200 Da, 300 Da, 500 Da, 750 Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 -35- WO 2010/138193 PCT/US2010/001560

Da, 7000 Da, 8000 Da, 9000 Da, 10,000 Da, or greater. In some embodiments, the polymershave a weight average molecular weight of about 800 Da to about 10,000 Da. The molecularweight 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 isconverted 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 thepresence of a base to give the desired conjugate (e.g., PLA-R848), for example, as shown in the following scheme:

<img img-format="tif" img-content="drawing" file="IL216548AD00021.tif" id="idf0001" />

PLA-CO2H

<img img-format="tif" img-content="drawing" file="IL216548AD00022.tif" id="idf0002" />

activiating agent

..........—.....PLA-COX

X = active moeity Activated PLA

<img img-format="tif" img-content="drawing" file="IL216548AD00023.tif" id="idf0003" />

Activating reagents that can be used to convert polymers or units thereof, such asPLA or PLGA, to an activated acylating form include, but are not limited to cyanuricfluoride, Ν,Ν-tetramethylfluoroformamidinium hexafluorophosphate (TFFH);Acylimidazoles, such as carbonyl diimidazole (GDI), 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); Ν,Ν’-disuccinimidyl carbonate (DSC); pentaflurophenol inthe 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, underargon) following activation, or may be used immediately. The activated polymer or unitthereof may be reacted with an immunomodulatory agent under any suitable conditions. In -36- WO 2010/138193 PCT/US2010/001560 some cases, the reaction is carried out in the presence of a base and/or catalyst. Non-limitingexamples 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 presenceof 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,

<img img-format="tif" img-content="drawing" file="IL216548AD00024.tif" id="idf0004" />

<img img-format="tif" img-content="drawing" file="IL216548AD00025.tif" id="idf0005" />

base/sotvent coupling agent PLA-R848orPLGA-R848

PLGA-CO2H

Coupling or activating agents include but are not limited to: activating agents used inthe 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, suchas O-Benzotriazol-l-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP), O-Benzotriazol-l-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-l-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP);uronium salts such as O-Benzotriazol-l-yloxytris-l,l,3,3-tetramethyluroniumtetrafluoroborate (TBTU) and hexafluorophosphate (HBTU), 0-(7-Azabenzotriazol-l-y 1)- 1.1.3.3- tetramethyluronium hexafluorophosphate (HATU), 0-(l,2-dihydro-2-oxo-l-pyridyΟ-Ι, 1,3,3-tetramethyl-uronium tetrafluoroborate (TPTU); Halouronium and halophosphoniumsalts such as bis(tetramethylene)fluoroformamidinium hexafluorophosphate (BTFFH),bromotris(dimethylamino) phosphonium hexafluoro-phosphate (BroP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP) and chlorotripyrrolidino phosphoniumhexafluorophosphate (PyClop); Benzotriazine derivatives such as 0-(3,4-Dihydro-4-oxo- 1.2.3- benzotriazine-3-yl)-N,N,N’,N’-tetramethyluronium tetrafluoro borate (TDBTU) and 3- -37- WO 2010/138193 PCT/US2010/001560 (diethyloxyphosphoryloxy )-1,2,3-benzotriazin-4(3H)-one (DEPBT). Non-limiting examplesof 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 unitsthereof that are terminated in a hydroxyl group. Such polymers or units thereof includepolyethylene glycol, polylactide, polylactide-co-glycolide, polycaprolactone, and other likepolyesters, or units thereof. In general, the reaction proceeds as follows where an imide ofthe general structure (IV) will react with the terminal hydroxyl of the aforementionedpolymers or units thereof using a catalyst used in lactone ring opening polymerizations. Theresulting reaction product (II) links the amide of the agent to the polymer or unit thereof viaan ester bond. The compounds of formula (IV) and (II) are as follows:

<img img-format="tif" img-content="drawing" file="IL216548AD00026.tif" id="idf0006" />

(IV)

<img img-format="tif" img-content="drawing" file="IL216548AD00027.tif" id="idf0007" />

Rg Rio R11 R12 wherein R| = H, OH, SH, NH2, or substituted or unsubstituted alkyl, alkoxy, alkylthio, oralkylamino; 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 thecarbon atoms of the pyridine ring to which they are connected if Y = C; R4 is H, orsubstituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylamino when not combined withR3 to form a carbocycle or heterocycle with the carbon atoms of the pyridine ring to which -38- WO 2010/138193 PCT/US2010/001560 they are connected; or is combined with R3 to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; R5 is a polymer or unitthereof; X is C, N, 0, or S; Re and R? are each independently H or substituted; and R9, Rio,Ri 1, and R12 are each independently H, a halogen, OH, thio, NH2, or substituted orunsubstituted alkyl, aryl, heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, or arylamino.

Catalysts include, but are not limited to, phosphazine bases, l,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD), and N-methyl-l,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:

<img img-format="tif" img-content="drawing" file="IL216548AD00028.tif" id="idf0008" />

<img img-format="tif" img-content="drawing" file="IL216548AD00029.tif" id="idf0009" />

wherein R5-OH contains two hydroxyl groups (e.g., a diol, HO-R5-OH), each of which arefunctionalized by reaction with an imide associated with R848. In some cases, HO-R5-OH isa 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 beprotected with a protecting group (e.g., /-butyloxycarbonyl), thus the poly-diol would be acompound of formula HO-R5-OP, wherein P is a protecting group. Following reaction withan immunomodulatory agent to form a immunomodulatory agent-Rs-OP conjugate, theprotecting group may be removed and the second diol group may be reacted with any suitablereagent (e.g., PLGA, PLA).

Method 4 A conjugate (e.g., R848-PLA) can be formed via a one-pot ring-openingpolymerization of an immunomodulatory agent (e.g., R848) with a polymer or unit thereof WO 2010/138193 -39- PCT/US2010/001560 (e.g., D/L-lactide) in the presence of a catalyst, for example, as shown in the followingscheme:

<img img-format="tif" img-content="drawing" file="IL216548AD000210.tif" id="idf0010" />

cff-iacticfe

<img img-format="tif" img-content="drawing" file="IL216548AD000211.tif" id="idf0011" />

O

<img img-format="tif" img-content="drawing" file="IL216548AD000212.tif" id="idf0012" />

R848-PLA (R848 loading 3 mg/g)

In a one-step procedure, the immunomodulatory agent and the polymer or unit thereofmay be combined into a single reaction mixture comprising a catalyst. The reaction mayproceed at a suitable temperature (e.g., at about 150 °C) and the resulting conjugate may beisolated using commonly known techniques. Non-limiting examples of suitable catalystsinclude DMAP and tin ethylhexanoate.

Method 5 A conjugate can be formed two-step ring opening polymerization of animmunomodulatory 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 followingscheme:

<img img-format="tif" img-content="drawing" file="IL216548AD000213.tif" id="idf0013" />

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 solutioncomprising the polymers or units thereof, followed by addition of a catalyst (e.g., tinethylhexanoate). The resulting conjugate may be isolated using commonly knowntechniques. Non-limiting examples of suitable catalysts include DMAP and tinethylhexanoate.

In some embodiments, the immunomodulatory agent, antigen, and/or targeting moietycan be covalently associated with a polymeric matrix. In some embodiments, covalentassociation 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 targetingmoiety can be encapsulated within, surrounded by, and/or dispersed throughout a polymeric WO 2010/138193 -40- PCT/US2010/001560 matrix. Alternatively or additionally, the immunomodulatory agent, antigen, and/or targetingmoiety can be associated with a polymeric matrix by hydrophobic interactions, chargeinteractions, van der Waals forces, etc.

The immunomodulatory agents can also be encapsulated within the nanocarriers. Thenanocarriers, therefore, can be of any material that is pH sensitive provided that the resultinginventive synthetic nanocarriers satisfy the dissociation relationships provided herein. Suchsynthetic nanocarriers are well known in the art and include polyketal nanocarriers, pHsensitive 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 uponcellular internalization transform to a hydrophilic structure (a hydrogel particle), andpolymeric nanoparticles, such as those of Griset, Dissertation entitled: Delivery of Paclitaxelvia pH-Responsive Polymeric Nanoparticles for Prevention of Lung Cancer andMesothelioma Recurrence, Ohio State University, 2003. The pH sensitive syntheticnanocarriers also include those that comprise polymers that dissolve at a pH below 6 orpolymers that swell at an acidic pH. In some embodiments, the synthetic nanocarriers are ofa non-polyketal material. In other embodiment, the synthetic nanocarriers are not micelles. A wide variety of polymers and methods for forming polymeric matrices therefromare known conventially. In general, a polymeric matrix comprises one or more polymers.Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymersor copolymers comprising two or more monomers. In terms of sequence, copolymers may berandom, 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 notlimited to polyethylenes, polycarbonates (e.g., poly(l,3-dioxan-2one)), polyanhydrides (e.g.,poly(sebacic anhydride)), polyhydroxyacids (e.g., poly(P-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 includepolymers which have been approved for use in humans by the U.S. Food and DrugAdministration (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(l,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., -41- PCT/US2010/001560 WO 2010/138193 polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; andpolycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymers maycomprise anionic groups (e.g., phosphate group, sulphate group, carboxylate group); cationicgroups (e.g., quaternary amine group); or polar groups (e.g., hydroxyl group, thiol group,amine group). In some embodiments, a synthetic nanocarrier comprising a hydrophilicpolymeric matrix generates a hydrophilic environment within the synthetic nanocarrier. Insome embodiments, polymers can be hydrophobic. In some embodiments, a syntheticnanocarrier comprising a hydrophobic polymeric matrix generates a hydrophobicenvironment within the synthetic nanocarrier. Selection of the hydrophilicity orhydrophobicity of the polymer may have an impact on the nature of materials that areincorporated (e.g., coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moieties and/orfunctional groups. A variety of moieties or functional groups can be used in accordance withthe present invention. In some embodiments, polymers may be modified with PEG, with acarbohydrate, 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. Insome 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 someembodiments, 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 comprisinglactic acid and glycolic acid units, such as poly(Iactic acid-co-glycolic acid) and poly(Iactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprisingglycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lacticacid, 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, exemplarypolyesters include, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEGcopolymers, 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, -42- WO 2010/138193 PCT/US2010/001560 poly(ethyleneimine), polyethylene imine)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyI)-L-g!ycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA arecharacterized 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 thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used in accordance withthe present invention is characterized by a lactic acid:glycolic acid ratio of approximately85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately40:60, approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. In certainembodiments, acrylic polymers include, for example, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acidanhydride), 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 methacrylicacid esters with a low content of quaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general, cationicpolymers 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 ak, 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 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 Haensleret al., 1993, Bioconjugate Chem., 4:372) are positively-charged at physiological pH, form ionpairs with nucleic acids, and mediate transfection in a variety of cell lines.

