Synthetic nanocarriers comprising polymers comprising multiple immunomodulatory agents   

Abstract: This invention relates to compositions, and related methods, of synthetic nanocarriers that comprise polymers that comprise at least two immunomodulatory agent moieties.

This application claims the benefit under 35 U.S.C. §119 of U.S. provisional application 61/513,496, 61/513,526 and 61/513,527, each filed Jul. 29, 2011, the entire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions, and related methods, of synthetic nanocarriers that comprise polymers that comprise at least two immunomodulatory agent moieties.

BACKGROUND OF THE INVENTION

Immunomodulatory agents, such as adjuvants, are useful in enhancing the effectiveness of vaccines. However, effective ways of delivering high concentrations of immunomodulatory agents to target cells, such as dendritic cells, are needed.

SUMMARY

In one aspect, a composition comprising synthetic nanocarriers comprising a first type of polymer that comprises at least two immunomodulatory agent moieties is provided. In one embodiment, the immunomodulatory agent moieties are between 2 and 100% of the polymer weight. In another embodiment, the immunomodulatory agent moieties are at least 4% of the polymer weight. In another embodiment, the immunomodulatory agent moieties are at least 8% of the polymer weight. In one embodiment, at least a portion of the immunomodulatory agent moieties are not present at the surface of the synthetic nanocarriers. In another embodiment, at least a portion of the immunomodulatory agent moieties are not at the terminus of a polymer.

In one embodiment, the first type of polymer comprises at least three, four, five, six, seven, eight, nine or ten immunomodulatory agent moieties. In another embodiment, the at least two immunomodulatory agent moieties are at at least one terminus of the first type of polymer. In yet another embodiment, the at least two immunomodulatory agent moieties are along the backbone of the first type of polymer.

In still another embodiment, the at least two immunomodulatory agent moieties are themselves polymerized and form the backbone or portion of the backbone of the first type of polymer. In one embodiment, the synthetic nanocarriers further comprise another material in addition to the immunomodulatory agent moieties. In another embodiment, the other material is another type of polymer. In a further embodiment, the polymer backbone comprises at least one type of monomeric residue that is not the immunomodulatory agent moiety.

In another embodiment, the synthetic nanocarriers comprise at least a second type of polymer.

In one embodiment, the first type of polymer and/or the second type of polymer is a linear polymer. In another embodiment, the first type of polymer and/or the second type of polymer is a branched polymer. In yet another embodiment, the first type of polymer and/or the second type of polymer is part of or forms a dendrimer. In still another embodiment, the first type of polymer and/or the second type of polymer is part of or forms a polymeric matrix.

In one embodiment, the immunomodulatory agent moieties are the same type of immunomodulatory agent moiety. In one embodiment, the immunomodulatory agent moieties comprise a toll-like receptor (TLR) agonist. In another embodiment, the TLR agonist is a TLR-7 and/or TLR-8 agonist. In yet another embodiment, the TLR agonist comprises an aminodiazepine, adenine derivative or an imidazoquinoline. In still another embodiment, the imidazoquinoline comprises resiquimod or imiquimod. In a further embodiment, the adenine derivative comprises PF-4171455 or SM-276001.

In another embodiment, the composition further comprises an antigen. In one embodiment, the antigen comprises a B cell or T cell antigen. In another embodiment, the T cell antigen is a T helper cell antigen.

In one embodiment, the first type of polymer and/or the second type of polymer comprises a polyester, polyether, polycarbonate or polyamino acid. In another embodiment, the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In yet another embodiment, the polyester is coupled to a hydrophilic polymer. In still another embodiment, the hydrophilic polymer comprises a polyether. In a further embodiment, the polyether comprises polyethylene glycol. In still a further embodiment, the polyamino acid comprises polyglutamic acid.

In one embodiment, the first type of polymer and/or the second type of polymer has a weight average or number average molecular weight of at least 2000 Da, at least 2500 Da, at least 3000 Da, at least 3500 Da, at least 4000 Da, at least 4500 Da or at least 5000 Da.

In one embodiment, the mean of a particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is a maximum dimension of from 20 nm to 500 nm. In another embodiment, the mean of a particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is a maximum dimension of from 20 nm to 400 nm. In another embodiment, the mean of a particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is a maximum dimension of from 20 nm to 300 nm. In another embodiment, the mean of a particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is a maximum dimension of from 20 nm to 250 nm.

