WO2015153632A1

WO2015153632A1 - Particles that enhance immune responses by increasing cytotoxic t-cell function or production of interferon gamma therefrom

Info

Publication number: WO2015153632A1
Application number: PCT/US2015/023623
Authority: WO
Inventors: Joseph Desimone; Chintan KAPADIA; Shaomin TIAN; Jillian PERRY
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2014-03-31
Filing date: 2015-03-31
Publication date: 2015-10-08

Abstract

The subject matter disclosed herein is directed to particle mediated vaccines. In an aspect, the subject matter disclosed herein relates to the enhancement of the frequency of IFN-gamma (γ) producing cells. In an aspect, the subject matter disclosed herein relates to the enhancement of function of killer T cells. In an aspect, the subject matter disclosed herein is an optimized particle mediated system for conjugation of similar amounts of an adjuvant and antigen agents to particles. The particles are particularly useful in vaccines.

Description

PARTICLES THAT ENHANCE IMMUNE RESPONSES BY INCREASING CYTOTOXIC T-CELL FUNCTION OR PRODUCTION OF INTERFERON

GAMMA THEREFROM

FIELD OF THE INVENTION

The subject matter herein is directed to micro- and nano-particles exhibiting size, charge and surface properties whereby the particles traffic towards the lymph nodes upon administration.

BACKGROUND

Vaccines are typically given through a tissue injection such as intra-muscular route; however, the site of action for the vaccine antigens, i.e. antigen presentation to T cells and B cells, and generation of antigen-specific adaptive immunity is the draining lymph nodes (dLN). Dendritic cells are the most professional antigen presentation cells. For efficient antigen presentation, antigens can either be taken up by dendritic cells (DCs) or macrophages and trafficked to the draining lymph nodes, or traffic there themselves to be taken up by LN-resident DCs or interact with B cells directly. Presence of adjuvants may greatly facilitate the activation/maturation of DCs and other cell types to excrete cytokines to recruit other immune cells to the area. The processing of antigens and maturation of DCs allow them to efficiently present the antigen to T cells, which are then able to produce antigen-specific helper T cells and/or killer T cells.

It is known that killer T cells, i.e., CD8 T cells directly kill cells and secrete cytokines that activate other immune cells. However, there is still a need in the art for vaccines and formulations that improved CD8 T cell function and production of cytokines.

SUMMARY OF THE INVENTION

The subject matter disclosed herein is directed to particle mediated vaccines. In an aspect, the subject matter disclosed herein relates to the enhancement of the frequency of IFN-gamma (γ) producing cells by antigens or adjuvants linked to a particle through linkers longer than about 50 angstroms, longer than about 100 angstroms, longer than about 200 angstroms, and preferably longer than about 400 angstroms.

In an aspect, the subject matter disclosed herein relates to the enhancement of function of killer T cells by antigens or adjuvants linked to a particle through linkers longer than about 50 angstroms, longer than about 100 angstroms, longer than about 200 angstroms, and preferably longer than about 400 angstroms.

In an aspect, the subject matter disclosed herein is an optimized particle mediated system for conjugation of similar amounts of an adjuvant and antigen agents to particles with linkers that substantially enhance the immune response thereto. The particles are particularly useful in vaccines.

In an aspect, the subject matter disclosed herein relates to a method of enhancing an immune response of a T cell, such as a CD8+ T-cell, by administering a particle as described herein.

In an aspect, the subject matter disclosed herein relates to a method of preparing the particles through the methods described herein.

DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a method for preparing the particles by mixing HP4A, PEG Diacrylate, AEM and TPO. HP4A is a monomer, PEG Diacrylate is a cross linker that is relatively inert, and the amine group from AEM is utilized. TPO is a cross linker. A film is drawn with PPS on a PET sheet. Laminate against the mold and pass through nip. Once the mold is filled, it is passed through UV light which polymerizes monomers in the cavity of mold. Polymerized nanoparticles are in the mold cavity. Laminate against PVOH layer and dissolve in water. The components of an exemplary particle are shown.

Fig. 2 shows the physical characteristics of particles. 1 unit of PEG is 3.5 angstrom long (flori radius of PEG); PEG molecular weight is 44 gm/mole; Molecular weight of OPSS PEG NHS is 2000 gm/mole; accordingly, the length of PEG can be determined as (2000/44)* 3.5 = 160 angstrom; alternatively, Creative PEG works discloses a 4.4 angstrom length of 1 PEG unit; this formula is (2000/44)*4.4 = 200 angstrom. Fig. 3 shows PEG hydrogel particles prepared with about equivalent amounts of conjugated antigen and adjuvant.

Fig. 4 shows numbers of theoretical amine groups on 1 mg of exemplified particles and incubated NPs with different moles ratio of linker to amine sites.

Fig. 5 shows hydrogel PRINT NP vaccine particles were fabricated to the conjugation linking strategies described herein. The vaccine formulations were evaluated for inducing antigen-specific CD8+ T cells that produce cytokine IFN-γ (interferon), which is an indicator for effector T cell functions. According to the data presented in Figures 5, the particle-mediated co-delivery of antigenic peptide and CpG is able to induce potent effector CD8+ T cell response. Furthermore, compared to the short linker (in some embodiments, for example, less than 50 angstrom in length) NP-SPDP-OVA/CpG, the long linker NP-PEG-OPSS-OVA/CpG (in some embodiments, for example, longer than 50 angstrom in length) is more effective at increasing the frequency of IFN-γ producing cells. In some embodiments, the long linker is twice as effective at increasing the frequency of IFN-γ producing cells. In some embodiments, the long linker is more than twice as effective at increasing the frequency of IFN-γ producing cells.

Fig. 6 shows vaccine formulations were evaluated for inducing antigen- specific CD8+ T cells that produce cytokine IFN-γ (interferon), which is an indicator for effector T cell functions. The left graph shows IFN-γ ELISPOT to measure the frequency of IFN-γ producing CD8+ T cells and the right shows IFN-γ ELISA to measure the total production of IFN-γ by bulk CD8+ T cells. All particles-mediated co-delivery of CpG and CD8 T cell epitope SIINFEKL promoted generation of IFN-γ producing CD8+ T cells toward statistical significance (p=0.1-0.2), in either cell frequency as shown by ELISOPT or total secretion of IFN-gamma as shown by ELISA.

Fig. 7 shows hydrogel PRINT NP vaccine particles were fabricated to the conjugation linking strategies described herein. The vaccine formulations were evaluated for inducing antigen-specific CD8+ T cells that produce cytokine IFN-γ (interferon), which is an indicator for effector T cell functions. According to the data presented in Figures 7, the particle-mediated co-delivery of antigenic peptide and CpG is able to induce potent effector CD8+ T cell response. Furthermore, compared to the short linker NP-SPDP-OVA/CpG (for example, less than about 50 angstrom in length), the long linker (for example, more than about 50 angstrom in length) NP- PEG-OPSS-OVA/CpG is more effective at increasing the frequency of IFN-γ producing cells. In some embodiments, the long linker is twice as effective at increasing the frequency of IFN-γ producing cells. In some embodiments, the long linker is more than twice as effective at increasing the frequency of IFN-γ producing cells.

Fig. 8 shows shows the basic particle composition for the trafficking and immunization studies exemplified herein.

Fig. 9 shows PEG-OVA particles with various length of linkers.