In some embodiments, polymers can be degradable polyesters bearing cationic sidechains (Putnam et ak, 1999, Macromolecules, 32:3658; Barrera et ak, 1993, J. Am. Chem.Soc., 115:11010; Kwon et ak, 1989, Macromolecules, 22:3250; Lim et ak, 1999, J. Am.Chem. Soc., 121:5633; and Zhou et ak, 1990, Macromolecules, 23:3399). Examples of these -43- PCT/US2010/001560 WO 2010/138193 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 wellknown in the art (see, for example, U.S. Patents 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 polymersare described in Concise Encyclopedia of Polymer Science and Polymeric Amines andAmmonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization byOdian, John Wiley &amp;amp; Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry byAllcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S. Patents6,506,577,6,632,922,6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. In someembodiments, polymers can be dendrimers. In some embodiments, polymers can besubstantially cross-linked to one another. In some embodiments, polymers can besubstantially free of cross-links. In some embodiments, polymers can be used in accordancewith the present invention without undergoing a cross-linking step. It is further to beunderstood that inventive compounds and synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any of the foregoing andother polymers. Those skilled in the art will recognize that the polymers listed hereinrepresent an exemplary, not comprehensive, list of polymers that can be of use in accordancewith the present invention.

In some embodiments, synthetic nanocarriers may comprise metal particles, quantumdots, ceramic particles, etc.

In some embodiments, synthetic nanocarriers may optionally comprise one or moreamphiphilic entities. In some embodiments, an amphiphilic entity can promote theproduction of synthetic nanocarriers with increased stability, improved uniformity, orincreased viscosity. In some embodiments, amphiphilic entities can be associated with theinterior surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many -44- WO 2010/138193 PCT/US2010/001560 amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers inaccordance with the present invention. Such amphiphilic entities include, but are not limitedto, phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC);dioleylphosphatidyl ethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA);dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol;diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols suchas 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 aciddiglycerides; fatty acid amides; sorbitan trioleate (Span®85) glycocholate; sorbitanmonolaurate (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 assorbitan 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; polyethylene glycol)5000-phosphatidylethanolamine;poly(ethylene glycol)400-monostearate; phospholipids; synthetic and/or natural detergentshaving high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion pairingagents; and combinations thereof. An amphiphilic entity component may be a mixture ofdifferent amphiphilic entities. Those skilled in the art will recognize that this is anexemplary, not comprehensive, list of substances with surfactant activity. Any amphiphilicentity may be used in the production of synthetic nanocarriers to be used in accordance withthe present invention.

In some embodiments, synthetic nanocarriers may optionally comprise one or morecarbohydrates. Carbohydrates may be natural or synthetic. A carbohydrate may be aderivatized natural carbohydrate. In certain embodiments, a carbohydrate comprisesmonosaccharide or disaccharide, including but not limited to glucose, fructose, galactose,ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronicacid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. Incertain embodiments, a carbohydrate is a polysaccharide, including but not limited topullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch,hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, WO 2010/138193 -45- PCT/US2010/001560 algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In certain embodiments, the carbohydrate is a sugaralcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, andlactitol.

Synthetic nanocarriers may be prepared using a wide variety of methods known in theart. For example, synthetic nanocarriers can be formed by methods as nanoprecipitation,flow focusing using fluidic channels, spray drying, single and double emulsion solventevaporation, solvent extraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, andother 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, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755, andalso US Patents 5578325 and 6007845).

In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitationprocess or spray drying. Conditions used in preparing synthetic nanocarriers may be alteredto yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing the synthetic nanocarriersand the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used maydepend on the materials to be coupled to the synthetic nanocarriers and/or the composition ofthe polymer matrix.

If particles prepared by any of the above methods have a size range outside of thedesired 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 syntheticnanocarrier. Elements of the inventive synthetic nanocarriers (such as moieties of which animmunofeature 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 becoupled by means of one or more linkers. Additional methods of functionalizing syntheticnanocarriers may be adapted from Published US Patent Application 2006/0002852 to -46- WO 2010/138193 PCT/US2010/001560

Saltzman et al., Published US Patent Application 2009/0028910 to DeSimone et al., orPublished International Patent Application WO/2008/127532 Al to Murthy et al.

Any suitable linker can be used in accordance with the present invention. Linkersmay be used to form amide linkages, ester linkages, disulfide linkages, etc. Linkers maycontain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.). In someembodiments, a linker is an aliphatic or heteroaliphatic linker. In some embodiments, thelinker is a polyalkyl linker. In certain embodiments, the linker is a polyether linker. Incertain embodiments, the linker is a polyethylene linker. In certain specific embodiments, thelinker 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 acidlinkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitivelinkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzymecleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavablelinkers, 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 syntheticnanocarrier with the synthetic nanocarrier. General strategies include passive adsorption(e.g., via electrostatic interactions), multivalent chelation, high affinity non-covalent bindingbetween 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 toassociate a material with a synthetic nanocarrier.

Non-covalent specific binding interactions can be employed. For example, either aparticle or a biomolecule can be functionalized with biotin with the other beingfunctionalized with streptavidin. These two moieties specifically bind to each othernoncovalently and with a high affinity, thereby associating the particle and the biomolecule.Other specific binding pairs could be similarly used. Alternately, histidine-taggedbiomolecules can be associated with particles conjugated to nickel-nitrolotriaceteic acid (Ni-NTA).

For additional general information on coupling, see the journal BioconjugateChemistry, published by the American Chemical Society, Columbus OH, PO Box 3337,Columbus, OH, 43210; “Cross-Linking,” Pierce Chemical Technical Library, available at thePierce web site and originally published in the 1994-95 Pierce Catalog, and references citedtherein; Wong SS, Chemistry of Protein Conjugation and Cross-linking, CRC Press -47- WO 2010/138193 PCT/US2010/001560

Publishers, Boca Raton, 1991; and Hermanson, G. T., Bioconjugate Techniques, AcademicPress, Inc., San Diego, 1996.

It is to be understood that the compositions of the invention can be made in anysuitable manner, and the invention is in no way limited to compositions that can be producedusing the methods described herein. Selection of an appropriate method may requireattention to the properties of the particular moieties being associated.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF USE

Compositions according to the invention comprise inventive synthetic nanocarriers incombination with pharmaceutically acceptable excipients. The compositions may be madeusing conventional pharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. In an embodiment, inventive synthetic nanocarriers are suspended insterile saline solution for injection together with a preservative.

In some embodiments, inventive synthetic nanocarriers are manufactured under sterileconditions or are terminally sterilized. This can ensure that resulting composition are sterileand non-infectious, thus improving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receiving synthetic nanocarriershave immune defects, are suffering from infection, and/or are susceptible to infection. Insome embodiments, inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulation strategy for extendedperiods 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 acombination 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 describedherein can be used in the diagnosis, prophylaxis and/or treatment of conditions such ascancers, infectious diseases, metabolic diseases, degenerative diseases, inflammatorydiseases, immunological diseases, or other disorders and/or conditions. The compositionsand methods described herein can also be used for the prophylaxis or treatment of anaddiction, such as an addiction to nicotine or a narcotic. The compositions and methodsdescribed herein can also be used for the prophylaxis and/or treatment of a condition resulting -48- WO 2010/138193 PCT/US2010/001560 from the exposure to a toxin, hazardous substance, environmental toxin, or other harmfulagent.

EXAMPLES

Example 1: Preparation of Activated Polymer PLA (dl-polylactide) (Resomer R202H from Boehringer-Ingelheim, KOH equivalentacid number of 0.21 mmol/g, intrinsic viscosity (iv): 0.21 dl/g) (10 g, 2.1 mmol, 1.0 eq) wasdissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2g, 10.5 mmol, 5 eq) were added. The solids were dissolved with the aid of sonication. Theresulting solution was stirred at room temperature for 6 days. The solution was concentratedto remove most of DCM and the residue was added to a solution of 250 mL of diethyl etherand 5 mL of MeOH to precipitate out the activated PLA-NHS ester. The solvents wereremoved and the polymer was washed twice with ether (2x200 mL) and dried under vacuumto give PLA-NHS activated ester as a white foamy solid (~ 8 g recovered, H NMR was usedto confirm the presence of NHS ester). The PLA-NHS ester was stored under argon in abelow -10C freezer before use.