In yet another embodiment, the composition further comprises a pharmaceutically acceptable excipient.

In another embodiment, the composition is sterile. In another embodiment, the compositions is in lyophilized form.

In another aspect, a dosage form comprising any of the compositions provided herein is provided.

In yet another aspect, a vaccine comprising any of the dosage forms provided herein is provided.

In still another aspect, a method comprising administering any of the compositions, dosage forms or vaccines to a subject is provided. In one embodiment, the subject is a human. In another embodiment, the method further comprises administering an antigen. In one embodiment, the antigen comprises a B cell or T cell antigen. In another embodiment, the T cell antigen is a T helper cell antigen. In still another embodiment, the subject has or is at risk of having cancer. In a further embodiment, the subject has or is at risk of having an infection or infectious disease. In yet another embodiment, the subject has or is at risk of having an autoimmune disease, an inflammatory disease, an allergy or graft versus host disease. In a further embodiment, the subject has undergone or will undergo transplantation.

In a further aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties at a terminus of a polymer, comprising preparing a ring-opened polyester polymer with polyalcohol, contacting the ring-opened polyester polymer with succinic anhydride, and reacting the polyester polymer with immunomodulatory agent moieties in the presence of a coupling agent and a base is provided. In one embodiment, the polyester comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In another embodiment, the coupling agent comprises TBTU, HBTU, EDC, DCC or PyBop. In still another embodiment, the base comprises DIPEA, DMAP or Et3N.

In another aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties along the polymer backbone, comprising preparing a polyamino acid polymer with a free side chain acid group, and coupling the polymer with immunomodulatory agent moieties in the presence of a coupling agent and a base is provided. In one embodiment, the polyamino acid polymer comprises polyglutamic acid. In another embodiment, the coupling agent comprises TBTU, HBTU, EDC, DCC or PyBop. In still another embodiment, the base comprises DIPEA, DMAP or Et3N.

In yet another aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties along the polymer backbone, the method comprising polymerizing a monomer in the presence of a polyol to provide a multi-armed polymer, functionalizing the multi-armed polymer with one or more carboxylic acid groups, and coupling the immunomodulatory agent moieties comprising an amino group with the multi-armed polymer in the presence of a coupling agent is provided. In one embodiment, the multi-armed polymer comprises a poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone. In another embodiment, the coupling agent comprises TBTU, HBTU, EDC, DCC or PyBop. In still another embodiment, the coupling is performed also in the presence of a base. In yet another embodiment, the base is DIPEA, DMAP or Et3N.

In yet a further aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties, the method comprising providing a linear polymer comprising two or more side chain groups comprising an electrophilic or nucleophilic chemical moiety attached thereto, and coupling the immunomodulatory agent moieties to the side chain group is provided. In one embodiment, the side chain chemical moiety comprises a carboxylic acid and the immunomodulatory agent moieties, comprising an amino group, are coupled to carboxylic acid group in the presence of a coupling agent. In another embodiment, the coupling agent is TBTU, HBTU, EDC, DCC or PyBop. In still another embodiment, the coupling is performed also in the presence of a base. In yet another embodiment, the base is DIPEA, DMAP or Et3N.

In another aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties along the polymer backbone, comprising functionalizing monomers of a polymer, coupling the functionalized monomers with immunomodulatory agent moieties, and polymerizing the coupled monomers is provided.

In yet another aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties along the polymer backbone, the method comprising providing a monomer functionalized with immunomodulatory agent moieties, and polymerizing the monomer is provided. In one embodiment, the monomer comprises lactide, glycolide or caprolactone monomer.

In still another aspect, a method of producing a polymer comprising at least two immunomodulatory agent moieties along the polymer backbone, comprising producing or obtaining reactive bifunctional immunomodulatory agent moieties, and reacting the bifunctional immunomodulatory agent moieties such that a polymer is formed is provided. In one embodiment, the immunomodulatory agent moieties comprise a TLR agonist. In another embodiment, the TLR agonist comprises a TLR-7 and/or TLR-8 agonist. In still another embodiment, the TLR agonist comprises an aminodiazepine, adenine derivative or an imidazoquinoline. In a further embodiment, the imidazoquinoline comprises resiquimod or imiquimod. In another embodiment, the adenine derivative comprises PF-4171455 or SM-276001.

In one embodiment, any of the methods provided further comprise producing a synthetic nanocarrier with the polymer.

