Timps (tissue inhibitors of metalloproteinase) encapsulating japanese cedar pollen epitopes

Abstract

To provide safe and effective means to induce tolerance to cedar pollen antigens (e.g., CRYJ1 and CRYJ2) in subjects that suffer from Japanese cedar pollinosis, or in subjects at risk for developing Japanese cedar pollinosis, taking account of the substantial prevalence of the disease, particularly in Japan and neighboring countries, and the likelihood of antibody cross-reactivity between conifer pollens.SOLUTION: The present invention provides compositions comprising particles with a negative zeta potential that encapsulate one or more epitopes associated with Japanese cedar pollen. Methods of inducing immunological tolerance to Japanese cedar pollen by administering the particles are also provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 293,261, filed February 9, 2016, the entire contents of which are incorporated herein by reference.

[0002] Description of text files submitted electronically The contents of the text file submitted electronically herewith are incorporated by reference in their entirety herein: Computer-readable copy of the Sequence Listing (Filename: COUR_014_02WO_SeqList_ST25.txt, Recorded on: February 9, 2017, File Size: 12 KB). [Background technology]

[0003] Japanese cedar pollen allergy is a common allergic disease in Japan caused by inhalation of Japanese cedar (Cryptomeria japonica) pollen. The prevalence of Japanese cedar pollen allergy in nonrandomized groups in schools, factories, and communities may be as high as 30%, and it is estimated that up to 20 million people may be affected by the disease. Thus, the disease is a significant public health problem in Japan, due in part to the severity of symptoms, high morbidity, poor spontaneous cure rate, and high medical costs associated with controlling the disease.

[0004] Japanese cedar pollen allergy is a severe type I allergic disease. Type I allergic disease is mediated by an abnormal Th2-polarized immune response to an otherwise harmless environmental antigen (e.g., Japanese cedar pollen) characterized by high systemic levels of allergen-specific immunoglobulin E (IgE), degranulation of mast cells, and release of histamine and other chemical mediators of allergy. The major clinical approaches to the treatment of allergic disease generally consist of antigen (Ag) avoidance (e.g., allergen avoidance) and control of symptoms by targeting acute effector molecules with drugs such as antihistamines, leukotriene inhibitors, or broadly acting glucocorticoids. However, such treatments fail to address the development of the underlying abnormal Th2-type immune response that drives allergic inflammation. Alternative approaches, such as specific immunotherapy (SIT), in which patients are exposed to slowly increasing doses of soluble Ag delivered intramucosally or subcutaneously, are also used clinically. SIT induces both regulatory and Th1 responses that inhibit established Th2 responses. Thus, SIT may result in a bias of the immune response to environmental allergens away from pathological Th2-type responses and toward protective or regulatory Th1 / T regulatory responses. However, administration of soluble Ag to sensitized patients poses a significant risk of adverse reactions, thereby necessitating slow dose escalation of the antigen or coadministration of the antigen with additional drugs such as omalizumab to minimize anaphylaxis.

[0005] Thus, improved methods for safely and effectively inducing Ag-specific tolerance continue to be sought as clinical avenues for the treatment of allergic diseases in pre-sensitized subjects. Nanoparticles encapsulating allergen peptides have previously been demonstrated to reduce allergen-induced Th2 responses in in vivo models (see US Patent Application Publication No. 2015 / 0209293, incorporated herein by reference in its entirety). Thus, pollen from various environmental sources can be encapsulated in nanoparticles for use in the treatment of allergic diseases such as Japanese cedar pollen allergy. Furthermore, the Japanese cedar pollen antigen CRYJ1 has high similarity to the Jun a 1 antigen of mountain cedar, the Cha o 1 antigen of Japanese cypress, and the Cup a 1 antigen of Cupressus arizonica, indicating that certain antigens from Japanese cedar pollen can mediate allergic antibody responses that are cross-reactive across multiple coniferous pollens.

[0006] However, attempts to encapsulate cedar pollen into particles suitable for the treatment of allergic diseases have only been partially successful. This is likely due to the fact that cedar pollen is highly sticky even at low concentrations. Therefore, the SIT treatment method for cedar pollen allergy remains limited. Considering the considerable prevalence of this disease, especially in Japan and neighboring countries, and the possibility of antibody cross-reactivity between coniferous pollens, there is a need in the art for a safe and effective means to induce tolerance to cedar pollen antigens (e.g., CRYJ1 and CRYJ2) in subjects suffering from cedar pollen allergy or at risk of developing cedar pollen allergy. Summary of the Invention

[0007] In some embodiments, the present invention provides compositions (e.g., for inducing antigen-specific tolerance) that include carrier particles (e.g., PLG particles) having embedded or attached thereto one or more epitopes derived from cedar pollen. In certain embodiments, the carrier particles are poly(lactide-co-glycolide) (PLG) particles having a negative zeta potential.

[0008] Certain embodiments of the present invention are directed to compositions comprising biodegradable particles comprising one or more encapsulated antigenic epitopes derived from Japanese cedar pollen, wherein the biodegradable particles have a negative zeta potential. In certain embodiments, the biodegradable particles comprise poly(lactide-co-glycolide) (PLG). In some embodiments, the biodegradable particles comprise PLG with a copolymer ratio of polylactic acid:polyglycolic acid of about 50:50. In certain embodiments, the surface of the biodegradable particles is carboxylated. In some embodiments, carboxylation is achieved by using poly(ethylene-maleic anhydride) (PEMA) or poly(acrylic acid) (PAA).

[0009] In certain embodiments, the biodegradable particles have a zeta potential of about -100 mV to about 0 mV. In certain embodiments, the biodegradable particles have a zeta potential of about -50 mV to about -40 mV. In some embodiments, the biodegradable particles have a zeta potential of about -75 mV to about -50 mV. In certain embodiments, the biodegradable particles have a zeta potential of about -50 mV.

[0010] In certain embodiments, the biodegradable particles have a diameter of about 0.1 μm to about 10 μm. In some embodiments, the biodegradable particles have a diameter of about 0.3 μm to about 5 μm. In certain embodiments, the biodegradable particles have a diameter of about 0.5 μm to about 3 μm. In certain embodiments, the biodegradable particles have a diameter of about 0.5 μm to about 1 μm. In some embodiments, the biodegradable particles have a diameter of about 0.2 μm to about 0.7 μm. In certain embodiments, the biodegradable particles have a diameter of about 0.5 μm.

[0011] Certain embodiments of the present invention are directed to compositions comprising biodegradable particles with negative zeta potential, comprising one or more encapsulated antigen epitopes derived from Japanese cedar pollen. In some embodiments, the one or more encapsulated antigen epitopes derived from Japanese cedar pollen comprise Cry j 1, Cry j 2, Cry j 3, Cry j 4, Cry j IFR, Cry j chitinase, Cry j Asp, Cry j LTP, and / or Cry j CPA9. In certain embodiments, the one or more encapsulated antigen epitopes derived from Japanese cedar pollen comprise CRYJ1, or a fragment or variant thereof. In some embodiments, CRYJ1 has the following amino acid sequence: MDNPIDSSWRGDSNWAQNRMKLADSAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPSVFIKRVSNVIIHGLHLYGSSTSVLGNVLINESFGVEPVHPQDGDALTLRTANIWIDHNSFSNSSDGLVDVTLSSTGVTISNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNSGQRMPRARYGLVHVANNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGSKTSSSSSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFN (SEQ ID NO: 1)

[0012] In some embodiments, a fragment of CRYJ1 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 contiguous amino acids having at least 90% sequence identity to SEQ ID NO: 1. In certain embodiments, a variant of CRYJ1 has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, a fragment of CRYJ1 is selected from the group consisting of p16-30, p81-95, p106-120, p111-125, p211-225, and p301-315.

[0013] In certain embodiments, the one or more encapsulated antigen epitopes from cedar pollen include CRYJ2 or a fragment or variant thereof. In some embodiments, CRYJ2 has the following amino acid sequence: VENGNATPQLTKNAGVLTSSSLSKRCRKVEHSRHDAINIFNVEKYGAVGDGKHDSTEAFSTAWQAASKXPSAMLLVPGNKKFVVNNLFFNGPSQPHFTFK VDGIIAAYQNPASWI^NRIWLQFAKITGFTLMGKGVIDGQGKQWWAGQSKXVVNGREISNDRPTAIKFDFSTGLIIQGLKMNSPEFHLVFGNSEGVKI IGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDSVAIGTGSSNIVIEDLISGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYSTSASASQNQRSAVQIQDVTYKNTIRGTSATAAAIQLKSSDSMPSKDIKLSDISLKLTSGKIASSL (Sequence number 2)

[0014] In some embodiments, a fragment of CRYJ2 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 contiguous amino acids having at least 90% sequence identity to SEQ ID NO: 2. In certain embodiments, a variant of CRYJ2 has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, a fragment of CRYJ2 is selected from the group consisting of p66-80, p81-95, p141-155, p186-200, p236-250, p346-360, p351-365, and p336-350.

[0015] In certain embodiments, the biodegradable particles described herein contain two or more encapsulation antigen epitopes derived from cedar pollen proteins. In some embodiments, two or more encapsulation epitopes are contained in a fusion protein, where the two or more encapsulation epitopes in the fusion protein are separated by a cleavable linker. In certain embodiments, the amino acid sequence of the cleavable linker is cleavable by a protease located in the phagolysosome of a cell and / or a protease located in the cytosol of a cell. In some embodiments, the amino acid sequence of the cleavable linker is cleavable by a protease located in the phagolysosome of a cell and a protease located in the cytosol of a cell. In certain embodiments, the cleavable linker is a furin-sensitive linker or a cathepsin-sensitive linker. In certain embodiments, the cleavable linker is a furin-sensitive linker. In some embodiments, the cleavable linker is a cathepsin-sensitive linker. In certain embodiments, the cathepsin-sensitive linker is susceptible to cleavage by one or more of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, and / or cathepsin Z. In some embodiments, the amino acid sequence of the linker is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 3).

[0016] In certain embodiments, one or more encapsulated antigen epitopes from cedar pollen are covalently bound to the biodegradable particles. In certain embodiments, one or more encapsulated antigen epitopes from cedar pollen are covalently bound to the biodegradable particles by a conjugate molecule. In some embodiments, the conjugate molecule comprises a carbodiimide compound. In certain embodiments, the carbodiimide compound comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0017] Certain embodiments are directed to pharmaceutical compositions comprising the biodegradable particles described herein. In some embodiments, the pharmaceutical compositions further comprise a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions further comprise a pharma- ceutically acceptable excipient. Certain embodiments are directed to lyophilized compositions comprising the biodegradable particles described herein.

[0018] Certain embodiments are directed to a method for inducing antigen-specific tolerance to cedar pollen in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.Certain embodiments are directed to a method for treating cedar pollen allergy in a subject in need of treatment, comprising administering to the subject a pharmaceutical composition as described herein.Some embodiments are directed to a method for preventing cedar pollen allergy in a subject in need of prevention, comprising administering to the subject a pharmaceutical composition as described herein.

[0019] Certain embodiments are directed to a method for inducing antigen-specific tolerance to cedar pollen in a subject, the method comprising reconstituting the freeze-dried particles described herein to obtain a reconstituted pharmaceutical composition, and administering the reconstituted pharmaceutical composition to the subject.Certain embodiments are directed to a method for treating cedar pollen allergy in a subject in need of treatment, the method comprises reconstituting the freeze-dried particles described herein to obtain a reconstituted pharmaceutical composition, and administering the reconstituted pharmaceutical composition to the subject.Some embodiments are directed to a method for preventing cedar pollen allergy in a subject in need of prevention, the method comprises reconstituting the freeze-dried particles described herein to obtain a reconstituted pharmaceutical composition, and administering the reconstituted pharmaceutical composition to the subject. [Brief description of the drawings]

[0020] [Figure 1] We present a model of how administration of biodegradable particles with negative zeta potential encapsulating antigenic epitopes derived from Japanese cedar pollen provides an effective treatment for Japanese cedar pollen allergy. [Diagram 2] Shown is the viscosity of the JCP extract (Figure 2A), an exemplary illustration of biodegradable particles with negative zeta potential encapsulating antigenic epitopes of cedar pollen (TIMP-JCP) (Figure 2B), and a diagram of the double emulsion process (Figure 2C). [Diagram 3] SDS-PAGE analysis of JCP extract and recombinant JCP protein is shown. [Figure 4] Particle characteristics of TIMP-encapsulated JCP extract are shown. [Diagram 5] Graphs of the acute inflammation mouse model (FIG. 5A) and antibody responses 21 days after sensitization (FIGS. 5B-5D) are shown. [Figure 6] The graph shows changes in body temperature after JCP sensitization, TIMP treatment, and JCP antigen administration. [Figure 7] Scratching, sneezing, and coughing scores following JCP sensitization, TIMP treatment, and JCP challenge are shown. [Figure 8] Cytokine production from splenocytes of JCP-sensitized and antigen-challenged mice treated with TIMP-JCP or TIMP-OVA is shown. [Figure 9] Antibody responses 30 days after immunization with JCP are shown. [Figure 10] Serum levels of histamine (FIG. 10A) and MCPT-1 (FIG. 10B) are shown. [Figure 11] Illustrates the effect of the route of administration on the resulting immune response. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The inventors of the present invention have discovered that antigen-embedded nanoparticles can induce tolerance and reduce immune responses to autoimmune diseases, and thus these particles may be useful in the treatment of any disease or condition characterized by an excessive inflammatory immune response, such as allergies to cedar pollen.