Fig. 10 shows conjugation and characterization of PEG-OVA particles with various length of linkers.

Fig. 11 show, 80nm xl80nm PEG500-OVA NPs are shown to be effective in targeting draining LNs (dLN): 1) 80x180 nm PEG500-OVA NPs appeared in PLNs minutes after footpad injections and increased with time, suggesting that this particle may self-drain to LN instead of relying on transportation via phagocytic cells (top figure). 2) Confocal microscopy confirms the accumulation of particles in dLNs over time for long time. In addition, numerous particles were observed both in the B cell follicles and the medullary area where the T cells are located and co-localized with DCs, showing great potential for both B cell nad T cell activation.

Fig. 12 compares the effect of antigen protein conjugation and length of PEG (500, 5k) and non PEG linkers (shown as PEG0, conjugated through EDC-NHS chemistry) on lymphatic trafficking of particles. Both OVA conjugation and short PEG linkers help improve total drainage of particles significantly.

Fig. 13 shows the effect of size of particle and linker length on the uptake of hydrogel particles by antigen presenting cells (dendritic cells and macrophages). Consistent with total trafficking of particles, uptake of particles by phagocytic DCs and macrophages were also affected by size of particles. Much lower percentage of 1 um particles resulted in these cell types at 48 h post injections.

Figs. 14 & 15 show drainage and retention of soluble antigen vs PEG hydrogel-conjugated antigen. C57BL/6 mice were injected in footpads 2 ug or 5 ug OVA-Alexa Fluor555. The draining popliteal lymph nodes (PLN) were harvested at indicated time points and 10 um sections were made and stained with B220 antibody (B cell marker). As seen, soluble OVA drains freely and rapidly and accumulates in draining LNs in 2 h. However, by 24 h it is barely detected. It is believed that soluble protein antigen is subject to rapid degradation by macrophages.

Fig. 16 depicts the immunogenicity of 80x180 nm PEG(500)-OVA in

C57BL/6 mice. 5 ug OVA was dosed each mouse via subcutaneous injections, following prime-boost dosing scheme as shown. Left graph: NP -mediated delivery of OVA (NP-OVA) produced lOx higher titers of total IgG than OVA alone or co- injected OVA + NPs. Right graph: although non-inflammatory PEG-based hydrogel NP-OVA induced ~5x lower IgG than inflammatory adjuvant Alum + OVA. NP- OVA combined with Alum induces 15x higher IgG than soluble OVA + Alum.

Fig. 17 shows, in the left figure: 80x180 nm PEG500-OVA increased IgG by 10 fold; 1 um PEG500-OVA, however, induced very low level of IgG. The IgG response correlated well to the drainage of these two sizes of particles, suggesting that direct delivery of antigen to the draining lymph node and sustained delivery to B follicles is important for humoral immunity.

Fig. 18 shows 80x180 nm hydrogels with three different lengths of linker (0, PEG500, PEG5k), elicited similar levels of IgG, all ~ 10 x higher than soluble OVA, which does not correlate to lymphatic drainage of particles.

Fig. 19 shows that at 1 ug dose level, particle-conjugated OVA can efficiently stimulate proliferation of antigen-specific CD4⁺ T cells, while soluble OVA does not.

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

In embodiments, a particle comprising an antigen and an adjuvant is described. Both are conjugated to the particle through a linker as described elsewhere herein. Typically, the antigen, e.g., a peptide is conjugated to the particle. Then, the adjuvant, e.g., CpG can be conjugated. Both conjugations are performed utilizing the methods described herein. After conjugation, the peptide and CpG removed from the nanoparticles can be evaluated, peptide via HPLC and CpG via absorbance. At higher amounts of linker and peptide, particles become negative (zeta potential or ZP). Aggregation of nanoparticles was also found at higher peptide conjugation. The linker and peptide to ZP are balanced to obtain a useful amount of peptide without changing ZP, which gives a monodisperse suspension of particles. Furthermore, theoretically, primary amines from AEM or Poly AEM, for example, help the PRINT nanoparticles (NP or NPs) to escape endosome. As will be appreciated by one of ordinary skill in the art, endosomal escape is necessary for MHC-I presentation and to activate strong CD8+T cell and eventually CTL.

According to another aspect, the particles were optimized for their ability to stimulate CD8+ T cells to kill target cells. Releatively long PEG-OPSS linked CSIINFEKL/CpG particle formulation (for example, linker length longer than about 50 angstrom, in some embodiments longer than about 100 angstrom, in other embodiments longer than about 200 angstrom, in further embodiments longer than about 300 angstrom and in still further embodiments longer than about 400 angstrom) is significantly more effective than soluble peptide/CpG formulation or relatively short SPDP linked peptide/CpG particle formulation (for example, less than about 50 angstrom in length) in eliciting antigen-specific cytotoxic T cells that kills peptide antigen pulsed target cells.

As used herein, the term "particle" or "particles" is intended to mean one or more molded particles. Preferably, the particles comprise a polymer. The particles may further comprise an active agent. Methods of preparing particles are described in US 2011/0182805; US 2009/0028910; US 2009/0061152; WO 2007/024323; US 2009/0220789; US 2007/0264481; US 2010/0028994; US 2010/0196277; WO 2008/106503; US 2010/0151031; WO 2008/100304; WO 2009/041652;

PCT/US2010/041797; US 2008/0181958; WO 2009/111588; and WO 2009/132206, each of which is hereby incorporated by reference in their entirety.

The particles are preferably molded wherein the molded particle further comprises a three-dimensional shape substantially mimicking the mold shape and a size less than about 50 micrometers in a broadest dimension. In further embodiments, the particles are preferably molded to have a three-dimensional shape substantially mimicking the mold shape and a size less than about 5 micrometers in a broadest dimension. Preferably, the molded particles have a first dimension of less than about 200 nanometers and a second dimension greater than about 200 nanometers. Other sizes are also contemplated including dimensions less than 100 nm, less than 75 nm, less than 50 nm and less than 25 nm. Particles that are about 80 nm x 80 nm; about 60 nm x 100 nm; about 40 nm x 200 nm are contemplated. Particles that are about 80 nm x 180 nm are particularly useful.

In an embodiment the particle is fabricated utilizing the PRINT® (Liquidia Technologies, Inc., North Carolina) particle replication in non-wetting template micro- and nano particle technology. According to such embodiments, the particles are formed by filling Fluorocur® (Liquidia Technologies, Inc., North Carolina) mold cavities of discrete predetermined shape and size with particle matrix compositions of the desired base particle.

The particle matrix comprises a polyethylene glycol (PEG) polymer. In embodiments, the polymers are water soluble. In embodiments, the matrix of the particle is a hydrogel. Compositions utilized in the forming of the particles include PEG500 or PEG5k as discussed herein in the figures and examples. Hydrogels are formed by crosslinking polymer chains through physical, ionic or covalent interactions. A hydrogel is formed from a network of polymer chains wherein the network is water-insoluble. Additionally, the hydrogel matrices are particularly useful for the complexation of the active agents. Hydrogels possess a high degree of flexibility that can be similar to natural tissue. Accordingly, the modulus of the hydrogel particles, in embodiments, is about 1 MPa or less.