Alternatively, the reaction can be performed in DMF, THF, dioxane, or CHC13instead of DCM. DCC can be used instead of EDC (resulting DCC-urea is filtered off beforeprecipitation of the PLA-NHS ester from ether). The amount of EDC or DCC and NHS canbe 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 orPLGA (Resomer RG653H, 65% lactide-35% glycolide, iv: 0.39 dl/g and acid number 0.08mmol/g) or PLGA (Resomer RG752H, 75% lactide-25% glycolide, iv: 0.19 dl/g and acidnumber of 0.22 mmol/g) is converted to the corresponding PLA-NHS or PLGA-NHSactivated ester and stored under argon in a below -10C 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 dissolvedin 10 mL of dry acetonitrile. Ν,Ν’-disuccinimidyl carbonate (DSC) (215 mg, 1.26 mmol, 3.0eq) and catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) were added. Theresulting mixture was stirred under argon for 1 day. The resulting solution was concentratedto almost dryness. The residue was then added to 40 mL of ether to precipitate out the -49- WO 2010/138193 PCT/US2010/001560 polymer which was washed twice with ether (2x30 mL) and dried under vacuum to givePLA-NHS activated ester (1H NMR showed the amount of NHS ester at about 80%).

Example 3: Preparation of Activated Polymer PL A (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous DCM and 2.5mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq) and pentafluorophenol (PFP) (580mg, 3.15 mmol, 5.0 eq) were added. The resulting solution was stirred at room temperaturefor 6 days and then concentrated to remove DCM. The resulting residue was added to 250mL of ether to precipitate out the activated PLA polymer which was washed with ether(2xl00mL) 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 resultingsolution was heated at 50-60 C for 2 days. The solution was cooled to rt and added to 40 mLof de-ionized (DI) water to precipitate out the polymer product. The polymer was thenwashed with DI water (40 mL) and ether (2x40 mL) and dried at 30 C under vacuum to giveR848-PLA conjugate as a white foamy solid (0.8 g, H NMR showed the conjugation of R848to PLA via the amide bond). The degree of conjugation (loading) of R848 on the polymerwas confirmed by HPLC analysis as follows: a weighed amount of polymer was dissolved inTHF/MeOH and treated with 15% NaOH. The resulting hydrolyzed polymer products wereanalyzed 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 mLof dry DMF under argon. The resulting solution was heated at 50-60 C for 2 days. Thesolution was cooled to rt and added to 40 mL of de-ionized (DI) water to precipitate out thepolymer product. The polymer was then washed with DI water (40 mL) and ether (2x40 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 and0.1 mL of 15% NaOH. The amount of R848 on the polymer was determined to be about 35 WO 2010/138193 -50- PCI7US2010/001560 mg/g by reverse phase HPLC analysis (Cl8 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. Themixture was heated at 50-55 C for 3 days. The mixture was cooled to rt and diluted withDCM. The DCC-urea was filtered off and the filtrate was concentrated to remove DCM. Theresulting residue in DMF was added to water (40 mL) to precipitate out the polymer productwhich was washed with water (40 mL), ether/DCM (40 mL/4 mL) and ether (40 mL). Afterdrying under vacuum at 30 C, the desired PLA-R848 conjugate was obtained as a whitefoamy 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 Cfor 2 days. The solution was cooled to rt and added to water (40 mL) to precipitate out thepolymer product which was washed with water (40 mL), ether/MeOH (40 mL/2 mL) andether (40 mL). The orange colored polymer was dissolved in 4 mL of DCM and the resultingsolution was added to 40 mL of ether to precipitate out the polymer without much of theorange color. The light colored polymer was washed with ether (40 mL). After drying undervacuum 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 heatedat 50-55 °C for 2 days. The solution was cooled to room temperature and added to water (40mL) to precipitate out the polymer product. The orange colored polymer was dissolved in 2mL of DCM and the resulting solution was added to 40 mL of ether to precipitate out thepolymer which was washed with water/acetone (40 mL/2 mL) and ether (40 mL). After -51- PCT/US2010/001560 WO 2010/138193 drying under vacuum at 30 °C, the desired PLA-R848 conjugate was obtained as an off-whitefoamy solid (1.0 g, loading of R848 on polymer was about 45 mg/g based on HPLC analysisand confirmed by JH NMR). In the same manner, PLGA (75% Lactide)-R848 and PLGA(50% lactide)-R848 were prepared.

<img img-format="tif" img-content="drawing" file="IL216548AD000214.tif" id="idf0014" />

To a round bottom flask equipped with a stir bar and condenser was added theimidazoquinoline, resiquimod (R-848, 218 mg, 6.93 X 10"4 moles), D/L lactide (1.0 g, 6.93 X10’3 moles) and anhydrous sodium sulfate (800 mg). The flask and contents were dried undervacuum at 55 °C for 8 hours. After cooling, the flask was then flushed with argon andtoluene (50 mL) was added. The reaction was stirred in an oil bath set at 120 °C until all ofthe lactide had dissolved and then tin ethylhexanoate (19 mg, 15pL) was added via pipette.Heating was continued under argon for 16 hours. After cooling, the reaction was diluted withether (200 mL) and the solution was washed with water (200 mL). The solution was driedover magnesium sulfate, filtered and evaporated under vacuum to give 880 mg. of crudepolylactic acid-R-848 conjugate. The crude polymer was chromatographed on silica using10% methanol in methylene chloride as eluent. The fractions containing the conjugate werepooled and evaporated to give the purified conjugate. This was dried under high vacuum toprovide the conjugate as a solid foam in a yield of 702 mg (57.6%). By integrating the NMRsignals for the aromatic protons of the quinoline and comparing this to the integrated intensityof the lactic acid CH proton it was determined that the molecular weight of the conjugate wasapproximately 2KD. GPC showed that the conjugate contained less than 5% of free R848.

Example 10: Preparation Of Low MW PLA-R848 Conjugate

<img img-format="tif" img-content="drawing" file="IL216548AD000215.tif" id="idf0015" />

PLA-CO2H -52- PCTAJS2010/001560 WO 2010/138193 A solution of PLA-CO2H (average MW: 950, DPI: 1.32; 5.0 g, 5.26 mmol) andHBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at room temperature under argonfor 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 15h. After cooling, the mixture was diluted with EtOAc (150 mL) and washed with 1% citricacid solution (2x40 mL), water (40 mL) and brine solution (40 mL). The solution was driedover Na2SO4 (10 g) and concentrated to a gel-like residue. Methyl /-butyl ether (MTBE) (150mL) was then added and the polymer conjugate precipitated out of solution. The polymerwas then washed with MTBE (50 mL) and dried under vacuum at room temperature for 2days as a white foam (5.3 g, average MW by GPC is 1200, PDI: 1.29; R848 loading is 20%by HPLC).

<img img-format="tif" img-content="drawing" file="IL216548AD000216.tif" id="idf0016" />

A solution of PLA-CO2H (average MW: 1800, DPI: 1.44; 9.5 g, 5.26 mmol) andHBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at room temperature under argonfor 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 15h. After cooling, the mixture was diluted with EtOAc (150 mL) and washed with 1 % citricacid solution (2 x 40 mL), water (40 mL) and brine solution (40 mL). The solution was driedover Na2SO4 (10 g) and concentrated to a gel-like residue. Methyl /-butyl ether (MTBE) (150mL) was then added and the polymer conjugate precipitated out of solution. The polymerwas then washed with MTBE (50 mL) and dried under vacuum at room temperature for 2days 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 amethod provided in Pulendran et al, WO 2008/127532, as illustrated in step 1 below. -53- WO 2010/138193 PCT/US2010/001560 PCADK is synthesized in a 50 mL two-necked flask, connected to a short-pathdistilling 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 benzenesolution (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 (10.94mL, 90.0 mmol, Aldrich) is added to the benzene solution, initiating the polymerizationreaction. Additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) aresubsequently added to the reaction every hour for 6 hours via a metering funnel tocompensate for 2,2-dimethoxypropane and benzene that is distilled off. After 8 hours, thereaction is stopped by addition of 500 pL of triethylamine. The polymer is isolated byprecipitation in cold hexane (stored at -20°C) followed by vacuum filtration. The molecularweight 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 rateof 1 ml/min. Polystyrene standards from Polymer Laboratories (Amherst, MA) are used toestablish a molecular weight calibration curve. This compound is used to generate thePCADK particles in all subsequent experiments. R848 may be conjugated to the terminal alcohol groups of the PCADK havingmolecular weight 6000 via imide ring opening, according to the step 2 shown below.

Step 1: Preparation of PCADK HO'

<img img-format="tif" img-content="drawing" file="IL216548AD000217.tif" id="idf0017" />

HO'

OH

Step 2: Conjugation of PCADK to R848 \ /—OEt

HO ΌΗ

HO

OH -54- WO 2010/138193 PCT/US2010/001560

In step 2, the polymer from step 1 (12 g, 2.0 x IO’3 moles) is dissolved in methylenechloride 100 mL, and the lactam of R848 (3.3 g, 8.0 x IO'3 moles) is added. This slurry isstirred as l,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g, 6 X 10“3 moles) is added in asingle portion. After stirring at room temperature overnight, a clear solution forms. Thesolution 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 evaporatedunder vacuum. After drying under high vacuum there is obtained 11.3 grams (81%) ofpolymer. A portion is hydrolyzed in acid and the R848 content is determined to be 9% byweight.