In a further aspect, a method comprising the steps of any of the methods exemplified herein is provided.

In one aspect, a polymer, synthetic nanocarrier or vaccine obtainable by a process comprising the steps of any method provided herein is provided.

In another aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in therapy or prophylaxis.

In yet another aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in any of the methods provided herein.

In still another aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in a method of treating or preventing cancer.

In a further aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in a method of treating or preventing infection or infectious disease.

In still a further aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in a method of treating or preventing an autoimmune disease, an inflammatory disease, an allergy or graft versus host disease.

In another aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for use in a method of treating a subject that has undergone or will undergo transplantation.

In yet another aspect, any of the compositions, dosage forms, vaccines, polymers or synthetic nanocarriers can be for the manufacture of a medicament for use in any of the methods provided herein.

DETAILED DESCRIPTION

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

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a polymer” includes a mixture of two or more such molecules or a mixture of differing molecular weights of a single polymer species, reference to “a synthetic nanocarrier” includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, reference to “a DNA molecule” includes a mixture of two or more such DNA molecules or a plurality of such DNA molecules, reference to “an adjuvant” includes mixture of two or more such adjuvant molecules or a plurality of such adjuvant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. The phrase “consisting essentially of” is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method/process steps or limitations) alone.

The inventors have unexpectedly discovered that it is possible to produce polymers comprising multiple immunomodulatory agent moietes (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more moieties of immunomodulatory agent per polymer) and use such polymers to form synthetic nanocarriers. These polymers can be useful for targeted delivery of high concentrations of immunomodulatory agents to cells, such as dendritic cells, and can be useful in the treatment and prevention of diseases and conditions, such as cancer, infection or infectious disease, addiction, allergy, autoimmune disease, inflammatory disease, etc. The increased functional-density of multiply-loaded polymers (e.g., moles of immunomodulatory agent per weight polymer) permit not only higher concentration dosage forms but, alternatively, equi-loaded dosage forms with reduced amount of volume taken up by the polymer conjugate.

Accordingly, a composition comprising synthetic nanocarriers comprising a first type of polymer that comprises at least two immunomodulatory agent moieties is provided. In embodiments, at least a portion of the immunomodulatory agent moieties are not present at the surface of the synthetic nanocarriers. In other embodiments, at least one of the immunomodulatory agent moieties of a polymer is not at the terminus (or end) of the polymer. In still other embodiments, wherein the polymer is made up of the immunomodulatory agent moieties forming its backbone, the synthetic nanocarrier comprises another material in addition to the immunomodulatory agent moieties or the polymer comprises at least one type of monomeric residue that is not the immunomodulatory agent moiety.

Dosage forms and vaccines comprising these compositions are further provided.

Additionally, methods of administering the compositions provided herein to a subject are also provided. In one embodiment, the subject is a human.

Finally, methods of producing the polymers and synthetic nanocarriers described herein are also provided.

The invention will now be described in more detail below.

“Adjuvant” means an agent that does not constitute a specific antigen, but boosts the strength and longevity of immune response to a concomitantly administered antigen. Such adjuvants may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), etc. In embodiments, adjuvants comprise agonists for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof. In other embodiments, adjuvants comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited adjuvants comprise aminodiazepine, such as those disldosed in WO 2010/054215, WO 2007/040840, US 2008/0306050, US 2008/0234251; imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company), US Published Patent Application 2010/0075995 to Biggadike et al., or WO 2010/018132 to Campos et al. In embodiment, the adenine derivative comprises SM-276001(9-benzyl-2-butoxy-8-hydroxyadenine) and its analogs with different substituents at C-9 and C-2 positions and 8-oxo-purine derivatives such as PF-4171455 (4-Amino-1-benzyl-6-trifluoromethyl-1,3-dihydroimidazol[4,5-c]pyridin-2-one) and its analogs with different substituents at C-6 position. In specific embodiments, synthetic nanocarriers incorporate as adjuvants compounds that are agonists for toll-like receptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8 agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al., including but not limited to imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. Preferred adjuvants comprise imiquimod and resiquimod (also known as R848). In specific embodiments, an adjuvant may be an agonist for the DC surface molecule CD40. In certain embodiments, to stimulate immunity rather than tolerance, a synthetic nanocarrier incorporates an adjuvant that promotes DC maturation (needed for priming of naive T cells) and the production of cytokines, such as type I interferons, which promote antibody immune responses. In some embodiments, an adjuvant may be a TLR-4 agonist. In some embodiments, adjuvants may comprise TLR-5 agonists. In specific embodiments, synthetic nanocarriers incorporate a ligand for Toll-like receptor (TLR)-9. Examples of TLR9 antagonists include hydroxychloroquine and its analogs as well as adenine derivatives.