In embodiments, a particle is formed from a hydrogel. PEG-based hydrogels are known. Useful PEG hydrogel particles are disclosed in US 8,465,775, herein incorporated by reference in its entirety. Hydrogels suitable for use in the particles disclosed herein are preferably biocompatible, by which is meant that they are suitable to be introduced into a subject, i.e. they will not leach unwanted substances. Suitable hydrogels include macromolecular and polymeric materials into which water and small molecules can easily diffuse and include hydrogels prepared through the cross linking, where crosslinking may be either through covalent, ionic or

hydrophobic bonds introduced through use of either chemical cross-linking agents or electromagnetic radiation, such as ultraviolet light, of both natural and synthetic hydrophilic polymers, including homo and co-polymers. Hydrogels of interest include those prepared through the cross-linking of: polyethers, e.g.

polyakyleneoxides such as poly(ethylene glycol), poly(ethylene oxide), poly(ethylene oxide)-co-(poly(propyleneoxide) block copolymers; poly( vinyl alcohol); poly(vinyl pyrrolidone); polysaccharides, e.g. hyaluronic acid, dextran, chondroitin sulfate, heparin, heparin sulfate or alginate; proteins, e.g. gelatin, collagen, albumin, ovalbumin or polyamino acids; and the like. Because of their high degree of biocompatibility and resistance to protein adsorption, polyether derived hydrogels are preferred, with poly(ethylene glycol) derived hydrogels being particularly preferred. In embodiments, the hydrogels can have molecular weight cutoffs of, e.g., 200,000 daltons or more; 100,000 daltons; 50,000 daltons; 15,000 daltons; etc.

As used herein, "PEG" or "poly(ethylene glycol)," is meant to encompass any water-soluble poly(ethylene oxide). Typically, PEGs for use in the present invention will comprise the following structure: "— (CH₂CH₂0) _n— ". The variable (n) is 3 to 3,000, or about 3 to about 30,000; about 3 to about 10,000 or about 3 to about 5,000. The variable "n" can also be from 1-200; from 1 to 100; from 1 to 50; and from 1 to 20. The terminal groups and architecture of the overall PEG may vary. PEGs having a variety of molecular weights, structures or geometries as is known in the art.

"Water-soluble" in the context of a water soluble polymer is any segment or polymer that is soluble in water at room temperature. Typically, a water-soluble polymer or segment will transmit at least about 75%, more preferably at least about 95% of light, transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer or segment thereof will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85%) (by weight) soluble in water. It is most preferred, however, that the water- soluble polymer or segment is about 95% (by weight) soluble in water or completely soluble in water.

The particle can comprise homo- and hetero-bifunctional and monofunctional PEG derivatives. Examples include: hydroxyl-terminated PEG-acrylate (HP4A); (COOH)_x-PEG-COOH; succinimidyl carboxymethyl ester (SCM)_X-PEG-SCM;

(Amine)_x-PEG-Amine; maleimide (MAL)_X-PEG-MAL; acrylate (ACL)_X-PEG-ACL, also referred to as PEG-DA (PEG diacrylate when x is one; thiol (HS)_X-PEG-HS; vinylsulfone (VS)_X-PEG-VS. In each derivative, x is one or zero. Each of these is commercially available in varying molecular weights from MW 200 to MW 8000. The nomenclature used for the MW of the PEG reagent designates the MW, e.g., PEG₇₀₀DA, is a PEG-diacrylate having a MW of about 700. Branched and multi-arm PEG derivatives can also be used. To prepare the matrix, it is preferred to use bifunctional PEG derivatives while the surface is modified with monofunctional derivatives.

An "end-capping" or "end-capped" group is an inert group present on a terminus of a polymer such as PEG. An end-capping group is one that does not readily undergo chemical transformation under typical synthetic reaction conditions. An end capping group is generally an alkoxy group,—OR, where R is an organic radical comprised of 1-20 carbons and is preferably lower alkyl (e.g., methyl, ethyl) or benzyl. "R" may be saturated or unsaturated, and includes aryl, heteroaryl, cyclo, heterocyclo, and substituted forms of any of the foregoing. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled, can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, calorimetric (e.g., dyes), metal ions, radioactive moieties, and the like.

The polymer matrix can comprise crosslinkers. TPO is a known crosslinker.

In some embodiments, the particles are composed of a crosslink density or matrix "mesh" density designed to allow slow release of the active agent. The term crosslink density means the mole fraction of prepolymer units that are crosslink points.

Prepolymer units include monomers, macromonomers and the like. In some embodiments, the particles are configured to degrade in the presence of an

intercellular stimulus. In some embodiments, the particles are configured to degrade in a reducing environment. In some embodiments, the particles contain crosslinking agents that are configured to degrade in the presence of an external stimulus. In some embodiments, the crosslinking agents are configured to degrade in the presence of a pH condition, a radiation condition, an ionic strength condition, an oxidation condition, a reduction condition, a temperature condition, an alternating magnetic field condition, an alternating electric field condition, combinations thereof, or the like. In some embodiments, the particles contain crosslinking agents that are configured to degrade in the presence of an external stimulus and/or a therapeutic agent. In some embodiments, the particles contain crosslinking agents that are configured to degrade in the presence of an external stimulus, a targeting ligand, and an active agent. In some embodiments, particles are configured to degrade in the cytoplasm of a cell. In some embodiments, particles are configured to degrade in the cytoplasm of a cell and release an active agent.

According to some embodiments, the composition can further include a plurality of particles, where the particles have a substantially uniform mass, are substantially monodisperse, are substantially monodisperse in size or shape, or are substantially monodisperse in surface area. Within a plurality of substantially monodisperse particles, the amount of PEG-ylation and the molecular weight of the PEG may vary independently or may independently be controlled. In some embodiments, the plurality of particles have a normalized size distribution of between about 0.80 and about 1.20, between about 0.90 and about 1.10, between about 0.95 and about 1.05, between about 0.99 and about 1.01, between about 0.999 and about 1.001. According to some embodiments, the normalized size distribution is selected from the group of a linear size, a volume, a three dimensional shape, surface area, mass, and shape. In yet other embodiments, the plurality of particles includes particles that are monodisperse in surface area, volume, mass, three-dimensional shape, or a broadest linear dimension.

According to an embodiment of the present invention, PEG hydrogel particles are prepared with about equivalent amounts of conjugated antigen and adjuvant by utilizing functional groups attached to the surface of the particle. As described herein, the linker on the particle surface can contain a PEG unit or repeating units. Thus, the linker can comprise 1 or more PEG monomer units. The variable "n" is shown to indicate the number of monomer units and its value is as described elsewhere herein. The linker does not always contain a PEG, e.g., the linker SPDP, having a length of about 6.8 A.

The linker can also be described in terms of length of the linker between the particle and the cargo, such as for example the antigen or adjuvant. As will be appreciated by one of ordinary skill in the art, according to some suppliers, 1 unit of PEG is about 3.5 angstrom long (flori radius of PEG); PEG molecular weight is about 44 gm/mole; Molecular weight of OPSS PEG NHS is 2000 gm/mole; accordingly, the length of PEG can be determined as (2000/44)* 3.5 = approximately 160 angstrom. Alternatively, according to a different supplier, Creative PEG works, discloses 1 PEG unit is about 4.4 angstrom in length; this formula is (2000/44)*4.4 = approximately 200 angstrom linker length for the OPSS linker disclosed herein. In other

embodiments herein, NHS PEG2k OPSS was measured to be roughly 200 angstrom. In other embodiments, linker of PEG500 comprises about 10 PEG repeat units and is about 45 angstrom in length. In other embodimetns, a PEG5k linker of the present invention comprises about 120 repeat PEG units and is about 420 angstrom in length. In embodiments, useful linker lengths less than about 50 angstrom include from 1 to about 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 30, 25, 20, 15, 10 or 5 angstrom. In embodiments, useful linker lengths longer than about 50 angstrom include from 51 to about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 angstrom.