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 fromAldrich Chemical Company, Cat. #189421 and has the following structure:

<img img-format="tif" img-content="drawing" file="IL216548AD000218.tif" id="idf0018" />

<img img-format="tif" img-content="drawing" file="IL216548AD000219.tif" id="idf0019" />

(CH2)5fOH

The polycaprolactone diol-R854 conjugate has the following structure: H3CSO2NHy^N/"°Et

<img img-format="tif" img-content="drawing" file="IL216548AD000220.tif" id="idf0020" />

NHSO2CH3

(CHalslO n

The polymer (5 g, 2.5 x IO’3 moles) is dissolved in methylene chloride 25 mL and thelactam of R854 (2.4 g, 5.0 x 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 X IO'3 moles) is added in a single portion. After stirring atroom temperature for 15 minutes, a clear pale yellow solution forms. The solution is dilutedwith methylene chloride (100 mL) and the solution is washed with 5% citric acid. Thissolution 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 ishydrolyzed in acid and the R848 content is determined to be 18.5% by weight.

Example 14: Conjugation Of R848 To Poly-(Hexamethylene Carbonate)DioI Via Imide

Ring Opening -55- WO 2010/138193 PCT/US2010/001560

Imide ring opening is used to attach R848 to the terminal alcohol groups of poly-(hexamethylene carbonate)diol of molecular weight 2000. The poly(hexamethylenecarbonate) diol is purchased from Aldrich Chemical Company, Cat # 461164, and has thefollowing structure:

HO—[CH2(CH2)4CH2OCO2]nCH2(CH2)4CH2-OH

The poly(hexamethylene carbonate) diol-R848 conjugate has the following structure:

y—OEtH

<img img-format="tif" img-content="drawing" file="IL216548AD000221.tif" id="idf0021" />

The polymer (5 g, 2.5 x 10'3 moles) is dissolved in methylene chloride 25 mL and thelactam of R848 (2.06 g, 5.0 X 10’3 moles) is added. This sluny is stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4 X 10"3 moles) is added in a single portion.

After stirring at room temperature overnight a clear pale yellow solution forms. The solutionis 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 TinEthylhexanoate Catalyst

<img img-format="tif" img-content="drawing" file="IL216548AD000222.tif" id="idf0022" />

To a two necked round bottom flask equipped with a stir bar and condenser wasadded the imidazoquinoline resiquimod (R-848, 100 mg, 3.18 X 10-4 moles), D/L lactide (5.6g, 3.89 X IO'2 moles) and anhydrous sodium sulfate (4.0 g). The flask and contents weredried 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 thelactide had dissolved and then tin ethylhexanoate (75 mg, 60 pL) was added via pipette.Heating was continued under argon for 16 hours. After cooling, water (20 mL) was addedand stirring was continued for 30 minutes. The reaction was diluted with additional toluene -56- PCT/US2010/001560 WO 2010/138193 (200 mL) and was then washed with water (200 mL). The toluene solution was then washedin turn with 10% sodium chloride solution containing 5% cone. Hydrochloric acid (200 mL)followed by saturated sodium bicarbonate (200 mL). TLC (silica, 10% methanol inmethylene chloride) showed that the solution contained no free R-848. The solution wasdried over magnesium sulfate, filtered and evaporated under vacuum to give 3.59 grams ofpolylactic acid-R-848 conjugate. A portion of the polymer was hydrolyzed in base andexamined by HPLC for R-848 content. By comparison to a standard curve of R-848concentration vs. HPLC response, it was determined that the polymer contained 4.51 mg ofR-848 per gram of polymer. The molecular weight of the polymer was determined by GPCto be about 19,000.

Example 16: Low Molecular Weight Polylactic Acid Conjugates Of AnImidazoquinoline h3c.HO—

H3C

<img img-format="tif" img-content="drawing" file="IL216548AD000223.tif" id="idf0023" />

•OPLAOH

To a round bottom flask equipped with a stir bar and condenser was added theimidazoquinoline, resiquimod (R-848, 218 mg, 6.93 X 10-4 moles), D/L lactide (1.0 g, 6.93 XIO"3 moles) and anhydrous sodium sulfate (800 mg). The flask and contents were dried undervacuum at 55 °C for 8 hours. After cooling, the flask was then flushed with argon andtoluene (50 mL) was added. The reaction was stirred in an oil bath set at 120 °C until all ofthe lactide had dissolved and then tin ethylhexanoate (19 mg, 15pL) was added via pipette.Heating was continued under argon for 16 hours. After cooling, the reaction was diluted withether (200 mL) and the solution was washed with water (200 mL). The solution was driedover magnesium sulfate, filtered and evaporated under vacuum to give 880 mg. of crudepolylactic acid-R-848 conjugate. The crude polymer was chromatographed on silica using10% methanol in methylene chloride as eluent. The fractions containing the conjugate werepooled and evaporated to give the purified conjugate. This was dried under high vacuum toprovide the conjugate as a solid foam in a yield of 702 mg (57.6%). By integrating the NMRsignals for the aromatic protons of the quinoline and comparing this to the integrated intensityof the lactic acid CH proton it was determined that the molecular weight of the conjugate wasapproximately 2KD. GPC showed that the conjugate contained less than 5% of free R848. -57- PCT/US2010/001560 WO 2010/138193

Example 17: Low Molecular Weight Poly lactic Acid Co-Glycolic Acid Conjugates Of

An Imidazoquinoline

-OPLAOH CH3

To a round bottom flask equipped with a stir bar and condenser was added theimidazoquinoline, resiquimod (R-848,436 mg, 1.39 X 10*3 moles), glycolide (402 mg, 3.46X 10‘3 moles), D/L lactide (2.0 g, 1.39 X IO’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, theflask was then flushed with argon and toluene (60 mL) was added. The reaction was stirredin an oil bath set at 120°C until all of the R848, glycolide and lactide had dissolved and thentin ethylhexanoate (50 mg, 39 pL) was added via pipette. Heating was continued under argonfor 16 hours. After cooling, the reaction was diluted with ethyl acetate (200 mL) and thesolution 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 crudepolymer was chromatographed on silica using 10% methanol in methylene chloride as eluent.The fractions containing the conjugate were pooled and evaporated to give the purifiedconjugate. This was dried under high vacuum to provide the conjugate as a solid foam in ayield of 1.55 g (54.6%). By integrating the NMR signals for the aromatic protons of thequinoline and comparing this to the integrated intensity of the lactic acid CH proton it wasdetermined that the molecular weight of the conjugate was approximately 2KD. GPCshowed that the conjugate contained no detectable free R848.

Example 18: Polylactic Acid Conjugates Of An Imidazoquinoline Using A LithiumDiisopropylamide Catalysis

The imidazoquinoline (R-848), D/L lactide, and associated glassware were all driedunder vacuum at 50 °C for 8 hours prior to use. To a round bottom flask equipped with a stirbar and condenser was added the R-848 (33 mg, 1.05 x 10"4 moles), and dry toluene (5 mL).This was heated to reflux to dissolve all of the R-848. The solution was stirred undernitrogen and cooled to room temperature to provide a suspension of finely divided R-848. Tothis suspension was added a solution of lithium diisopropyl amide (2.0 M in THF, 50pL, 1.0x 10"4 moles) after which stirring was continued at room temperature for 5 minutes. The pale -58- WO 2010/138193 PCT/US2010/001560 yellow solution that had formed was added via syringe to a hot (120 °C) solution of D/Llactide (1.87 g, 1.3 x IO’2 moles) under nitrogen. The heat was removed and the pale yellowsolution was stirred at room temperature for one hour. The solution was diluted withmethylene chloride (200 mL) and this was then washed with 1% hydrochloric acid (2 x 50mL) followed by saturated sodium bicarbonate solution (50 mL). The solution was driedover 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 solutioncontained no free R-848. The polymer was dissolved in methylene chloride (10 mL) and thesolution was dripped into stirred hexane (200 mL). The precipitated polymer was isolated bydecantation and was dried under vacuum to give 1.47 grams of the polylactic acid - R-848conjugate as a white solid. A portion of the polymer was hydrolyzed in base and examinedby 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 gramof 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 indichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2 g, 10.5mmol, 5 eq) are added. The resulting solution is stirred at room temperature for 3 days. Thesolution is concentrated to remove most of DCM and the residue is added to a solution of 250mL of diethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHS ester. Thesolvents are removed and the polymer is washed twice with ether (2 x 200 mL) and driedunder vacuum to give PLA-NHS activated ester as a white foamy solid (~ 8 g recovered, HNMR can be used to confirm the presence of NHS ester). The PLA-NHS ester is storedunder argon in a below -10 °C freezer before use.