“Administering” or “administration” means providing a material, such as a drug to a subject in a manner that is pharmacologically useful.

An “allergy” also referred to herein as an “allergic condition,” is any condition where there is an undesired (e.g., a Type 1 hypersensitive) immune response (i.e., allergic response or reaction) to a substance. Such substances are referred to herein as allergens. Allergies or allergic conditions include, but are not limited to, allergic asthma, hay fever, hives, eczema, plant allergies, bee sting allergies, pet allergies, latex allergies, mold allergies, cosmetic allergies, food allergies, allergic rhinitis or coryza, topic allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions and other allergic conditions. The allergic reaction may be the result of an immune reaction to any allergen. In some embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergies, egg allergies, nut allergies, fish allergies, shellfish allergies, soy allergies or wheat allergies.

“Amount effective” is any amount of a composition provided herein that produces one or more desired responses, such as one or more desired immune responses. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be one that a clinician would believe may have a clinical benefit for a subject in need thereof. In embodiments, clinically effective amounts are effective amounts that can be helpful in the treatment of a subject with a disease or condition. Such subjects include, in some embodiments, those that have or are at risk of having cancer, an infection or infectious disease, a non-autoimmune or degenerative disease or an addiction. In other embodiments, the subjects include those that have or are at risk of having an autoimmune disease, an inflammatory disease, an allergy or graft versus host disease or has undergone or will undergo transplantation.

A subject\'s immune response can be monitored by routine methods. An amount that is effective to produce the desired immune responses as provided herein can also be an amount of a composition provided herein that produces a desired therapeutic endpoint or a desired therapeutic result. Amounts effective will depend, of course, on the particular subject being treated; the severity of a condition, disease or disorder; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason.

In general, doses of the compositions of the invention can range from about 10 μg/kg to about 100,000 μg/kg. In some embodiments, the doses can range from about 0.1 mg/kg to about 100 mg/kg. In still other embodiments, the doses can range from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50 mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100 mg/kg. Alternatively, the dose can be administered based on the number of synthetic nanocarriers. For example, useful doses include greater than 106, 107, 108, 109 or 1010 synthetic nanocarriers per dose. Other examples of useful doses include from about 1×106 to about 1×1010, about 1×107 to about 1×109 or about 1×108 to about 1×109 synthetic nanocarriers per dose.

“Antigen” means a B cell antigen or T cell antigen. In embodiments, antigens are coupled to the synthetic nanocarriers. In other embodiments, antigens are not coupled to the synthetic nanocarriers. “Type(s) of antigens” means molecules that share the same, or substantially the same, antigenic characteristics.

An “at risk” subject is one in which a health practitioner believes has a chance of having a disease or condition as provided herein.

An “autoimmune disease” is any disease where the immune system mounts an undesired immune response against self (e.g., one or more autoantigens). In some embodiments, an autoimmune disease comprises an aberrant destruction of cells of the body as part of the self-targeted immune response. In some embodiments, the destruction of self manifests in the malfunction of an organ, for example, the colon or pancreas. Examples of autoimmune diseases are described elsewhere herein. Additional autoimmune diseases will be known to those of skill in the art and the invention is not limited in this respect.

“Average”, as used herein, refers to the arithmetic mean unless otherwise noted.