One such functional group on the surface of a particle is an amine group. In particular embodiments, the AEM to conjugate with SPDP (N-Succinimidyl 3-(2- pyridyldithio)-propionate) (SPDP or "short" linker, for example in certain

embodiments, a linker less than about 50 angstrom in length) or NHS-PEG-OPSS (Ortho-Pyridyldisulfide-PEG-N-Hydroxylsuccinimide ester) (OPSS or "long" linker, for example in certain embodiments, a linker longer than about 50 angstrom in length, longer than about 100 angstrom in length, longer than about 200 angstrom in length, longer than about 300 angstrom in length, or longer than about 400 angstrom in length, such as for example the length of PEG500 to PEG5k as shown herein) which has pyridine ring with disulfide bond on one end and NHS ester on other end, as shown in Figure 1. We calculated the numbers of theoretical possible amine groups on 1 mg of nanoparticles and incubated NPs with different 'moles ratio of linker to amine sites', as shown in Figure 2. Reaction is performed in IX PBS + 0.1% PVA (ph is around 7, because NHS ester form amide bond to amine from pH 7 to 9, but at higher pH rate of hydrolysis goes high. (PVA prevents the aggregation of nanoparticles in PBS buffer, used where needed).

As used herein, the term "antigen" refers to a composition that elicits an immune response in a subject, in partilcur, polypetides. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et ah, Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, California, p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvant include, but are not limited to, a CpG

oligodeoxynucleotide, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

The particles can comprise an active agent. The particles can be formulated into pharmaceutical compositions. In particular, the particles can be formulated into vaccine for administration to a subject. The administration can be accomplished through any route known in the art, e.g., injection or inhalation. These vaccines can further contain any excipient and/or known vaccine components including adjuvants.

Compositions comprising a hydrogel particle conjugated to an antigen and an adjuvant in combination with a pharmaceutically acceptable carrier are provided. In particular, the particles can be formulated and administered as vaccines. The hydrogel particles presented herein can be prepared in an admixture with an adjuvant to prepare a vaccine. Pharmaceutically acceptable carriers and adjuvants are well known in the art. Methods for formulating pharmaceutical compositions and vaccines are generally known in the art. A thorough discussion of formulation and selection of pharmaceutical acceptable carriers, stabilizers, and isomolytes can be found in

Remington 's Pharmaceutical Sciences (18^th ed.; Mack Publishing Company, Eaton, Pennsylvania, 1990), herein incorporated by reference. As provided herein, a vaccine may comprise, for example, at least one of the hydrogel particles provided herein.

The vaccines provided herein can be administered via any parenteral route, including, but not limited, to intramuscular, intraperitoneal, intravenous, and the like. Preferably, since the desired result of vaccination is to elucidate an immune response to the antigen, administration directly, or indirectly, to lymphoid tissues, e.g., lymph nodes or spleen, is desirable. Further, as used herein "pharmaceutically acceptable carrier" are well known to those skilled in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such

pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

A subject in whom administration of an active component as set forth above is preferably a human, but can be any animal. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods and pharmaceutical compositions provided herein are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., for veterinary medical use.

In the therapeutic methods and compositions provided herein, a therapeutically effective dosage of the active component is provided. A therapeutically effective dosage can be determined by the ordinary skilled medical worker based on patient characteristics (age, weight, sex, condition, complications, other diseases, etc.), as is well known in the art. Furthermore, as further routine studies are conducted, more specific information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age and general health of the recipient, is able to ascertain proper dosing. Generally, for intravenous injection or infusion, dosage may be lower than for intraperitoneal, intramuscular, or other route of administration. The dosing schedule may vary, depending on the circulation half-life, and the formulation used. The compositions are administered in a manner compatible with the dosage formulation in the therapeutically effective amount. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The term "PEG" or polyethylene glycol refers to an oligomer or polymer of ethylene oxide. PEG is often described by the molecular weight of the polymer chain. Useful chain lengths are described herein using common terminology.

The term "therapeutically effective amount" as used herein refers to an amount of the plurality of monodisperse particles sufficient to achieve a certain outcome, such as to elicit an immune response in the subject. By "eliciting an immune response" is intended the generation of a specific immune response (or immunogenic response) in a subject. In some embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the vertebrate animal to better resist infection or disease progression from the organism or tumor cell against which the immunogenic composition is directed. The effective amount and dosage of such active agents required to be administered for effective treatment are known in the art or can be readily determined by those of skill in this field. Where active agents do not have a known dosage for certain diseases, the effective amount of active agent and the amount of a particular dosage form required to be administered for effective treatment can be readily determined by those of skill in this field.

By an "effective" amount or a "therapeutically effective amount" of an active agent is also meant a nontoxic but sufficient amount of the agent to provide the desired effect. Of course, the amount of active agent administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, and the like. Determination of an effective dosage is well within the capabilities of those skilled in the art coupled with the general and specific examples disclosed herein.

As used herein "a subject in need thereof may be a subject whom could have been but is not required to have been diagnosed as suffering from the condition intended to be treated. In one aspect, the present method is directed to conditions that are noticeable to the subject and the subject wishes to treat or ameliorate the condition without a formal diagnosis. Alternatively, a subject could be diagnosed with a condition and seek treatment or amelioration by a method disclosed herein. Clearly, one who suffers from a condition has an acute awareness of a problem and is in need thereof of treatment with or without a formal diagnosis by medical personnel.

Alternatively, one may be aware of symptoms associated with a condition described herein, without knowing that the condition may be causing the symptoms.

Additionally, in embodiemnts, a subject in need thereof is one who has been diagnosed as having a condition and is in need of specific treatment.

As used herein the terms "treating" and "ameliorating" are intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the condition or symptoms and does not necessarily indicate a total elimination of the underlying condition.

The term "increasing Interferon-gamma (IFN-γ) producing CD8 T cells" refers to the increase in frequency of IFN-γ producing CD8 T cells after

administration of a particle composition described herein as compared to the frequency of IFN-γ producing CD 8 T cells in the absence of an admisnitration of a particle composition as described herein. This increase can be by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to soluble antigen and adjuvant alone.

The term "enhanced cytotoxic T cell activity" and "enhancing a CD8+ T-cell immune response" refer to an increase in the killing of target cells by a cytotoxic T cell. This increase can be by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to soluble antigen and adjuvant alone. In an embodiment, the subject matter disclosed herein is directed to a method of treating a subject comprising administering an inventive pharmaceutical formulation as disclosed herein to the subject.

The term "subject" refers to a mammal, which means humans as well as all other warm-blooded mammalian animals. As used herein, the term "mammal" includes a "patient." As used herein "a mammal in need thereof may be a subject whom could have been but is not required to have been diagnosed as suffering from the condition intended to be treated.

The term "treating" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, for example, "treating" a patient involves prevention of a particular disorder or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting or causing regression of a disorder or disease. As used herein the terms "treating" includes "ameliorating," which refers to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the condition or symptoms and does not necessarily indicate a total elimination of the underlying condition.