Alternatively, the reaction can be performed in DMF, THF, dioxane, or CHC13instead of DCM. DCC can be used instead of EDC (resulting DCC-urea is filtered off beforeprecipitation of the PLA-NHS ester from ether). The amount of EDC or DCC and NHS canbe 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 with50% to 75% glycolide is converted to the corresponding PLGA-NHS activated ester and isstored under argon in a below -10 °C freezer before use. -59- PCT/US2010/001560 WO 2010/138193

Example 21: One-Pot Ring-Opening Polymerization Of R848 With D/L-Lactide In The

Presence Of A Catalyst

<img img-format="tif" img-content="drawing" file="IL216548AD000224.tif" id="idf0024" />

<img img-format="tif" img-content="drawing" file="IL216548AD000225.tif" id="idf0025" />

odl-lactide

<img img-format="tif" img-content="drawing" file="IL216548AD000226.tif" id="idf0026" />

R848-PLA (R848 loading 3 mg/g) 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 washeated 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 theresulting 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 puffysolid (5.0 g). Polymeric structure was confirmed by NMR in CDCI3. A small sample ofthe polymer was treated with 2 N NaOH aq in THF/MeOH to determine the loading of R848on 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

<img img-format="tif" img-content="drawing" file="IL216548AD000227.tif" id="idf0027" />

<img img-format="tif" img-content="drawing" file="IL216548AD000228.tif" id="idf0028" />

PLGA

Sn(C8H 15)2)2 ------------------------------------------------------------------1 heat A mixture of D/L-lactide (10.8 g, 0.075 moles) and glycolide (2.9 g, 0.025 moles) washeated to 135 °C under argon. Once all of the materials had melted and a clear solution hadresulted, R848 (1.08 g, 3.43 X 10"3 moles) was added. This solution was stirred at 135 °Cunder a slow stream of argon for one hour. Tin ethylhexanoate (150 pL) was added andheating was continued for 4 hours. After cooling, the solid pale brown mass was dissolved inmethylene chloride (250 mL) and the solution was washed with 5% tartaric acid solution (2 x200 mL). The methylene chloride solution was dried over magnesium sulfate, filtered, and -60- PCT/US2010/001560 WO 2010/138193 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 isolatedby decantation of the 2-propanol and was dried under high vacuum. NMR showed that thepolymer was 71.4% lactide and 28.6% glycolide with a molecular weight of 4000. Theloading of R848 was close to theoretical by NMR.

Example 23: Preparation Of PLGA-R848 Conjugate

<img img-format="tif" img-content="drawing" file="IL216548AD000229.tif" id="idf0029" />

<img img-format="tif" img-content="drawing" file="IL216548AD000230.tif" id="idf0030" />

A mixture of PLGA (Lakeshores Polymers, MW -5000,7525DLG1A, acid number0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc (160 mL) wasstirred 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 wasstirred at room temperature for 6 h and then at 50-55 °C overnight (about 16 h). Aftercooling, the mixture was diluted with EtOAc (200 mL) and washed with saturated NH4CIsolution (2 x 40 mL), water (40 mL) and brine solution (40 mL). The solution was dried overNa2SO4 (20 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) wasthen added and the polymer conjugate precipitated out of solution. The polymer was thenwashed with IP A (4 x 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% byHPLC).

Example 24: Preparation Of PLGA-854A Conjugate

<img img-format="tif" img-content="drawing" file="IL216548AD000231.tif" id="idf0031" />

<img img-format="tif" img-content="drawing" file="IL216548AD000232.tif" id="idf0032" />

A mixture of PLGA (Lakeshores Polymers, MW -5000,7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (20 mL) was -61- PCT/US2010/001560 WO 2010/138193 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 mixturewas stirred at room temperature for 6 h and then at 50-55 °C overnight (about 15 h). Aftercooling, the mixture was diluted with EtOAc (100 mL) and washed with saturated NH4C1solution (2 x 20 mL), water (20 mL) and brine solution (20 mL). The solution was dried overNa2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (40 mL) wasthen added and the polymer conjugate precipitated out of solution. The polymer was thenwashed with IP A (4 x 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% byHPLC).

Example 25: Preparation Of PLGA-BBHA Conjugate

<img img-format="tif" img-content="drawing" file="IL216548AD000233.tif" id="idf0033" />

HBTU/DIPEA -

EtOAc

<img img-format="tif" img-content="drawing" file="IL216548AD000234.tif" id="idf0034" />

PLGA A mixture of PLGA (Lakeshores Polymers, MW -5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) wasstirred at room temperature under argon for 30 minutes. Compound BBHA (0.22 g, 0.7mmol) in 2 mL of dry DMSO was added, followed by diisopropylethylamine (DIPEA) (0.73mL, 4.2 mmol). The mixture was stirred at room temperature for 20 h. Additional amountsof HBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) were added and the mixturewas heated at 50-55 °C for 4 h. After cooling, the mixture was diluted with EtOAc (100 mL)and washed with saturated NH4C1 solution 20 mL), water (2 x 20 mL) and brine solution (20mL). 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 conjugateprecipitated out of solution. The polymer was then washed with IPA (2 x 20 mL) to removeresidual reagents and dried under vacuum at 35-40 °C for 2 days as a brownish powder (1.1g)·

Example 26: Conjugation Of R848 To Polyglycine, A Polyamide -62- PCT/US2010/001560 WO 2010/138193

<img img-format="tif" img-content="drawing" file="IL216548AD000235.tif" id="idf0035" />

CH3EtO—&lt; __i-OH / X CH3

CH3 Ο Ο H

The ί-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid (I) is preparedby ring opening polymerization of glycine N-carboxyanhydride (Aldrich cat #369772) using6-aminohexanoic acid benzyl ester (Aldrich cat &amp;amp;S33465) by the method of Aliferis et al.{Biomacromolecules, 5,1653, (2004)). Protection of the end amino group as the /-BOCcarbamate followed by hydrogenation over palladium on carbon to remove the benzyl estercompletes the synthesis of BOC protected polyglycine carboxylic acid (I). A mixture of BOC-protected polyglycine carboxylic acid (5 gm, MW = 2000,2.5 xIO'3 moles) and HBTU (3.79 gm, 1.0 x 10’2 moles) in anhydrous DMF (100 mL) is stirred atroom temperature under argon for 50 minutes. Then R848 (1.6 gm, 5.0 X 10’3 moles) isadded, followed by diisopropylethylamine (4 mL, 2.2 x 10‘2 moles). The mixture is stirred atRT for 6 h and then at 50-55 °C overnight (16 h). After cooling, the DMF is evaporatedunder vacuum and the residue is triturated in EtOAc (100 mL). The polymer is isolated byfiltration and the polymer is then washed with 2-propanol (4 x 25 mL) to remove residualreagents and dried under vacuum at 35-40 °C for 3 days. The polymer is isolated as an offwhite solid in a yield of 5.1 g (88%). The R848 loading can be determined by NMR is10.1%.

The /-BOC protecting group is removed using trifluoroacetic acid and the resultingpolymer 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 intrifluoroacetic acid (25 mL) and this solution is wanned 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. Afterdrying under vacuum there is obtained 4.5 grams of polymer as an off white solid. -63- WO 2010/138193 PCT/US2010/001560

Step 2: A mixture of PLGA (Lakeshores Polymers, MW ~5000,7525DLG1A, acidnumber 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous DMF (100mL) 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). Aftercooling, the DMF is evaporated under vacuum, and the residue is dissolved in methylenechloride (50 mL). The polymer is precipitated by the addition of 2-propanol (200 mL). Thepolymer is isolated by decantation and is washed with 2-propanol (4 x 50 mL) to removeresidual 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

<img img-format="tif" img-content="drawing" file="IL216548AD000236.tif" id="idf0036" />

HBTU/DIPEA -

EtOAc

<img img-format="tif" img-content="drawing" file="IL216548AD000237.tif" id="idf0037" />

PLGA A mixture of PLGA (Lakeshores Polymers, MW ~5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) isstirred at RT under argon for 30 minutes. Compound (I) (0.22 g,0.7 mmol) in 2 mL of dryDMSO is added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). Themixture is stirred at room temperature for 20 h. Additional amounts of HBTU (0.53 g, 1.4mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at 50-55 °C for 4h. After cooling, the mixture is diluted with EtOAc (100 mL) and washed with saturatedNH4CI solution 20 mL), water (2 x 20 mL) and brine solution (20 mL). The solution is driedover 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 polymeris then washed with IPA (2 x 20 mL) to remove residual reagents and dried under vacuum at35-40 °C for 2 days as a brownish powder (1.0 g).

Example 29: Preparation Of PLGA-2,9-Dibenzyl-8-Hydroxyadenine Conjugate -64- PCI7US2010/001560 WO 2010/138193

<img img-format="tif" img-content="drawing" file="IL216548AD000238.tif" id="idf0038" />

HBTU/DIPEA

EtOAc

<img img-format="tif" img-content="drawing" file="IL216548AD000239.tif" id="idf0039" />

PLGA A mixture of PLGA (Lakeshores Polymers, MW ~5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrous EtOAc (30 mL) isstirred at RT under argon for 30 minutes. Compound (II) (0.24 g,0.7 mmol) in 2 mL of dryDMSO is added, followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). Themixture is stirred at RT for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol) andDIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at 50-55 °C for 4 h. Aftercooling, the mixture is diluted with EtOAc (100 mL) and washed with saturated NH4CIsolution 20 mL), water (2 x 20 mL) and brine solution (20 mL). The solution is dried overNa2SO4 (10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) isthen added and the brownish polymer conjugate precipitated out of solution. The polymer isthen washed with IPA (2 x 20 mL) to remove residual reagents and dried under vacuum at35-40 °C for 2 days as a brownish powder (1.2 g).

Example 30: Imide Ring Opening Used To Attach 2-PentyI-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 ChemicalCompany, Cat #461164.