[0022] "Particle" as used herein refers to any composition that is not tissue-derived, which may be a sphere or spherical entity, a bead, or a liposome. The terms "particle", "immunomodified particle", "carrier particle", and "bead" may be used interchangeably depending on the context. Furthermore, the term "particle" may be used to encompass beads and spheres.

[0023] "Negatively charged particles," as used herein, refer to particles that have been modified to carry a net surface charge that is less than zero.

[0024] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups enhances phagocyte / monocyte uptake of the particle from the bloodstream, for example, through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be accomplished using any compound that adds or incorporates carboxyl groups, including, but not limited to, poly(ethylene-alt-maleic anhydride) (PEMA).

[0025] "Antigenic moiety" as used herein refers to any moiety, e.g., a peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, enzymes, and / or bacterial or viral proteins, peptides, drugs or components. Without wishing to be bound by theory, while carboxylated beads themselves may be recognized by the immune system, carboxylated beads that are not attached to anything are not considered "antigenic moieties" for the purposes of the present invention.

[0026] "Bare bead" or "bare particle" or "bare sphere" as used herein refers to a bead, particle, or sphere that is not carboxylated.

[0027] "Pro-inflammatory mediator" or "pro-inflammatory polypeptide" as used herein refers to a polypeptide or fragment thereof that induces, maintains, or prolongs inflammation in a subject. Examples of pro-inflammatory mediators include, but are not limited to, cytokines and chemokines.

[0028] As used herein, the term "inflammatory monocyte" refers to any myeloid cell that expresses any combination of CD14 / CD26 and CCR2.

[0029] As used herein, the term "inhibitory neutrophils" refers to immune suppressive cells derived from neutrophils and / or monocytes.

[0030] As used herein, the term "Th cells" or "helper T cells" refers to CD4 + Refers to cells. CD4 + T cells help other white blood cells through immunological processes including maturation of B cells to plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. T cells become activated when presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs).

[0031] As used herein, the term "Th1 cells" refers to a subset of Th cells that produce proinflammatory mediators. Th1 cells secrete cytokines to facilitate immune responses and play a role in host defense against pathogens, in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines including IFN-γ, IL-2, IL-10, and TNF α / β to function as defenses against intracellular pathogens such as viruses and some bacteria.

[0032] As used herein, the term "Th2 cells" refers to a subset of Th cells that mediate the activation and maintenance of antibody-mediated immune responses to extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing a variety of cytokines, such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25), which are responsible for antibody production, eosinophil activation, and inhibition of multiple macrophage functions, thus providing a phagocyte-independent protective response.

[0033] As used herein, the term "Th17 cells" refers to a subset of Th cells. Th17 cells secrete cytokines to facilitate immune responses and mediate the recruitment of neutrophils and macrophages to infected tissues, thereby playing a role in host defense against pathogens. TH17 cells secrete cytokines such as IL-17, IL-21, IL-22, IL-24, IL-26, and TNFα to function as defense against extracellular pathogens, including fungi and bacteria.

[0034] "Bound" as used herein refers to an antigen that is immobilized on the outside of the particle or encapsulated within the particle. Thus, an antigen that is bound to a particle includes both surface attachment to the particle as well as encapsulation within the particle.

[0035] The term "IMP" as used herein refers to an immune-modified particle that is not bound to an antigen. The term "TIMP" as used herein refers to a tolerized immune-modified particle that is bound to an antigen. In some embodiments, the antigen is attached to the surface of the TIMP. In other embodiments, the antigen is encapsulated within the TIMP.

[0036] The particles may have any particle shape or configuration. However, in some embodiments, it is preferred to use particles that are less prone to aggregation in vivo. Exemplary particles in these embodiments are those having a spherical shape.

[0037] Another aspect of the invention relates to a composition comprising an immunomodified particle having a negative zeta potential and free from an antigen moiety. In a further embodiment, the invention provides a composition comprising an immunomodified particle having a negative zeta potential bound to an antigen. In a further embodiment, the antigen is bound to the outside of the particle. In a preferred embodiment, the antigen is encapsulated within the particle.

[0038] Yet another aspect of the invention relates to a method for the preparation of immune-modified particles having a negative zeta potential and free from antigen moieties. The method involves contacting immune-modified particle precursors with a buffer solution under conditions effective to form immune-modified particles having a negative zeta potential. In some embodiments of the invention, the immune-modified particle precursors are formed via copolymerization. The microstructure of the particles may depend on the method of copolymerization.

[0039] In some embodiments, the antigenic peptide molecules are bound to the carrier particles (e.g., immune-modified particles) by conjugate molecules and / or linker groups. In some embodiments, the binding of the antigenic peptide and / or apoptosis signaling molecule to the carrier (e.g., PLG particles) comprises one or more covalent and / or non-covalent interactions. In some embodiments, the antigenic peptide is attached to the surface of the carrier particles having a negative zeta potential. In some embodiments, the antigenic peptide is encapsulated within the carrier particles having a negative zeta potential.

[0040] In one embodiment, the buffer solution contacting the immunomodified particles may have a basic pH. Suitable basic pH for the basic solution includes 7.1, 7.5, 8.0, 8.5, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5. The buffer solution may also be made with any suitable base and its conjugates. In some embodiments of the present invention, the buffer solution may include, but is not limited to, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate, and conjugates thereof.

[0041] In one embodiment of the present invention, the immunomodified particle contains copolymers.These copolymers can have various molar ratios.The suitable copolymer ratio of the immunomodified particle of the present invention can be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In another embodiment, the copolymer can be a periodic, statistical, linear, branched (including star, brush, or comb copolymers). In some embodiments, the copolymer ratio can be, but is not limited to, polystyrene:poly(vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90:10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90:10, polylactic acid:polyglycolic acid / 50:50, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90:10.

[0042] In one embodiment, the particles of the present invention are made by adding a composition comprising a polymer (e.g., PLGA) to a solution of poly(ethylene-maleic anhydride) (PEMA). The concentration of PEMA in the solution can be about 0.1% to about 10%. In one embodiment, the concentration of PEMA in the solution is about 0.2% to about 5%. In another embodiment, the concentration of PEMA in the solution is about 0.1% to 4%. In another embodiment, the concentration of PEMA in the solution is about 0.1% to 2%. In another embodiment, the concentration of PEMA in the solution is about 0.5% to 1%. In one embodiment, the percentage of PEMA in the solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, the percentage of PEMA in the solution is about 0.5%. In another embodiment, the percentage of PEMA in the solution is about 1.0%. Other compounds that may be used include, but are not limited to, poly(ethylene-alt-maleic anhydride), poly(isobutylene-co-maleic acid), poly(methyl vinyl ether-alt-maleic acid), poly(methyl vinyl ether-alt-maleic acid monoethyl ester), poly(methyl vinyl ether-alt-maleic anhydride), poly(methyl vinyl ether-alt-maleic anhydride) crosslinked with 1,9-decadiene powder, poly(styrene-alt-maleic acid) sodium salt, poly(vinyl alcohol), poly(acrylic acid), and / or sodium deoxycholate.

[0043] In one embodiment, the particle is a liposome. In a further embodiment, the particle is a liposome composed of the following lipids in the following molar ratio - 30:30:40 phosphatidylcholine:phosphatidylglycerol:cholesterol. In yet a further embodiment, the particle is encapsulated within a liposome.

[0044] Although each particle need not be of uniform size, the particles should generally be of sufficient size to be sequestered in the spleen or liver and to induce phagocytosis or uptake by antigen presenting cells, including endothelial cells, or other MPS cells through receptor or non-receptor mediated mechanisms. Preferably, the particles are of microscopic or nanoscale size to enhance solubility, avoid complications that may be caused by aggregation in vivo, and facilitate pinocytosis. Particle size may be a factor in uptake from the interstitial space to areas of lymphocyte maturation. Particles having a diameter of about 0.1 μm to about 10 μm can induce phagocytosis. Thus, in one embodiment, the particles have a diameter within these limits. In another embodiment, the particles have a diameter of about 0.3 μm to about 5 μm on average. In yet another embodiment, the particles have a diameter of about 0.5 μm to about 3 μm on average. In another embodiment, the particles have a diameter of about 0.2 μm to about 2 μm on average. In further embodiments, the particles have an average size of about 0.1 μm, or about 0.2 μm, or about 0.3 μm, or about 0.4 μm, or about 0.5 μm, or about 1.0 μm, or about 1.5 μm, or about 2.0 μm, or about 2.5 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm. In certain embodiments, the particles have an average size of about 0.5 μm. In some embodiments, the particles have an average diameter of about 0.5 μm to about 0.95 μm. For example, in such embodiments, the particles have an average diameter of about 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, or about 0.95 μm. In certain embodiments, the particles have an average diameter of about 0.7 μm. In some embodiments, the total weight of the particles is at least about 1000 kDa. In some embodiments, the total weight of the particles is about 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, or more.

[0045] In some embodiments, the total weight of the particles is less than about 10,000 kDa, less than about 5,000 kDa, or less than about 1,000 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa, 50 kDa, 20 kDa, 10 kDa. It is not necessary that the particles in the composition be of uniform diameter. As an example, a pharmaceutical formulation may contain a plurality of particles, some of which have a diameter of about 0.5 μm, while other particles have a diameter of about 1.0 μm. As an additional example, a pharmaceutical formulation may contain a plurality of particles, some of which have a diameter of about 0.7 μm, while other particles have a diameter of about 0.5 μm to about 0.95 μm. Any mixture of particle sizes within these given ranges is useful.

[0046] The particles of the present invention may have a particular zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than (e.g., more negative than) about -100 mV. In one embodiment, the zeta potential is less than (e.g., more negative than) about -50 mV. In certain embodiments, the particles possess a zeta potential between -100 mV and 0 mV. In further embodiments, the particles possess a zeta potential between -75 mV and 0 mV. In further embodiments, the particles possess a zeta potential between -60 mV and 0 mV. In further embodiments, the particles possess a zeta potential between -50 mV and 0 mV. In yet further embodiments, the particles possess a zeta potential between -40 mV and 0 mV. In further embodiments, the particles possess a zeta potential between -30 mV and 0 mV. In further embodiments, the particles possess a zeta potential between -20 mV and +0 mV. In further embodiments, the particles possess a zeta potential of -10 mV and -0 mV. In further embodiments, the particles possess a zeta potential of -100 mV and -50 mV. In another particular embodiment, the particles possess a zeta potential of -75 mV and -50 mV. In a particular embodiment, the particles possess a zeta potential of -50 mV and -40 mV. In another particular embodiment, the particles possess a zeta potential of less than (e.g., more negative than) about -40 mV. In another particular embodiment, the particles possess a zeta potential of at least about -30 mV. In another particular embodiment, the particles possess a zeta potential of less than (e.g., more negative than) about -30 mV.

[0047] The particles of the invention may possess a ratio of antigen to polymer (e.g., μg of antigen / mg of polymer). In some embodiments, the ratio of antigen to polymer is about 1 μg / mg to at least about 5 μg / mg. For example, in some embodiments, the ratio of antigen to polymer is about 1 μg / mg, 1.5 μg / mg, 2 μg / mg, 2.5 μg / mg, 3.0 μg / mg, 3.5 μg / mg, 4 μg / mg, 4.5 μg / mg, or about 5 μg / mg. In some embodiments, the ratio of antigen to polymer is at least about 5 μg / mg. For example, in some embodiments, the ratio of antigen to polymer is at least about 5 μg / mg, 5.5 μg / mg, 6 μg / mg, 6.5 μg / mg, 7 μg / mg, 7.5 μg / mg, 8 μg / mg, 8.5 μg / mg, 9.0 μg / mg, 9.5 μg / mg, 10 μg / mg, 10.5 μg / mg, 11 μg / mg, 11.5 μg / mg, 12 μg / mg, 12.5 μg / mg, 13 μg / mg, 13.5 μg / mg, 14 μg / mg, 14.5 μg / mg, or about 15 μg / mg.

[0048] In some embodiments, the charge of the carrier (e.g., positive, negative, neutral) is selected to confer an inherent benefit to the application (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier has a net neutral or negative charge (e.g., to reduce non-specific binding to cell surfaces that generally bear a net negative charge). In certain embodiments, the carrier can be conjugated, either directly or indirectly, to an antigen (also referred to herein as an antigen-specific peptide, antigenic peptide, autoantigen, inducible antigen, or tolerizing antigen) for which tolerance is desired. In some cases, the carrier has multiple binding sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10..., 20..., 50..., 100, or more) to expose multiple copies of an antigen-specific peptide or multiple different peptides on the surface (e.g., to increase the likelihood of a tolerance response). In some embodiments, the carrier presents a single type of antigenic peptide. In some embodiments, the carrier presents multiple different antigenic peptides on the surface. In some embodiments, the carrier surface presents functional groups for covalent attachment of a selected moiety (e.g., an antigenic peptide). In some embodiments, the functional groups on the carrier surface provide sites for non-covalent interaction with a selected moiety (e.g., an antigenic peptide). In some embodiments, the carrier has a surface to which a conjugate moiety can be adsorbed without forming a chemical bond.