Described herein are several advantages and unexpected properties of particles comprising an antigen and an adjuvant conjugated to the particle. The length and size of the linker that conjugates the antigen and/or adjuvant to the particle can be tailored. As shown herein, the length or size of the linker can have surprising and unexpected functionality to the particles.

The particle can also contain an active agent. By "active agent" is intended an agent that may find use in the treatment, diagnosis and/or management of a disease state. Such agents include but are not limited to small molecule pharmaceuticals, therapeutic and diagnostic proteins, immunogenic components, antibodies, DNA and RNA sequences, imaging agents, and other active pharmaceutical ingredients.

Exemplary active agents include, without limitation, analgesics, anti-inflammatory agents (including NSAIDs), anticancer agents, antimetabolites, antineoplastic agents, immunosuppressants, antiviral agents, astringents, beta-adrenoceptor blocking agents, blood products and substitutes, contrast media, corticosteroids, diagnostic agents, diagnostic imaging agents, haemostatics, immunological agents, therapeutic proteins, enzymes, lipid regulating agents, prostaglandins, radio-pharmaceuticals, sex hormones (including steroids), anti-allergic agents, stimulants and anoretics, sympathomimetics, xanthines, antibiotics, and antiviral agents. The amount of active agent present in the pharmaceutical composition will depend on the agent. Most useful agents are indicated for certain diseases and conditions and the dose amount of active agent can be readily determined and a pharmaceutical composition comprising the desired amount can be prepared as disclosed herein. Useful values of active agents are from about 1 mg to about 1,500 mg active agent per dosage form of the pharmaceutical composition. Preferred values are from about 100 mg to about 800 mg.

The particles can be formulated into pharmaceutical compositions as described herein.

The administration of the particles and compositions comprising the particles can be accomplished through any route known in the art. Routes of administration include intravenous or parenteral administration, oral administration, topical administration, transmucosal administration and transdermal administration. For intravenous or parenteral administration, i.e., injection or infusion, the composition may also contain suitable pharmaceutical diluents and carriers, such as water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative, or synthetic origin. It may also contain preservatives, and buffers as are known in the art. When a therapeutically effective amount is administered by intravenous, cutaneous or subcutaneous injection, the solution can also contain components to adjust pH, isotonicity, stability, and the like, all of which is within the skill in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

In particular, aerosolized medicaments are used to deliver particles to the lungs by having the patient inhale the aerosol through a tube and/or mouthpiece coupled to the aerosol generator. By inhaling the aerosolized medicament, the patient can quickly receive a dose of medicament in the lungs. In this way, the particles are delivered in a manner that can be the most efficient for licensing immunity. Aerosols of solid particles may be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the anti-malarial compound, a suitable powder diluent, such as lactose, and an optional surfactant. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the anti-malarial compound in a liquified propellant. During use these devices discharge the formulation through a valve, adapted to deliver a metered volume, from 10 to 22 microliters to produce a fine particle spray containing the antimalarial compound.

Suitable propellants include certain chlorofluorocarbon (compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more co- solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents. Any propellant may be used in carrying out the present invention, including both chlorofluorocarbon-containing propellants and non-chlorofluorocarbon-containing propellants. Fluorocarbon aerosol propellants that may be employed in carrying out the present invention including fluorocarbon propellants in which all hydrogen are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing

chlorofluorocarbon propellants. A stabilizer such as a fluoropolymer may optionally be included in formulations of fluorocarbon propellants, such as described in U.S. Pat. No. 5,376,359 to Johnson. In pulmonary delivery in particular, therapeutics must circumvent the lung's particle clearance mechanisms such as mucociliary transport, phagocytosis by macrophages and rapid absorption of drug molecules into the systemic circulation. Mucociliary clearance can be reduced by avoiding particle deposition in the tracheobronchial region which contains the cilia and goblets cells that make up the mucociliary escalator. Upon delivery to the pulmonary region, particles can be rapidly cleared by alveolar macrophages.

Typically, compositions for intravenous or parenteral administration comprise a suitable sterile solvent, which may be an isotonic aqueous buffer or

pharmaceutically acceptable organic solvent. The compositions can also include a solubilizing agent as is known in the art if necessary. Compositions for intravenous or parenteral administration can optionally include a local anesthetic to lessen pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form in a hermetically sealed container such as an ampoule or sachette. The pharmaceutical compositions for administration by injection or infusion can be dispensed, for example, with an infusion bottle

containing, for example, sterile pharmaceutical grade water or saline. Where the pharmaceutical compositions are administered by injection, an ampoule of sterile water for injection, saline, or other solvent such as a pharmaceutically acceptable organic solvent can be provided so that the ingredients can be mixed prior to administration.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the condition being treated or ameliorated and the condition and potential idiosyncratic response of each individual mammal. The duration of each infusion is from about 1 minute to about 1 hour. The infusion can be repeated as necessary.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection. Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions also can contain solubilizing agents, formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and can contain added preservatives. For prophylactic administration, the compound can be administered to a patient at risk of developing one of the previously described conditions or diseases. Alternatively, prophylactic administration can be applied to avoid the onset of symptoms in a patient suffering from or formally diagnosed with the underlying condition.

The amount of compound administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the

bioavailability of the particular active compound, and the like. Determination of an effective dosage is well within the capabilities of those skilled in the art coupled with the general and specific examples disclosed herein.

Oral administration of the composition or vehicle can be accomplished using dosage forms including but not limited to capsules, caplets, solutions, suspensions and/or syrups. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts, e.g., in Remington: The Science and Practice of Pharmacy (2000), supra.

The dosage form may be a capsule, in which case the active agent-containing composition may be encapsulated in the form of a liquid. Suitable capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, with gelatin capsules preferred. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like. See, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra, which describes materials and methods for preparing encapsulated pharmaceuticals.

Capsules may, if desired, be coated so as to provide for delayed release.

Dosage forms with delayed release coatings may be manufactured using standard coating procedures and equipment. Such procedures are known to those skilled in the art and described in the pertinent texts (see, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra). Generally, after preparation of the capsule, a delayed release coating composition is applied using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Delayed release coating compositions comprise a polymeric material, e.g., cellulose butyrate phthalate, cellulose hydrogen phthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethyl ethylcellulose, hydroxypropyl

methylcellulose acetate succinate, polymers and copolymers formed from acrylic acid, methacrylic acid, and/or esters thereof.

Sustained-release dosage forms provide for drug release over an extended time period, and may or may not be delayed release. Generally, as will be appreciated by those of ordinary skill in the art, sustained-release dosage forms are formulated by dispersing a drug within a matrix of a gradually bioerodible (hydrolyzable) material such as an insoluble plastic, a hydrophilic polymer, or a fatty compound. Insoluble plastic matrices may be comprised of, for example, polyvinyl chloride or

polyethylene. Hydrophilic polymers useful for providing a sustained release coating or matrix cellulosic polymers include, without limitation: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate,

hydroxypropylcellulose phthalate, cellulose hexahydrophthalate, cellulose acetate hexahydrophthalate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, with a terpolymer of ethyl acrylate, methyl methacrylate and trimethylammonioethyl methacrylate chloride (sold under the tradename Eudragit RS) preferred; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; zein; and shellac, ammoniated shellac, shellac- acetyl alcohol, and shellac n-butyl stearate. Fatty compounds for use as a sustained release matrix material include, but are not limited to, waxes generally (e.g., carnauba wax) and glyceryl tristearate.