Poly(hexamethylene carbonate) diol:

HO—[CH 2(CH 2)4CH 2OCO 2]nCH 2(CH 2)4CH 2-OH

Poly (hexamethylene carbonate) diol - 8-oxoadenine conjugate: -65- PCT/US2010/001560 WO 2010/138193

<img img-format="tif" img-content="drawing" file="IL216548AD000240.tif" id="idf0040" />

TBD

<img img-format="tif" img-content="drawing" file="IL216548AD000241.tif" id="idf0041" />

The polymer (5 g, 2.5 x 10‘3 moles) is dissolved in methylene chloride 25 mL and thelactam of 2-pentyl-8-hydroxy-9-benzyladenine (2.05 g, 5.0 x 10*3 moles) is added. Thisslurry is stirred as l,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4 x 10'3 moles) isadded in a single portion. After stirring at room temperature overnight a clear pale yellowsolution forms. The solution is diluted with methylene chloride (100 mL), and the solution iswashed with 5% citric acid. This solution is dried over sodium sulfate after which it isfiltered and evaporated under vacuum. After drying under high vacuum there is obtained 5.5grams (78%) of polymer. NMR is used to determine the benzyladenine content which is18%.

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 of3.5KD (0.20 gm, 5.7 X 10-5moles) and an excess of 4-carboxycotinine (0.126 gm, 5.7 X 10-4 moles) were dissolved in dimethylformamide (5.0 mL). The solution was stirred anddicyclohexylcarbodiimide (0.124 gm, 6.0 X 10-4 moles) was added. This solution wasstirred overnight at room temperature. Water (0.10 mL) was added and stirring wascontinued for an additional 15 minutes. The precipitate of dicyclohexylurea was removed byfiltration and the filtrates were evaporated under vacuum. The residue was dissolved inmethylene chloride (4.0 mL) and this solution was added to diethyl ether (100 mL). Thesolution was cooled in the refrigerator for 2 hours and the precipitated polymer was isolatedby filtration. After washing with diethyl ether, the solid white polymer was dried under highvacuum. The yield was 0.188 gm. This polymer was used without further purification forthe next step. -66- PCT/US2010/001560 WO 2010/138193

The cotinine/PEG polymer (0.20 gm, 5.7 X 10-5 moles) was dissolved in drytetrahydroforan (10 mL) under nitrogen and the solution was stirred as a solution of lithiumaluminum hydride in tetrahydrofuran (1.43 mL of 2.0M, 2.85 X 10-3 moles) was added. Theaddition of the lithium aluminum hydride caused the polymer to precipitate as a gelatinousmass. The reaction was heated to 80°C under a slow stream of nitrogen and thetetrahydrofuran 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 hadstopped, 10% methanol in methylene chloride (50 mL) was added and the reaction mixturewas stirred until the polymer had dissolved. This mixture was filtered through Celite® branddiatomaceous earth (available from EMD Inc. as Celite® 545, part # CX0574-3) and thefiltrates were evaporated to dryness under vacuum. The residue was dissolved in methylenechloride (4.0 mL) and this solution was slowly added to diethyl ether (100 mL). The polymerseparated as a white flocculent solid and was isolated by centrifugation. After washing withdiethyl 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 wascharged with the PEG/nicotine polymer (0.081 gm, 2.2 X 10-5 moles), D/L lactide (0.410gm, 2.85 X 10-3 moles) and anhydrous sodium sulfate (0.380 gm). This was dried undervacuum at 55°C for 8 hours. The flask was cooled and flushed with argon and then drytoluene (10 mL) was added. The flask was placed in an oil bath set at 120°C, and once thelactide had dissolved, tin ethylhexanoate (5.5 mg, 1.36 X 10-5 moles) was added. Thereaction 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 tosettle. The methylene chloride layer was isolated and dried over anhydrous magnesiumsulfate. After filtration to remove the drying agent, the filtrates were evaporated undervacuum to give the polymer as a colorless foam. The polymer was dissolved intetrahydrofuran (10 mL) and this solution was slowly added to water (150 mL) with stirring.The precipitated polymer was isolated by centrifugation and the solid was dissolved inmethylene chloride (10 mL). The methylene chloride was removed under vacuum and theresidue 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 synthesizedaccording to the synthesis provided in Example 99 of US Patent 5,389,640 to Gerster et al. -67- PCT/US2010/001560 WO 2010/138193 R848 was conjugated to PL A by a method provided above, and the PL A structure wasconfirmed by NMR. PLA-PEG-nicotine conjugate was prepared according to Example 31. PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 North NormandyDrive, Petersburg, VA 23805). The polyvinyl alcohol (Mw = 11 KD - 31 KD, 85-89%hydrolyzed) was purchased from VWR scientific. Ovalbumin peptide 323-339 was obtainedfrom Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505. Part # 4064565).

The above materials were used to prepare the following solutions: 1. Resiquimod (R848) @10 mg/mL and PLA @100 mg/mL in methylenechloride 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 addedsolution #5 (2.0 mL) and sonication at 35% amplitude for 40 seconds using the BransonDigital Sonifier 250 forms the second emulsion. This was added to a beaker containingphosphate buffer solution (30 mL) and this mixture was stirred at room temperature for 2hours to form the nanoparticles.

To wash the particles a portion of the nanoparticle dispersion (7.4 mL) wastransferred to a centrifuge tube and spun at 5,3OOg for one hour, supernatant was removed,and the pellet was re-suspended in 7.4 mL of phosphate buffered saline. The centrifugeprocedure was repeated and the pellet was re-suspended in 2.2 mL of phosphate bufferedsaline 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 ofOvalbumin protein, was purchased from Bachem Americas Inc. (3132 Kashiwa Street,Torrance CA 90505.) -68- PCT/US2010/001560 WO 2010/138193

Resiquimod (aka R848) was synthesized according to a method provided in US Patent6,608,201. PLA-R848, resiquimod, was conjugated to PLA with a molecular weight ofapproximately 2,500 Da according to a method provided above. PLGA-R848, resiquimod, was conjugated to PLGA with a molecular weight ofapproximately 4,100 Da according to a method provided above. PS-1826 DNA oligonucleotide with fully phosphorothioated backbone havingnucleotide sequence 5'-TCC ATG ACG TTC CTG ACG TT-3' with a sodium counter-ionwas purchased from Oligos Etc (9775 SW Commerce Circle C-6, Wilsonville, OR 97070.) PO-1826 DNA oligonucleotide with phosphodiester backbone having nucleotidesequence 5'-TCC ATG ACG TTC CTG ACG TT-3’ with a sodium counter-ion waspurchased from Oligos Etc. (9775 SW Commerce Circle C-6, Wilsonville, OR 97070.)\ PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL2A.) PLA with an inherent viscosity of 0.71 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211. Product Code 100 DL7A.) PLA with an inherent viscosity of 0.19 dL/g was purchased from BoehringerIngelheim Chemicals, Inc. (Petersburg, VA. Product Code R202H.) PLA-PEG-nicotine with a molecular weight of approximately 18,500 to 22,000 Dawas prepared according to a method provided above. PLA-PEG-R848 with a molecular weight of approximately 15,000 Da wassynthesized 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 primaryemulsions. The table below references the solution suffix (e.g., B in Solution #1 columnindicates Solution #1B was used) and volume of solution used.

SampleNumber Solution #1(Volume) Solution #2 (Volume) Solution #3 (Volume) Solution #4(Volume) Solution #5(Volume) 1 B (0.1 ml) C (1.0 ml) A (0.1 ml) C (1.0 ml) A (2.0 ml) -69- WO 2010/138193 PCT/US2010/001560 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 mi)

Solution 1A: Ovalbumin peptide 323 - 339 @ 35 mg/mL in dilute hydrochloric acidaqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13Nhydrochloric acid solution at room temperature.

Solution IB: Ovalbumin peptide 323 - 339 @ 70 mg/mL in dilute hydrochloric acidaqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13Nhydrochloric acid solution at room temperature.

Solution 2A: 0.21-IV PLA @75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml inmethylene chloride. The solution was prepared by first preparing two separate solutions atroom 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 byadding 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 inmethylene chloride. The solution was prepared by first preparing two separate solutions atroom 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 byadding 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 inmethylene chloride. The solution was prepared by first preparing two separate solutions atroom 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 byadding 3 parts PLA solution for each part of PLA-PEG-nicotine solution.

Solution 3 A: Oligonucleotide (either PS-1826 or PO-1826) @ 200 mg/ml in purifiedwater. The solution was prepared by dissolving oligonucleotide in purified water at roomtemperature.

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 bycombining solution 1 and solution 2 in a small pressure tube and sonicating at 50% amplitude -70- PCT/US2010/001560 WO 2010/138193 for 40 seconds using a Branson Digital Sonifier 250. W3/O4 was prepared by combiningsolution 3 and solution 4 in a small pressure tube and sonicating at 50% amplitude for 40seconds 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 primaryemulsion (W1/O2 and W3/O4) and solution 5 and sonicating at 30% amplitude for 40 to 60seconds using the Branson Digital Sonifier 250.

The third emulsion was added to a beaker containing 70mM phosphate buffer solution(30 mL) and stirred at room temperature for 2 hours to allow for the methylene chloride toevaporate and for the nanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube and spinning at 13,823g for onehour, 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 bufferedsaline for a final nanocarrier dispersion of about 10 mg/mL.

The amounts of oligonucleotide and peptide in the nanocarrier were determined byHPLC analysis.

Example 34: Standard Double Emulsion

Materials

As provided in Example 33 above.