“B cell antigen” means any antigen that is recognized by or triggers an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell or a receptor thereon). In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is not also a B cell antigen. B cell antigens include, but are not limited to proteins, peptides, small molecules, oligosaccharides and carbohydrates. In some embodiments, the B cell antigen comprises a non-protein antigen (i.e., not a protein or peptide antigen). In some embodiments, the B cell antigen comprises a carbohydrate associated with an infectious agent. In some embodiments, the B cell antigen comprises a glycoprotein or glycopeptide associated with an infectious agent. The infectious agent can be a bacterium, virus, fungus, protozoan, or parasite. In some embodiments, the B cell antigen comprises a poorly immunogenic antigen. In some embodiments, the B cell antigen comprises an abused substance or a portion thereof. In some embodiments, the B cell antigen comprises an addictive substance or a portion thereof. Addictive substances include, but are not limited to, nicotine, a narcotic, a cough suppressant, a tranquilizer, and a sedative. In some embodiments, the B cell antigen comprises a toxin, such as a toxin from a chemical weapon or natural sources. The B cell antigen may also comprise a hazardous environmental agent. In some embodiments, the B cell antigen comprises a self antigen. In other embodiments, the B cell antigen comprises an alloantigen, an allergen, a contact sensitizer, a degenerative disease antigen, a hapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an autoimmune disease antigen, a non-autoimmune disease antigen, an addictive substance, a xenoantigen, or a metabolic disease enzyme or enzymatic product thereof.

“Couple” or “Coupled” or “Couples” (and the like) means to chemically associate one entity (for example a moiety) with another. In some embodiments, the coupling is covalent, meaning that the coupling occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent coupling is mediated by non-covalent interactions including but not limited to charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. In embodiments, encapsulation is a form of coupling.

“Dosage form” means a pharmacologically and/or immunologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject.

“Encapsulate” means to enclose at least a portion of a substance within a synthetic nanocarrier. In some embodiments, a substance is enclosed completely within a synthetic nanocarrier. In other embodiments, most or all of a substance that is encapsulated is not exposed to the local environment external to the synthetic nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of a substance on a surface of a synthetic nanocarrier, and leaves the substance exposed to the local environment external to the synthetic nanocarrier.

“Immunomodulatory agent” means an agent that modulates an immune response to an antigen but is not the antigen or derived from the antigen. “Modulate”, as used herein, refers to inducing, enhancing, suppressing, directing, or redirecting an immune response. Such agents include immunostimulatory agents, such as adjuvants, that stimulate (or boost) an immune response to an antigen but is not an antigen or derived from an antigen. There are several distinct types of immunomodulatory agents, which include, but are not limited to, Toll-like Receptor (TLR) agonists and Toll-like Receptor (TLR) antagonists. Such agents also include immunosuppressants. “Moieties” are the active portions of a molecule of the immunomodulatory agents and are useful in the practice of the invention. Such moieties can be chemically modified, e.g., for coupling to the synthetic nanocarrier, and still remain immunologically active. In certain embodiments, multiple moieties of the immunomodulatory agent (e.g., two, three, four, five, six, seven, eight, nine, ten, or more moieties) are coupled to a polymer, e.g., at an end (or terminus) of a polymer, to a polymer backbone, or form at least part of the polymer backbone.

Preferably, at least a portion or all of the immunomodulatory agent moieties are incorporated within the synthetic nanocarriers. Some of the immunomodulatory agent moieties may be present at the surface of the synthetic nanocarriers. In some embodiments, not all of the immunomodulatory agent moieties are present at the surface of the synthetic nanocarriers. In some embodiments, all of the immunomodulatory moieties that are attached to or form part of the polymer, or synthetic nanocarrier that comprises the polymer, are the same type of immunomodulatory agent moiety (i.e., are identical to one another in chemical structure). In other embodiments, a polymer, or synthetic nanocarrier that comprises the polymer, as provided herein comprises one or more moieties of a number of different types of immunomodulatory agents (e.g., two, three, four, five, six, seven, eight, nine, or ten different types of immunomodulatory agent moieties). In still other embodiments, a polymer, or synthetic nanocarrier that comprises the polymer, as provided herein comprises exactly one type of immunomodulatory agent moiety. In some embodiments, a polymer, or synthetic nanocarrier that comprises the polymer, as provided herein comprises exactly two distinct types of immunomodulatory agent moieties. In some embodiments, a polymer, or synthetic nanocarrier that comprises the polymer, comprises three or more distinct types of immunomodulatory agent moieties (e.g., three, four, five, six, seven, eight, nine, or ten distinct types of immunomodulatory agent moieties).

“Immunosuppressant” means a compound that causes an immunosuppressive (e.g., tolerogenic) effect. An immunosuppressive effect generally refers to the production or expression of cytokines or other factors by immune cells, such as antigen-presenting cells, that reduce, inhibit or prevent an undesired immune response Immunosuppressants include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors, such as Trichostatin A; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio, Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors; G-protein coupled receptor agonists; G-protein coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3KB inhibitors, such as TGX-221; autophagy inhibitors, such as 3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers. Immunosuppressants also include IDO, vitamin D3, cyclosporins, such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol and triptolide. Other exemplary immunosuppressants include, but are not limited, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506), etc. Further immunosuppressants, are known to those of skill in the art, and the invention is not limited in this respect.