Topical administration of a particle can be accomplished using any

formulation suitable for application to the body surface, and may comprise, for example, an ointment, cream, gel, lotion, solution, paste or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres and/or

microneedles. Preferred topical formulations herein are ointments, creams, and gels. Ointments, as is well known in the art of pharmaceutical formulation, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of

Pharmacy (2000), supra, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.

Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight (See, e.g., Remington: The Science and Practice of Pharmacy (2002), supra).

Creams, as also well known in the art, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

As will be appreciated by those working in the field of pharmaceutical formulation, gels-are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil. Preferred "organic macromolecules," i.e., gelling agents, are crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark. Also preferred are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.

Various additives, known to those skilled in the art, may be included in the topical formulations. For example, solubilizers may be used to solubilize certain active agents. For those drugs having an unusually low rate of permeation through the skin or mucosal tissue, it may be desirable to include a permeation enhancer in the formulation; suitable enhancers are as described elsewhere herein.

Transmucosal administration of an agent can be accomplished using any type of formulation or dosage unit suitable for application to mucosal tissue. For example, the particles can be administered to the buccal mucosa in an adhesive patch, sublingually or lingually as a cream, ointment, or paste, nasally as droplets or a nasal spray, or by inhalation of an aerosol formulation or a non-aerosol liquid formulation.

Preferred buccal dosage forms will typically comprise a bioerodible

(hydrolyzable) polymeric carrier that may also serve to adhere the dosage form to the buccal mucosa. The buccal dosage unit is fabricated so as to erode over a

predetermined time period, wherein drug delivery is provided essentially throughout. The time period is typically in the range of from about 1 hour to about 72 hours. Preferred buccal delivery preferably occurs over a time period of from about 2 hours to about 24 hours. Buccal drug delivery for short-term use should preferably occur over a time period of from about 2 hours to about 8 hours, more preferably over a time period of from about 3 hours to about 4 hours. As needed buccal drug delivery preferably will occur over a time period of from about 1 hour to about 12 hours, more preferably from about 2 hours to about 8 hours, most preferably from about 3 hours to about 6 hours. Sustained buccal drug delivery will preferably occur over a time period of from about 6 hours to about 72 hours, more preferably from about 12 hours to about 48 hours, most preferably from about 24 hours to about 48 hours. Buccal drug delivery, as will be appreciated by those skilled in the art, avoids the

disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver.

The "therapeutically effective amount" of an agent in the buccal dosage unit will of course depend on the potency and the intended dosage, which, in turn, is dependent on the particular individual undergoing treatment, the specific indication, and the like. The buccal dosage unit will generally contain from about 1.0 wt. % to about 60 wt. % active agent, preferably on the order of from about 1 wt. % to about 30 wt. % active agent. With regard to the bioerodible (hydrolyzable) polymeric carrier, it will be appreciated that virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with any other components of the buccal dosage unit. Generally, the polymeric carrier comprises a hydrophilic (water-soluble and water-swellable) polymer that adheres to the wet surface of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and co, e.g., those known as "carbomers" (Carbopol®, which may be obtained from B. F. Goodrich, is one such polymer). Other suitable polymers include, but are not limited to: hydro lyzed polyvinyl alcohol; polyethylene oxides (e.g., Sentry Polyox® water soluble resins, available from Union Carbide); polyacrylates (e.g., Gantrez®, which may be obtained from GAF); vinyl polymers and copolymers; polyvinylpyrrolidone; dextran; guar gum; pectins; starches; and cellulosic polymers such as hydroxypropyl methylcellulose, (e.g., Methocel®, which may be obtained from the Dow Chemical Company), hydroxypropyl cellulose (e.g., Klucel®, which may also be obtained from Dow), hydroxypropyl cellulose ethers (see, e.g., U.S. Pat. No. 4,704,285 to Alderman), hydroxyethyl cellulose,

carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate phthalate, cellulose acetate butyrate, and the like.

Other components may also be incorporated into the buccal dosage forms described herein. The additional components include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. Examples of disintegrants that may be used include, but are not limited to, cross- linked polyvinylpyrrolidones, such as crospovidone (e.g., Polyplasdone® XL, which may be obtained from GAF), cross-linked carboxylic methylcelluloses, such as croscarmelose (e.g., Ac-di-sol®, which may be obtained from FMC), alginic acid, and sodium carboxymethyl starches (e.g., Explotab®, which may be obtained from Edward Medell Co., Inc.), methylcellulose, agar bentonite and alginic acid. Suitable diluents are those which are generally useful in pharmaceutical formulations prepared using compression techniques, e.g., dicalcium phosphate dihydrate (e.g., Di-Tab®, which may be obtained from Stauffer), sugars that have been processed by

cocrystallization with dextrin (e.g., co-crystallized sucrose and dextrin such as Di- Pak®, which may be obtained from Amstar), calcium phosphate, cellulose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar and the like. Binders, if used, are those that enhance adhesion. Examples of such binders include, but are not limited to, starch, gelatin and sugars such as sucrose, dextrose, molasses, and lactose. Particularly preferred lubricants are stearates and stearic acid, and an optimal lubricant is magnesium stearate.

Sublingual and lingual dosage forms include creams, ointments and pastes. The cream, ointment or paste for sublingual or lingual delivery comprises a therapeutically effective amount of the selected active agent and one or more conventional nontoxic carriers suitable for sublingual or lingual drug administration. The sublingual and lingual dosage forms of the present invention can be manufactured using conventional processes. The sublingual and lingual dosage units are fabricated to disintegrate rapidly. The time period for complete disintegration of the dosage unit is typically in the range of from about 10 seconds to about 30 minutes, and optimally is less than 5 minutes.

Other components may also be incorporated into the sublingual and lingual dosage forms described herein. The additional components include, but are not limited to binders, disintegrants, wetting agents, lubricants, and the like. Examples of binders that may be used include water, ethanol, polyvinylpyrrolidone; starch solution gelatin solution, and the like. Suitable disintegrants include dry starch, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic monoglyceride, lactose, and the like. Wetting agents, if used, include glycerin, starches, and the like. Particularly preferred lubricants are stearates and polyethylene glycol. Additional components that may be incorporated into sublingual and lingual dosage forms are known, or will be apparent, to those skilled in this art (See, e.g., Remington: The Science and Practice of Pharmacy (2000), supra).

Other preferred compositions for sublingual administration include, for example, a bioadhesive; a spray, paint, or swab applied to the tongue; or the like. Increased residence time increases the likelihood that the administered invention can be absorbed by the mucosal tissue.

Transdermal administration of a particle through the skin or mucosal tissue can be accomplished using conventional transdermal drug delivery systems, wherein the agent is contained within a laminated structure (typically referred to as a transdermal "patch") that serves as a drug delivery device to be affixed to the skin.

Transdermal drug delivery may involve passive diffusion or it may be facilitated using electrotransport, e.g., iontophoresis. In a typical transdermal "patch," the drug composition is contained in a layer, or "reservoir," underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one type of patch, referred to as a "monolithic" system, the reservoir is comprised of a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.

The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active agent and any other materials that are present, the backing is preferably made of a sheet or film of a flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, polyesters, and the like.

During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the drug reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin. The release liner should be made from a drug/vehicle impermeable material.