Batches were produced using a standard double emulsion process. The table belowreferences the solution suffix (e.g., B in Solution #1 column indicates Solution #1B was used)and volume of solution used.

Sample Number Solution #1(Volume) Solution #2 (Volume) Solution #3(Volume) Solution #4 (Volume) Solution #5 (Volume) 1 A (0.1 ml) A (0.75 ml) A (0.25 m!) 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) -71- PCT/US2010/001560 WO 2010/138193

Solution 1 A: Ovalbumin peptide 323 - 339 @ 69 mg/mL in de-ionized water. Thesolution was prepared by slowly adding ovalbumin peptide to the water while mixing at roomtemperature.

Solution IB: Ovalbumin peptide 323 - 339 @ 70 mg/mL in dilute hydrochloric acidaqueous solution. The solution was prepared by dissolving ovalbumin peptide in 0.13Nhydrochloric acid solution at room temperature.

Solution 1C: Oligonucleotide (PS-1826) @50 mg/ml in purified water. The solutionwas prepared by dissolving oligonucleotide in purified water at room temperature.

Solution ID: Ovalbumin peptide 323 - 339 @17.5 mg/mL in dilute hydrochloric acidaqueous solution. The solution was prepared by dissolving ovalbumin peptide @ 70 mg/mlin 0.13N hydrochloric acid solution at room temperature and then diluting the solution with 3parts purified water per one part of starting solution.

Solution 2A: R848 @10 mg/ml and 0.19-IV PLA @ 100 mg/mL in pure methylenechloride prepared at room temperature.

Solution 2B: PLA-R848 @ 100 mg/ml in pure methylene chloride prepared at roomtemperature.

Solution 2C: PLGA-R848 @100 mg/ml in pure methylene chloride prepared at roomtemperature.

Solution 2D: PLA-PEG-R848 @ 100 mg/ml in pure methylene chloride prepared atroom temperature.

Solution 3A: PLA-PEG-nicotine @ 100 mg/ml in pure methylene chloride prepared atroom temperature.

Solution 4A: 0.19-IV PLA @100 mg/mL in pure methylene chloride prepared atroom 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 andsolution 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 andsonicating 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 (30mL) and stirred at room temperature for 2 hours to allow for the methylene chloride toevaporate and for the nanocarriers to form. A portion of the nanocarriers were washed by -72- PCT/US2010/001560 WO 2010/138193 transferring the nanocarrier suspension to a centrifuge tube and spinning at 5,000 to 9,500RPM for one hour, removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated and the pellet was re-suspended inphosphate 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) wasmeasured 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-C18column (3.5pm. 75 x 4.6 mm. Column Temp = 40°C (part no. 866953-902)) using MobilePhase 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% Bto 9 min; decrease back to 5% B to 9.5 min and kept equilibrating to end. Total run time was13 minute with flow rate of 1 mL/min).

Method for CpG

The amount of CpG (immunostimulatory agent) was measured using reverse phaseHPLC on Agilent 1100 system at 260 nm equipped with Waters XBridge C-18 (2.5 micronparticle, 50x 4.6 mm ID (part No. 186003090), column temp. 600C) using mobile phase A of2% 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, thenramped to 90% B to 12 minutes. Strength of B was rapidly decreased to 5% in one minuteand equilibrated until stop time, 16 minutes. The flow rate was 1 mL/min until end of themethod, 16 minutes).

Method for Nicotine analog

Nicotine analog was measured using reverse phase HPLC on Agilent 1100 system at254 nm equipped with Waters X-Bridge C-18 (5 micron particle, 100 x 4.6 mm ID, columntemp at 400C) using Mobile Phase A (MPA) of 95% water/5% acetonitrile/0.1% TFA andMobile Phase B (MPB) of 90 % acetonitrile/10% water/0.09% TFA (gradient: column wasequilibrated at 5% B increased to 45% B in 14 minutes. Then ramped up to 95% B from 14to 20 minutes. Mobile B strength was quickly decreased back to 5% and requilibrated until -73- PCT/US2010/001560 WO 2010/138193 the end of the method. The flow rate of the method was maintained at 0.5 ml/min with totalrun time of 25 minutes. The NC suspension was centrifuged @14000 rpm for about 15-30minutes depending on particle size. The collected pellets were treated with 200 uL of cone.NH4OH (8 M) for 2h with agitation until the solution turns clear. A 200 uL of 1% TFA wasadded to neutralize the mixture solution, which brought the total volume of the pellet solutionto 200 uL. An aliquot of 50 uL of the solution was diluted with MPA(or water) to 200 uLand 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 @14000rpm for about 15 minutes. Thecollected pellet was dissolved with 0.3 mL of acetonitrile and centrifuged briefly @14000rpm to remove any residual insolubles. The clear solution was further diluted with 4times 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 14000rpm for 15 to 30 minutes depending on particle size. The collectedpellets were re-suspended with 500 uL of water and sonicated for 30 minutes to fully dispersethe particles. The NC was then heated at 600°C for 10 minutes. Additional 200 uL of 1 NNaOH was added to the mixture, heated for another 5 minutes where the mixture becomesclear. The hydrolyzed NC solution was centrifuged briefly at 14000 rpm. A final 2x dilutionof the clear solution using water was then made and assayed on the reverse HPLC describedabove.

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 14000rpm for 15 to 30 minutes. 100 uL of acetonitrile was added to thepellets to dissolve the polymer components of the NC. The mixture was vortexed andsonicated for 1 to 5 minutes. 100 uL 0.2% TFA was added to the mixture to extract thepeptides and sonicated for another 5 minutes to ensure the break down of the aggregates. Themixture was centrifuged at 14000rpm 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) wastaken and assayed on the reverse phase HPLC as described above. -74- WO 2010/138193 PCT/US2010/001560

Amount of conjugated nicotine analog (B cell antigen) in nanocarriers 1.5 mL of NC suspension was spun down @ 14000rpm for about 15 minutes, thepellets were hydrolyzed using 150 uL of concentrated NH4OH (8M) for about 2-3 h until thesolution turns clear. A 150 uL of 2% TFA(aq) solution was added to the pellet mixture toneutralize the solution. A 100 uL aliquot of the mixture was diluted with 200 uL of waterand assayed on reverse phase HPLC described above and quantified based on the standardcurve established using the precursor (PEG-nicotine) of the PLA-PEG-nicotine used in themanufacture.

Example 36: Release Rate Testing

The release of T-cell antigen, ova peptide and adjuvant, R848 from the syntheticnanocarrier (nanoparticles) in PBS (lOOmM, 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 usingreverse phase HPLC on a Agilent 1100 system at λ = 215 nm equipped with an AgilentZorbax SB-C18 column (3.5pm. 75 x 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 MobilePhase B (MPB) of 90 % acetonitrile/10% water/0.09% TFA with Gradient: B = 5 to 45 % in7 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 shownin Table 1. An aqueous suspension of the tested synthetic nanocarriers was then diluted to afinal stock volume of 4.4 mL with PBS. (A) In vitro release rate measurement in PBS (pH=7.4):

For TO sample, a 200 pL aliquot was immediately removed from each of the NPsample and centrifuged @ 14000 rpm in a microcentrifuge tubes using a Microcentrifuge (Model: Galaxy 16). 100 pL of supernatant was removed and diluted to 200 pL in HPLCMobile Phase A (MPA) and assayed for the amount of R848 and ova peptide released on thereverse phase HPLC.

For time point measurements: 9 x 200 pL of each of the samples were added tomicrocentrifuge tubes (3 x 200 for unconjugated) and 300 pL of 37C PBS was added to eachabove aliquot and the samples were placed immediately in 37 °C oven. At the following timepoints: 24 hr, 48 hr, 96 hr and 144 hr (for conjugated R848) or 2 h, 16h and 24 h (forunconjugated (encapsulated) R848), the samples were centrifuged and assayed for the amountof R848 and ova peptide released as above for TO sample. -75- WO 2010/138193 PCT/US2010/001560 (B) In vitro release rate measurement in Citrate Buffer (pH=4.5):

For TO sample, a 200 pL aliquot was removed from each of the samples andcentrifuged @ 6000 rpm for 20 minutes and the supernatant was removed. The residuenanoparticles was resuspended in 200 uL of citrate buffer and centrifuged @ 14000 rpm for15 minutes. 100 uL of the supernatant was removed and diluted to 200 uL with MPA andassayed for R848 and peptide as above.

For time point measurements: 9 x 200 uL of each of the samples were added tomicrocentrifuge tubes (3 x 200 for unconjugated) and centrifuged for 20 minutes @ 6000 rpmand the supernatants were removed. The residue NPs were then resuspended in 500 uL ofcitrate buffer and placed in 37 °C oven. At the following time points: 24 hr, 48 hr, 96 hr and144 hr (for conjugated R848) or 2 h, 16h and 24 h (for unconjugated (encapsulated) R848),the samples were centrifuged and assayed for the amount of R848 and ova peptide released asabove for TO sample.

In order to complete the mass balance from above measurements in PBS and Citratebuffer, the remaining pellets (conjugated R848 samples only) from each sample was treatedwith 200 uL of cone. NH4OH (8 M) for 3h with mixing. After the mixture was settled, 200uL of 1% TFA was added to bring total volume of the pellet to 400 uL. An aliquot of 50 uLof the solution was diluted with MPA to 200 uL and analyzed on HPLC as above todetermine the amount of R848 and ova peptide that remained in the pellet after in vitrorelease to close the mass balance. For unconjugated samples, the sample was diluted withTFA 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, 3.5pm. 75 x 4.6 mm. (part no. 866953-902)

Cl8 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 - lOOmM, pH=7.4.

