KR20180105243A - TIMPS encapsulating Japanese cedar pollen epitope - Google Patents

Abstract

The present invention provides a composition comprising particles having negative zeta potential encapsulating at least one epitope associated with Japanese cedar pollen. A method of inducing immunological tolerance to Japanese cedar pollen by administering the particles is also provided.

Description

TIMPS encapsulating Japanese cedar pollen epitope

Cross-reference to related application

This application claims priority from U.S. Provisional Application No. 62 / 293,261, filed Feb. 9, 2016, the entire contents of which are incorporated herein by reference.

Description of the electronic submission text file

The contents of an electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable formatted copy of the sequence listing (filename: COUR_014_02WO_SeqList_ST25.txt, Record Date: Feb. 9, 2017, File Size : 12kb).

Japanese cedar pollen hypersensitivity is a common allergic diseases in Japan, which is caused by inhaling the pollen of Japanese cedar (crypto-Almeria artillery NIKA (Cryptomeria japonica)). The prevalence of Japanese cedar pollen hypersensitivity can be up to 30% in randomized populations, not individuals in schools, factories and communities, and up to 20 million people are estimated to be infected with this disease. Thus, the disease is a major public health problem in Japan in part because of the high severity of symptoms, high prevalence, reduced spontaneous recovery and high medical costs associated with disease control.

Japanese cedar pollen hypersensitivity is a severe type I allergic disease. Type I allergic diseases are harmless environmental antigens characterized by elevated systemic levels of allergen-specific immunoglobulin E (IgE), mast cell degranulation and release of other chemical mediators of histamine and allergy Cedar pollen) by the abnormal Th2-polarized immune response. The major clinical approaches to the treatment of allergic diseases generally include antigen (Ag) avoidance (e. G., Allergy antigen avoidance) by targeting acute effector molecules to drugs such as antihistamines, leukotriene inhibitors or broad-acting glucocorticoids Symptom control. However, these therapies do not address the development of underlying abnormal Th2-biased immune responses that cause allergic inflammation. Alternative approaches such as specific immunotherapy (SIT) in which patients are exposed to a slowly increasing dose of soluble Ag delivered mucosally or subcutaneously are also used clinically. SIT induces both Th1 responses and regulatory inhibition of Th2 responses. Thus, SIT may result in deviations in the immune response to environmental allergens from a pathological, Th2-deflected response toward a protective or regulated Th1 / T regulatory response. However, the administration of soluble Ag to sensitized patients is associated with a high risk of side effects, and concomitant use of antipsychotic drugs or slow dose escalation of additional drugs to minimize hypersensitivity such as omalizumab is needed.

Thus, an improved method of safely and effectively inducing Ag-specific resistance remains a clinical tool for the treatment of allergic diseases in pre-sensitized subjects. Nanoparticles encapsulating allergenic peptide have already been demonstrated to reduce allergen-induced Th2 responses in vivo models (see US Publication No. 2015-0209293, which is incorporated herein by reference in its entirety). Thus, it is possible to encapsulate pollen derived from various environmental sources into nanoparticles for use in treating allergic diseases such as Japanese cedar pollen hypersensitivity. In addition, Japanese cedar pollen antigen CRYJ1 represents a mountain cedar Jun a 1 antigen, Cha o Japanese cypress (cypress) 1 antigen and cue presentation suspension ahrijo NIKA high similarity with respect to the Cup a 1 antigen (Cupressus arizonica), which Suggesting that certain antigens extracted from Japanese cedar pollen may mediate cross-reactive allergic antibody responses across multiple conifer pollen.

However, attempts to encapsulate Japanese cedar pollen as particles suitable for the treatment of allergic diseases were only partially successful. This may be because Japanese cedar pollen is very viscous at low concentrations. Therefore, the treatment of SIT for cedar pollen hypersensitivity is limited. In particular, considering the possibility of antibody cross-reactivity between conifer pollen, as well as the actual prevalence of the disease in Japan and neighboring countries, there is a possibility that cedar pollen antigen (eg, For example, there is a need for safe and effective means of inducing tolerance to CRYJ1 and CRYJ2.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides compositions (e. G., For induction of antigen-specific resistance) that comprise one or more epitopes from Japanese cedar pollen or that contain carrier particles (e. G., PLG particles) . In certain embodiments, the carrier particle is a poly (lactide-co-glycolipid) (PLG) particle having a negative zeta potential.

Certain embodiments of the invention are compositions comprising biodegradable particles comprising at least one encapsulated antigenic epitope 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 having 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, the carboxylation is accomplished using poly (ethylene-maleic anhydride) (PEMA) or poly (acrylic acid) (PAA).

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.

In certain embodiments, the biodegradable particles have a diameter of from about 0.1 microns to about 10 microns. In some embodiments, the biodegradable particles have a diameter of about 0.3 microns to about 5 microns. In certain embodiments, the biodegradable particles have a diameter of about 0.5 [mu] m to about 3 [mu] m. In certain embodiments, the biodegradable particles have a diameter of about 0.5 [mu] m to about 1 [mu] m. In some embodiments, the biodegradable particles have a diameter of about 0.2 [mu] m to about 0.7 [mu] m. In certain embodiments, the biodegradable particles have a diameter of about 0.5 占 퐉.

Certain embodiments of the invention are directed to compositions comprising biodegradable particles having negative zeta potential comprising one or more encapsulated antigenic epitopes from Japanese cedar pollen. In some embodiments, the at least one encapsulated antigenic epitope from Japanese cedar pollen is selected from the group consisting of 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 at least one encapsulated antigenic epitope from Japanese cedar pollen comprises CRYJ1 or a fragment or variant thereof. In some embodiments, CRYJ1 is

Lt; / RTI >

In some embodiments, the fragment of CRYJ1 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 1. In certain embodiments, a variant of CRYJ1 has 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: ≪ / RTI > In some embodiments, the fragment of CRYJ1 is selected from the group consisting of p16-30, p81-95, p106-120, p111-125, p211-225, and p301-315.

In certain embodiments, the at least one encapsulated antigenic epitope from Japanese cedar pollen comprises CRYJ2 or a fragment or variant thereof. In some embodiments, CRYJ2 is

Lt; / RTI >

In some embodiments, the fragment of CRYJ2 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 2. In certain embodiments, a variant of CRYJ2 has 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: ≪ / RTI > In some embodiments, the 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.

In certain embodiments, the biodegradable particles described herein comprise two or more encapsulated antigenic epitopes from Japanese cedar pollen protein. In some embodiments, two or more encapsulated epitopes are contained in the fusion protein, wherein the two or more encapsulated 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 phagocytic lysosome of the cell and / or in the cytosol of the cell. In some embodiments, the amino acid sequence of the cleavable linker is cleavable by a protease located in the phagocytic lysosome of the cell and a cytosol in the cytosol of the cell. In certain embodiments, the cleavable linker is a purine susceptible linker or a cathepsin susceptible linker. In certain embodiments, the cleavable linker is a purine susceptible linker. In some embodiments, the cleavable linker is a cathepsin-sensitive linker. In certain embodiments, the cathepsin-sensitive linker is selected from the group consisting of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, It is sensitive to cleavage by one or more of 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).

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

Certain embodiments relate to pharmaceutical compositions comprising the biodegradable particles described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Certain embodiments relate to lyophilized compositions comprising the biodegradable particles described herein.

Certain embodiments relate to methods of inducing antigen-specific tolerance to Japanese cedar pollen in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described herein. A particular embodiment relates to a method of treating Japanese cedar pollen allergy in a subject in need of treatment comprising administering a pharmaceutical composition as described herein. Some embodiments relate to a method of preventing Japanese cedar pollen allergy in a subject in need of treatment comprising administering a pharmaceutical composition as described herein.

Certain embodiments are directed to a method of inhibiting antigen-antibody interaction in Japanese cedar pollen from a subject, comprising reconstituting the lyophilized particles described herein to obtain a reconstituted pharmaceutical composition, and administering the reconstituted pharmaceutical composition to the subject. Lt; RTI ID = 0.0 > resistance. ≪ / RTI > Certain embodiments relate to a method of treating a subject in need of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of formula The present invention relates to a method for treating cedar pollen allergy. Some embodiments relate to a method of treating a subject in need of treatment, comprising reconstituting the lyophilized particles described herein to obtain a reconstituted pharmaceutical composition and administering the reconstituted pharmaceutical composition to a subject, This invention relates to a method for preventing allergy to cedar pollen.

Figure 1 shows a model of how the administration of biodegradable particles with negative zeta potential to encapsulate an antigenic epitope from Japanese cedar pollen provides effective treatment of Japanese cedar pollen allergy.
Figure 2 shows the viscosity of the JCP extract (Figure 2a), an illustrative illustration of biodegradable particles (TIMP-JCP) with negative zeta potential encapsulating the antigenic epitope of Japanese cedar pollen (Figure 2b), and the dual- (Fig. 2C).
Figure 3 shows the results of SDS-PAGE analysis of JCP extract and recombinant JCP protein.
Figure 4 shows particle characteristics for the TIMP encapsulated JCP extract.
Figure 5 shows a schematic (Figure 5a) of an acute inflammatory mouse model and an antibody response (Figures 5b-5d) on day 21 after sensitization.
Figure 6 shows body temperature changes after JCP sensitization, TIMP treatment and JCP challenge.
Figure 7 shows scratching, sneezing and cough scores after JCP sensitization, TIMP treatment and JCP challenge.
Figure 8 shows cytokine production from spleen cells from JCP sensitized and challenged mice treated with TIMP-JCP or TIMP-OVA.
Figure 9 shows the antibody response on day 30 after sensitization by JCP.
Figure 10 shows serum levels of histamine (Figure 10a) and MCPT-1 (Figure 10b).
Figure 11 illustrates the effect of the route of administration on the obtained immune response.

The present inventors have found that antigen-embedded nanoparticles can induce tolerance to autoimmune diseases and reduce the immune response. Thus, these particles may be useful in the treatment of any disease or disorder characterized by an excessive inflammatory immune response, such as allergy to Japanese cedar pollen.

As used herein, "particle" refers to any non-tissue-derived composition of matter and may be spherical or spheroid-like independent, bead or liposome. The term " particle ", the term "immunostimulatory particle ", the term" carrier particle "and the term" bead "may be used interchangeably depending on the context. In addition, the term "particle" may be used including beads and spheres.

As used herein, "negatively charged particle" refers to a modified particle having a net surface charge of less than zero.

"Carboxylated particle" or "carboxylated bead" or "carboxylated spheroid" includes any particle modified to contain a carboxyl group on its surface. In some embodiments, the addition of a carboxyl group improves the phagocyte / mononuclear absorption of the particle from the circulation through interaction with, for example, capture agent receptors such as MARCO. Carboxylation of the particles can be accomplished using any compound that adds or incorporates a carboxyl group, including, but not limited to, poly (ethylene-alt-maleic anhydride) (PEMA).

As used herein, "antigenic moiety" refers to any moiety, e. G., A peptide that is recognized by the immune system of the host. Examples of antigenic moieties include, but are not limited to, autoantigens, enzymes and / or bacterial or viral proteins, peptides, drugs or components. Without being bound by theory, the carboxylated bead itself can be recognized by the immune system, but the carboxylated bead to which nothing is attached is not considered an "antigenic moiety" for purposes of the present invention.