[0049] Particle size and particle charge are important in tolerance induction. Although particle size and particle charge vary based on the antigens encapsulated therein, generally, the particles of the present invention are effective in inducing tolerance when they are about 100 nanometers to about 1500 nanometers and have a charge of 0 to about -70 mV, and are most effective in inducing tolerance when they are 400 to 800 nanometers and have a charge of about -25 mV to -70 mV. Furthermore, due in part to the concentration of particles in the lyophilization process, as well as the presence of sucrose and D-mannitol, the average particle size and particle charge may be slightly altered in the lyophilization process. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to the particle size and particle charge before lyophilization. The terms "post-lyophilization size" and "post-lyophilization charge" refer to the particle size and particle charge after lyophilization.

[0050] In some embodiments, the particles are non-metallic. In those embodiments, the particles may be formed from polymers. In preferred embodiments, the particles are biodegradable in the individual. In this embodiment, the particles may be provided in the individual over multiple doses without accumulation of the particles in the individual. Examples of suitable particles include polystyrene particles, PLGA particles, PLURIONICS stabilized polypropylene sulfide particles, and diamond particles. Preferably, the particle surface is made of a material that minimizes non-specific or undesirable biological interactions. Interactions between the particle surface and the interstitium may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material to prevent or reduce non-specific interactions. Steric stabilization by coating the particles with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS® (including copolymers of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)), may reduce non-specific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All these facts show the significance of the particle's physical properties in relation to lymphatic uptake. Biodegradable polymers can be used to make up all or part of the polymer and / or particle and / or layer. Biodegradable polymers can undergo degradation, for example, as a result of functional groups reacting with water in solution. The term "degradation" as used herein refers to becoming soluble by reducing molecular weight or by converting hydrophobic groups to hydrophilic groups. Polymers with ester groups, such as polylactide and polyglycolide, are generally subject to spontaneous hydrolysis.

[0051] The particles of the present invention may also contain additional components. For example, the carrier may have a contrast agent incorporated or conjugated to the carrier. An example of a carrier nanosphere with a contrast agent that is currently commercially available is Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals known as quantum dots (QDs) have emerged as ideal donors in FRET applications, and their high quantum yield and tunable size-dependent Stokes shift allow different sizes to emit from blue to infrared when excited with a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew.Chem.Int.Ed.2003, 42, 5796; Waggoner, A.Methods Enzymol.1995, 246, 362; Brus, LEJChem.Phys.1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the synthesis of traditional inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or toxic or unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0052] The particles can be formed from a wide range of materials. The particles are preferably made of materials suitable for biological use. For example, the particles may be made of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles may be made of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. In addition, the carrier particles may be quantum dots or may be made of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006); Langmuir 22:1810-6). Carrier particles containing a mixture of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, the carrier particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG, the terms are interchangeable), [rho]oly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, and copolymers thereof with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxycarboxylic acids or dicarboxylic acids.In addition, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers, can be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugating to antigen peptides and proteins or conjugate moieties. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, non-biodegradable polymers of acrylate, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon can be used.

[0053] The particles of the present invention can be manufactured by any means generally known in the art. Exemplary methods for manufacturing particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, precipitation polymerization, emulsion evaporation, emulsion diffusion, solvent displacement, and salting out (Astete and Sabliov, J.Biomater.Sci.Polymer Edn.,17:247-289(2006)). The manipulation of the manufacturing process for PLGA particles can control particle characteristics (e.g., size, size distribution, zeta potential, morphology, hydrophobicity / hydrophilicity, polypeptide entrapment, etc.). Particle size is influenced by multiple factors, including, but not limited to, the concentration of PLGA, the solvent used in the manufacturing of the particles, the nature of the organic phase, the surfactant used in the manufacturing, the viscosity of the continuous and discontinuous phases, the nature of the solvent used, the water temperature used, sonication, evaporation rate, additives, shear stress, sterilization, and the nature of any encapsulated antigen or polypeptide.

[0054] Particle size is affected by polymer concentration, with larger particles being formed from higher polymer concentrations. For example, when the solvent propylene carbonate is used, increasing the PLGA concentration from 1% to 4% (w / v) can increase the average particle size from about 205 nm to about 290 nm. Alternatively, in ethyl acetate and 5% Pluronic F-127, increasing the PLGA concentration from 1% to 5% (w / v) increases the average particle size from 120 nm to 230 nm.

[0055] The viscosity of the continuous and discontinuous phases is also an important parameter that affects the diffusion process, which is a key step in forming smaller particles. Particle size increases with increasing viscosity of the dispersed phase, while particle size decreases when the continuous phase is more viscous. Generally, the lower the organic to aqueous solvent phase ratio, the smaller the particle size.

[0056] Homogenizer speed and agitation also affect particle size, generally with faster speeds and agitation particle size will decrease, but there is a point where further increases in speed and agitation will not result in any further particle size reduction. When an emulsion is homogenized with a high pressure homogenizer, there is a beneficial effect on size reduction compared to simply agitating faster. For example, at a phase ratio of 20% in 5% PVA, the average particle size when agitated is 288 nm and when homogenized (high pressure at 300 bar) the average particle size is 231 nm.

[0057] Significant particle size reduction can be achieved by varying the temperature of the added water to improve the diffusion of the solvent. The average particle size decreases with increasing water temperature.

[0058] The nature of the polypeptide encapsulated in the particle also influences particle size. In general, encapsulation of hydrophobic polypeptides leads to the formation of smaller particles compared to encapsulation of more hydrophilic polypeptides. In the double emulsion process, the entrapment of more hydrophilic polypeptides is improved by using high molecular weight PLGA and high molecular weight first surfactant, which causes higher internal phase viscosity. The interaction between the solvent, polymer and polypeptide affects the efficiency of incorporation of polypeptide into particles.

[0059] The PLGA molecular mass affects the final average particle size. Generally, the higher the molecular mass, the higher the average particle size. For example, as the composition and molecular mass of PLGA change (e.g., from 12 to 48 kDa for 50:50 PLGA and from 12 to 98 kDa for 75:25 PLGA), the average particle size also changes (approximately 102 nm to 154 nm and 132 nm to 152 nm, respectively). Even if the particles have the same molecular mass, their composition can affect the average particle size, e.g., particles with a 50:50 ratio generally form smaller particles than those with a 75:25 ratio. The end groups of the polymer also affect the particle size. For example, particles prepared with ester end groups form particles with an average size of 740 nm (PI=0.394) compared to an average size of 240 nm (PI=0.225) for acid PLGA end groups.

[0060] The solvent used can also affect particle size; solvents that reduce the surface tension of the solution will also reduce the particle size.

[0061] The organic solvent is removed by evaporation in vacuum to avoid damage to the polymer and polypeptide and to facilitate the reduction of the final particle size. Evaporation of the organic solvent under vacuum is more efficient in forming smaller particles. For example, evaporation under vacuum produces an average particle size approximately 30% smaller than that produced under normal speed evaporation.

[0062] The amplitude of the sonication wavelength also influences the particle characteristics. To form a very black miniemulsion without further droplet size changes, the amplitude of the wavelength should be above 20% for 600-800 s of sonication. However, the main drawback of sonication is the lack of monodispersity of the formed emulsions.

[0063] Organic phases that may be used in the production of the particles of the present invention include, but are not limited to, ethyl acetate, methyl ethyl ketone, propylene carbonate, and benzyl alcohol. Continuous phases that may be used include, but are not limited to, the surfactant poloxamer 188.

[0064] A variety of surfactants can be used in the preparation of the particles of the present invention. The surfactants can be anionic, cationic, or nonionic. Poloxamer and poloaxamine family surfactants are commonly used in particle synthesis. Surfactants that can be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, PVA, methylcellulose, lecithin, and DMAB. In addition, surfactants that are biodegradable and biocompatible include, but are not limited to, Vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are required (e.g., in the double emulsion evaporation method). These two surfactants can include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

[0065] Solvents that can be used in the production of the particles of the present invention include, but are not limited to, acetone, tetrahydrofuran (THF), chloroform, and methyl chloride, a member of the chlorinate family. The selection of an organic solvent requires two selection criteria: the polymer must be soluble in the solvent, and the solvent must be completely immiscible with the aqueous phase.

[0066] Salts that may be used in producing the particles of the present invention include, but are not limited to, magnesium chloride hexahydrate, magnesium acetate tetrahydrate.

[0067] Common salting out agents include, but are not limited to, electrolytes (eg, sodium chloride, magnesium acetate, magnesium chloride), or non-electrolytes (eg, sucrose).

[0068] The stability and particle size of the particles of the present invention can be improved by adding compounds including, but not limited to, fatty acids or short carbon chains.Addition of longer carbon chains of lauric acid is associated with improved particle characteristics.Furthermore, addition of hydrophobic additives can improve particle size, incorporation of polypeptides into particles, and release profile.Particle preparations can be stabilized by lyophilization.Addition of cryoprotectants such as trehalose can reduce particle aggregation during lyophilization.

[0069] Suitable beads currently available commercially include polystyrene beads, such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[0070] In some embodiments, the present invention provides a system comprising: (a) a delivery scaffold configured for delivery of a chemical and / or biological agent to a subject; and (b) antigen-conjugated poly(lactide-co-glycolide) particles for induction of antigen-specific tolerance. In some embodiments, at least a portion of the delivery scaffold is microporous. In some embodiments, the antigen-conjugated poly(lactide-co-glycolide) particles are encapsulated within the scaffold. In some embodiments, the chemical and / or biological agent is selected from the group consisting of proteins, peptides, small molecules, nucleic acids, cells, and particles. In some embodiments, the chemical and / or biological agent comprises cells, and the cells comprise pancreatic islet cells.

[0071] Physical properties are also relevant to the usefulness of the nanoparticles after uptake and retention in areas with immature lymphocytes. These include mechanical properties such as rigidity or rubberiness. Some embodiments are based on a rubbery core, e.g., poly(propylene sulfide) (PPS) core, with a hydrophilic overlayer, e.g., as in PEG, as in the PPS-PEG system recently developed and characterized for systemic (rather than targeted or immune) delivery. The rubbery core is in contrast to a substantially rigid core, as in polystyrene or metal nanoparticle systems. The term rubbery refers to certain elastic materials other than natural or synthetic rubber, and rubbery is a term well known to those skilled in the polymer art. For example, crosslinked PPS can be used to form a hydrophobic rubbery core. PPS is a polymer that degrades under oxidative conditions to polysulfoxide and ultimately polysulfone, transitioning from a hydrophobic rubber to a hydrophilic, water-soluble polymer. Other sulfide polymers may be adapted for use, and the term sulfide polymer refers to polymers with sulfur in the polymer backbone. Other rubbery polymers that can be used are polyesters that have a glass transition temperature of less than about 37° C. under hydration conditions. Hydrophobic cores can be advantageously used with hydrophilic overlayers, since the core and overlayer tend not to mix, and therefore the overlayer tends to expand sterically away from the core. Core refers to a particle having a layer thereon. Layer refers to a material that covers at least a portion of the core. The layer can be adsorbed or covalently bonded. The particle or core can be solid or hollow. Rubbery hydrophobic cores have an advantage over rigid hydrophobic cores, such as crystalline or glassy (as in polystyrene) cores, in that particles with rubbery hydrophobic cores can be loaded with more hydrophobic drugs.

[0072] Another physical property is the hydrophilicity of the surface. A hydrophilic material may have a water solubility of at least 1 gram per liter when not crosslinked. Steric stabilization of the particles with hydrophilic polymers may improve uptake from the interstitium by reducing non-specific interactions, but the high stealth of the particles may also reduce internalization by phagocytes in areas with immature lymphocytes. The challenge of balancing these competing characteristics has been met, and the present application demonstrates the creation of nanoparticles for effective lymphatic delivery to DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic component, e.g., a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers. The molecular weight of the polymers in the layer can be adjusted to provide a degree of steric hindrance that is useful in vivo, for example, from about 1,000 to about 100,000, or even higher, and one of ordinary skill in the art will immediately appreciate that all ranges and values ​​within the explicitly stated ranges, e.g., 10,000 to 50,000, are contemplated.

[0073] The nanoparticles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles, which are convenient for reacting with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

[0074] Various means well known in the art can be used to conjugate antigenic peptides and proteins to carriers. These methods include any standard chemistry that does not destroy or significantly limit the biological activity of antigenic peptides and proteins, and allows a sufficient number of antigenic peptides and proteins to be conjugated to the carrier in an orientation that allows the antigenic peptide or protein to interact with its cognate T cell receptor. Generally, methods that conjugate the C-terminal region of the antigenic peptide or protein, or the C-terminal region of an antigenic peptide or protein fusion protein to the carrier are preferred. The exact chemistry will of course depend on the nature of the carrier material, the presence or absence of a C-terminal fusion to the antigenic peptide or protein, and / or the presence or absence of a conjugated moiety.

[0075] Functional groups can be located on the particle as needed due to availability. One location can be a side group or terminal on the core polymer, or on a polymer that is a layer on the core, or on a polymer that is otherwise anchored to the particle. For example, examples are included herein that describe PEG stabilizing nanoparticles that can be easily functionalized for specific cell targeting or protein and peptide drug delivery.

[0076] Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propylene glycol diglycidyl ether containing two epoxy residues, and epichlorohydrin can be used to immobilize peptides or proteins on the carrier surface. Without being bound by theory, ECDI is believed to perform two main functions to induce tolerance: (a) chemically bind proteins / peptides to cell surfaces through catalysis of peptide bond formation between free amino and free carboxyl groups, and (b) induce carriers to mimic apoptotic cell death, so that they are selected by host antigen-presenting cells (which may include endothelial cells) in the spleen and induce tolerance. It is this presentation to host T cells in a non-immunogenic manner that results in the direct induction of anergy in autoreactive cells. In addition, ECDI serves as a strong stimulus to induce specific regulatory T cells.