Transdermal drug delivery systems may in addition contain a skin permeation enhancer. That is, because the inherent permeability of the skin to some drugs may be too low to allow therapeutic levels of the drug to pass through a reasonably sized area of unbroken skin, it is necessary to coadminister a skin permeation enhancer with such drugs. Suitable enhancers are well known in the art and include, for example, those enhancers listed below in transmucosal compositions.

Formulations can comprise one or more anesthetics. Patient discomfort or phlebitis and the like can be managed using anesthetic at the site of injection. If used, the anesthetic can be administered separately or as a component of the composition. One or more anesthetics, if present in the composition, is selected from the group consisting of lignocaine, bupivacaine, dibucaine, procaine, chloroprocaine, prilocaine, mepivacaine, etidocaine, tetracaine, lidocaine and xylocaine, and salts, derivatives or mixtures thereof.

Some of the subject matter disclosed herein is set forth in the following embodiments.

1. A particle for enhancing IFN-γ producing cells, comprising a hydrogel particle conjugated to an antigen and an adjuvant, wherein the antigen or adjuvant are conjugated to the hydrogel particle with a linker.

2. The particle of embodiment 1, wherein the antigen or adjuvant are conjugated to the hydrogel particle with a long linker.

3. The particle of embodiment 2, wherein the length of the linker is from about 100 to about 300 A.

4. The particle of embodiment 2, wherein the length of the linker is from about 150 to about 200 A.

5. The particle of embodiment 2, wherein the length of the linker is about

160 A.

6. The particle of embodiments 1 or 2, wherein the linker has a molecular weight of greater than 2000.

7. The particle of embodiments 1 or 2, wherein the linker has a molecular weight of about 5000.

8. The particle of any one of embodiments 1-7, wherein the linker is OPSS.

9. The particle of embodiment 1, wherein the antigen or adjuvant are conjugated to the hydrogel particle with a short linker. 10. The particle of embodiment 9, wherein the length of the linker is from about 3 to about 15 A.

11. The particle of embodiment 2, wherein the length of the linker is from about 5 to about 12 A.

12. The particle of embodiment 2, wherein the length of the linker is about

7 A.

13. The particle of embodiments 1 or 2, wherein the linker has a molecular weight of less than 2000.

14. The particle of embodiments 1 or 2, wherein the linker has a molecular weight of about 500.

15. The particle of any one of embodiments 1-7, wherein the linker is

SPDP.

16. The particle of any one of embodiments 1-15, wherein the particle with the linker increases the frequency of IFN- γ producing cells as compared to soluble antigen and adjuvant alone.

17. The particle of any one of embodiments 1-16, wherein the IFN-γ producing cell comprises a T cell.

18. The particle of embodiment 17, wherein the T cell comprises a CD8+

T cell.

19. A particle for enhancing the cytotoxic activity of CD8+ T cells, comprising a hydrogel particle conjugated to an antigen and an adjuvant, wherein the antigen or adjuvant are conjugated to the hydrogel particle with a linker.

20. The particle of embodiment 19, wherein the antigen or adjuvant are conjugated to the hydrogel particle with a long linker.

21. The particle of embodiment 20, wherein the length of the linker is from about 100 to about 300 A.

22. The particle of embodiment 20, wherein the length of the linker is from about 150 to about 200 A.

23. The particle of embodiment 20, wherein the length of the linker is about 160 A.

24. The particle of embodiments 19 or 20, wherein the linker has a molecular weight of greater than 2000. 25. The particle of embodiments 19 or 20, wherein the linker has a molecular weight of about 5000.

26. The particle of any one of claims 19-25, wherein the linker is OPSS.

27. The particle of any one of embodiments 19-26, wherein the particle with the linker enhances the cytotoxic activity of CD8+ T cells as compared to soluble antigen and adjuvant alone.

28. A method of enhancing a CD8+ T-cell immune response, comprising: administering to a subject in need thereof an antigen and an adjuvant linked to a particle through a linker, wherein the linker is greater than 50 angstrom in length.

29. The particle of embodiment 28, wherein the linker is a PEG linker and the antigen or adjuvant are conjugated to a hydrogel particle through the linker.

30. The particle of embodiment 28, wherein the length of the linker is from about 100 to about 300 A.

31. The particle of embodiment 28, wherein the length of the linker is from about 150 to about 200 A.

32. The particle of embodiment 28, wherein the length of the linker is greater than about 400 A.

33. The particle of embodiment 28, wherein the linker has a molecular weight of greater than 500.

34. The particle of embodiment 28, wherein the linker has a molecular weight of about 2000.

35. The particle of embodiment 28, wherein the linker is OPSS.

36. The particle of embodiment 28, wherein the linker is SPDP.

37. The particle of embodiment 36, wherein the linker comprises more than 5 ethylene glycol repeat units.

38. The particle of embodiment 37, wherein the particle with the linker increases the frequency CD8+ T-cells as compared to particles with antigen and adjuvant conjugated to the particle through a linker less than 50 angstrom in length.

The present subject matter is further described herein by the following non- limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention. EXAMPLES

1. Vaccines for Inducing CD8+ T cells that produce cytokine IFN-g

(interferon)

Figure 6 shows vaccine formulations were evaluated for inducing antigen- specific CD8+ T cells that produce cytokine IFN-g (interferon), which is an indicator for effector T cell functions. The left graph shows IFN-g ELISPOT to measure the frequency of IFN-g producing CD8+ T cells and the right shows IFN-g ELISA to measure the total production of IFN-g by bulk CD8+ T cells. All particles-mediated co-delivery of CpG and CD8 T cell epitope SIINFEKL promoted generation of IFN-γ producing CD8+ T cells toward statistical significance (p=0.1-0.2), in either cell frequency as shown by ELISOPT or total secretion of IFN-gamma as shown by ELISA.

On day 0, C57B1/6 mice were injected either with CFA+CSIINFEKL or 80X320 nm PEG Hydrogel NPs conjugated to CpG and C SIINFEKL via short cleavable linker or 80X320 nm PEG Hydrogel NPs conjugated to CpG and CSIINFEKL via long cleavable linker or 55X70 nm PEG Hydrogel NPs conjugated to CpG and CSIINFEKL via short cleavable linker. The dose of peptide was lOOug and CpG was around 20ug. On day 7 mice were dissected, spleens were harvested. Blood cells were separated from splenocytes. One million splenocytes were seeded in duplicate in 96 well plate that were previously coated with INF-Y capturing antibody. Cells supplemented with media and OVA peptide and kept for 18 hrs to induce production of INF-Y. Then, Cells were removed and production of INF-Y was detected using secondary antibody-enzyme conjugate and an enzyme substrate. Evaluation of antigen specific CD+8 T cell were done by counting red color spot formed after reduction of substrate.

2. Vaccine formulations evaluated for inducing antigen-specific CD8+ T cells that produce cytokine IFN-γ (interferon)

As shown in Figures 5 and 7, hydrogel PRINT NP vaccine particles were fabricated according to the above conjugation linking strategies. The vaccine formulations were evaluated for inducing antigen-specific CD8+ T cells that produce cytokine IFN-g (interferon), which is an indicator for effector T cell functions. According to Figures 5 and 7, the particle-mediated co-delivery of antigenic peptide and CpG is able to induce potent effector CD8+ T cell response. Furthermore, compared to the short linker NP-SPDP-OVA/CpG, the long linker NP-PEG-OPSS- OVA/CpG is more effective at increasing the frequency of IFN-g producing cells. In some embodiments, the long linker is twice as effective at increasing the frequency of IFN-g producing cells. In some embodiments, the long linker is more than twice as effective at increasing the frequency of IFN-g producing cells.