As used herein, "naked beads" or "naked particles" or "naked spheres" refers to non-carboxylated beads, particles or spheres.

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

As used herein, the term "inflammatory mononuclear" refers to any bone marrow cell expressing any combination of CD14 / CD26 and CCR2.

As used herein, the term "inhibitory neutrophil" refers to neutrophils and / or monocytes that induce inhibitory cells.

As used herein, the term "Th cell" or "helper T cell" refers to a CD4 ⁺ cell. CD4 ⁺ T cells help other leukocytes through immune processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. T cells are activated when presented by peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

As used herein, the term "Th1 cell" refers to a subset of Th cells that produce a pre-inflammatory mediator. Th1 cells partly play a role in host defense against pathogens by secreting cytokines that stimulate the immune response and mediating the mobilization of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines including IFN-y, IL-2, IL-10 and TNFa / beta to modulate defense against intracellular pathogens such as viruses and some bacteria.

As used herein, the term "Th2 cell " refers to a subset of Th cells that mediate the activation and maintenance of an antibody-mediated immune response to extracellular parasites, bacteria, allergenic antigens and toxins. Th2 cells produce various cytokines such as IL-4, IL-5, IL-6, IL-9, IL-13 and IL-17E Thereby providing a phagocyte-independent, protective response mediated by the function so that inhibition of function is possible.

As used herein, the term "Th17 cell" refers to a subset of Th cells. Th17 cells play a role in host defense against pathogens by secreting cytokines to stimulate the immune response and mediating mobilization of neutrophils and macrophages into infected tissues. TH17 cells secrete cytokines such as IL-17, IL-21, IL-22, IL-24, IL-26 and TNFα to regulate defense against extracellular pathogens including fungi and bacteria.

As used herein, "coupled" refers to an antigen immobilized outside the particle or encapsulated within the particle. Thus, the antigen coupled to the particle includes both intra-particle encapsulation as well as surface coupling.

As used herein, the term "IMP " refers to immunostimulatory particles that are not coupled to an antigen. As used herein, the term "TIMP" refers to tolerating immunostimulatory particles coupled to an antigen. In some embodiments, the antigen is attached to the surface of the TIMP. In another embodiment, the antigen is encapsulated within a TIMP.

The particles may have any particle shape or shape. However, in some embodiments, it is desirable to use less agglomerated particles in vivo. Examples of particles of these embodiments are particles having a spherical shape.

Another aspect of the invention relates to a composition comprising an immunostimulatory particle having a negative zeta potential and no antigenic moiety. In a further embodiment, the invention provides a composition comprising an immunostimulatory particle having a negative zeta potential coupled to an antigen. In a further embodiment, the antigen is coupled to the outside of the particle. In a preferred embodiment, the antigen is encapsulated in a particle.

Another aspect of the present invention relates to a method for producing an immunostimulated particle having a negative zeta potential and no antigenic moiety. The method comprises contacting the immunostimulated particle precursor with a buffer under conditions effective to form an immunostimulated particle having a negative zeta potential. In some embodiments of the invention, the immunostimulated particle precursor is formed through copolymerization. The particle microstructure can depend on the method of copolymerization.

In some embodiments, the antigenic peptide molecule is coupled to a carrier particle (e. G., An immunostimulated particle) by a conjugate molecule and / or a linker group. In some embodiments, the step of coupling the antigenic peptide and / or the apoptotic signaling molecule to a carrier (e. G., A PLG particle) comprises one or more covalent and / or non-covalent interactions. In some embodiments, the antigenic peptide is attached to the surface of the carrier particle having a negative zeta potential. In some embodiments, the antigenic peptide is encapsulated within a carrier particle having a negative zeta potential.

In one embodiment, the buffer in contact with the immunostimulated particles may have an alkaline pH. Suitable basic pHs for basic solutions include 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. Buffers can also be prepared with any suitable base and its conjugates. In some embodiments of the invention, the buffer solution includes, but is not limited to, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogenphosphate, sodium dihydrogenphosphate or lithium dihydrogenphosphate and conjugates thereof.

In one embodiment of the invention, the immunostimulated particles contain a copolymer. These copolymers can have various molar ratios. Suitable copolymer ratios of the immunostimulated particles of the present invention are 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: In another embodiment, the copolymer may be a periodic, statistical, linear, branched (including star, brush or comb copolymer) copolymer. In some embodiments, the copolymer ratio is selected from the group consisting of polystyrene: poly (vinylcarboxylate) / 80:20, polystyrene: poly (vinylcarboxylate) / 90:10, poly (vinylcarboxylate): polystyrene / Vinyl carboxylate): polystyrene / 90:10, polylactic acid: polyglycolic acid / 50: 50, polylactic acid: polyglycolic acid / 80: 20, or polylactic acid: polyglycolic acid / 90: , But is not limited thereto.

In one embodiment, the particles of the present invention are prepared 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 may be from about 0.1% to about 10%. In one embodiment, the concentration of PEMA in solution is from about 0.2% to about 5%. In another embodiment, the concentration of PEMA in solution is from about 0.1% to 4%. In another embodiment, the concentration of PEMA in solution is from about 0.1% to 2%. In another embodiment, the concentration of PEMA in solution is from about 0.5% to 1%. In one embodiment, the percentage of PEMA in solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2% %, 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 solution is about 0.5%. In another embodiment, the percentage of PEMA in solution is about 1.0%. Other compounds which may be used are poly (ethylene - alt - maleic anhydride), poly (isobutylene - co - maleic acid), poly (methyl vinyl ether - alt - maleic acid), poly (methyl vinyl ether - alt - maleic acid diethyl ester), poly (methyl vinyl ether - alt - maleic anhydride), 1,9-decadiene powder and crosslinked poly (methyl vinyl ether - alt - maleic anhydride), poly (styrene - alt - maleic But are not limited to, sodium salt, poly (vinyl alcohol), poly (acrylic acid), and / or sodium deoxycholate.

In one embodiment, the particle is a liposome. In a further embodiment, the particles are liposomes composed of the following lipids in the molar ratio: 30:30:40 phosphatidylcholine: phosphatidylglycerol: cholesterol. In another embodiment, the particles are encapsulated within a liposome.

The particles should generally be large enough to isolate from the spleen or liver, but each particle need not be uniform in size, and may be a receptor or non-receptor mediated mechanism by antigen presenting cells, including endothelial cells or other MPS cells Lt; RTI ID = 0.0 > and / or < / RTI > Preferably, the particles are of microscopic or nano-range size to enhance solubility, avoid complications that may be caused by in vivo aggregation, and facilitate pinocytosis. Particle size may be a factor for absorption from the interstitial space into the lymphocyte maturation region. Particles having diameters of from about 0.1 [mu] m to about 10 [mu] m can cause phagocytosis. Thus, in one embodiment, the particle has a diameter within this limiting range. In yet another embodiment, the particles have an average diameter of about 0.3 [mu] m to about 5 [mu] m. In yet another embodiment, the particles have an average diameter of about 0.5 [mu] m to about 3 [mu] m. In yet another embodiment, the particles have an average diameter of about 0.2 [mu] m to about 2 [mu] m. In further embodiments, the particles have a particle size of about 0.1 microns, or about 0.2 microns, or about 0.3 microns, or about 0.4 microns, or about 0.5 microns, or about 1.0 microns, or about 1.5 microns, or about 2.0 microns, or about 2.5 microns, Mu m or about 4.0 mu m or about 4.5 mu m or about 5.0 mu m. In certain embodiments, the particles have an average size of about 0.5 [mu] m. In some embodiments, the particles have an average diameter of about 0.5 [mu] m to about 0.95 [mu] m. For example, in this embodiment, 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 mu m. 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.

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 . The particles in the composition need not have a uniform diameter. For example, the pharmaceutical formulation may contain multiple particles, some of which have a diameter of about 0.5 占 퐉, and other particles have a diameter of about 1.0 占 퐉. As a further example, the pharmaceutical formulation may contain a plurality of particles, some of which have a diameter of about 0.7 microns, while others have diameters of about 0.5 microns to about 0.95 microns. Mixtures of any particle size within these given ranges will be useful.

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

The particles of the present invention may have a ratio of antigen to polymer (e.g., mg of antigen / mg of polymer). In some embodiments, the ratio of antigen to polymer is from about 1 [mu] g / mg to at least about 5 [mu] g / mg. For example, in some embodiments, the ratio of antigen to polymer is about 1 μg / mg, about 1.5 μg / mg, about 2 μg / mg, about 2.5 μg / mg, about 3.0 μg / mg, 4.5 / / mg, or about 5 / / mg. In some embodiments, the ratio of antigen to polymer is at least about 5 [mu] g / 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 / mg, 8.5 μg / mg, 9.5 μg / mg, 10 μg / mg, 10.5 μg / mg, 11 μg / mg, 11.5 μg / / mg, 13.5 μg / mg, 14 μg / mg, 14.5 μg / mg, or about 15 μg / mg.

In some embodiments, the charge (e.g., positive, negative, neutral) of the carrier is selected to confer application-specific advantages (e.g., physiological compatibility, beneficial surface-peptide interaction, etc.). In some embodiments, the carrier has a net neutrality or negative charge (e.g., to reduce nonspecific binding to a cell surface that generally has a net negative charge). In certain embodiments, the carrier can be conjugated directly or indirectly to an antigen (herein referred to as an antigen-specific peptide, an antigenic peptide, an autoantigen, an inducible antigen or a resistant antigen) for which resistance is desired. In some instances, the carrier can have multiple copies of an antigen-specific peptide or a number of different peptides exposed on the surface, (e.g., to increase the probability of an immune response), (e.g., 3, 4, 5, 6, 7, 8, 9, 10 ... 20 ... 50 ... 100). In some embodiments, the carrier represents a single type of antigenic peptide. In some embodiments, the carrier exhibits a number of different antigenic peptides on the surface. In some embodiments, the carrier surface represents a functional group for the covalent attachment of a selected moiety (e. G., An antigenic peptide). In some embodiments, the carrier surface functional group provides sites for non-covalent interactions with a selected moiety (e. G., An antigenic peptide). In some embodiments, the carrier has a surface on which the conjugating moiety can be adsorbed without chemical bond formation.

Particle size and charge are important in inducing tolerance. The particle will vary in size and charge based on the antigen encapsulated therein, but in general the particles of the present invention are from about 100 nanometers to about 1500 nanometers and have a charge of from 0 to about -70 mV to induce tolerance Effective, and most effective in inducing resistance when having a charge of about 400 mV to about 800 mV and a charge of about -25 mV to -70 mV. Also, in part in the freeze-drying process, due to the concentration of the particles and the presence of sucrose and D-mannitol, the average particle size and charge of the particles may be slightly changed in the lyophilization process. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to particle size and charge prior to lyophilization. The terms "post-lyophilization size" and "post-lyophilized charge" refer to particle size and charge after lyophilization.