[0077] In one series of embodiments, the antigen peptide and protein are bound to the carrier via a covalent chemical bond. For example, a reactive group or moiety near the C-terminus of the antigen (e.g., the C-terminal carboxyl group, or the hydroxyl, thiol, or amine group of an amino acid side chain) can be directly conjugated to a reactive group or moiety on the surface of the carrier by direct chemical reaction (e.g., the hydroxyl or carboxyl group of a PLA or PGA polymer, the terminal amine or carboxyl group of a dendrimer, or the hydroxyl, carboxyl, or phosphate group of a phospholipid). Alternatively, there can be a conjugation moiety that covalently conjugates both the antigen peptide and the protein to the carrier, thereby binding them together.

[0078] Reactive carboxyl groups on the surface of the carrier can be coupled to free amines (e.g., from Lys residues) on antigenic peptides or proteins by reacting them with, for example, 1-ethyl-3-[3,9-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). Similarly, the same chemistry can be used to conjugate free amines on the surface of the carrier with free carboxyls (e.g., from the C-terminus, or Asp or GIu residues) on antigenic peptides or proteins. Alternatively, free amine groups on the surface of the carrier can be covalently coupled to antigenic peptides and proteins, or antigenic peptide or protein fusion proteins, using sulfo-SIAB chemistry essentially as described in Arano et al. (1991) Chem. 2:71-6.

[0079] In another embodiment, the antigen may be conjugated to the carrier by non-covalent binding between a ligand bound to the antigen peptide or protein and an anti-ligand attached to the carrier. For example, a biotin ligase recognition sequence tag may be attached to the C-terminus of the antigen peptide or protein, and the tag may be biotinylated by biotin ligase. Biotin may then serve as a ligand for non-covalently conjugating the antigen peptide or protein to avidin or streptavidin adsorbed or otherwise bound to the surface of the carrier as an anti-ligand. Alternatively, when the antigen peptide and protein are fused to an immunoglobulin domain carrying an Fc region, the Fc domain may act as a ligand, as described above, and protein A covalently or non-covalently bound to the surface of the carrier may serve as an anti-ligand for non-covalently conjugating the antigen peptide or protein to the carrier. Metal ion chelation techniques (e.g., a poly-His tag at the C-terminus of the antigen peptide or protein or antigen peptide or protein fusion protein, and Ni +Other means that can be used to non-covalently conjugate antigenic peptides and proteins to carriers are well known in the art, including the use of carriers coated with .alpha.-methylcellulose (MgSO 4 ) and these methods can be substituted for the methods described herein.

[0080] Conjugation of the nucleic acid moiety to the platform molecule can be accomplished in any number of ways, but typically involves one or more crosslinkers and functional groups on the nucleic acid moiety and the platform molecule. The linking group is added to the platform using standard synthetic chemistry techniques. The linking group can be added to the nucleic acid moiety using standard synthetic chemistry techniques. The practitioner has several options regarding the antigen used in the combination of the present invention. The inducing antigen present in the combination contributes to the specificity of the immune tolerogenic response induced. It may or may not be the same as the target antigen, which is the target of the undesired immunological response and is the antigen present or given to the subject undergoing treatment to which tolerance is desired.

[0081] The inducing antigens of the present invention may be polypeptides, polynucleotides, carbohydrates, glycolipids, or other molecules isolated from biological sources, or may be chemically synthesized small molecules, polymers, or derivatives of biological materials, provided that they have the ability to induce tolerance according to the present invention when combined with a mucosal-binding component.

[0082] In some embodiments, the present invention provides carriers (e.g., immune-modified particles) coupled to one or more peptides, polypeptides, and / or proteins. In some embodiments, carriers such as those described herein (e.g., PLG carriers) are effective in inducing antigen-specific tolerance and / or preventing the onset of immune-related diseases (such as EAE in mouse models) and / or reducing the severity of existing immune-related diseases. In some embodiments, the compositions and methods of the present invention can cause T cells to initiate early events associated with T cell activation, but fail to cause the T cells to acquire effector functions. For example, administration of the compositions of the present invention can result in T cells with a quasi-activated phenotype, such as CD69 and / or CD44 upregulation, but do not display effector functions, as indicated by a lack of IFN-γ or IL-17 synthesis. In some embodiments ...naive antigen-specific T cell, such as CD25 + / Foxp3 + The present invention provides a method for the treatment of T cells with a subactivated phenotype without converting to a regulatory phenotype, such as those with a phenotype.

[0083] In some embodiments, the surface of the carrier (e.g., particle) comprises chemical moieties and / or functional groups that allow for attachment (e.g., covalently, non-covalently) of antigenic peptides and / or other functional elements to the carrier. In some embodiments, the number, orientation, spacing, etc. of the chemical moieties and / or functional groups on the carrier (e.g., particle) will vary according to the carrier chemistry, desired application, etc.

[0084] In some embodiments, the carrier includes one or more biological or chemical agents attached to, adsorbed onto, encapsulated in, and / or contained throughout the carrier. In some embodiments, the chemical or biological agents are encapsulated in the particles and / or contained throughout the particles. The present invention is not limited by the nature of the chemical or biological agents. Such agents include, but are not limited to, proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cellular components, and the like. In some embodiments, two or more (e.g., three, four, five, etc.) different chemical or biological agents are included on or within the carrier. In some embodiments, the agent is configured for a specific release rate. In some embodiments, a plurality of different agents are configured for different release rates. For example, a first agent may be released over a period of hours and a second agent may be released over a longer period of time (e.g., days, weeks, months, etc.). In some embodiments, the carrier or a portion thereof is configured for sustained release of the biological or chemical agent. In some embodiments, sustained release provides release of a biologically active amount of agent over a period of at least 30 days (e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 180 days, etc.). In some embodiments, the carrier or a portion thereof is configured to be sufficiently porous to allow ingrowth of cells into the pores. The size of the pores can be selected for the particular cell type of interest and / or the amount of ingrowth desired. In some embodiments, the particles contain an antigen of interest without other non-peptide activators such as drugs or immunomodulators. Furthermore, in some embodiments, the particles of the present invention do not contain immunostimulatory or immunosuppressive peptides in addition to the antigen of interest. Furthermore, in some embodiments, the particles do not contain other proteins or peptides (e.g., costimulatory molecules, MHC molecules, immunostimulatory peptides, or immunosuppressive peptides) on the surface of the particle or encapsulated within the particle.

[0085] Surprisingly, it has been found that the inclusion of antigens, biological agents, and / or chemical agents in the particles of the present invention induces immune tolerance and has several advantages. First, the encapsulated particles have a slower cytokine response. Second, when multiple antigens, biological agents, and / or chemical agents are used, the encapsulation eliminates the competition between these various molecules that may occur when the agents are attached to the surface of the particles. Third, the encapsulation allows more antigens, biological agents, and / or chemical agents to be incorporated into the particles. Fourth, the encapsulation makes it easier to use complex protein antigens or organ homogenates (e.g., pancreatic homogenates in type 1 diabetes or peanut extracts in peanut allergy). Finally, instead of conjugation to the surface of the particles, the encapsulation of antigens, biological agents, and / or chemical agents within the particles maintains a net negative charge on the surface of the particles. The encapsulation of antigens, biological agents, and / or chemical agents in the particles of the present invention can be carried out by any method known in the art. In one embodiment, the polypeptide antigen is encapsulated within the particles by a double emulsion process, hi a further embodiment, the polypeptide antigen is water soluble.

[0086] In another embodiment, the polypeptide antigen is encapsulated in the particle by the single emulsion process. In a further embodiment, the polypeptide antigen is more hydrophobic. In some cases, the double emulsion process leads to the formation of large particles that can lead to leakage of hydrophilic active ingredients and low entrapment efficiency. Coalescence and Ostwald ripening are two mechanisms that can destabilize the droplet size of double emulsions, while diffusion of hydrophilic active ingredients through the organic phase is the main mechanism responsible for low levels of entrapped active ingredients. In some embodiments, it can be beneficial to reduce the nanoparticle size. One way to achieve this is to apply a second intense shear rate. The leakage effect can be reduced by increasing the viscosity of the internal aqueous phase, increasing the molecular mass of the surfactant, and using high polymer concentration and high polymer molecular mass.

[0087] In certain embodiments, the present invention provides a carrier having cells therein (or thereon), or other biological or chemical agents. When cells are used, the carrier is not limited to a particular cell type. In some embodiments, the carrier has pancreatic islet cells thereon. In some embodiments, the microporous carrier additionally has ECM proteins and / or exendin-4 thereon. The carrier is not limited to a particular type. In some embodiments, the carrier has regions of variable porosity (e.g., variable pore sizes, pore depths, and / or pore densities). In some embodiments, the carrier has pharmaceuticals, DNA, RNA, extracellular matrix proteins, exendin-4, etc. thereon (or thereon). In certain embodiments, the present invention provides a method for transplanting pancreatic islet cells with such a carrier. In certain embodiments of the present invention, the inducing antigen is a single isolated molecule or a recombinantly produced molecule. To treat a condition in which the target antigen is scattered in various locations in the host, it is generally necessary that the inducing antigen is identical to or immunologically related to the target antigen. Most examples of such antigens are polynucleotide antigens, but also carbohydrate antigens (such as blood group antigens).

[0088] Any suitable antigen may find use within the scope of the present invention. In some embodiments, the inducing antigen contributes to the specificity of the induced tolerogenic response. The inducing antigen may or may not be the same as the target antigen, which is the target of the undesired immunological response and is present or given to the subject undergoing the treatment to which tolerance is desired.

[0089] In certain embodiments of the present invention, the induced antigen is not in the same form as found in pollen, for example, the same form as the polypeptide from cedar pollen, but in the same form as its fragment or derivative.The induced antigen of the present invention includes peptides based on molecules of appropriate specificity, but adapted by fragmentation, residue replacement, labeling, conjugation, and / or fusion with peptides having other functional properties.Adaptation can be performed for any desired purpose, including, but not limited to, eliminating any undesirable properties, such as toxicity or immunogenicity; or enhancing any desired properties, such as mucosal binding, mucosal penetration, or stimulating the tolerogenic arm of immune response. Terms such as CRYJ1 or CRYJ2, as used herein, refer not only to intact subunits, but also to allotypic and synthetic variants, fragments, fusion peptides, conjugates, and other derivatives that contain regions that are homologous to at least 10, and preferably 20, consecutive amino acids of the respective molecules that are analogs (preferably 70% identical, more preferably 80% identical, and even more preferably 90% identical at the amino acid level), where the homologous regions of the derivatives share with the respective parent molecules the ability to induce tolerance to a target antigen.

[0090] It is recognized that the tolerogenic regions of an inducing antigen are often distinct from the immunodominant epitope for stimulation of an antibody response. Tolerogenic regions are generally regions that can be presented in specific cellular interactions involving T cells. Tolerogenic regions may be present and can induce tolerance when the intact antigen is presented. Some antigens contain cryptic tolerogenic regions in that processing and presentation of native antigens does not normally induce tolerance. Details of cryptic antigens and their identification can be found in International Patent Application Publication No. WO 94 / 27634.

[0091] In certain embodiments of the present invention, two, three or more inducing antigens are used. In the case of multiple target antigens, it may be desirable to realize these embodiments or provide multiple bystanders for the target. It may also be desirable to provide a cocktail of antigens to cover multiple possible alternative targets. For example, a cocktail of CRYJ1 and CRYJ2 fragments, as well as other epitopes derived from cedar pollen, can be used to tolerize subjects. In another example, a mixture of allergens serves as an inducing antigen for the treatment of atopy.

[0092] Inducing antigens can be prepared by several techniques known in the art, depending on the nature of the molecule. Polynucleotide, polypeptide, and carbohydrate antigens can be isolated from cells of the species to be treated in which they are abundant. Short peptides are conveniently prepared by amino acid synthesis. Longer proteins of known sequence can be prepared by synthesizing the coding sequence or by PCR amplification of the coding sequence from a natural source or vector, and then expressing the coding sequence in a suitable bacterial or eukaryotic host cell.

[0093] In certain embodiments of the invention, the combination comprises a complex mixture of antigens obtained from pollen grains, one or more of which act as inducing antigens. The antigens can be in the form of whole cells, either intact or treated with fixatives such as formaldehyde, glutaraldehyde, or alcohol. The antigens can be in the form of lysates created by detergent solubilization or mechanical disruption of cells or tissues followed by clarification. The antigens can also be obtained by subcellular fractionation, in particular enrichment of plasma membranes by techniques such as differential centrifugation, optionally followed by detergent solubilization and dialysis. Other separation techniques such as affinity or ion exchange chromatography of solubilized membrane proteins are also suitable.

[0094] The allergen is another antigen for which tolerance of the immune response is also desired. In one embodiment, the antigen is a polypeptide associated with cedar pollen. In some embodiments, the antigen is CRYJ1. In certain embodiments, the antigen is CRYJ2.