An “infection” or “infectious disease” is any condition or disease caused by a microorganism, pathogen or other agent, such as a bacterium, fungus, prion or virus. “An infection or infectious disease antigen” is an antigen associated with an infection or infectious disease. Such antigens include antigens that can be used to generate an immune response against a pathogen or other infectious agent, or component thereof, or that can generate an immune response against infected cells.

“Inflammatory disease” means any disease, disorder or condition in which undesired inflammation occurs.

“Load” is the amount of a component (e.g., immunomodulatory agent) of a synthetic nanocarrier based on the total weight of materials in an entire synthetic nanocarrier (weight/weight). Generally, the load is calculated as an average across a population of synthetic nanocarriers. In one embodiment, the load of the immunomodulatory agent on average across the synthetic nanocarriers is between 0.0001% and 50%. In another embodiment, the load of the immunomodulatory agent on average across the synthetic nanocarriers is between 0.001% and 50%. In yet another embodiment, the load of the immunomodulatory agent is between 0.01% and 20%. In a further embodiment, the load of the immunomodulatory agent is between 0.1% and 10%. In still a further embodiment, the load of the immunomodulatory agent is between 1% and 10%.

In embodiments of any of the compositions and methods provided, the load is calculated as follows: Approximately 3 mg of synthetic nanocarriers are collected and centrifuged to separate supernatant from synthetic nanocarrier pellet. Acetonitrile is added to the pellet, and the sample is sonicated and centrifuged to remove any insoluble material. The supernatant and pellet are injected on RP-HPLC and absorbance is read at 278 nm. The μg found in the pellet is used to calculate % entrapped (load), μg in supernatant and pellet are used to calculate total μg recovered.

“Maximum dimension of a synthetic nanocarrier” means the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of a synthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spheroidal synthetic nanocarrier, the maximum and minimum dimension of a synthetic nanocarrier would be substantially identical, and would be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the minimum dimension of a synthetic nanocarrier would be the smallest of its height, width or length, while the maximum dimension of a synthetic nanocarrier would be the largest of its height, width or length. In an embodiment, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm. In an embodiment, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 μm. Preferably, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably still greater than 150 nm. Aspects ratios of the maximum and minimum dimensions of synthetic nanocarriers may vary depending on the embodiment. For instance, aspect ratios of the maximum to minimum dimensions of the synthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yet more preferably from 1:1 to 10:1. Preferably, a maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample is equal to or less than 3 μm, more preferably equal to or less than 2 μm, more preferably equal to or less than 1 μm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In preferred embodiments, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90%, of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm. Measurement of synthetic nanocarrier dimensions (e.g., diameter) is obtained by suspending the synthetic nanocarriers in a liquid (usually aqueous) media and using dynamic light scattering (DLS) (e.g. using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers can be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepared directly inside, or transferred to, a suitable cuvette for DLS analysis. The cuvette may then be placed in the DLS, allowed to equilibrate to the controlled temperature, and then scanned for sufficient time to acquire a stable and reproducible distribution based on appropriate inputs for viscosity of the medium and refractive indicies of the sample. The effective diameter, or mean of the distribution, is then reported. “Dimension” or “size” or “diameter” of synthetic nanocarriers means the mean of a particle size distribution obtained using dynamic light scattering.

“Not present at the surface of synthetic nanocarriers” refers to an entity that is not exposed to the environment that is external to the synthetic nanocarrier.

“Pharmaceutically acceptable excipient” means a pharmacologically inactive material used together with the recited synthetic nanocarriers to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.

“Polymeric monomer” refers to a monomeric unit of a polymer, the polymer generally being made up of a series of linked monomeric residues.

“Subject” means animals, including warm blooded mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like.