In some embodiments, the particles are non-metals. In these embodiments, the particles may be formed from a polymer. In a preferred embodiment, the particles are biodegradable in the individual. In this embodiment, the particles may be provided to the subject over multiple doses without the particles accumulating within the subject. Examples of suitable particles include polystyrene particles, PLGA particles, PLURIONICS stabilized polypropylene sulfide particles, and diamond particles. Preferably, the particle surface is composed of a material that minimizes non-specific or unwanted biological interactions. The interaction between particle surface and epilepsy may be an important factor in lymphatic absorption. The particle surface can be coated with a material that prevents or reduces non-specific interactions. Hydrophilic layers such as poly (ethylene glycol) (PEG) and copolymers thereof such as PLURONICS® (including copolymers of poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) Stability stabilization by coating the particles can reduce non-specific interactions of epilepsy with proteins as evidenced by improved lymphatic absorption after subcutaneous injection. All these facts point to the significance of the physical properties of the particles in relation to lymphatic absorption. The biodegradable polymer may be used to produce all or part of the polymer and / or particles and / or layers. The biodegradable polymer can be degraded, for example, by the result of functional groups that react with water in the solution. As used herein, the term "degradation " refers to a decrease in molecular weight or availability of a hydrophobic group by conversion to a hydrophilic group. The polymer having an ester group is generally applied to spontaneous hydrolysis, for example, polylactide and polyglycolide.

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 nanocrystal with a currently commercially available contrast agent is the Kodak X-site nanosphere. Inorganic quantum-limited luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: high quantum yields and variable size-dependent Stokes shifts are observed in blue (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, C.M. Angew. Chem. Int. Ed., 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ Chem. Quantum dots, such as hybrid organic / inorganic quantum dots based on polymer classes known as dendrimers, can be used in biological labeling, imaging and optical biosensor systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperature or highly toxic labile reagents. (Etienne, et al., Appl. Phys. Lett., 87, 181913, 2005).

The particles can be formed from a wide range of materials. The particles are preferably composed of a material suitable for biological use. For example, the particles may comprise glass, silica, a polyester of a hydroxycarboxylic acid, a polyanhydride of a dicarboxylic acid, or a copolymer of a hydroxycarboxylic acid and a dicarboxylic acid. More generally, the carrier particles may be linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or bridged alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, Substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, alkenyl, alkynyl, alkenyl, , Aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxydicarboxylic acid. Further, the carrier particles can be quantum dots, or they can be composed of quantum dots such as quantum dot polystyrene particles (Joumaa et al. (2006); Langmuir 22: 1810-6). Carrier particles comprising 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 can be selected from the group consisting of a polyglycolic acid polymer (PGA), a polylactic acid polymer (PLA), a polysaccharide polymer (PSA), a poly (lacto-co-glycol) (PGSA), polypropylene sulfide polymers, poly (capro) copolymers (PLSA), poly (lactone-co-succinate) acid copolymers Lactones), chitosan, and the like. Other biocompatible biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate and degradable urethane as well as straight or branched, substituted or unsubstituted Alkenyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acid. In addition, biologically important amino acids or their enantiomers with reactive side chains such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine provide reactive groups for conjugating to antigenic peptides and proteins or conjugating moieties , May be included in the copolymer with any of the above-mentioned materials. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the carrier particles of the present invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonate olefin, the vinyl ^{alcohol, TEFLON ® (DuPont, Wilmington,} Del) and nylon can be used.

The particles of the present invention can be prepared by any means commonly known in the art. Exemplary methods of preparing the 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 modulate particle properties (e.g., size, size distribution, zeta potential, shape, hydrophobicity / hydrophilicity, polypeptide capture, etc.). The particle size depends on the concentration of PLGA, the solvent used in the preparation of the particles, the nature of the organic phase, the surfactant used in the preparation, the viscosity of the continuous and discontinuous phase, the nature of the solvent used, the temperature of the water used, But are not limited to, a variety of factors including, but not limited to, the nature of the additive, shear stress, sterilization, and any encapsulated antigen or polypeptide.

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

The viscosity of continuous and discontinuous phases is an important parameter affecting the diffusion process, which is a major step in forming smaller particles. The size of the particles increases as the viscosity of the dispersed phase increases, while the size of the particles decreases to a more viscous continuous phase. Generally, the lower the phase ratio of the organic solvent to the aqueous solvent, the smaller the particle size.

Homogenizer speed and agitation also affect particle size; Generally, there is a point where the particle size decreases when the speed is increased and stirred, but the particle size does not decrease even when the speed and the stirring further increase. There is a good effect on the size reduction when the emulsion is homogenized with a high pressure homogenizer, compared to only high agitation. For example, at 20% phase ratio at 5% PVA, the average particle size upon agitation is 288 nm and the average particle size by homogenization (high pressure of 300 bar) is 231 nm.

The significant size reduction of the particles can be achieved by varying the temperature of the water added to improve the diffusion of the solvent. The average particle size decreases with increasing water temperature.

The nature of the polypeptide encapsulated within the particle also affects the particle size. In general, encapsulation of hydrophobic polypeptides leads to the formation of smaller particles as compared to encapsulation of polypeptides that are more hydrophilic. In double emulsion processes, the capture of more hydrophilic polypeptides is improved by using high molecular weight PLGA and higher molecular weight of the first surfactant resulting in a higher internal phase viscosity. The interaction between the solvent, the polymer and the polypeptide affects the efficiency of incorporating the polypeptide into the particle.

The PLGA molecular weight affects the final average particle size. Generally, the higher the molecular weight, the larger the average particle size. For example, because of the variety of PLGA composition and molecular weight (e.g., 12-48 kDa for 50:50 PLGA; 12-128 kDa for 75:25 PLGA), the average particle size varies (about 102 nm - 154 nm, about 132 nm to 152 nm). Although the particles have the same molecular weight, their composition can affect the average particle size; For example, a 50:50 ratio of particles generally forms particles smaller than a 75:25 ratio. The end groups on the polymer also affect the particle size. For example, particles made with ester end groups form particles with an average size of 740 nm (PI = 0.394) compared to an average size of acid PLGA end groups of 240 nm (PI = 0.225).

The solvent used may also affect particle size; Solvents that reduce the surface tension of the solution also reduce particle size.

The organic solvent is removed by evaporation in vacuo to avoid polymer and polypeptide damage and to reduce final particle size. The evaporation of the organic solvent under vacuum is more efficient in forming smaller particles. For example, evaporation in a vacuum produces an average particle size that is about 30% smaller than the average particle size produced under normal evaporation rates.

The amplitude of the ultrasound wavelength also affects particle characteristics. The amplitude of the wavelength should be greater than 20% by ultrasonic treatment for 600 to 800 seconds to form a sable mini emulsion in which the droplet size no longer changes. However, the main disadvantage of ultrasonic treatment is the lack of monodispersibility of the formed emulsion.

Organic phases that can be used in the preparation of the particles of the present invention include, but are not limited to, ethyl acetate, methyl ethyl ketone, propylene carbonate and benzyl alcohol. The continuous phase that may be used includes, but is not limited to, the surfactant poloxamer 188.

A variety of surfactants can be used in the preparation of the particles of the present invention. The surfactant may be anionic, cationic or nonionic. Poloxamer and poloxamine surfactants are typically used in particle synthesis. Surfactants that may be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, pluronic L-63, PVA, methylcellulose, lecithin and DMAB. Also biodegradable and biocompatible surfactants, including, but not limited to, vitamin E TPGS (D-alpha-tocopheryl polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are required (e. G., In a dual emulsion evaporation process). These two surfactants may comprise a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

Solvents that may be used in the preparation of the particles of the present invention include, but are not limited to, acetone, tetrahydrofuran (THF), chloroform, and methyl chloride, which is a member of the chlorate series. The choice of organic solvent requires two selection criteria: the polymer must be soluble in this solvent and the solvent must be completely immiscible with the aqueous phase.

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

Typical salting agents include, but are not limited to, electrolytes (e.g., sodium chloride, magnesium acetate, magnesium chloride), or non-electrolytes (e.g., sucrose).

The stability and size of the particles of the present invention can be improved by the addition of compounds including, but not limited to, short chains of fatty acids or carbons. The addition of longer carbon chains of lauric acid is associated with an improvement in particle characteristics. In addition, the addition of hydrophobic additives can improve particle size, incorporation and release profiles of polypeptides within the particles. The preparation of the particles by freeze-drying can be stabilized. Addition of a cryoprotectant such as trehalose may reduce particle agglomeration during lyophilization.

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

In some embodiments, the invention provides a method of treating a subject comprising: (a) a delivery scaffold configured to deliver a chemical and / or biological agent to a subject; And (b) antigen-coupled poly (lactide-co-glycolipid) particles for induction of antigen-specific resistance. In some embodiments, at least a portion of the transfer scaffold is microporous. In some embodiments, the antigen-coupled poly (lactide-co-glycolipid) 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 a cell, wherein the cell comprises pancreatic islet cells.

Physical properties are also related to the availability of nanoparticles after absorption and maintenance in areas with immature lymphocytes. This includes mechanical properties such as stiffness or rubberiness. Some embodiments include a gum core, such as an overlay, such as a hydrophilic core, such as a hydrophilic core, such as in a recently developed and characterized PPS-PEG system for systemic (but not targeted or immune) delivery, (Propylene sulfide) (PPS) core with an overlay layer. Gum cores are in contrast to cores that are substantially rigid, such as in polystyrene or metal nanoparticle systems. The term rubber refers to certain resilient materials other than natural or synthetic rubber, and rubber is a term familiar to people in the polymer arts. For example, cross-linked PPS can be used to form hydrophobic gum cores. PPS is a polymer that is decomposed to polysulfoxide under oxidative conditions and finally polysulfone, and then transferred from the hydrophobic rubber to a hydrophilic, water-soluble polymer. Other sulfide polymers may be suitable for use, and the term sulfide polymer refers to a polymer having sulfur at the skeleton of the mer. Other rubbery polymers that may be used are polyesters having a glass transition temperature under hydrated conditions below about < RTI ID = 0.0 > 37 C. < / RTI > Because the core and overlay tend not to mix, hydrophobic cores can be advantageously used with hydrophilic overlayers, so that the overlayers tend to expand in three dimensions from the core. The core refers to a particle having a layer thereon. The layer refers to a material that covers at least a portion of the core. The layer can be adsorbed or covalently bonded. The particles or cores may be rigid or hollow. A gummy hydrophobic core is advantageous over a rigid hydrophobic core such as crystalline or glassy (e.g., in the case of polystyrene) cores and is advantageous in that a high load of hydrophobic drug can be delivered by particles having a gummy hydrophobic core.

Another physical property is the hydrophilicity of the surface. The hydrophilic material may have a water solubility in water of at least 1 g / L when it is not crosslinked. Stabilization of particles with hydrophilic polymers can improve absorption from the epilepsy by reducing non-specific interactions; The increased stealth properties of the particles can also reduce the internalization by the phagocyte cells at sites with immature lymphocytes. However, the problem of balancing these competing functions has been met and this document documents the production of nanoparticles for lymphatic delivery that is effective on DC and other APCs of the lymph nodes. Some embodiments include a layer of a hydrophilic component, for example a hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxide, polyethylene oxide, polysaccharide, polyacrylic acid and polyether. The molecular weight of the polymer in the layer can be adjusted to provide a useful degree of steric hindrance in vivo, for example, from about 1,000 to about 100,000 or more; Those skilled in the art will readily recognize that all ranges and values within the explicitly stated ranges, e.g., 10,000 to 50,000, are contemplated.