[0095] In one embodiment, the particles of the present invention are bound to an antigen that comprises one or more epitopes associated with allergy, autoimmune disease, and / or inflammatory disease or disorder. The antigen may comprise one or more copies of the epitope. In one embodiment, the antigen comprises a single epitope associated with one disease or disorder. In a further embodiment, the antigen comprises more than one epitope associated with the same disease or disorder. In yet a further embodiment, the antigen comprises more than one epitope associated with different diseases or disorders. In a further embodiment, the antigen comprises one or more epitopes associated with one or more allergies.

[0096] Further non-limiting examples of epitopes associated with allergy to cedar pollen, Table 1. [Table 1-1] [Table 1-2]

[0097] Any suitable antigen related to allergy can find use within the scope of the present invention.In some embodiments, the particles described herein are encapsulated with one or more antigens or epitopes related to allergy.In certain embodiments, the antigen or epitope is related to dust, pet allergen, tree pollen, grass pollen, mold, vegetable allergen, fruit allergen, nut allergen, seed allergen, spice allergen, grain allergen, seafood allergen, meat allergen, nematode allergen, latex allergen, and / or venom allergen.

[0098] In some embodiments, the antigenic epitope derived from cedar pollen comprises Cry j 1, Cry j 2, Cry j 3, Cry j 4, Cry j IFR, Cry j chitinase, Cry j Asp, Cry j LTP, and / or Cry j CPA9, or a fragment or variant thereof. In certain embodiments, the antigenic epitope derived from cedar pollen comprises a CRYJ1 polypeptide, or a fragment or variant thereof. In some embodiments, the CRYJ1 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the antigenic epitope derived from cedar pollen is a fragment of CRYJ1. In some embodiments, the fragment of CRYJ1 comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive amino acids having at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In certain embodiments, the antigenic epitope is a variant of CRYJ1. In certain embodiments, the variant comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity to SEQ ID NO: 1.

[0099] In some embodiments, the antigenic epitope derived from cedar pollen is a fragment of CRYJ1 selected from the group consisting of p16-30, p81-95, p106-120, p111-125, p211-225, and p301-315, which are described in Sone et al., J. Immunol, vol. 161(1) 448-457 (1998), the entire contents of which are incorporated herein by reference.

[0100] In some embodiments, the antigenic epitope from cedar pollen comprises a CRYJ2 polypeptide, or a fragment or variant thereof. In some embodiments, the CRYJ2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the antigenic epitope from cedar pollen is a fragment of CRYJ2. In some embodiments, the fragment of CRYJ2 comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive amino acids having at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In certain embodiments, the antigenic epitope is a variant of CRYJ2. In certain embodiments, the variant comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity to SEQ ID NO:2.

[0101] In some embodiments, the antigenic epitope derived from cedar pollen is a fragment of CRYJ2 selected from the group consisting of p66-80, p81-95, p141-155, p186-200, p236-250, p346-360, p351-365, and p336-350, which are described in Sone et al. (1998).

[0102] In certain embodiments, the particles described herein encapsulate 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more antigenic epitopes derived from cedar pollen.In certain embodiments, 2 or more antigenic epitopes are contained in a fusion protein.In some embodiments, the antigenic epitopes contained in the fusion protein are connected by a cleavable linker.Cleavable linker, as used herein, refers to an amino acid sequence that contains a specific cleavage site.

[0103] In some embodiments, the cleavable linker is 5, 6, 7, 8, 9, 10, more than 10, more than 15, more than 20, more than 25 amino acids in length. In some embodiments, the cleavable linker is cleaved by a protease at a specific site. In certain embodiments, the cleavable linker contains more than one specific site that is cleaved by a protease. In some embodiments, the cleavable linker contains more than one specific site, where the specific sites are cleaved by different proteases. In some embodiments, the cleavable linker contains more than one specific site, where the specific sites are cleaved by the same protease.

[0104] In some embodiments, the cleavable linker is a furin-sensitive linker and / or a cathepsin-sensitive linker. In certain embodiments, the cleavable linker is cleaved at a specific site on the linker by one or more of furin, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, and / or cathepsin Z. In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:3.

[0105] In some embodiments, the fusion protein contains two or more epitopes from the same protein. In certain embodiments, the fusion protein contains two or more epitopes from different proteins. In certain embodiments, the fusion protein contains two or more epitopes from CRYJ1. In certain embodiments, the fusion protein contains two or more epitopes from CRYJ2. In some embodiments, the fusion protein contains epitopes from CRYJ1 and CRYJ2.

[0106] Combinations of antigens and / or epitopes can be tested for their ability to promote tolerance by performing experiments with isolated cells or in animal models.

[0107] In some embodiments, the tolerance-inducing composition of the present invention contains an apoptosis signaling molecule (e.g., in addition to the antigen peptide or other antigen molecule). In some embodiments, the apoptosis signaling molecule is bound and / or associated with the surface of the carrier. In some embodiments, the apoptosis signaling molecule enables the carrier to be recognized as an apoptotic body by antigen-presenting cells of the host, e.g., cells of the host reticuloendothelial system, and thus to present relevant peptide epitopes in a tolerance-inducing manner. Without being bound by theory, it is presumed that this prevents the upregulation of molecules involved in immune cell stimulation, e.g., MHC class I / II, and costimulatory molecules. These apoptosis signaling molecules may also serve as phagocytosis markers. For example, apoptosis signaling molecules suitable for the present invention are described in U.S. Patent Application Publication No. 2005 / 0113297, which is incorporated by reference in its entirety. Molecules suitable for the present invention include molecules that target phagocytes, including macrophages, dendritic cells, monocytes, granulocytes, and neutrophils.

[0108] In some embodiments, molecules suitable as apoptosis signaling molecules act to enhance the tolerance of related peptides. In addition, carriers bound to apoptosis signaling molecules can be bound by Clq in apoptotic cell recognition (Paidassi et al., (2008) J.Immunol.180:2329-2338, incorporated herein by reference in its entirety). For example, molecules that can be useful as apoptosis signaling molecules include phosphatidylserine, annexin-1, annexin-5, milk fat globule-EGF-factor 8 (MFG-E8), or thrombospondin family (e.g., thrombospondin-(TSP-1)). A variety of molecules suitable for use as apoptosis signaling molecules with the present invention are discussed, for example, in US Patent Publication No. 2012 / 0076831, incorporated herein by reference in its entirety).

[0109] In some embodiments, the apoptosis signaling molecule may be conjugated to an antigen-specific peptide. In some instances, the apoptosis signaling molecule and the antigen-specific peptide are conjugated by creating a fusion protein. For example, the fusion protein may comprise at least one antigen-specific peptide (or a fragment or variant thereof) bound to at least one molecule of the apoptosis signaling molecule (or a fragment or variant thereof). With respect to the creation of a fusion protein, the terms "fusion protein", "fusion peptide", "fusion polypeptide", and "chimeric peptide" are used interchangeably. Suitable fragments of antigen-specific peptides include any fragment of the full-length peptide that retains the function of generating the desired antigen-specific tolerance function of the present invention. Fusion proteins may be created by a variety of means understood in the art (e.g., gene fusion, chemical conjugation, etc.). The two proteins may be fused either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically joined C-terminus to N-terminus, but they may also be joined C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein may be in any order. A peptide linker sequence may be used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Amino acid sequences that may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985), Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986), U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180, which are incorporated herein by reference in their entireties. Linker sequences may generally be from 1 to about 50 amino acids in length. In some embodiments, for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that may be used to separate functional domains and prevent steric hindrance, a linker sequence is not necessary and / or is not used.

[0110] A surrogate for tolerogenic activity is the ability of an intact antigen or fragment to stimulate the production of the appropriate cytokine at the target site. The immunoregulatory cytokine released by T immunosuppressive cells at the target site is thought to be TGF-β (Miller et al., Proc. Natl. Acad. Sci. USA 89:421, 1992). Other factors that may be produced during tolerance are the cytokines IL4 and IL-10, as well as the transfer factor PGE. In contrast, lymphocytes in tissues undergoing active immune destruction secrete cytokines such as IL-1, IL-2, IL-6, and IFN-γ. Thus, the efficacy of a candidate inducing antigen can be evaluated by measuring its ability to stimulate the appropriate type of cytokine.

[0111] With this in mind, rapid screening tests for tolerogenic epitopes of inducing antigens, effective mucosally binding components, effective combinations, or effective modes and schedules of mucosal administration can be performed using syngeneic animals as donors for in vitro cell assays. Animals may be treated with the test composition at the mucosal surface and challenged with parenteral administration of the target antigen in complete Freund's adjuvant. Spleen cells are isolated and cultured in vitro in the presence of the target antigen at a concentration of about 50 μg / mL. The target antigen can be replaced with a candidate protein or subfragment, and the location of the tolerogenic epitope can be mapped. Cytokine secretion into the medium can be quantified by standard immunoassays.

[0112] The ability of cells to suppress the activity of other cells can be determined using cells isolated from animals immunized with the target antigen or by creating cell lines that respond to the target antigen (Ben-Nun et al., Eur. J. Immunol. 11:195, 1981, incorporated herein by reference in its entirety). In one variation of this experiment, immunosuppressant cell populations are lightly irradiated (approximately 1000-1250 rads) to prevent proliferation, and the immunosuppressant cells are co-cultured with killer cells, and then tritium-labeled thymidine incorporation (or MTT) is used to quantify the proliferative activity of the killer cells. In another variation, immunosuppressant and killer cell populations are cultured in a dual chamber transwell culture system (Costar, Cambridge Mass.) at higher and lower levels, allowing the populations to coincubate with each other within 1 mm separated by a polycarbonate membrane (WO93 / 16724). In this approach, irradiation of the immunosuppressant cell population is not necessary since the proliferative activity of the killer cells can be measured separately.

[0113] In the embodiment of the present invention where the target antigen is already present in an individual, it is not necessary to isolate the antigen or combine it with the mucosal-binding component beforehand. For example, the antigen may be expressed in a certain manner in an individual as a result of a pathological condition (such as inflammatory bowel disease or celiac disease) or by digestion of a food allergen. The test is carried out by providing the mucosal-binding component in one or more doses or formulations and determining its ability to promote tolerization to the antigen in situ.

[0114] The efficacy and mode of administration of the composition for the treatment of a particular disease can also be detailed in the corresponding animal disease model. The ability of the treatment to reduce or delay the symptomatology of the disease is monitored at the level of circulating biochemical and immunological characteristics of the disease in the model used, immunohistology of the affected tissues, and optionally the overall clinical characteristics. Non-limiting examples of animal models that can be used for testing are included in the following sections. The present invention contemplates the modulation of tolerance by modulating TH1, TH2, TH17 responses, or a combination of these responses. Modulating TH1 responses includes, for example, altering the expression of interferon-gamma. Modulating TH2 responses includes, for example, altering the expression of any combination of IL-4, IL-5, IL-10, and IL-13. Typically, an increase (decrease) in a TH2 response includes an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10, or IL-13, more typically, an increase (decrease) in a TH2 response includes an increase (decrease) in the expression of at least two of IL-4, IL-5, IL-10, or IL-13, and most typically, an increase (decrease) in a TH2 response includes an increase (decrease) in the expression of at least three of IL-4, IL-5, IL-10, or IL-13, but ideally, an increase (decrease) in a TH2 response includes an increase (decrease) in the expression of all of IL-4, IL-5, IL-10, and IL-13. Modulating TH17 includes, for example, changing the expression of TGF-beta, IL-6, IL-21, and IL23, as well as the effect levels of IL-17, IL-21, and IL-22. In some embodiments, the present invention contemplates modulating tolerance by promoting one type of immune response over another type of immune response (e.g., immune switching or immune deviation). In such embodiments, an established immune response of one phenotype is suppressed or reduced and an immune response of a different phenotype is increased or enhanced, for example, immune switching from a Th2 to a Th1 response in an allergic immune response promotes the generation of an IgG2a antibody response and the production of IFNγ and / or IL-12, resulting in a decrease in allergen-specific IgE responses and a decrease in T cell polarization towards Th2 cells.Other suitable methods for assessing the effectiveness of the compositions and methods of the invention will be understood in the art, for example, as discussed in U.S. Patent Application Publication No. 2012 / 0076831, which is incorporated herein by reference in its entirety.

[0115] Certain embodiments of the invention relate to the priming of immune tolerance in individuals not previously tolerized by therapeutic intervention. These embodiments generally involve multiple administrations of a combination of antigen and mucosal binding component. Typically, at least three administrations, frequently at least four administrations, and sometimes at least six administrations are administered during priming to achieve long-lasting results, although the subject may show signs of tolerance early in the treatment course. In most cases, each dose is administered as a bolus administration, although sustained release formulations capable of mucosal release are also suitable. When multiple administrations are administered, the time between administrations is generally between 1 day and 3 weeks, and typically between about 3 days and 2 weeks. Generally, the same antigen and mucosal binding component are present in the same concentration, and administrations are to the same mucosal surface, although variations of any of these variables may be applied during the treatment course. Other embodiments of the invention relate to boosting or extending the persistence of previously established immune tolerance. These embodiments generally involve a single administration or short-term treatment when established tolerance is waning or at risk of waning. Boosting generally occurs one month to one year, and typically two to six months after the prime or previous boost. The invention also includes embodiments involving periodic maintenance of tolerance on a schedule of administration that occurs twice weekly, once weekly, every other week, or on any other regular schedule.