The method for conducting this experiment follows. On day 0, C57B1/6 mice were injected either with CF A+C S IINFEKL or 80X320 nm PEG Hydrogel NPs conjugated to CpG and CS IINFEKL via short cleavable linker or 80X320 nm PEG Hydrogel NPs conjugated to CpG and CSIINFEKL via long cleavable linker or 55X70 nm PEG Hydrogel NPs conjugated to CpG and CSIINFEKL via short cleavable linker.

The dose of peptide was lOOug and CpG was around 20ug. On day 7 mice were dissected, spleen were harvested. Blood cells were separated from splenocytes. One million splenocytes were seeded in duplicate in 96 well plate that were previously coated with INF- Y capturing antibody. Cells supplemented with media and OVA peptide and kept for 18 hrs to induce production of INF- Y. Then, Cells were removed and production of INF- Y was detected using secondary antibody- enzyme conjugate and an enzyme substrate. Evaluation of antigen specific CD+8 T cell were done by counting red color spot formed after reduction of substrate.

3. Nanoparticle effective for Targeting Draining LNs

According to Figure 11, 80nm xl80nm PEG500-OVA NPs are shown to be effective in targeting draining LNs (dLN):

1) 80x180 nm PEG500-OVA NPs appeared in PLNs minutes after footpad injections and increased with time, suggesting that this particle may self-drain to LN instead of relying on transportation via phagocytic cells (top figure).

2) Confocal microscopy confirms the accumulation of particles in dLNs over time for long time. In addition, plenty of particles were observed both in the B cell follicles and the medullary area where the T cells are located and co -localized with DCs, showing great potential for both B cell nad T cell activation. Figure 12 compares the effect of antigen protein conjugation and length of PEG (500, 5k) and non PEG linkers (shown as PEGO, conjugated through EDC-NHS chemistry) on lymphatic trafficking of particles. Both OVA conjugation and short PEG linkers help improve total drainage of particles significantly. 4. Effect of Particle Size and Linker Length

Figure 13 examines the effect of size of particle and linker length on the uptake of hydrogel particles by antigen presenting cells (dendritic cells and

macrophages). Consistent with total trafficking of particles, uptake of particles by phagocytic DCs and macrophages were also affected by size of particles. Much lower percentage of 1 um particles resulted in these cell types at 48 h post injections.

For 80x180 nm hydrogels, conjugation of antigen via long PEG5k linker apparently retarded uptake by DCs but not macrophages. While OVA directly linked to hydrogels through EDC-NHS chemistry greatly enhanced uptake by macrophage, DC cells did not take up as much, which may increase degradation and clearance of antigens. Together with total drainage result in Figure 12, 80x180 hydrogel with PEG500 particles with short linker is an optimized formulation to target DCs for antigen delivery.

For B cells, surprisingly only blank anionic particle showed significant uptake. Our explanation is that chemical groups on the surface makes the difference. For blank particles the surface was full of -OH but no real negatively charged groups. But all OVA-conjugated particles started with AEM-containing cationic particles and were quenched with anhydrides later, which added carboxylates to the surface. For non-phagocytic cells like B cells, the negative charge would definitely retard cell uptake. 5. Retention of Soluble Antigen

Figures 14 and 15 show drainage and retention of soluble antigen vs PEG hydrogel-conjugated antigen. C57BL/6 mice were injected in footpads 2 ug or 5 ug OVA-Alexa Fluor555. The draining popliteal lymph nodes (PLN) were harvested at indicated time points and 10 um sections were made and stained with B220 antibody (B cell marker). As seen, soluble OVA drains freely and rapidly and accumulates in draining LNs in 2 h. However, by 24 h it is barely detected. Moreover, C57BL/6 mice were injected in footpads particles loaded with 2 ug OVA-Alexa Fluor555. The draining popliteal lymph nodes (PLN) were harvested at indicated time points and 10 um sections were made and stained with B220 antibody (B cell marker). The particle-loaded OVA also drains rapidly and accumulates in draining LNs in 2 h (red OVA and green particles shows yellow overlapping), like soluble OVA. As time goes by, however, particle-loaded OVA decreases at a slower pace. By 48 h there is still protein antigen present. More importantly, the OVA antigen mostly shows up in the B cell follicles. This retention of OVA in B cell zones lasts up to 15 d. Similar effect was observed for particles conjugated with OVA by two lengths of linkers, PEG500 and PEGO (EDC-NHS chemistry).

Thus PEG hydrogel-based vaccine vector not only realized sustained delivery of antigen but also is able to deliver antigen directly to B cell area, which would greatly favor generation of humoral response.

6. In vivo Immunogenicity

Figure 16 examines the immunogenicity of 80x180 nm PEG(500)-OVA in

Figure 17 shows, in the left figure: 80x180 nm PEG500-OVA increased IgG by 10 fold; 1 um PEG500-OVA, however, induced very low level of IgG. The IgG response correlated well to the drainage of these two sizes of particles, suggesting that direct delivery of antigen to the draining lymph node and sustained delivery to B follicles is important for humoral immunity.

Figure 18 shows 80x180 nm hydrogels with three different lengths of linker (0, PEG500, PEG5k), elicited similar levels of IgG, all ~ 10 x higher than soluble OVA, which does not correlate to lymphatic drainage of particles. It is likely that even though PEG5k-OVA particle showed several fold lower lymph node drainage, like the other two short linker particles it can also deliver antigen to B follicles in a sustained way which, rather than total drainage, is more crucial for antibody production. Microscopy studies are needed to confirm.

Figure 19 shows that at 1 ug dose level, particle-conjugated OVA can efficiently stimulate proliferation of antigen-specific CD4⁺ T cells, while soluble OVA does not. This is likely due to more efficient uptake of particle-loaded antigens by dendritic cells/macrophages.

Throughout this specification and the claims, the words "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%), and in some embodiments ± 0.1 %> from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

That which is claimed: 1. A method of enhancing a CD8+ T-cell immune response, comprising: administering to a subject in need thereof an antigen and an adjuvant linked to a particle through a linker, wherein the linker is greater than 50 angstrom in length.

2. The particle of claim 1, wherein the linker is a PEG linker and the antigen or adjuvant are conjugated to a hydrogel particle through the linker.

3. The particle of claim 1, wherein the length of the linker is from about 100 to about 300 A.

4. The particle of claim 1, wherein the length of the linker is from about 150 to about 200 A.

5. The particle of claim 1, wherein the length of the linker is greater than about 400 A.

6. The particle of claim 1 , wherein the linker has a molecular weight of greater than 500.

7. The particle of claim 1 , wherein the linker has a molecular weight of about 2000.

8. The particle of claim 1, wherein the linker is OPSS.

9. The particle of claim 1, wherein the linker is SPDP.

10. The particle of claim 1, wherein the linker comprises more than 5 ethylene glycol repeat units.

11. The particle of claim 10, wherein the particle with the linker increases the frequency CD8+ T-cells as compared to particles with antigen and adjuvant conjugated to the particle through a linker less than 50 angstrom in length.