Nanoparticles can incorporate functional groups for further reaction. The functional group for the further reaction comprises an electrophile or a nucleophile; They are convenient for reacting with other molecules. Examples of nucleophiles are primary amines, thiols and hydroxyls. Examples of electrophiles include succinimidyl esters, aldehydes, isocyanates and maleimides.

Many means well known in the art can be used to conjugate antigenic peptides and proteins to carriers. This method does not disrupt or severely limit the biological activity of the antigenic peptides and proteins, and it is contemplated that a sufficient number of antigenic peptides and proteins will be delivered to the carrier in an orientation that allows interaction of the antigenic peptides or proteins with the corresponding T- Lt; RTI ID = 0.0 > chemistry < / RTI > Generally, a method of conjugating the C-terminal region of an antigenic peptide or protein, or the C-terminal region of an antigenic peptide or protein fusion protein, to an earner is preferred. Will depend on the precise chemistry as well as the nature of the parent material, the presence or absence of C-terminal fusion to the antigenic peptide or protein, and / or the presence or absence of the conjugating moiety.

The functional groups may be located on the particles for availability as needed. One location may be a layer on the core or polymer, or else a side group or end on the core polymer or polymers bound to the particles. This includes, for example, describing PEGs that stabilize nanoparticles that can be readily functionalized for specific cell targeting or protein and peptide drug delivery.

In order to fix the peptide or protein to the carrier surface, a conjugate such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propylene glycol di-glycidyl ether containing two epoxy moieties and epichlorohydrin are used . Without being bound by theory, it is suspected that ECDI performs two major functions for the induction of resistance: (a) catalyzes the formation of peptide bonds between free amino and free carboxyl groups to form proteins / peptides on the cell surface Chemically coupled; And (b) the antibody is induced to mimic cell apoptotic cell death and is captured by a splenic host antigen presenting cell (which may include endothelial cells) to induce tolerance. Is an indication of host T-cells in a non-immunogenic manner that directly induces anergy in autoreactive cells. In addition, ECDI acts as a powerful stimulant for the induction of specific regulatory T cells.

In a series of embodiments, antigenic peptides and proteins are bound to the carrier via covalent chemical bonds. For example, a reactive group or moiety near the C-terminus of the antigen (e.g., a hydroxyl, thiol, or amine group of a C-terminal carboxyl group or an amino acid side chain) is reacted with a reactive group on the surface of the carrier Or may be directly conjugated to a moiety (e.g., a hydroxyl or carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of a dendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid). Alternatively, there may be conjugation moieties covalently linked to both antigenic peptides, proteins and carriers, thereby linking them.

The reactive carboxyl group on the carrier surface can be reacted with a free amine on the antigenic peptide or protein (e. G., From the Lys residue), for example 1-ethyl-3- [3,9-dimethylaminopropyl] carbodiimide hydrochloride ) Or N-hydroxysuccinimide ester (NHS). Similarly, the same chemistry can be used to conjugate the free amines on the surface of the carrier with the free carboxyl (e.g., from the C-terminus or from the Asp or GIu residue) on the antigenic peptide or protein. Alternatively, the free amine groups on the surface of the carrier are essentially as described in Arano et al. (1991) Chem. Can be covalently linked to antigenic peptides and proteins, or antigenic peptide or protein fusion proteins, using sulfo-SIBA chemistry, as described by Wang et al.

In another embodiment, a non-covalent bond between a ligand bound to an antigenic peptide or protein and an anti-ligand attached to the carrier can conjugate the antigen to the carrier. For example, a biotinylated sequence tag may be linked to the C-terminus of an antigenic peptide or protein, which tag may be biotinylated with biotinylated. Biotin can then act as a ligand that non-covalently conjugates the antigenic peptide or protein as an anti-ligand to the surface of the carrier, or to an otherwise conjugated avidin or streptavidin. Alternatively, when an antigenic peptide and protein are fused to an immunoglobulin domain having an Fc region as described above, the Fc domain may act as a ligand and the protein A that is covalently or non-covalently bound to the surface of the carrier , An antigenic peptide, or an anti-ligand that non-covalently binds the protein to the carrier. Other methods are well known in the art and can be used to non-covalently conjugate antigenic peptides and proteins to carriers, and include metal ion chelation techniques (e. G., Antigenic peptides or proteins, or antigenic peptides Or a poly-His tag at the C-terminus of a protein fusion protein, and a Ni ⁺ -coated carrier), and these methods can be replaced by those described herein.

Conjugation of the nucleic acid moiety to the platform molecule may be effected in any number of ways, typically including one or more cross-linking agents and functional groups on the nucleic acid moiety and the platform molecule. Connectors are added to the platform using standard synthetic chemistry. The linker may be added to the nucleic acid moiety using standard synthetic techniques. The practitioner has numerous options for the antigens used in the combinations of the present invention. Induced antigens present in the combination contribute to the specificity of the induced immunogenic response. The target and immunity of the unwanted immunological response may be the desired target and may or may not be the same as the target antigen that is an antigen present or located in the subject to be treated.

The inducing antigen of the present invention may be a polypeptide, polynucleotide, carbohydrate, glycolipid, or other molecule isolated from a biological source, or may be a chemically synthesized small molecule, polymer or derivative of a biological material, And have the ability to induce tolerance according to this description.

In some embodiments, the invention provides carriers (e. G., Immunostimulatory particles) coupled to one or more peptides, polypeptides, and / or proteins. In some embodiments, a carrier (e. G., A PLG carrier) such as those described herein is capable of inducing antigen-specific resistance and / or preventing the onset of an immune-related disorder (e. And / or to reduce the severity of existing immune-related diseases. In some embodiments, the compositions and methods of the invention may allow T-cells to perform initial events associated with T-cell activation, but do not allow T-cells to acquire effector function. For example, administration of a composition of the present invention can induce T-cells with quasi-activated phenotypes such as CD69 and / or CD44 upregulation, but may be induced by a deficiency of IFN-gamma or by IL-17 synthesis And does not exhibit the effector functions such as. In some embodiments, the administration of the compositions of the present invention does not convert naive antigen-specific T-cells into a regulated phenotype such as having a CD25 ⁺ / Foxp3 ⁺ phenotype, but T-cells with a quasi-activated phenotype .

In some embodiments, the surface of a carrier (e.g., a particle) is a chemical moiety capable of attaching (e.g., covalently, non-covalently) an antigenic peptide and / And / or functional groups. In some embodiments, the number, orientation, spacing, etc. of chemical moieties and / or functional groups on a carrier (e.g., a particle) varies with carrier chemistry, desired application, and the like.

In some embodiments, the carrier comprises one or more biological or chemical agents that are attached to, adhered to, encapsulated within, and / or contained throughout the carrier. In some embodiments, a chemical or biological agent is encapsulated throughout the particle and / or is contained throughout the particle. The invention is not limited by the nature of the chemical or biological agent. 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 in the carrier. In some embodiments, the formulation is configured with a specific release rate. In some embodiments, a number of different agents are configured at different release rates. For example, the first agent can release over a period of time while the second agent is 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 a biological or chemical agent. In some embodiments, the sustained release is a biological activity of the 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.) Provides positive release. In some embodiments, the carrier or a portion thereof is configured to be porous enough to allow cells to enter the interior with voids. The size of the pores may be selected for the particular cell type of interest and / or the desired amount of internalization. In some embodiments, the particle comprises an antigen of interest without a drug or other non-peptide activator such as an immunomodulatory agent. Further, in some embodiments, the particles of the present invention do not contain immunostimulatory or immunosuppressive peptides other than the antigen of interest. Further, in some embodiments, the particles do not contain other proteins or peptides (e.g., co-stimulatory molecules, MHC molecules, immunostimulatory peptides, or immunosuppressive peptides) encapsulated on or in the surface.

The encapsulation of antigenic, biological and / or chemical agents in the particles of the invention has surprisingly been found to induce immunological tolerance and has several advantages. First, the encapsulated particles have a slower cytokine response. Second, when multiple antigens, biological and / or chemical agents are used, encapsulation eliminates the competition between the various molecules that can occur when the agent is attached to the particle surface. Third, encapsulation allows more antigen, biological and / or chemical agents to be incorporated with the particles. Fourth, encapsulation allows for easier use of complex protein antigens or long-term homogenates (eg, pancreatic homogenate in the case of type 1 diabetes or peanut extract of peanut allergy). Finally, instead of conjugation to the surface of the particles, an antigen, biological and / or chemical agent is encapsulated within the particle to maintain a net negative charge on the surface of the particle. The encapsulation of the intra-particle antigens, biological and / or chemical agents 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 particle by a dual-emulsion process. In a further embodiment, the polypeptide antigen is water-soluble.

In another embodiment, the polypeptide antigen is encapsulated within the particle by a single-emulsion process. In a further embodiment, the polypeptide antigen is more hydrophobic. Occasionally, dual emulsion processes can lead to formation of large particles, resulting in leakage of hydrophilic active ingredients and low capture efficiency. Adhesion and Ostwald aging are two mechanisms by which dual-emulsion droplets can be destabilized, and diffusion through the organic phase of the hydrophilic active ingredient is a major mechanism leading to low levels of entrapped active ingredients. In some embodiments, it may be beneficial to reduce the nanoparticle size. One strategy to achieve this is to apply a second strong shear rate. Leakage effects can be reduced by increasing the viscosity of the internal aqueous phase and increasing the surfactant molecular weight using higher polymer concentrations and higher polymer molecular weights.

In certain embodiments, the invention provides a carrier having (in or on) a cell or other biological or chemical agent. When a cell is used, the carrier is not limited to a particular type of cell. In some embodiments, the carrier has pancreatic islet cells thereon. In some embodiments, the microporous carrier further has an ECM protein and / or exendin-4. Carriers are not limited to any particular type. In some embodiments, the carrier has various areas of porosity (e.g., varying pore size, pore depth and / or pore density). In some embodiments, the carrier has (on or in) a drug, DNA, RNA, extracellular matrix protein, exendin-4, and the like. In certain embodiments, the invention provides a method of transplanting pancreatic islet cells into the carrier. In certain embodiments of the invention, the inducing antigen is a single isolated or recombinantly produced molecule. In order to treat a condition in which the target antigen is spread to various locations in the host, it is generally necessary that the inducing antigen be identical or immunologically related to the target antigen. Examples of such antigens are most polynucleotide antigens and some carbohydrate antigens (e. G., Blood group antigens).

Any suitable antigen may be used within the scope of the present invention. In some embodiments, the inducing antigen contributes to the specificity of the induced immunogenic response. An induced antigen may be the same or different from a target antigen that is a target for an unwanted immune response and is an antigen that is present or placed on the subject to be treated that requires resistance.

In certain embodiments of the invention, the inducing antigen is not the same form found in pollen, the polypeptide from Japanese cedar pollen, but is a fragment or derivative thereof. The inducing antigens of the present invention include peptides that are based on molecules of appropriate specificity but are adapted by fusion with peptides with fragmentation, residue substitution, labeling, conjugation, and / or other functional properties. Adaptation may include the removal of any undesirable properties such as toxicity or immunogenicity; Or any desired property improvement, such as, but not limited to, mucosal binding, mucosal penetration, or stimulation of an immunogenic arm of an immune response. As used herein, terms such as CRYJ1 or CRYJ2 refer to homologous subunits for homologous subunits for at least 10 and preferably 20 consecutive amino acids of each molecule of similarity, homology (amino acid level preferably 70% identical, Refers to homologous and synthetic variants, fragments, fusion peptides, conjugates and other derivatives containing a region that is at least 80% identical, preferably at least 90% identical, and even more preferably 90% identical, Lt; RTI ID = 0.0 > resistance < / RTI > to each parent molecule.