[0116] The particles of the present invention may be provided in any dose effective to attenuate an inflammatory immune response in a subject in need thereof or to treat a bacterial or viral infection in a subject in need thereof. In certain embodiments, the particles of the present invention may be provided in a dose of about 10 2 ~about 10 20 In a further embodiment, about 10 particles are provided to the individual. 3 ~about 10 15In yet a further embodiment, about 10 particles are provided. 6 ~about 10 12 In yet a further embodiment, about 10 particles are provided. 8 ~about 10 10 particles are provided. In a preferred embodiment, the preferred dosage is 0.1% solids / ml. Thus, for 0.5 μm beads, the preferred dosage is approximately 4×10 9 beads, for 0.05 μm beads the preferred dose is approximately 4×10 12 beads, for 3 μm beads the preferred dose is 2×10 7 beads. However, any dosage that is effective to treat the particular condition being treated is encompassed by the present invention.

[0117] The present invention is useful for the treatment of immune-related disorders such as autoimmune diseases, transplant rejection, enzyme deficiencies, and allergic reactions. The substitution of synthetic biocompatible particulate systems to induce immune tolerance may result in ease of manufacturing, wide availability of therapeutic agents, increased uniformity between samples, an increased number of possible treatment sites, and a dramatic reduction in the likelihood of allergic responses to the carrier cells.

[0118] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production, and cytotoxicity. Additionally, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages. Immune cells involved in an immune response include lymphocytes, e.g., B cells and T cells (CD4 + , CD8 +, Th1 cells, and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the modified particles of the present invention are effective in reducing the trafficking of inflammatory cells to sites of inflammation.

[0119] As used herein, the terms "anergy", "tolerance", or "antigen-specific tolerance" refer to the insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen-specific and persists after exposure to antigenic peptides has ceased. For example, anergy in T cells is characterized by the lack of cytokine production, e.g., IL-2. T-cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen results in an inability to produce cytokines (even if re-exposure occurs in the presence of costimulatory molecules) and subsequently in an inability to proliferate. Thus, the inability to produce cytokines prevents proliferation. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells cannot initiate transcription of the DL-2 gene induced by a heterologous promoter under the control of the 5'IL-2 gene enhancer or by a multimer of API sequences that can be found within the enhancer (Kang et al. 1992 Science. 257:1134).

[0120] As used herein, the term "immunological tolerance" in the case of a) reducing the level of a specific immunological response (believed to be at least partially mediated by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or their equivalents), b) delaying the onset or progression of a specific immunological response, or c) reducing the risk of onset or progression of a specific immunological response refers to a method that is performed based on the proportion of treated subjects compared to untreated subjects. "Specific" immunological tolerance occurs when immunological tolerance is preferentially elicited to a certain antigen compared to others. "Non-specific" immunological tolerance occurs when immunological tolerance is elicited indiscriminately to an antigen that leads to an inflammatory immune response. "Semi-specific" immunological tolerance occurs when immunological tolerance is elicited semi-discriminately to an antigen that leads to a pathogenic immune response, but not to other antigens that lead to a protective immune response.

[0121] Tolerance to self-antigens and autoimmune diseases is achieved by various mechanisms, including negative selection of self-reactive T cells in the thymus, and peripheral tolerance to self-reactive T cells that avoid thymic deletion and are found in the periphery. Examples of mechanisms that provide peripheral T cell tolerance include "ignoring" self-antigens, anergy or non-responsiveness to self-antigens, cytokine immune deviation, and activation-induced cell death of self-reactive T cells. In addition, regulatory T cells have been shown to be involved in mediating peripheral tolerance. See, for example, Walker et al. (2002) Nat. Rev. Immunol. 2:11-19; Shevach et al. (2001) Immunol. Rev. 182:58-67. In some situations, peripheral tolerance to self-antigens is lost (or destroyed), resulting in an autoimmune response. For example, in animal models of EAE, activation of antigen-presenting cells (APCs) by TLR innate immune receptors has been shown to break self-tolerance and lead to the induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113:990-997).

[0122] Thus, in some embodiments, the invention provides methods for increasing antigen presentation while suppressing or reducing TLR7 / 8, TLR9, and / or TLR7 / 8 / 9 dependent cellular stimulation. As described herein, administration of certain modified particles results in antigen presentation by DCs or APCs while suppressing TLR7 / 8, TLR9, and / or TLR7 / 8 / 9 dependent cellular responses associated with immunostimulatory polynucleotides. Such suppression can include a reduction in the levels of one or more TLR-associated cytokines.

[0123] As discussed above, the present invention provides novel compounds that have biological properties useful in the treatment of Mac-1 and LFA-1 mediated disorders.

[0124] Thus, in another aspect of the present invention, pharmaceutical compositions are provided that include immune modified particles and, optionally, include a pharma- ceutically acceptable carrier. In certain embodiments, these compositions optionally further include one or more additional therapeutic agents. Alternatively, the modified particles of the present invention can be administered to a patient in need of the modified particles in combination with the administration of one or more other therapeutic agents. For example, the additional therapeutic agent for co-administration with the compounds of the present invention or for inclusion in a pharmaceutical composition having the compounds of the present invention can be an approved anti-inflammatory agent or any one of several drugs in the Food and Drug Administration approval stage that will eventually be approved for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or bacterial or viral infection. It will also be understood that certain modified particles of the present invention can be present in free form for treatment or, if necessary, as a pharma- ceutical acceptable derivative thereof.

[0125] In addition, the pharmaceutical composition of the present invention, as used herein, includes pharma- ceutically acceptable carriers, including any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surface-active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses a variety of carriers used to formulate pharmaceutical compositions and known techniques for their preparation. Use of any conventional carrier medium is contemplated within the scope of the present invention, except insofar as it is incompatible with the compounds of the present invention, such as by producing any undesirable biological effects or interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Some examples of materials which may serve as pharma- ceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propyl alcohol, glycerol, glyceryl stearate, sorbitol ... are included in the composition, but are not limited to, ethyl glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, and coloring agents, releasing agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0126] Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and aromatic agents.

[0127] The particles of the invention may be administered orally, intranasally, intravenously, intramuscularly, intraocularly, transdermally, intraperitoneally, or subcutaneously, hi one embodiment, the particles of the invention are administered intravenously.

[0128] The effective amount and method of administration of the present invention in modulating an immune response can vary based on the individual, the condition being treated, and other factors that will be apparent to one of skill in the art. Factors to consider include the route of administration and the number of doses administered. Such factors are known in the art and can be determined by one of skill in the art without undue experimentation. A suitable dose range is one that provides the desired immune control. A useful dose range of the carrier, as indicated by the amount of carrier delivered, can be, for example, any of the following, about 0.5-10 mg / kg, 1-9 mg / kg, 2-8 mg / kg, 3-7 mg / kg, 4-6 mg / kg, 5 mg / kg, 1-10 mg / kg, 5-10 mg / kg. Alternatively, the dose can be administered based on the number of particles. For example, a useful dose of the carrier, as indicated by the amount of carrier delivered, can be, for example, about 10 6 , 10 7 , 10 8 , 10 9, 10 10 , 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 19 ... th Edition, 1990, Mack Publishing Co., Easton, Pa., USA, which is incorporated by reference in its entirety.

[0129] The effective amount and method of administration of a particular carrier formulation may vary based on the individual patient, the type of outcome and / or disorder desired, the stage of the disease, and other factors evident to one of skill in the art. The route(s) of administration useful in a particular application will be evident to one of skill in the art. Routes of administration include, but are not limited to, topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal, and nasopharyngeal and pulmonary, e.g., transbronchial and transalveolar administration. A suitable dose range is one that provides sufficient IRP-containing composition to achieve a tissue concentration of about 1-50 μM, as measured by blood levels. The absolute amount given to each patient will depend on pharmacological properties such as bioavailability, clearance rate, and route of administration.

[0130] The present invention provides suitable carrier formulations for topical application, including, but not limited to, physiologically acceptable implants, ointments, creams, rinses, and gels. Exemplary routes of dermal administration are the least invasive routes, such as transdermal penetration, epidermal administration, and subcutaneous injection.

[0131] Transdermal administration is accomplished by application of creams, rinses, gels, etc., which allow the carrier to penetrate the skin and enter the bloodstream. Suitable compositions for transdermal administration include, but are not limited to, pharma- ceutically acceptable suspensions, oils, creams, and ointments that are applied directly to the skin or incorporated into a protective carrier, such as a transdermal device (a so-called "patch"). Examples of suitable creams, ointments, etc. can be found, for example, in the Physician's Package Insert. Transdermal permeation can also be accomplished by iontophoresis, for example, using commercially available patches that continuously deliver their products through intact skin over a period of several days or longer. The use of this method allows for controlled permeation of pharmaceutical compositions at relatively high concentrations, allows for infusion of combined drugs, and allows for the simultaneous use of absorption enhancers.

[0132] Parenteral routes of administration include, but are not limited to, direct injection, such as electroinjection (iontophoresis) or direct injection into a central venous catheter, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Formulations of carriers suitable for parenteral administration are generally formulated in USP water or water for injection, and may further include pH buffers, salt bulking agents, preservatives, and other pharmaceutically acceptable excipients. Immunomodulatory polynucleotides for parenteral injection may be formulated in pharmaceutically acceptable sterile isotonic solutions, such as saline and phosphate buffered saline for injection.

[0133] Gastrointestinal routes of administration include, but are not limited to, oral ingestion and rectal routes, and may include, for example, the use of a pharma- ceutically acceptable powder, pill, or liquid for oral ingestion, and suppositories for rectal administration.

[0134] Nasopharyngeal and pulmonary administration are included and are accomplished by inhalation, including delivery routes such as nasal, transbronchial, and transalveolar. The present invention includes formulations of carriers suitable for administration by inhalation, including, but not limited to, liquid suspensions for forming aerosols, and powder forms for dry powder inhalation delivery systems. Devices suitable for administration by inhalation of carrier formulations include, but are not limited to, atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.

[0135] Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents.Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol.Among the acceptable vehicles and solvents that can be used, among others, are water, Ringer's solution, USP, and isotonic sodium chloride solution.In addition, sterile fixed oils are conventionally used as solvents or suspending media.For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides.In addition, fatty acids such as oleic acid are used in injectable preparations.

[0136] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0137] To prolong the effect of a drug, it may be desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by using liquid suspensions or crystalline or amorphous materials with poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0138] In some embodiments, the synthetic biodegradable particles of the invention offer ease of manufacture, broad availability of therapeutic agents, and increased treatment sites. In certain embodiments, surface-functionalized biodegradable poly(lactide-co-glycolide) particles with a high density of surface carboxylate groups, synthesized using the surfactant poly(ethylene-alt-maleic anhydride), provide a carrier that offers numerous advantages over other carrier particles and / or surfaces. Experiments performed during the development of embodiments of the invention have demonstrated that the incorporation of peptides (e.g., PLP) into these particles is highly effective. 139-151 Conjugation of peptides (e.g., SJL / J PLP for multiple sclerosis) has been demonstrated. Such peptide-bound particles have shown that they are effective in preventing disease onset and in inducing immunological tolerance (e.g., SJL / J PLP for multiple sclerosis). 139-151 100% glycerol- ... In one embodiment, the particles of the present invention have a D,L-lactide:glycolide ratio of about 50:50.

[0139] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate and dicalcium phosphate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar-agar, calcium carbonate, , potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0140] Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may optionally contain opacifying agents, and may also have compositions that release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0141] The modified particles may also be in microencapsulated form with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, and starch. Such dosage forms may also contain additional substances other than inert diluents, such as tablet lubricants, and other tableting aids, such as magnesium stearate and microcrystalline cellulose, in normal practice. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents, and may also have a composition that allows them to release only or preferentially the modified particles in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0142] The present invention encompasses pharma- ceutically acceptable topical formulations of the modified particles of the present invention. The term "pharma- ceutically acceptable topical formulation" as used herein means any formulation that is pharma- ceutically acceptable for intradermal administration of the modified microparticles of the present invention by application of the formulation to the epidermis. In certain embodiments of the present invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffer solutions (e.g., hypotonic saline or buffered saline), or any other carriers known in the art for topical administration of pharmaceuticals. A more complete list of carriers known in the art is provided by standard reference texts in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both of which are published by Mack Publishing Company, Easton, Pa., the disclosures of which are incorporated herein by reference in their entirety. In certain other embodiments, the topical formulation of the present invention may include an excipient. Any pharma- ceutically acceptable excipient known in the art may be used to prepare the pharma-ceutically acceptable topical formulation of the present invention. Examples of excipients that may be included in the topical formulation of the present invention may include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, other penetrants, skin protectants, surfactants, and propellants, and / or additional therapeutic agents used in combination with the modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, or organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable moisturizers include, but are not limited to, glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol.Suitable buffers for use with the present invention include, but are not limited to, citric acid, hydrochloric acid, and lactic acid buffers.Suitable solubilizers include, but are not limited to, quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin, and polysorbate.Suitable skin protectants that can be used in the topical formulation of the present invention include, but are not limited to, vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0143] In certain embodiments, the pharma- ceutically acceptable topical formulation of the present invention comprises at least the modified particles of the present invention and a penetration enhancer. The choice of topical formulation depends on several factors, including the condition to be treated or the physicochemical characteristics presented by the compounds of the present invention and other excipients, their stability in the formulation, available manufacturing equipment, and cost constraints. As used herein, the term "penetration enhancer" refers to an agent that can transport pharmacologically active compounds through the stratum corneum and preferably into the epidermis or dermis with little or no systemic absorption. A wide variety of compounds have been evaluated for their effectiveness in enhancing the penetration rate of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which reviews and examines the use of a variety of skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum SI (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplary embodiments, penetrating agents for use with the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0144] In certain embodiments, the composition may be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant, or patch. In certain exemplary embodiments, the formulation of the composition according to the invention is a cream, which may further contain saturated or unsaturated fatty acids, such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl alcohol, or oleyl alcohol, with stearic acid being particularly preferred. The cream of the present invention may also contain a non-ionic surfactant, such as polyoxy-40-stearate. In certain embodiments, the active ingredient is mixed under sterile conditions with a pharma- ceutical acceptable carrier, and any required preservatives or buffers, if required. Ophthalmic formulations, ear drops, and eye drops are also contemplated to be within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the added advantage of providing a controlled delivery of the compound into the body. Such dosage forms are made by dissolving or dispensing the compound in a suitable medium. As discussed above, penetration enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0145] The modified particles can be administered by aerosol. This can be accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the modified particles. Non-aqueous (e.g., fluorocarbon propellant) suspensions can be used.