Citrate Buffer - 100 mM, pH = 4.5.

Oven - -76- PCT7US2010/001560 WO 2010/138193

Microcentrifuge - Galaxy 16

Microcentrifuge tubes

Sonicator

Pipets - 20, 200,1000 pL adjustable HPLC grade water - EMD - #WX0008-l. NH4OH—8M. Mallinkcrodt. TFA, 0.2%. Prep 4/27/09. TFA, 1%. Prep 5/13/09.

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:

Sample ID Estimated R848 in NPs (pg/mL) Estimated Ova inNPs (gg/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 isavailable for all time points, the following volumes of PBS were added to the samples tobring them all to 4.4 mL.

Table 3

Sample ID SampleVolume (mL) Volume PBS added(mL) 1 4.35 0.05 2 4.23 0.17 3 4.21 0.19 -77- PCT/US2010/001560 WO 2010/138193 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 pL aliquot from each of the samples.Microcentrifuge @ 14000 rpm. Remove supernatant. ii. Dilute supernatant 100 pL &gt; 200 pL in MPA. (DF=2). iii. Assay for peptide and R848. b. Citrate i. Remove a 200 pL aliquot from each of the samples.Microcentrifuge @ 6000 rpm for 20 minutes. Remove supernatant. ii. Add 200 uL of citrate buffer and thoroughly resuspend. iii. Microcentrifuge @ 14000 rpm for 15 minutes. Removesupernatant. iv. Dilute supernatant lOOpL &gt; 200 pL in MPA. (DF=2) v. Assay for peptide and R848.

2) PBSIVR a. Add 9 x 200 pL of each of the samples to microcentrifuge tubes. (3 x 200for unconjugated) b. To each aliquot add 300 pL of 37C PBS. c. Immediately place samples in 37C oven.

3) Citrate IVR a. Add 9 x 200 uL of each of the samples to microcentrifuge tubes. (3 x 200 for unconjugated) b. Centrifuge for 20 minutes @ 6000 rpm. c. Remove the supernatants. d. To each tube, add 500 pL of citrate buffer and resuspend thoroughly. e. Place samples in 37C 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) -78- PCT/US2010/001560 WO 2010/138193 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 pL to 200 pL in MPA. (DF=2) 7) Assay for peptide and R848. This will provide the amount released at each timepoint. TO COMPLETE MASS BALANCE, PERFORM THE FOLLOWING: 8) To the remaining pellets (conjugated only) add 200uL NH4OH. 9) Vortex briefly and sonicate to disperse. 10) Add stir bar. Allow to sit until clear (at least 3 hours). 11) Add 200uL of 1% TFA (total pellet volume = 400 pL). 12) Dilute 50pL to 200 pL in MPA. Analyze by HPLC to determine peptide andR848 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 immunostimulatoryagents (e.g., R848, CpG) from synthetic nanocarriers in phosphate buffered saline solution(PBS) (lOOmM, pH=7.4) and citrate buffer (100 mM, pH=4.5) at 37 °C was determined asfollows:

The release of R848 from the nanocarrier composed of conjugated R848 and the ovapeptide was achieved by exchanging desired amount of the aqueous suspension of the testedsynthetic nanocarriers obtained from the manufacture (e.g., about 10 mg/mL in PBS) into thesame volume of the appropriate release media (Citrate buffer lOOmM) via centrifugation andre-suspension.

In vitro release rate measurement in PBS (pH=7.4) 1 mL of the PBS suspension NC was centrifuged @ 14000 rpm in microcentrifugetubes generally from 15-30 minutes depending on particle size. The collected supernatant WO 2010/138193 PCT/US2010/001560 -79- was then diluted with equal volume of the mobile phase A (MPA) or water and assayed onreverse phase HPLC for the amount of the R848 release during the storage. The remainingpellet was re-suspended to homogeneous suspension in ImL of PBS and placed to 37°Cthermal chamber with constant gentle agitation

For TO sample, a 150 pL aliquot was immediately removed from NC suspension priorplacing the NC suspension to 37°C thermal chamber and centrifuged @ 14000 rpm inmicrocentrifuge tubes using a microcentrifuge (Model: Galaxy 16). 100 pL of thesupernatant was removed and diluted to 200 pL with HPLC Mobile Phase A (MPA) or waterand assayed for the amount of R848 and ova peptide released on the reverse phase HPLC.

For time point measurements, 150 pL aliquot was removed from the 37°C NC samplesuspension, and the samples were centrifuged and assayed for the amount of R848 and ovapeptide released in the same manner as for TO sample. The R848 and ova peptide releasedwas tested at 6h, 24h for routine monitoring with additional 2h, 48h, 96h and 144h forcomplete release profile establishment. s

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 NCstorage solution (e.g., PBS) instead of the PBS buffer, pH= 7.4. In order to complete themass balance from above measurements in PBS and Citrate buffer, the remaining pellets fromeach time point were treated with 100 uL of NH4OH (8 M) for 2h (or more) with agitationuntil solution turn clear. A 100 uL of 1% TFA was added to neutralize the mixture, whichbrought the total volume of the pellet solution to 200 uL. An aliquot of 50 uL of the mixturewas diluted with MPA (or water) to 200 uL and analyzed on HPLC as above to determine theamount of unreleased R848 remaining in the pellets after in vitro release to close the massbalance. For unconjugated samples, the sample was diluted with TFA in acetonitrile andassayed as above for R848.

The release of CpG was determined similar to the measurement of R848 and ovapeptide in terms of sample preparation and monitored time points. However, the amount ofthe CpG in the release media was assayed by the reverse phase HPLC method describedabove.

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 pg of NC-Nic. NC-Nic was a composition of -80- PCT/US2010/001560 WO 2010/138193 nanocarriers exhibiting nicotine on the outer surface and, for all groups of mice except forGroup 1, carrying CpG-1826 (thioated) adjuvant, which was released from the nanocarriers atdifferent 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-nicotineantibodies 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 peptideand polymers, 75% of which were PLA and 25% were PLA-PEG-Nic. The Group 2 micewere administered NC-Nic containing ova peptide, polymers, 75% of which were PLA and25% were PLA-PEG-Nic, and 3.2% CpG-1826; release rate at 24 hours: 4.2 pg CpG per mgof NC. The Group 3 mice were administered NC-Nic containing polymers, 75% of whichwere PLA and 25% were PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours: 15 pgCpG 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 nanocarriersis beneficial for the immune response against NC-associated antigen, and, furthermore, thatthe higher release rate of entrapped CpG adjuvant from within the nanocarriers (NC) at 24hours produced an immune response, which was elevated compared to one induced by NCwith 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 pg of NC-Nic and serum anti-nicotine antibodieswere then measured on days 12,24 and 40. NC-Nic was a composition of nanocarriersexhibiting 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 inFig. 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); releaserate at 24 hours: 16.6 pg CpG per mg of NC. The Group 2 mice were administered NC-Niccontaining 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 pg CpG per mg of NC. TheGroup 3 mice were administered NC-Nic containing ova peptide, polymers, 75% of whichwere PLA and 25% were PLA-PEG-Nic, and 7.9% CpG-1826 (phosphodiester or PO, non-thioated); release rate at 24 hours: 19.6 pg CpG per mg of NC. The Group 4 mice were -81- PCT/US2010/001560 WO 2010/138193 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 pgCpG 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-boundantigen (nicotine) with the nanocarrier exhibiting higher release rate at 24 hours inducedstronger humoral immune response (group 1 &gt; group 2 and group 3 &gt; group 4). This was trueirrespective 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 pg of NC-Nic and serum anti-nicotine antibodieswere then measured on days 26, 40 and 54. The nanocarriers were prepared according to amethod provided above. EC50 for anti-nicotine antibodies as measured in standard ELISAagainst 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 2mice were administered NC-Nic containing ova peptide, polymers, 75% of which were PLAand 25% were PLA-PEG-Nic, and 1.0% R848; of which 92% is released at 2 hours and morethan 96% is released at 6 hours. The Group 3 mice were administered NC-Nic containingova peptide, polymers, 75% of which were PLA-R848 and 25% were PLA-PEG-Nic, and1.3% R848, of which 29.4% is released at 6 hours and 67.8% is released at 24 hours. TheGroup 4 mice were administered NC-Nic containing ova peptide, polymers, 75% of whichwere PLA-R848 and 25% were PLA-PEG-Nic, and 1.4% of R848, of which 20.4% isreleased at 6 hours and 41.5% is released at 24 hours. The Group 5 mice were administeredNC-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 24hours. 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 ofR848 was elevated an immune response to the same level as NC releasing R848 atintermediate rate (group 3 ~ group 4 &gt; group 2 ~ group 5). -82- PCT/US2010/001560 WO 2010/138193

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 pg of NC-Nic (nanocarrier exhibiting nicotine onthe outer surface) with entrapped ΡΟ-CpG or not containing entrapped ΡΟ-CpG admixedwith free ΡΟ-CpG. The synthetic nanocarriers were prepared according to methods providedabove. 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 ΡΟ-CpG and MHC-IIhelper peptide from ovalbumin (Ov-II) encapsulated (6.6% PO-CpG; 2.3% Ov-II). Thegroup 2 mice were immunized with a NC-Nic with 0.7% of entrapped Ov-II admixed with 20pg 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 titerin group 1 &gt; antibody titer in group 2).