“Synthetic nanocarrier(s)” means a discrete object that is not found in nature, and that possesses at least one dimension that is less than or equal to 5 microns in size. Albumin nanoparticles are generally included as synthetic nanocarriers, however in certain embodiments the synthetic nanocarriers do not comprise albumin nanoparticles. In embodiments, synthetic nanocarriers do not comprise chitosan. In certain other embodiments, the synthetic nanocarriers do not comprise chitosan. In other embodiments, synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, synthetic nanocarriers do not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or a plurality of lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles where the majority of the material that makes up their structure are lipids), polymeric nanoparticles, metallic nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), peptide or protein-based particles (also referred to herein as protein particles, i.e., particles where the majority of the material that makes up their structure are peptides or proteins) (such as albumin nanoparticles) and/or nanoparticles that are developed using a combination of nanomaterials such as lipid-polymer nanoparticles. Synthetic nanocarriers may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Synthetic nanocarriers according to the invention comprise one or more surfaces. Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention comprise: (1) the biodegradable nanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric nanoparticles of Published US Patent Application 20060002852 to Saltzman et al., (3) the lithographically constructed nanoparticles of Published US Patent Application 20090028910 to DeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosed in Published US Patent Application 2008/0145441 to Penades et al., (6) the protein nanoparticles disclosed in Published US Patent Application 20090226525 to de los Rios et al., (7) the virus-like particles disclosed in published US Patent Application 20060222652 to Sebbel et al., (8) the nucleic acid coupled virus-like particles disclosed in published US Patent Application 20060251677 to Bachmann et al., (9) the virus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al., “Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853 (2010) or (11) apoptotic cells, apoptotic bodies or the synthetic or semisynthetic mimics disclosed in U.S. Publication 2002/0086049. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface with hydroxyl groups that activate complement or alternatively comprise a surface that consists essentially of moieties that are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that substantially activates complement or alternatively comprise a surface that consists essentially of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention that have a minimum dimension of equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not comprise a surface that activates complement or alternatively comprise a surface that consists essentially of moieties that do not activate complement. In embodiments, synthetic nanocarriers exclude virus-like particles. In embodiments, when synthetic nanocarriers comprise virus-like particles, the virus-like particles comprise non-natural adjuvant (meaning that the VLPs comprise an adjuvant other than naturally occurring RNA generated during the production of the VLPs). In embodiments, synthetic nanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

“T cell antigen” means any antigen that is recognized by and triggers an immune response in a T cell (e.g., an antigen that is specifically recognized by a T cell receptor on a T cell or an NKT cell via presentation of the antigen or portion thereof bound to a Class I or Class II major histocompatability complex molecule (MHC), or bound to a CD1 complex). In some embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is not also a B cell antigen. T cell antigens generally are proteins or peptides. T cell antigens may be an antigen that stimulates a CD8+ T cell response, a CD4+ T cell response, or both. The nanocarriers, therefore, in some embodiments can effectively stimulate both types of responses.

In some embodiments the T cell antigen is a T helper cell antigen (i.e. one that can generate an enhanced response to a B cell antigen, preferably an unrelated B cell antigen, through stimulation of T cell help). In embodiments, a T helper cell antigen may comprise one or more peptides obtained or derived from tetanus toxoid, Epstein-Barr virus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, or a PADRE peptide (known from the work of Sette et al. U.S. Pat. No. 7,202,351). In other embodiments, a T helper cell antigen may comprise one or more lipids, or glycolipids, including but not limited to: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols (from Borrelia burgdorferi), lypophosphoglycan (from Leishmania donovani), and phosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae). For additional lipids and/or glycolipids useful as a T helper cell antigen, see V. Cerundolo et al., “Harnessing invariant NKT cells in vaccination strategies.” Nature Rev Immun, 9:28-38 (2009). In embodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cell antigen that is obtained from a source, such as a natural source. In such embodiments, CD4+ T-cell antigen sequences, such as those peptides that bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity to the antigen obtained from the source. In embodiments, the T cell antigen, preferably a T helper cell antigen, may be coupled to, or uncoupled from, a synthetic nanocarrier. In some embodiments, the T cell antigen is encapsulated in the synthetic nanocarriers of the compositions.

A “terminus of a polymer” is an end of a polymer chain or branch.

“Vaccine” means a composition of matter that improves the immune response to a particular pathogen or disease. A vaccine typically contains factors that stimulate a subject\'s immune system to recognize a specific antigen as foreign and eliminate it from the subject\'s body. A vaccine also establishes an immunologic ‘memory’ so the antigen will be quickly recognized and responded to if a person is re-challenged. Vaccines can be prophylactic (for example to prevent future infection by any pathogen), or therapeutic (for example a vaccine against a tumor specific antigen for the treatment of cancer). In embodiments, a vaccine may comprise dosage forms according to the invention.