[0146] Usually, aqueous aerosols are made by formulating aqueous solutions or suspensions of drugs together with conventional pharma- ceutically acceptable carriers and stabilizers.Carriers and stabilizers vary according to the requirements of a particular compound, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycols), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols.Aerosols are generally prepared from isotonic solutions.

[0147] It will also be understood that the modified particles and pharmaceutical compositions of the present invention can be formulated and used in combination therapy, i.e., the compounds and pharmaceutical compositions can be formulated with one or more other desired therapeutic agents or medical procedures, or can be administered simultaneously, before, or after.The combination of specific therapies (e.g., therapies or procedures) to be used in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures, and the desired therapeutic effect to be achieved.It will also be understood that the treatments used can achieve the desired effect on the same disorder (e.g., the compounds of the present invention can be administered simultaneously with another anti-inflammatory agent), or they can achieve different effects (e.g., control of any adverse effects).

[0148] In certain embodiments, the pharmaceutical composition containing the modified particles of the present invention further comprises one or more additional therapeutically active ingredients (e.g., anti-inflammatory and / or palliative drugs). For the purposes of the present invention, the term "palliative" refers to treatment that focuses on reducing the symptoms of a disease and / or the side effects of a treatment regimen, but is not curative. For example, palliative treatment includes analgesics, antiemetics, and antiemetics.

[0149] The present invention provides a method of regulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual a modified particle as described herein. Methods of immune regulation provided by the present invention include methods of suppressing and / or inhibiting innate or adaptive immune responses, including but not limited to immune responses stimulated by immunostimulatory polypeptides, or viral or bacterial components.

[0150] The modified particles are administered in an amount sufficient to regulate an immune response, as described herein, which may be humoral and / or cellular, and is measured as described herein using standard techniques in the art.

[0151] In some embodiments, compositions described herein are administered in conjunction with (e.g., simultaneously with, prior to, or after) implants (e.g., devices) and / or grafts (e.g., tissues, cells, organs) to mediate, negate, control, and / or reduce immune responses associated therewith.

[0152] In certain embodiments, the individual suffers from a disorder associated with undesired immune activation, such as an allergic disease or condition, allergy, and asthma.An individual with an allergic disease or asthma is an individual with a recognizable symptom of an existing allergic disease or asthma.Tolerance can be induced in such an individual, for example, by a particle complex inhalant (e.g., cedar pollen protein) that induces an allergic reaction.

[0153] In some embodiments, the present invention relates to the use of the compositions of the present invention before the onset of allergies, such as allergies to cedar pollen. In certain embodiments, the present invention relates to the use of the compositions of the present invention to inhibit ongoing or existing allergies. In some embodiments, the present invention relates to alleviating allergies or allergic responses in a subject. Alleviating is meant to include treating, preventing, or suppressing allergies or allergic responses in a subject.

[0154] In some embodiments, the compositions of the invention (e.g., PLG carriers bound to antigen molecules associated with cedar pollen allergy) find use with one or more scaffolds, matrices, and / or delivery systems (see, e.g., U.S. Patent Application Publication No. 2009 / 0238879, U.S. Patent Nos. 7,846,466, 7,427,602, 7,029,697, 6,890,556, 6,797,738, and 6,281,256, which are incorporated herein by reference in their entireties). In some embodiments, the particles (e.g., antigen-bound PLG particles) are associated with, adsorbed to, embedded in, or conjugated to a scaffold, matrix, and / or delivery system (e.g., for delivery of chemical / biological materials, cells, tissues, and / or organs to a subject). In some embodiments, scaffolds, matrices, and / or delivery systems (e.g., for delivery of chemical / biological materials, cells, tissues, and / or organs to a subject) comprise and / or are made from materials described herein (e.g., PLG conjugated to one or more antigenic peptides).

[0155] In some embodiments, a microporous scaffold is provided (e.g., for implantation of biological material (e.g., cells, tissues, etc.) into a subject). In some embodiments, a microporous scaffold is provided having thereon an agent (e.g., extracellular matrix protein, exendin-4) and a biological material (e.g., pancreatic islet cells). In some embodiments, the scaffold is used in the treatment of disease (e.g., type 1 diabetes) and related methods (e.g., diagnostic methods, research methods, drug screening). In some embodiments, a scaffold is provided having thereon and / or therein a carrier conjugated to an antigen as described herein. In some embodiments, the scaffold is generated from a material conjugated to an antigen (e.g., PLG conjugated to an antigen).

[0156] In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or has one or more chemical and / or biological entities / agents (e.g., particles conjugated to proteins, peptides, small molecules, cells, tissues, etc.), see, e.g., US Patent Application Publication No. 2009 / 0238879, incorporated herein by reference in its entirety. In some embodiments, antigen-bound particles are co-administered with the scaffold delivery system to induce induction of immunological tolerance to the scaffold and associated materials. In some embodiments, a microporous scaffold is administered to a subject with particles described herein on or in the scaffold. In some embodiments, the antigen-bound particles are bound to the scaffold delivery system. In some embodiments, the scaffold delivery system comprises antigen-bound particles.

[0157] Various modifications, rearrangements, and variations of the described features and embodiments will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes and embodiments that are obvious to those skilled in the relevant arts are intended to be within the scope of the following claims. See, for example, U.S. Application No. 62 / 221,504, PCT Application Nos. PCT / US2016 / 068423, and PCT / US2017 / 012173 (each of which is incorporated by reference herein in its entirety), U.S. Patent Application Publication Nos. 2012 / 0076831, 2002 / 0045672, 2005 / 0090008, 2006 / 0002978, 2009 / 0238879, 2012 / 0076831, 2015 / 02092, and 2016 / 02093. Nos. 93, 2015 / 0283218 (each of which is incorporated by reference herein in its entirety), as well as U.S. Pat. Nos. 7,846,466, 7,427,602, 7,029,697, 6,890,556, 6,797,738, and 6,281,256 (each of which is incorporated by reference herein in its entirety) provide details, modifications, and variations that find use in the various embodiments described herein.

[0158] All publications and patents mentioned in this application and / or listed below are hereby incorporated by reference in their entirety. EXAMPLES

[0159] The following examples are provided to further illustrate the advantages and features of the present invention and are not intended to limit the scope of the disclosure.

[0160] Example 1: Encapsulation of recombinant cedar pollen proteins into TIMPs As mentioned above, the use of particles encapsulating Japanese cedar pollen (JCP) extracts in the treatment of Japanese cedar pollen allergy is limited due to the challenge of encapsulating JCP, which remains highly viscous in solution even at low concentrations. TIMP particles encapsulating JCP (TIMP-JCP) using a double emulsion-solvent evaporation method have been developed. P ) was performed (Figure 2C). Briefly, depending on the batch of JCP extract being tested, 200 μL of 5 mg / mL JCP solution was added to 0.5 mL of 20% w / v PLGA in DCM or 400 μL of 5 mg / mL JCP solution was added to 1.0 mL of 20% w / v PLGA in DCM. Each solution was emulsified by sonication to generate a primary emulsion. PEMA solution (10 mL of 1% w / v aqueous PEMA) was then added to the emulsion and re-emulsified by sonication to generate a secondary emulsion. The emulsion was poured into 200 mL of 0.5% w / v aqueous PEMA while stirring. The particles were purified, lyophilized, and stored at -20 degrees for later use. The antigen dose of the particles was determined as previously described by dissolving the particles in DMSO, followed by CBQCA analysis. Nanoparticle size and zeta potential in water were measured using dynamic light scattering with a Zetasizer Nano ZSP (Malvern Instruments, Westborough, MA). Lot release criteria were TIMP-JCP 700 nm ± 250 nm. P diameter, charged at least -30 mV, and containing at least 5 μg antigen / 1 mg PLGA. The conditions for particle fabrication and the results of JCP encapsulation are shown in FIG.

[0161] Initial attempts to encapsulate whole JCP extracts into TIMPs were only partially successful, as the maximum antigen loading achieved was approximately 1 μg protein / mg TIMP (Figure 4). This could be due to the high viscosity of JCP even at low concentrations (Figure 2A). Therefore, experiments were performed to determine the optimal conditions for encapsulating recombinant JCP proteins (CRY1 and CRY2, Lifeome) into TIMPs (TIMP-JCP®, diagram shown in Figure 2B).

[0162] TIMP-JCP® particles were produced using low molecular weight PLGA (0.17 dL / g), cedar basic protein (CRYJ1, 55,180 Da), and polygalacturonase (CRYJ2, 59,070 Da). The purity of the recombinant protein was assessed by SDS-PAGE (Figure 3C) and compared with the results previously determined (Y. Mitobe et al., Regulatory toxicology and pharmacology: RTP 2015 71). The conditions for TIMP-JCP® particle production are shown in Table 2. [Table 2]

[0163] CRYJ1 or CRYJ2 were encapsulated using the double emulsion protocol for particle formulation and described above (scheme shown in FIG. 2C). Protein concentration and burst release were determined for each sample by CBQCA. The results of these analyses are shown below in Table 3. Particle size and particle charge were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS or ZSP, and the results are shown below in Table 4. [Table 3] [Table 4]

[0164] Example 2: Mouse model of JCP allergy Mouse models with acute allergic responses to inhaled allergens have been widely used to elucidate the mechanisms underlying the immunological and inflammatory responses in asthma and to identify and investigate novel targets for controlling allergic inflammation.

[0165] The quality of the acute inflammation model can be influenced by the choice of mouse strain, allergen, and sensitization and challenge protocol (Figure 5A). Briefly, cedar pollen extract (cedar pollen extract-Cj, LSL-LG5280, Lo#153101, Cosmo Bio USA) was resuspended in MilliQ water at 1 mg / mL. JCP solution was diluted 1:1 in Imject Alum (Thermo Scientific, Cat.#77161) to a final concentration of alum of 20 mg / mL. On day 0, Balb / c mice were intraperitoneally injected with 100 μL of JCP+alum solution (i.e., 50 μg JCP adsorbed to 2 mg / mL alum, n=10). Control mice were injected with alum diluted in PBS (n=10). On day 14, mice were administered a second equivalent dose. On day 21, mice were bled retro-orbitally and serum was collected for antibody analysis (Figures 5B-5D). On day 22, some mice from each group were tolerized with a single 2.5 mg dose of either TIMP-OVA (n=5) or TIMP-JCP (n=4) administered intravenously via the tail vein. In addition, mice were tolerized with TIMP-OVA or TIMP-JCP and then sensitized with JCP.

[0166] On days 28 and 29, mice were challenged intranasally with 100 μg of JCP extract in 20 μL of MilliQ water. On day 29, mice were monitored for scratching (FIG. 7) and temperature changes (FIG. 6) for 1 hour after challenge. After 1 hour, blood was collected to assess serum levels of MCPT-1 (FIG. 10B) and histamine (FIG. 10A).

[0167] On day 30, mice were bled again for analysis of antibodies in serum (Figures 9A-9C). Mice were then sacrificed and spleens were collected from each mouse and homogenized for ex vivo cytokine analysis and proliferation (Figure 8). Spleens were collected, processed into red blood cell-free single cell suspensions, and incubated in 1, 25, or 50 μg / mL JCP complete RPMI culture medium for 48 hours at 37°C. 50 μL of supernatant was removed for cytokine analysis, and cells were then pulsed with 1 μCi / well [3H]TdR for the last 24 hours of culture. Proliferation was determined by incorporation of [3H]TdR, detected by a Topcount Microplate Scintillation Counter (PerkinElmer, Waltham, MA).

[0168] These data show that treatment with JCP-TIMP after JCP sensitization significantly reduced the production of IFNγ, IL-17, and IL-5 in ex vivo assays. Similar results were observed for IL-4, IL-10, and IL-13 levels when splenocytes were stimulated with higher doses of JCP. Antibody levels were unchanged after TIMP treatment.

[0169] Example 3: Mouse model of JCP allergy and treatment with CRYJ1 and CRYJ2 TIMPs Allergic airway inflammation model Mice were immunized intraperitoneally (ip) with two doses of 10 μg of CRYJ1 and CRYJ2, a large amount of cedar pollen allergen in alum (3 mg), or alum and PBS alone. Mice were then challenged with aerosolized CRYJ1 and CRYJ2 (10 mg / ml) for 20 min on three consecutive days, after which tissues were collected.