It is recognized that the immunogenic region of an induced antigen is often different from an immunogenic epitope for stimulation of an antibody response. Immunogenic regions are regions that may appear in certain cell interactions, including T cells in general. Immunogenic regions may be present and induce tolerance in the presentation of intact antigens. Some antigens contain a latent immunogenic region in that the treatment and presentation of the virion antigens do not normally result in resistance. Elucidation of the latent antigen and its identification are found in International Patent Publication WO 94/27634.

In certain embodiments of the invention, two, three, or more inducible antigens are used. It may be desirable to perform these embodiments when a plurality of target antigens are present, or to provide a plurality of bystanders for the target. It may also be desirable to provide an antigenic cocktail to handle as many different possible targets as possible. For example, the subject could be tolerated using cocktails of CRYJ1 and CRYJ2 fragments as well as other epitopes derived from Japanese cedar pollen. In another example, a mixture of allergic antigens may serve to induce an antigen for atopic treatment.

Inducible antigens can be produced by a number of techniques known in the art depending on the nature of the molecule. Polynucleotides, polypeptides and carbohydrate antigens can be isolated from cells of an abundant target species. Short peptides are conveniently prepared by amino acid synthesis. The longer protein of known sequence may be prepared by synthesizing an encoding sequence, PCR-amplifying the encoding sequence from a natural source or vector, and then expressing the encoding sequence in a suitable bacterial or eukaryotic host cell.

In certain embodiments of the invention, the combination comprises a complex mixture of the antigens obtained from the pollen particles, one or more of which serves to induce the antigen. The antigen may be intact or in the form of whole cells treated with a fixative such as formaldehyde, glutaraldehyde or alcohol. The antigen may be in the form of a purified lysate after it has been produced by formulation or mechanical rupture of a cell or tissue formulation. Antigens may also be obtained by enrichment of the plasma membrane by techniques such as subcellular fractionation, particularly differential centrifugation, optionally by formulation solubilization and dialysis. Other separation techniques such as affinity of solubilized membrane proteins or ion exchange chromatography are also suitable.

Allergy antigens are other antigens that are also desirable for their immunogenicity. In one embodiment, the antigen is a polypeptide associated with Japanese cedar pollen. In some embodiments, the antigen is CRYJ1. In certain embodiments, the antigen is CRYJ2.

In one embodiment, the particles of the invention are coupled to an antigen comprising at least one epitope associated with an allergy, an autoimmune disease, and / or an inflammatory disease or disorder. An antigen may comprise a replica of one or more epitopes. In one embodiment, the antigen comprises a single epitope associated with a disease or disorder. In further embodiments, the antigen comprises more than one epitope associated with the same disease or disorder. In another embodiment, the antigen comprises more than one epitope associated with a different disease or disorder. In a further embodiment, the antigen comprises one or more epitopes associated with one or more allergies.

Additional non-limiting examples of allergy-related epitopes for Japanese cedar pollen are shown in Table 1.

Any suitable antigen associated with allergy can be used within the scope of the present invention. In some embodiments, the particles described herein are encapsulated in one or more antigens or epitopes associated with allergy. In certain embodiments, the antigen or epitope is selected from the group consisting of dust, pet allergenic antigen, plant pollen, grass pollen, fungus, plant allergen, fruit allergen, nut allergen, seed allergen, cereal allergen, Antigens, allergenic antigens, nematode allergens, latex allergens, and / or allergic antigens.

In some embodiments, the antigenic epitopes from Japanese cedar pollen are 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 from Japanese 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 from Japanese cedar pollen is a fragment of CRYJ1. In some embodiments, a fragment of CRYJ1 has a sequence identity of at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, such as 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 contiguous amino acids. In certain embodiments, the antigenic epitope is a variant of CRYJ1. In certain embodiments, the variant is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% sequence identity.

In some embodiments, the antigenic epitope from Japanese 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, Et al., J. Immunol, vol. 161 (1) 448-457 (1998); Which is incorporated herein by reference.

In some embodiments, the antigenic epitope from Japanese 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 Japanese cedar pollen is a fragment of CRYJ2. In some embodiments, the fragment of CRYJ2 has a sequence identity of at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, such as 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 contiguous amino acids. In certain embodiments, the antigenic epitope is a variant of CRYJ2. In certain embodiments, the variants comprise at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% sequence identity.

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

In certain embodiments, the particles described herein may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, Or encapsulate an antigenic epitope from 20 or more Japanese cedar pollen. In certain embodiments, two or more antigenic epitopes are contained in the fusion protein. In some embodiments, the antigenic epitope contained in the fusion protein is linked by a cleavable linker. As used herein, a cleavable linker refers to an amino acid sequence containing a specific cleavage site.

In some embodiments, cleavable linkers are 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, cleavable linkers are cleaved by proteases at specific sites. In certain embodiments, cleavable linkers contain one or more specific sites that are cleaved by a protease. In some embodiments, the cleavable linker contains one or more specific sites, wherein the specific site is cleaved by a different protease. In some embodiments, the cleavable linker contains one or more specific sites, wherein the specific sites are cleaved by the same protease.

In some embodiments, the cleavable linker is a purine susceptible linker and / or a cathepsin susceptible linker. In certain embodiments, the cleavable linker is selected from the group consisting of purine, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, 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.

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 comprises two or more epitopes of CRYJ1. In certain embodiments, the fusion protein comprises two or more epitopes from CRYJ2. In some embodiments, the fusion protein comprises an epitope from CRYJ1 and CRYJ2.

The combination of antigen and / or epitope can be tested for its ability to enhance immunogenicity by performing experiments with isolated cells or animal models.

In some embodiments, an immunogenic inducing composition of the invention (including, for example, in addition to an antigenic peptide or other antigenic molecule) contains apoptotic signaling molecules. In some embodiments, the apoptotic signaling molecule is coupled and / or associated with the surface of the carrier. In some embodiments, the apoptotic signaling molecule causes the carrier to be detected as an apoptotic cell by an antigen presenting cell of the host, e. G., Cells of the host retinal endothelial system; This allows the presentation of the associated peptide epitope in an immunogenicity-inducible manner. Without being bound by theory, it is believed that this prevents the upregulation of molecules involved in immune cell stimulation, such as MHC class I / II and co-stimulatory molecules. Such apoptotic signaling molecules may also act as host cell markers. For example, apoptotic signaling molecules suitable for the present invention are described in U.S. Patent Application No. 20050113297, which is incorporated herein by reference. Molecules suitable for the present invention include molecules targeting macrophage cells, including macrophages, dendritic cells, monocytes, granulocytes and neutrophils.

In some embodiments, molecules suitable as apoptotic signaling molecules serve to enhance the immunogenicity of the associated peptides. In addition, carriers bound to apoptotic signaling molecules can be bound by Clq in apoptotic cell recognition (Paidassi et al., (2008) J. Immunol. 180: 2329-2338; ≪ / RTI > For example, molecules that may be useful as apoptotic signaling molecules include, but are not limited to, phosphatidylserine, annexin-1, annexin-5, endosperm globulomer-EGF factor 8 (MFG-E8), or thrombospondin family For example, thrombospondin-1 (TSP-1). Various molecules suitable for use as apoptotic signaling molecules by the present invention are discussed, for example, in U.S. Patent Publication No. 2012/0076831; The entirety of which is incorporated herein by reference).

In some embodiments, the apoptotic signaling molecule may be conjugated to an antigen-specific peptide. In some instances, apoptotic signaling molecules and antigen-specific peptides are conjugated by the production of fusion proteins. For example, the fusion protein may comprise at least one antigen-specific peptide (or fragment or variant thereof) coupled to at least one molecule of a cell apoptotic signaling molecule (or fragment or variant thereof). For the production of a fusion protein, the terms "fusion protein", "fusion peptide", "fusion polypeptide" and "chimeric peptide" are used interchangeably. Suitable fragments of an antigen-specific peptide include any fragment of a full length peptide that retains the ability to produce the desired antigen-specific immunogenic function of the present invention. Fusion proteins can be produced by a variety of means as understood in the art (e. G., Genetic fusion, chemical conjugation, etc.). Both proteins can be fused directly or via an amino acid linker. Polypeptides that form fusion proteins can link the C-terminus to the C-terminus, the N-terminus to the N-terminus, or the N-terminus to the C-terminus, but generally the C- do. The polypeptides of the fusion protein may be present in any order. The peptide linker sequence may be used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide is folded into its secondary and tertiary structure. Amino acid sequences that may be usefully employed as linkers are described 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; The entire contents of which are incorporated herein by reference. Linker sequences can generally be from 1 to about 50 amino acids in length. In some embodiments, for example, when the first and second polypeptides have a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference, the linker sequence is not required and / Or not used.

Proxies for immunogenic activity are the ability of intact antigens or fragments to stimulate the production of appropriate cytokines at the target site. The immunomodulatory cytokine released by T suppressor cells at the target site is thought to be TGF-beta (Miller et al., Proc. Natl. Acad Sci USA 89: 421, 1992). Other factors that may be generated during the tolerance period are cytokines IL4 and IL-10 and the mediator PGE. In contrast, lymphocytes in tissues undergoing active immunodeficiency secrete cytokines such as IL-1, IL-2, IL-6 and IFN-y. Thus, the efficacy of a candidate agent to induce an antigen can be assessed by measuring its ability to stimulate an appropriate type of cytokine.

With this in mind, a rapid screening test for an effective method and schedule of an immunogenic epitope of an inducible antigen, an effective mucosal binding component, an effective combination, or mucosal administration can be made using a winter animal as a donor for in vitro cell assays Can be performed. Animals are treated with the test composition at the mucosal surface and sometimes parenteral administration of the target antigen is attempted in complete Freund's adjuvant. Splenocytes are isolated and cultured in vitro in the presence of the target antigen at a concentration of about 50 μg / mL. The target antigen may be replaced with a candidate protein or sub-fragment to map the location of the immunogenic epitope. The secretion of cytokines to the medium can be quantified by standard immunoassays.

The ability of a cell to inhibit the activity of other cells can be determined by using isolated cells from an animal immunized with the target antigen or by generating a cell line that responds to the target antigen (Ben-Nun et al., Eur. J. Immunol. 11: 195, 1981, which is incorporated herein by reference in its entirety). In one variation of this experiment, the inhibitor cell population is lightly irradiated (about 1000-1250 rads) to prevent proliferation, the inhibitor co-cultured with the response group cells, and then the tritiated thymidine incorporation (or MTT) To quantify the proliferative activity of the reaction group. In another variation, inhibitor cell populations and reaction group cell populations are cultured at the upper and lower levels of a dual chamber transwell culture system (Costar, Cambridge Mass.), Population is synchronized within 1 mm of each other, and the polycarbonate membrane (WO 93/16724). In this approach, irradiation of the inhibitor cell population is unnecessary since the proliferative activity of the response group can be measured separately.