“Weight”, as used herein, refers to mass unless otherwise noted. When a molecular weight of a polymer is measured, it can be measured as the weight average molecular weight or a number average molecular weight. “Weight average molecular weight” for the polymers of the compositions provided herein is calculated by the following formula:

M _ w = ∑ i  N i   M i 2 ∑ i  N i  M i

Formula I, where Ni is the number of molecules of molecular weight Mi. The weight average molecular weight can be determined by a variety of methods including light scattering, small angle neutron scattering (SANS), X-ray scattering, Nuclear Magnetic Resonance (NMR) and sedimentation velocity. An example of an alternative for weight average molecular weight is to perform gel permeation chromatography using suitable traceable-weight standards to establish a retention-time versus weight curve, and calculating the mean weight-averaged molecular weight of a sample polymer from the mean of the integrated sample peak as compared to the calibration curve. The “number average molecular weight” can be determined by NMR. For example, number average molecular weight can be determined by proton NMR wherein the ratio of the polymer repeating units to the end group is established and then multiplied by theoretical repeating unit molecular weight. Alternatively, in the case of a titratable (e.g., acid or base) end group polymer, a known weight concentration may be established and then titrated in the presense of an indicator dye with an appropriate neutralizing agent of known molar concentration to provide moles of end group per mass of polymer. Any of the weights of a polymer as provided herein can be a weight average molecular weight or a number average molecular weight.

As generally described herein, the inventors have discovered that it is possible to couple multiple molecules of immunomodulatory agent to a polymer backbone, e.g., coupling two, three, four, five, six, seven, eight, nine, ten, or more moieties of immunomodulatory agent to the polymer. Such immunomodulatory agent moieties are, for example, coupled to the terminus (or end) of a polymer and/or or to its backbone and/or are coupled to monomers and/or themselves are monomers used in the preparation of a polymer. The present invention, therefore, also provides synthetic nanocarriers comprising such polymers. Further provided are synthetic nanocarriers that further comprise other components coupled thereto, such as additional immunomodulatory agents, antigens, etc.

The immunomodulatory agent moieties can be coupled to or form the polymers by a variety of methods. For example, the immunomodulatory agent moieties (e.g., adjuvant moieties) can be coupled to the polymers at the end (or terminus) of a linear, branched or dendrimeric polymeric structure as shown below in Scheme 1 or can be coupled to a polymer along its backbone as shown below in Scheme 2.

More specifically, in certain embodiments, the polymer is a branched polymer prepared from the reaction of a polyol with one or more different types of monomers to provide a multi-armed polymer (see, e.g., Scheme 3). In the embodiments described in Scheme 3, the polyol, wherein n is an integer between 2 and 100, is reacted with lactide, glycolide, or caprolactam monomers, any of which may be functionalized with other groups, such as optionally substituted alkyl groups, to provide a multi-armed polymer. It is envisioned that not all of the hydroxyl groups of the polyol may react, or are desirable to react, under the polymerization conditions employed, thus, in certain embodiments, R1 is selected from hydrogen and a polymer, wherein at least two R1 groups comprise a polymer arm. The polymer arm of the multi-armed polymer may be further functionalized with other monomers, e.g., lactide, glycolide, or caprolactam monomers, to provide a block-co-polymer arm. The polymer arm may then be further functionalized with one or more groups useful for conjugation with an appropriately functionalized immunomodulatory agent moiety. In this way, a polymer is designed such that multiple immunomodulatory agent moieties may be installed along the branch points of the polymer.

For example, in Scheme 3, the polymer is treated with succinic anhydride to provide a polyacid, e.g., a polymer comprising two or more carboxylic acid groups. Scheme 3 depicts only one exemplary way of installing a carboxylic acid via a linking group L1, and many other ways are contemplated, e.g., wherein L1 may be any group linking the polymer to one or more carboxylic acids. Exemplary L1 groups include, but are not limited to, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted carbocyclene, optionally substituted heterocyclene, optionally substituted arylene, and optionally substituted heteroarylene. In the case wherein the multi-armed polymer is treated with succinic anhydride, L1 is a substituted alkylene group, i.e., —C(═O)CH2CH2—, linking the polymer arm to a terminal carboxylic acid —CO2H to provide a polyacid.

Monomer(s) R1 polymer arm (R3 = H) R4 acid functionalized arm

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