[0170] Analysis of eosinophils in bronchoalveolar lavage fluid Lungs are flushed with 1 mL of bronchoalveolar lavage fluid (BALF, 1 mM EDTA and 10% FBS in PBS). Total cell counts are determined and samples are cytospun onto slides and stained with DiffQuik (Siemens, Newark, Del.) for differential cell counts.

[0171] Airway histology Lungs are harvested, formalin fixed, and processed into paraffin. Paraffin sections are stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS).

[0172] JCP-specific IgE After sacrifice, serum is collected from the mice and CRYJ1 / 2-specific IgE is quantified by sandwich ELISA using anti-mouse IgE (BD Biosciences) as the capture antibody and biotinylated CRYJ1 / 2 (prepared using the EZ-Link Sulfo-NHS-LC-Biotin kit from Pierce, Rockford, Ill.) as the secondary reagent. The amount of CRYJ1 / 2-IgE is determined by a standard curve generated using purified mouse CRYJ1 / 2-IgE.

[0173] Cytokine quantification BAL fluids and supernatants from re-response cultures (collected at 48 hours) will be assayed for IL-4, IL-5, IL-13, IL-10, IL-17, and IFNγ by magnetic Milliplex MAP multiplex assays (Millipore, Billerica, Mass.).

[0174] Production and / or source of cedar pollen antigens Purified cedar pollen extract is sourced from the Japanese Aerology Society. In addition, recombinant cedar pollen antigens are produced as previously described (Fujimura T, Int. Arch. Allergy Immunol. 2015; 168(1): 32-43). The amino acid sequences of the immunodominant epitopes in JCP are CRYJ1 (Accession No. AB081309, SEQ ID NO: 1) and CRYJ2 (Accession No. AB211810, SEQ ID NO: 2).

[0175] Production of research batches of TIMP-JCP Recombinant JCP antigen is encapsulated in TIMP as described above. For efficacy testing in a mouse model of JCP allergy, 200 mg of TIMP-CRYJ1 and / or CRYJ2 (TIMP-JCP®) and 200 mg of TIMP-OVA control are used. Six groups of 5-10 mice are treated with TIMP-OVA or TIMP-JCP®, given either a one-dose (groups 1-3) or two-dose (groups 3-6) regimen. Animals are sensitized to JCP antigen (either before or after TIMP treatment). On days 28 and 29, airway challenge is performed and efficacy of treatment is determined using the primary readout and success metrics described below. Note that mice in group 7 are challenged with OVA instead of JCP to sensitize these mice to OVA and serve as positive allergy controls.

[0176] Animal POC: TIMP-JCP treatment before sensitization As shown in Table 5, six groups of 5-10 mice are treated with TIMP-OVA or TIMP-JCP®, given either a one dose (groups 1-3) or two dose (groups 4-6) regimen. The animals are then sensitized to JCP antigen. On days 28 and 29, airway challenge is performed and efficacy of treatment is determined using the primary readout and success metrics described below. Note that mice in group 7 are challenged with OVA instead of JCP to sensitize these mice to OVA and serve as positive allergy controls. [Table 5]

[0177] Animal POC-TIMP-JCP treatment after sensitization Six groups of mice are treated with TIMP-JCP® or TIMP-OVA after sensitization to JCP, as shown in Table 6 below. Two dose regimens are tested, including a single TIMP dose regimen (groups 1-3) and a double dose regimen (groups 4-6), separated by 7 days. Air challenge is performed on days 28 and 29, and efficacy of treatment is determined using the primary readout and success metrics described below. [Table 6]

[0178] Primary readouts and success metrics in animal models Primary readout and success metrics are measured in mice treated with TIMP-JCP® before sensitization. Mice treated with TIMP-JCP® have no temperature change after air challenge compared to baseline temperature. There is no change in blood eosinophil and mast cell counts compared to baseline metrics in mice treated with TIMP-JCP®. Mice treated with TIMP-JCP® have significantly reduced JCP-specific IgE compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® also have significantly reduced systemic IL-4, IL-5, and IL-13 compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® are compared to mice treated with TIMP-OVA.

[0179] Primary readout and success metrics are measured in mice treated with TIMP-JCP® after sensitization. Mice treated with TIMP-JCP® have no temperature change compared to baseline temperature after air challenge. There is no change in blood eosinophil and mast cell counts compared to baseline metrics in mice treated with TIMP-JCP®. Mice treated with TIMP-JCP® have significantly reduced JCP-specific IgE compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® also have significantly reduced JCP-specific systemic IL-4, IL-5, and IL-13 compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® also have reduced specific antigen production compared to mice treated with TIMP-OVA.

[0180] Example 4: Clinical study on TIMP-JCP A clinical study will be conducted to evaluate the efficacy and safety of TIMP-JCP in a single-center, open-label, placebo-controlled study of Japanese patients with seasonal allergic rhinitis (SAR) exposed to quantitative high-density cedar pollen.

[0181] The first objective of the clinical study was to analyze and evaluate the changes in total nasal symptom score (TNSS) in Japanese SAR patients treated with oral TIMP-JCP during high-intensity exposure to Japanese cedar pollen.

[0182] The secondary objectives were to analyze and evaluate the changes in total symptom score (TSS), symptom score, amount of nasal discharge, number of sneezes, and patient impression in patents with allergic rhinitis, as well as to evaluate the safety of TIMP-JCP administered via intravenous route in patients with high-density exposure to pure cedar pollen.

[0183] General Research Plan An Environmental Exposure Unit will be used to determine the safety and efficacy of TIMP-JCP in patients allergic to Japanese cedar pollen. Although many Environmental Exposure Units (EEUs) exist, this study will be conducted in collaboration with the Japan Health Support Network Unit in Wakayama Prefecture. Unconstrained by many of the limitations of traditional methods, EEUs allow for rigorous controlled studies to be designed and carried out (a similar study design is described by Enomoto, et al., 2009). The diagram below outlines the dosing and event schedule.

[0184] subject There will be a total of 25 subjects in the clinical study. 5 subjects will be treated with placebo. 20 subjects will be treated with TIMP-JCP.

[0185] Diagnosis and inclusion criteria Inclusion Criteria: Japanese patients with SAR. -Patients with a history of cedar pollen allergy symptoms for at least 2 years. Positive IgE (against cedar pollen antigens): determined by fluorescent enzyme immunoassay (FEIA) or chemiluminescent enzyme immunoassay (CLEIA) within 1.5 years prior to the screening exposure test date. Patients with a TNSS score of 8 or greater and a nasal congestion score of 2 (moderate) or greater at at least one assessment point 90-150 minutes after the start of the screening exposure. (TNSS) and nasal congestion scores may be at any other assessment point (if criteria are met). Age ≥ 20 and ≤ 65 years (any sex). Patients who signed an informed consent form.

[0186] Exclusion criteria -Patients with perennial allergic rhinitis symptoms. Patients with severe asthma, bronchiectasis, severe hepatic, renal or cardiac dysfunction, blood disease, endocrine disease, and other severe coexisting conditions. Patients with nasal diseases (hypertrophic rhinitis, sinusitis, nasal polyps, deviated nasal septum, etc.) or eye diseases that may interfere with the assessment of the efficacy of TIMP-JCP. Patients with evidence of upper and / or lower respiratory tract infection (acute rhinitis in the presence of a cold, etc., chronic rhinitis, congestive rhinitis, atrophic rhinitis, purulent nasal discharge, sinusitis) on the day of treatment exposure. Patients taking any of the following medications (including anti-contestants and immunomodulatory agents) that may affect the evaluation of TIMP-JCP.

[0187] Additional exclusion criteria:

[0188] Within 2 weeks prior to the first dose: Antiallergic drugs, antihistamines (H1 and H2 blockers: oral, nasal, eye, injection, and topical), anticholinergics, vasoconstrictor nasal sprays, antihistamine-containing cold medicines, medicines that may be expected to have antiallergic / antihistamine effects (including herbal medicines and glycyrrhizin), and other medicines indicated for allergic symptoms (such as sneezing, rhinorrhea, stuffy nose, and itchy eyes). Steroids (oral, inhaled, nasal, eye drops, or topical), immunosuppressants (oral, topical, or injectable), azole fungicides, and histamine-containing gamma globulin preparations. Preparations containing azole fungicides, macrolide antibiotics, and aluminum hydroxide / magnesium hydroxide.

[0189] Within 4 weeks prior to the screening exposure test date: Depot steroid preparations.

[0190] Within 6 months prior to the screening exposure test: steroid injection. Steroid injections

[0191] Within one year prior to the screening exposure date: Patients receiving specific desensitization maintenance therapy or non-specific alternative therapy. Patients who are participating in another study or who have previously participated in another study within 6 months prior to informed consent. Patients who are deemed by the Investigator / Sub-Investigator to be unsuitable for participation in this study with respect to any other criteria. ·Patients with a history of hypersensitivity to antihistamines or antihistamine drugs (including fexofenadine HCI) and pseudoephedrine hydrochloride. Patients participating in another study or who have previously participated in another study within 6 months prior to the screening exposure test date. Women who are pregnant, may be pregnant or are currently breastfeeding.

[0192] Safety and Stopping Criteria

[0193] The study may be placed on hold at the discretion of the Data Safety Monitoring Board if any of the following parameters are met: a) One or more unexpected drug-related serious adverse events (SAEs) are reported to the Data Safety Monitoring Board. b) Excessive and / or unexpected adverse events not clearly related to the study drug. c) Unexpected patient death(s).

[0194] Dose, duration, and mode of administration: Drug: TIMP-JCP, poly(lactic-co-glycolic acid) tolerogenic immune modulation

[0195] Dosage and Form: TIMP-JCP will be provided as a sterile, lyophilized white powder in vials. Each vial will contain 500 mg of TIMP-JCP. TIMP-JCP will be given at a dose of [adjusted for Phase 1 results - initial assumption is 10 mg / kg].

[0196] Treatment / Study Duration: Patients are enrolled after patient screening and informed consent. Medication begins after recruitment and entry into the study. Medication is administered on days 0 and 14. The first high-density Japanese cedar pollen exposure occurs on days 18-21 (patients are exposed to 8,000 grains / m2 in an environmental exposure unit). 3 The second high-density cedar pollen exposure will occur on days 32-35 (patients will be exposed to 8,000 grains / m3 of JCP for 5 hours in an environmental exposure unit). Patients will be visited for follow-up on days 42, 49, and 56. A final follow-up visit will be made on day 63, 4 weeks after the second exposure.

[0197] Mode of administration: Reconstituted TIMP-JCP is diluted in normal saline (NS, 0.9% sodium chloride, NaCl) by slow IV infusion over 30 minutes. TIMP-JCP can be stopped at any time if the investigator determines that an alternative therapy should be administered.

[0198] Evaluation criteria Safety: Incidence and severity of AEs and AEs of special interest (including standard reporting criteria for hypersensitivity and immune-mediated reactions). Vital signs (axillary temperature, blood pressure in sitting position, and pulse rate in sitting position) Standard laboratory evaluation 12-lead ECG Lab Tests (Hematology, Biochemistry, and Urine Analysis) Cedar pollen prick test · Parameters generated by antibody and histamine release tests.

[0199] Primary Endpoint:

[0200] TNSS: Total score for sneezing, rhinorrhea, nasal congestion, and nasal itching. Each symptom is rated by the patient in five categories: 1=none (no symptoms), 2=mild (symptoms present, easily tolerated), 3=moderate (symptoms recognized, bothersome but tolerable), 4=severe (symptoms clearly recognized, intolerable but not interfering with daily activities), and 5=very severe (intolerable, interfering with daily activities).

[0201] Secondary Efficacy Endpoints:

[0202] Immune surveillance: ·Antibodies: IgG antibody, specific IgG antibody (anti-JCP, anti-CRYJ1, and anti-CRYJ2), specific IgG4 antibody (anti-JCP), IgE antibody, specific IgE antibody (anti-JCP) Cytokines (IFN-gamma, IL-4, IL-5, IL-10, IL-12, and IL-13) Immune cell phenotype (Tregs, effector T cells, and monocytes) Peripheral B and T cell responses to Japanese cedar pollen. Changes in TSS, changes in each symptom score, amount of nasal discharge, number of sneezes, and patient impression. Amount of nasal mucus: The weight of the tissue after the patient blows their nose is measured every hour. The difference in weight from before blowing the nose is calculated as the amount of nasal mucus. Number of sneezes: The patient should record the number of sneezes they have per hour.

[0203] Pharmacokinetics (PK): Serum concentrations of TIMP-JCP measured daily in a small sampling schedule during study drug administration.

[0204] result:

[0205] Subjects treated with TIMP-JCP show a reduction in the occurrence and severity of allergic reactions to cedar pollen exposure. This is demonstrated by the reduction in TNSS in subjects treated with TIMP-JCP compared to subjects treated with placebo. Subjects treated with JCP also have reduced levels of specific IgG antibodies (anti-JCP, anti-CRYJ1, and anti-CRYJ2), specific IgG4 antibodies (anti-JCP), IgE antibodies, specific IgE antibodies (anti-JCP), reduced peripheral B and T cell responses to cedar pollen, and lower TSS scores, symptom scores, amount of nasal discharge, and number of sneezes compared to placebo group.

[0206] While particular embodiments of the present invention have been described and illustrated, such embodiments should be considered merely as illustrative of the invention and not as limiting the invention, as interpreted according to the appended claims.

[0207] All patents, applications, and other references cited herein are incorporated by reference in their entirety.

Claims

[Claim 1] The invention described in this specification.

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