In embodiments of the invention where the target antigen is already present in the subject, it is not necessary to isolate the antigen or combine it with the mucosal binding component in advance. For example, the antigen may be expressed in a subject in a particular manner, either as a result of pathological conditions (e.g., inflammatory bowel disease or pediatric adiposity) or through digestion of a food allergy antigen. The test is carried out by providing the mucosal binding component in one or more doses or formulations and measuring the ability to promote tolerance to the in situ antigen.

The efficacy of compositions and modes of administration for the treatment of certain diseases can also be elaborated in corresponding animal disease models. The ability of the treatment to reduce or delay the manifestation of disease is monitored at the level of circulation of the total clinical characteristics appropriate to the biochemical and immunological characteristics of the disease, the immunohistology of the infected tissue and the model used. Non-limiting examples of animal models that can be used for testing are included in the next section. The present invention contemplates modulation of immunogenicity by modulating TH1, TH2, TH17 or combinations of these reactions. Control of the TH1 response includes, for example, altering the expression of interferon-gamma. Modulating the TH2 response includes, for example, altering the expression of any combination of IL-4, IL-5, IL-10 and IL-13. Typically, an increase (decrease) in the TH2 response will involve an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10 or IL-13; More typically, the increase (decrease) in the TH2 response will involve an increase in expression of at least two of IL-4, IL-5, IL-10 and IL-13 and most typically the increase IL-5, IL-10, IL-4, IL-5, IL-10 or IL-13, while ideally the increase (decrease) Or increase (decrease) in expression of both IL-13. Modulation of TH17 includes, for example, changes in the expression of TGF-beta, IL-6, IL-21 and IL23 and the effects of IL-17, IL-21 and IL-22 levels. In some embodiments, the invention contemplates modulation of resistance by promoting one type of immune response to another type (e.g., immuno-switching or immune deviation). In such embodiments, the established immune response of one phenotype is suppressed or reduced, and the immune response of the different phenotype is increased or enhanced. For example, in the context of an allergic immune response, immune-switching from a Th1 response to a Th1 response stimulates the production of an IgG2a antibody response and the production of IFNγ and / or IL-12 leading to a reduction of allergen- Leading to a decrease in T-cell polarization. Other suitable methods for evaluating the effectiveness of the compositions and methods of the present invention are described in, for example, U.S. Patent Publication No. 2012/0076831, which is incorporated herein by reference in its entirety, have.

Certain embodiments of the invention relate to priming of immune tolerance in an individual not previously tolerated by treatment intervention. These embodiments generally involve multiple administrations of a combination of antigen and mucosal binding components. At the beginning of treatment, the subject may exhibit indications of tolerance, but typically at least 3 doses, often at least 4 doses, sometimes at least 6 doses, are performed during priming to obtain long lasting results. In most cases, each dose is given in bolus doses, but sustained formulations capable of mucosal release are also suitable. When multiple administrations are performed, the administration time interval is generally from 1 day to 3 weeks, typically from about 3 days to 2 weeks. Generally, although the same antigen and mucosal binding components are present at the same concentration and administration is given to the same mucosal surface, any change in these parameters can be accommodated during the course of treatment. Another embodiment of the present invention relates to increasing or extending the persistence of previously established immunity tolerance. These embodiments generally involve a single administration or short term treatment, at a time when the established immunogenicity is at risk of diminishing or decreasing. Boosting is generally performed between one month and one year, and is typically performed between two months and six months after priming or previous boost. The invention also encompasses embodiments that include the regular maintenance of immunogenicity to the dosing schedules being made in accordance with twice weekly, weekly, biweekly or any other regular schedule.

The particles of the present invention may be provided in any volume 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, about 10 ² to about 10 ²⁰ particles are provided to an individual. In a further embodiment, about 10 ³ to about 10 ¹⁵ particles are provided. In another embodiment, from about 10 ⁶ to about 10 ¹² particles are provided. In yet another embodiment, about 10 ⁸ to about 10 ¹⁰ particles are provided. In a preferred embodiment, the preferred dose is 0.1% solids / ml. Thus, for a 0.5 탆 bead, a preferred volume is about 4 x 10 ⁹ beads for a 0.05 μm bead, and a preferred volume is about 4 × 10 ¹² beads for a 3 μm bead, with a preferred volume being 2 × 10 ⁷ Beads. However, any dose effective for the treatment of the particular condition being treated is included in the present invention.

The present invention is useful for the treatment of immune related disorders such as autoimmune diseases, graft rejection, enzyme deficiency and allergic reactions. Substitution of synthetic biocompatible particle systems to induce immunotolerance has led to a dramatic reduction in the ease of manufacture, widespread availability of therapeutic agents, increased uniformity between samples, increased number of potential therapeutic sites, and potential for allergic reactions to carrier cells .

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, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral responses) and cytokine reactive cellularization, e.g., activation of macrophages. Immune cells associated with the immune response include lymphocytes such as B cells and T cells (CD4 ⁺ , CD8 ⁺ , Th1 and Th2 cells); The antigen presenting cells (e. G., Specialized antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells and non-progenitor antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, ); Natural killer cells; Bone marrow cells such as macrophages, eosinophils, mast cells, basophils and granulocytes. In some embodiments, the modified particles of the present invention are effective in reducing inflammatory cell migration regulation to the site of inflammation.

As used herein, the term "anergy", "resistance" or "antigen-specific resistance" refers to the T cell insensitivity to T cell receptor-mediated stimulation. This anesthesia is generally antigen-specific and persists after exposure to the antigenic peptide has ceased. For example, anergy in T cells is characterized by a deficiency of cytokines, such as IL-2 production. T-cell anergy occurs when a T cell is exposed to an antigen and a second signal (a co-stimulatory signal) receives a first signal (T cell receptor or CD-3 mediated signal) in the absence. Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of co-stimulatory molecules) fails to produce cytokines and consequently fails to proliferate. Thus, if cytokine production fails, proliferation can be prevented. However, anergen T cells can be proliferated when cultured with a cytokine (e.g., IL-2). For example, T cell anergy can also be observed as a deficiency of IL-2 production by T lymphocytes as measured by proliferation assays using ELISA or indicator cell lines. Alternatively, reporter gene constructs may be used. For example, anergen T cells do not initiate DL-2 gene transcription induced by heterologous promoters under the control of the 5 ' IL-2 gene enhancer or by oligomers of the API sequences that can be found in this enhancer (Kang et al., 1992 Science 257: 1134).

As used herein, the term "immunological tolerance " refers to a method performed on a proportion of a treated subject as compared to an untreated subject, such as: a) a reduced level of a particular immunological response At least in part, antigen-specifically, effector T lymphocytes, B lymphocytes, antibodies, or equivalents thereof); Delaying the onset or progression of a particular immunological response; Or c) reducing the risk of initiation or progression of a particular immunological response. "Specific" immunological tolerance occurs when immunological tolerance is preferentially triggered for a particular antigen compared to other immune systems. "Non-specific" immunological tolerance occurs when immunological tolerance is induced indiscriminately against an antigen causing an inflammatory immune response. "Semi-specific" immunological tolerance occurs when immunological tolerance occurs semi-differentially for antigens that induce a pathogenic immune response, but not against other antigens that induce a protective immune response.

Resistance to autoantigens and autoimmune diseases is achieved by a variety of mechanisms, including negative selection of thymic autoreactive T cells, and avoidance of thymic deletions and the establishment of peripheral tolerance to autoreactive T cells found around. Examples of mechanisms that provide for peripheral T cell resistance include "ignorance" of self-antigen, anergy or non-reactivity to autoantigens, cytokine immunity deviations, and activation-induced cell death of self-reactive T cells. In addition, regulatory T cells are involved in mediating peripheral resistance. For example, Walker et al. (2002) Nat. Rev. Immunol. 2: 11-19; Shevach et al. (2001) Immunol. Rev. 182: 58-67. In some situations, the peripheral immunity to autoantigens is lost (or destroyed), resulting in autoimmune reactions. For example, in the EAE animal model, activation of antigen presenting cells (APCs) via the TLR congenital immunoreceptor has been shown to destroy self-resistance and lead to induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113: 990-997).

Thus, in some embodiments, the invention provides methods of increasing antigen delivery while inhibiting or reducing TLR7 / 8, TLR9, and / or TLR 7/8/9 dependent cell stimulation. As described herein, administration of certain modified particles results in antigen delivery by DC or APC while inhibiting TLR 7/8, TLR9 and / or TLR7 / 8/9 dependent cellular responses associated with immunostimulatory polynucleotides. Such inhibition may include a reduced level of one or more TLR-associated cytokines.

As discussed above, the present invention provides novel compounds having biological properties useful in the treatment of Mac-1 and LFA-I mediated disorders.

Thus, in another aspect of the present invention, there is provided a pharmaceutical composition comprising an immunostimulatory particle and optionally a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the modified particles of the present invention may be administered to a patient in need thereof in combination with administration of one or more other therapeutic agents. For example, additional therapeutic agents for co-administration or inclusion in a pharmaceutical composition with a compound of the present invention may be an approved anti-inflammatory agent, or may be an unregulated inflammatory immune response or an infection characterized by a bacterial or viral infection May be any one of a number of drugs approved by the Food and Drug Administration which ultimately obtain approval for the treatment of the disorder of the present invention. It is also to be understood that the specification of the modified particles of the present invention may exist in free form or, where appropriate, as a pharmaceutically acceptable derivative thereof for treatment.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and may be any and all solvents, diluents or other liquid vehicles, dispersants or suspending aids, as appropriate for the particular dosage form desired, Surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. If any conventional carrier media is incompatible with the compounds of the present invention by creating any undesirable biological effects or otherwise interacting in a deleterious manner with any other component (s) of the pharmaceutical composition, Are considered to be within the scope of the present invention. Some examples of materials that can act as pharmaceutically 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; Propylene glycol; Esters such as ethyl oleate and ethyl laurate; Agar; Buffers such as magnesium hydroxide and aluminum hydroxide; Alginic acid; Water without heat source; Isotonic saline; Ringer's solution; Ethyl alcohol and phosphate buffers as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavoring and fragrances, preservatives and antioxidants , And may be present in the composition according to the judgment of the preparer.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, (Especially cotton seed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol Cole and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

The particles of the present invention may be administered orally, nasally, intravenously, intramuscularly, intraocularly, transdermally, intraperitoneally, or subcutaneously. In one embodiment, the particles of the present invention are administered intravenously.

The effective amount and method of administration of the invention for the modulation of the immune response may vary based on the subject, the condition being treated, and other factors evident to those skilled in the art. Factors to consider include the route of administration and the number of doses to be administered. These factors are well known in the art and it is within the skill of the art to make such determinations without undue experimentation. Suitable dosage ranges are those that provide the desired modulation of immunity. A useful dosage range of the carrier given by the amount of carrier delivered is, for example, about any of the following ranges: about 0.5 to 10 mg / kg, 1 to 9 mg / kg, 2 to 8 mg / kg, mg / kg, 4 to 6 mg / kg, 5 mg / kg, 1 to 10 mg / kg, 5 to 10 mg / kg. Alternatively, the dosage can be administered based on the number of particles. For example, a useful dose of the carrier given by the amount of carrier delivered may be, for example, about 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ or more particles per dose. The absolute amount given to each patient depends on the pharmacological properties such as bioavailability, clearance rate and route of administration. The pharmaceutically acceptable carrier available, details of excipients, diluents, and details and methods of preparing pharmaceutical compositions and formulations. Remmingtons Pharmaceutical Sciences 18 ^th Edition, 1990, Mack Publishing Co., Easton, Pa, is provided in the USA, which in its entirety Are incorporated herein by reference.

The effective amount and mode of administration of the particular carrier formulation will depend on the particular patient, the desired outcome and / or type of disorder, the stage of the disease, and other factors evident to those skilled in the art. The route (s) of administration useful in certain applications will be apparent to those skilled in the art. Routes of administration include but are not limited to topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal and non-pharyngeal, and lungs (including, but not limited to, transbronchial and transalveolar) It is not limited. A suitable dosage range is a dosage range that provides an IRP-containing composition sufficient to achieve a tissue concentration of about 1-50 [mu] M as measured by blood concentration. The absolute amount given to each patient depends on the pharmacological properties such as bioavailability, clearance rate and route of administration.

The present invention provides a carrier formulation suitable 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 pathways such as transdermal, epidermal, and subcutaneous injection.

Percutaneous administration is achieved by the application of creams, rinses, gels, etc., which allow the carrier to penetrate through the skin into the blood stream. Compositions suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams and ointments, either directly applied to the skin or incorporated into a protective carrier such as a transdermal device (so-called "patch"). Examples of suitable creams, ointments, and the like can be found in the Physician's Desk Reference. Percutaneous delivery can also be accomplished by iontophoresis, for example using commercially available patches that continuously deliver its product through undamaged skin for days or longer. By using this method, controlled delivery of relatively large concentrations of the pharmaceutical composition is made possible, permitting the infusion of compound drugs and allowing simultaneous use of absorption promoters.

Parenteral routes of administration include, but are not limited to, direct injection, such as by electrical (iontophoretics) or direct injection into the central venous line, 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 additionally comprise a pH buffering agent, a salt extender, a preservative and other pharmaceutically acceptable excipients. Parenteral injectable immunoregulatory polynucleotides may be formulated into pharmaceutically acceptable sterile isotonic solutions for injection, such as saline and phosphate buffered saline.

The route of gastrointestinal administration may include, but is not limited to, ingestion and rectal routes, for example, the use of pharmaceutically acceptable powders, pills or liquids for ingestion, and suppositories for rectal administration.

Non-pharyngeal and pulmonary administration is achieved by inhalation and includes delivery routes such as intranasal, transbronchial, and transalveolar routes. The present invention includes formulations of carriers suitable for administration by inhalation, including, but not limited to, powder forms for dry powder inhalation delivery systems as well as liquid suspensions for forming aerosols. Suitable devices for administration by inhalation of carrier formulations include, but are not limited to, an atomizer, an evaporator, a sprayer, and a dry powder inhalation delivery device.

Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be solutions in sterile injectable solutions, suspensions or emulsions, for example, 1,3-butanediol in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be used include water, Ringer's solution, U.S.P. And isotonic sodium chloride solution. In addition, sterilized fixed oils are conventionally employed as a solvent or dispersion medium. For this purpose, any unstiffened stationary oil may be used including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-fixed filter, or by incorporating the sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use have.

To prolong the effect of the drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by the use of liquid suspensions or crystalline or amorphous materials with low solubility in water. The rate of absorption of the drug depends on the dissolution rate, which in turn can depend on the crystal size and on the crystal form. Alternatively, delayed uptake of the parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are prepared by forming microcapsule 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 are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

In some embodiments, the synthetic biodegradable particles of the present invention provide ease of manufacture, broad availability of therapeutic agents, and increased site of treatment. In certain embodiments, surface-functionalized biodegradable poly (lactide-co-glycolide) particles with high density of surface carboxylate groups, synthesized using a surfactant poly (ethylene- alpha -maleic anhydride) Provides a carrier that provides many advantages over other carrier particles and / or surfaces. Experiments performed during the development of an embodiment of the present invention demonstrated conjugation of these particles with peptides (e. _G. , PLP _139-151 peptides). These peptide-coupled particles have been shown to be effective in the prevention of disease progression and induction of immunological tolerance (e.g., in the SJL / J PLP _139-151 / CFA-induced R-EAE murine model of multiple sclerosis) gave. Carriers coupled to the peptides of the invention provide many advantages over other resistance inducing structures. In some embodiments, the particles are biodegradable and therefore will not remain in the body for an extended period of time. The complete decomposition time can be controlled. In some embodiments, the particles are functionalized to facilitate internalization without cellular activation (e.g., phosphatidylserine charged to PLG microspheres). In some embodiments, the particle comprises a target ligand for a particular cell population. In some embodiments, anti-inflammatory cytokines such as IL-10 and TGF- [beta] inhibit the activation of cell types that internalize particles and induce tolerance through energy and / or deletion and promote activation of regulatory T cells Is contained on or in the particle. The composition of the particles has been shown to affect the length of time the particles last in the body, and resistance requires rapid particle absorption and cleavage / degradation. Since the lactide: glycolide slows down the rate of degradation in excess of 50:50, the particles of the present invention have a lactide: glycolide ratio of about 50:50 or less. In one embodiment, the particles of the present invention have a ratio of about 50:50 D, L-lactide: glycolide.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles may contain at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, Mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) wetting agents such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca G) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) sorbents, such as sorbitan monolaurate, sorbitan monolaurate, sorbitan monolaurate, sorbitan monolaurate, Kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, Body polyethylene glycol, a sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose 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 pharmaceutical formulation arts. They may optionally contain opacifying agents and may optionally be a composition which releases only the active ingredient (s) in a delayed manner or preferentially in certain parts of the intestine. Examples of embedding compositions that may be used include polymeric materials and waxes. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose as well as high molecular weight polyethylene glycols and the like.

The modified particles may also be in micro-encapsulated form with one or more excipients as described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings, release-controlled coatings and other coatings well known in the pharmaceutical formulating arts. 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 include additional substances other than inert diluents, such as tabletting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose, as in conventional practice. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. They may optionally contain opacifying agents, and may be a composition that releases only the modified particles, optionally in a delayed manner, or preferably in certain areas of the intestine. Examples of embedding compositions that may be used include polymeric materials and waxes.

The present invention includes pharmaceutically acceptable topical formulations of the modified particles of the present invention. As used herein, the term "pharmaceutically acceptable topical formulation" means any formulation that is pharmaceutically acceptable for intradermal administration of the modified microparticles of the present invention by applying the formulation to the epidermis. In certain embodiments of the invention, topical formulations comprise 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, Saline solution) or any other carrier known in the art for topical administration of the medicament. A more complete list of carriers known in the art is provided in reference texts standard in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both published by Mack Publishing Company, Easton, Pa. , The disclosure of which is incorporated herein by reference in its entirety. In certain other embodiments, topical formulations of the present invention may include excipients. Any pharmaceutically acceptable excipient known in the art may be used to prepare the pharmaceutically acceptable topical formulations of the present invention. Examples of excipients which may be included in topical formulations of the invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, other penetrants, skin protectants, surfactants and propellants and / But are not limited to, therapeutic agents. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium hydrogen sulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol and chelating agents such as EDTA and citric acid. Suitable moisturizing agents include, but are not limited to, glycerin, sorbitol, polyethylene glycol, urea and propylene glycol. Suitable buffers for use in the present invention include, but are not limited to, citric acid, hydrochloric acid and lactic acid buffers. Suitable solubilizing agents include, but are not limited to, quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin and polysorbate. Suitable skin protectants that may be used in the topical formulations of the present invention include, but are not limited to, vitamin E oil, allatoin, dimethicone, glycerin, vaseline and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topical formulations of the present invention comprise at least the modified particles of the present invention and penetration enhancers. The choice of topical formulation will depend on several factors including the condition to be treated, the physicochemical properties of the compounds of the present invention and other excipients, stability within the formulation, available manufacturing facilities and cost constraints. As used herein, the term " penetration enhancer "preferably means an agent capable of transporting a pharmacologically active compound to the epidermis or dermis, with little or no systemic absorption. Various compounds were evaluated for their efficacy in enhancing the rate of drug penetration through the skin. For example, transdermal skin enhancers, investigated for use and testing of various skin penetration enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995) and Buyuktimkin et al., Buyuktimkin et al., Chemical means of improving transdermal drug penetration of transdermal and topical drug delivery systems, Gosh TK, Pfister WR, Yum SI (Eds.), Interpharm Press Inc., Buffalo Grove, 111 (1997). In certain exemplary embodiments, the penetrants for use in the present invention are selected from the group consisting of triglycerides (e.g., soybean oil), aloe compositions (e.g., Aloe-Vera gel), ethyl alcohol, isopropyl alcohol, octoly, Propylene glycol, N-decyl methyl sulfoxide, fatty acid esters (for example, isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol ≪ / RTI > colomooleate) and N-methylpyrrolidone.

In certain embodiments, the composition may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In certain exemplary embodiments, the formulation of the composition according to the present invention is a cream, and particularly preferred are saturated or unsaturated fatty acids, such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleyl alcohol, May be further contained. 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 pharmaceutically acceptable carrier and any necessary preservatives or buffers that may be required. Ophthalmic formulations, drops and eye drops are also contemplated as falling within the scope of the present invention. The present invention also contemplates the use of transdermal patches and the transdermal patch has the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms are prepared by dissolving or dispensing the compound in a suitable medium. As discussed above, penetration enhancers can also be used to increase the flux of a compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The modified particles may be administered by an aerosol. This is achieved by preparing solid particles containing an aqueous aerosol, a liposome preparation or modified particles. Non-aqueous (e. G., Fluorocarbon propellants) suspensions may be used.

Typically, aqueous aerosols are prepared by formulating aqueous solutions or suspensions of the agents together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers will vary depending on the requirements of the particular compound but will generally include non-ionic surfactants (Tweens, Pluronics® or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine , Buffers, salts, saccharides or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

In addition, the modified particles and pharmaceutical compositions of the present invention may be formulated and used in combination therapy, i.e., the compound and pharmaceutical composition may be administered prior to, or subsequent to, one or more other desired therapeutic or medical treatments, Or administered. The particular combination of therapies (therapeutic agents or procedures) for use in combination therapies will allow for the interchangeability of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It is contemplated that the treatment employed may achieve the desired effect for the same disorder (e.g., the compound of the invention may be administered concurrently with other anti-inflammatory agents), or it may achieve a different effect (e. It will be appreciated.

In certain embodiments, the pharmaceutical composition containing modified particles of the invention further comprises one or more additional therapeutically active ingredients (e.g., anti-inflammatory and / or emollient). For the purposes of the present invention, the term "palliative " refers to a treatment that is focused on the relief of the symptoms of the disease and / or the side effects of therapeutic regimens, but which is not curable. For example, palliative care includes analgesics, anti-nausea drugs and anti-psychotic drugs.

The invention provides a method comprising administering to a subject a modified particle described herein as a method for modulating an immune response in a subject, preferably a mammal, more preferably a human. The immunomodulatory methods provided by the present invention include the steps of inhibiting and / or inhibiting the innate or adaptive immune response, including, but not limited to, immunostimulatory polypeptides or immune responses that are stimulated by viral or bacterial components .

The modified particles are administered in an amount sufficient to modulate the immune response. As described herein, modulation of the immune response may be humoral and / or cellular and is measured using standard techniques in the art as described herein.

In some embodiments, the compositions described herein may be administered in conjunction with an implant (e.g., a device) and / or with an implant (e.g., a tissue, a cell, a organ) ), Mediates, nullifies, modulates and / or reduces the immune response associated therewith.

In certain embodiments, the subject suffers from a disorder associated with unwanted immune activation such as allergic diseases or conditions, allergies and asthma. An individual with an allergic disease or asthma is an individual with an existing allergic disease or a recognizable symptom of asthma. For example, immunogenicity may be induced in such an individual by particles that form complexes with an inhaling substance that induces an allergic response (e. G., Japanese cedar pollen protein).

In some embodiments, the invention relates to the use of the composition of the present invention prior to the onset of an allergic reaction, for example, an allergic reaction to Japanese cedar pollen. In certain embodiments, the present invention relates to the use of a composition of the present invention to inhibit ongoing or existing allergies. In some embodiments, the invention relates to alleviating an allergic or allergic response in a subject. Mitigation means treating, preventing or inhibiting the allergic or allergic response of a subject.

In some embodiments, a composition of the invention (e.g., a PLG carrier coupled to an antigenic molecule associated with Japanese cedar pollen allergy) is used in conjunction with one or more scaffolds, matrices, and / or delivery systems , U.S. Patent Nos. 2009/0238879, 7,846,466, 7,427,602, 7,029,697, 6,890,556, 6,797,738, and 6,281,256, incorporated herein by reference). In some embodiments, the particles (e. G., Antigen-coupled PLG particles) may be conjugated to a scaffold, a matrix, and / or a substrate (e. G., To deliver a chemical / biological material, Or conjugated, adsorbed, embedded, conjugated with a delivery system. In some embodiments, a scaffold, matrix and / or delivery system (e.g., for delivering a chemical / biological material, cells, tissue and / or organ to a subject) PLG conjugated to an antigenic peptide) and / or are prepared therefrom.

In some embodiments, a microporous scaffold is provided (e.g., for implanting a biological material (e.g., a cell, tissue, etc.) into a subject). In some embodiments, a microporous scaffold is provided having a formulation (e.g., extracellular matrix protein, exendin-4) and a biological material (e.g., pancreatic islet cells) thereon. In some embodiments, the scaffold is used for the treatment of diseases (e. G., Type 1 diabetes) and related methods (e. G., Diagnostic methods, study methods, drug screening). In some embodiments, the scaffold has the antigen-conjugated carrier described herein on the scaffold and / or in the scaffold. In some embodiments, the scaffold is produced from an antigen-conjugated material (e. G., Antigen-conjugated PLG).

In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or one or more chemical and / or biological entities / agents (e.g., proteins, peptide-conjugated particles, small molecules, Tissue, etc.), see, for example, U.S. Patent Publication No. 2009/0238879; The entirety of which is incorporated by reference. In some embodiments, the antigen-coupled particles are administered with a scaffold delivery system to induce induction of immunological tolerance to the scaffold and related materials. In some embodiments, the microporous scaffold is administered on a scaffold or in a scaffold, along with the particles described herein. In some embodiments, the antigen-coupled particles are coupled to a scaffold delivery system. In some embodiments, the scaffold delivery system comprises antigen-coupled particles.

Various modifications, rearrangements and modifications 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. While specific embodiments have been described, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the modes and embodiments, which will be apparent to those skilled in the relevant art, are intended to be within the scope of the following claims. See, for example, U.S. Patent Application 62 / 221,504, PCT Application Nos. PCT / US2016 / 068423 and PCT / US2017 / 012173, each of which is incorporated herein by reference in its entirety; U.S. Patent Application Publication Nos. 2012/0076831, 2002/0045672, 2005/0090008, 2006/0002978, 2009/0238879, 2012/0076831, 2015/0209293, 2015/0283218 (Each of which is incorporated herein by reference in its entirety); And U.S. Patent No. 7,846,466; 7,427,602; 7,029,697; 6,890,556; 6,797,738; And 6,281,256, each of which is incorporated herein by reference in its entirety, provide details, modifications and variations that may be used in the various embodiments described herein.

All publications and patents cited herein and / or hereinafter are incorporated herein by reference in their entirety.

Example

The following examples are provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention.

Example 1: Encapsulation of recombinant Japanese cedar pollen protein with TIMPs

As mentioned above, the use of particles to encapsulate Japanese cedar pollen (JCP) extracts in the treatment of cedar pollen hypersensitivity is limited due to the problem of encapsulating JCPs which remain very viscous in solution at low concentrations. Experiments were conducted to prepare TIMP particles (TIMP-JCP ^P ) encapsulating JCP using the dual emulsion-solvent evaporation method (FIG. 2C). Briefly, according to a batch of the tested JCP extract, 200 μL of a 5 mg / mL JCP solution was added to 0.5 mL of 20% w / v PLGA in DCM, or 400 μL of a 5 mg / mL JCP solution was added to DCM Was added to 1.0 mL of 20% w / v PLGA. Each solution was emulsified by ultrasonic treatment to produce a primary emulsion. PEMA solution (10 mL 1% w / v aqueous PEMA) was 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 under agitation. The particles were purified, lyophilized and stored at -20 degrees for future use. The antigenic capacity of the particles was determined as previously described by dissolving the particles in DMSO prior to CBQCA analysis. The nanoparticle size and zeta potential of the water were measured using dynamic light scattering in a Zetasizer Nano ZSP (Malvern Instruments, Westborough, Mass.). Lot-release criteria are that the TIMP-JCP ^P diameter is 700 nm ± 250 nm, requires a charge of at least -30 mV, and should contain at least 5 μg antigen / mg PLGA. The conditions for particle preparation and the results of JCP encapsulation are shown in Fig.

Early attempts to encapsulate total JCP extracts into TIMPs were only partially successful (Figure 4), since the maximum antigen load achieved was about 1 [mu] g protein / mg TIMP or less. This was due to the high viscosity of JCP even at potentially low concentrations (Figure 2a). Therefore, the optimal conditions for encapsulating the recombinant protein JCP (CRYJ1 and CRY2, Lifeome) a TIMP (TIMP-JCP ^R, the schematic diagram shown in Figure 2b) experiments were performed to determine.

TIMP-JCP ^R particles were produced using low molecular weight PLGA (0.17 dL / g), Sugi basic protein (CRYJ1, 55,180 Da) and polygalacturonase (CRYJ2, 59,070 Da). The purity of the recombinant protein was assessed by SDS-PAGE (Fig. 3c) and compared with previously determined results (Y. Mitobe et al., Regulatory Toxicology and Pharmacology : RTP 2015 71). The conditions for preparing the TIMP-JCP ^R particles are shown in Table 2.

CRYJ1 or CRYJ2 was encapsulated using the dual-emulsion protocol described above for the particle formulation (schematic diagram shown in Figure 2C). Protein concentration and burst release were determined for each sample by CBQCA. The results of these analyzes are shown in Table 3 below. Size and particle charge were measured by dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS or ZSP and the results are shown in Table 4 below.

Example 2: Murine model of JCP allergy

A mouse model of an acute allergic response to inhaled allergens has been widely used to identify the mechanism of immunological and inflammatory responses in asthma and to identify and investigate novel targets to control allergic inflammation.

The characteristics of the acute inflammatory model can be influenced by the selection of mouse strains, allergenic antigens and sensitization and challenge protocols (Fig. 5A). Briefly, cedar pollen extract-Cj (LSL-LG5280, Lo # 153101, Cosmo Bio USA) was resuspended in 1 mg / mL MilliQ water. 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 JCP + alum solution (ie, 50 μg JCP adsorbed to 2 mg / mL albumin, n = 10). Control mice were injected with alum diluted with PBS (n = 10). A second, equivalent dose was administered to the mice on day 14. On day 21, mice were collected from abdominal endothelial cells and serum was collected for antibody analysis (Fig. 5b-5d). On day 22, a portion of mice from each group were tolerated with a single 2.5 mg dose of TIMP-OVA (n = 5) or TIMP-JCP (n = 4) administered intravenously via the tail vein. In addition, mice are tolerated with TIMP-OVA or TIMP-JCP prior to sensitization with JCP.

On days 28 and 29, mice were administered intranasally with 100 μg of JCP extract in 20 μL MilliQ water. 29 days after administration, the mice were monitored for scratching (Fig. 7) and body temperature changes (Fig. 6) for 1 hour. One hour later, blood was collected and blood was collected to assess serum levels of MCPT-1 (Figure 10b) and histamine (Figure 10a).

On day 30, the mice were bled again for antibody analysis in serum (Figures 9a-9c). Mice were then sacrificed and spleens from each mouse were collected and homogenized for in vitro cytokine analysis and proliferation (Figure 8). The spleen was harvested, processed into a single cell suspension without red blood cells, and incubated with 1, 25 or 50 μg / mL JCP complete RPMI culture medium for 48 hours at 37 ° C. 50 [mu] l of supernatant was removed for cytokine analysis and the cells were pulsed with 1 [mu] Ci / well [< 3 > H] TdR for the last 24 hours of culture. Proliferation was measured by [3H] TdR incorporation detected by Topcount Microplate Scintillation Counter (PerkinElmer, Waltham, Mass.).

The data show that treatment with JCP-TIMP after JCP sensitization significantly reduces IFNγ, IL-17 and IL-5 production in in vitro assays. Similar effects were also observed at IL-4, IL-10 and IL-13 levels when stimulated with splenocytes by high dose of JCP. Antibody levels did not change after treatment with TIMP.

Example 3: Murine model of JCP allergy and treatment with CRYJ1 and CRYJ2 TIMPs

Allergy airway inflammation model

Mice were immunized intraperitoneally (i.p.) with PBS alone with the major Japanese cedar pollen allergen or alum in the aliquot of 10 μg CRYJ1 and CRYJ2, alum (3 mg). Mice were then dosed for 20 minutes with CRYJ1 and CRYJ2 (10 mg / ml) aerosolized for three consecutive days prior to tissue harvest.

Analysis of bronchoalveolar lavage eosinophils

The lungs are flushed with 1 mL of bronchoalveolar lavage (BALF; 1 mM EDTA and 10% FBS in PBS). Total cell counts are determined, samples are stained for differential cell counts and cytospun on DiffQuik (Siemens, Newark, Del.).

Airway histology

The lungs are collected, fixed in formalin and processed into paraffin. Paraffin sections are stained with hematoxylin and eosin (H & E) or periodic acid-Schiff (PAS).

JCP-specific IgE

Serum is collected from the mice at sacrifice and the CRYJ1 / 2-specific IgE is quantitated by sandwich ELISA. (Prepared using EZ-Link sulfo-NHS-LC-biotin kit of Pierce [Rockford, Rock]) with anti-mouse IgE (BD Biosciences) as capture antibody and biotinylated CRYJ1 / It is used as a reagent. The amount of CRYJ1 / 2-IgE should be measured by a standard curve generated using the purified mouse CRYJ1 / 2-IgE.

Quantification of cytokines

BAL fluids and supernatants (harvested at 48 hours) from recalled reaction media were analyzed by magnetic Milliplex MAP multiplex assay (Millipore, Billerica, MA) for IL-4, IL-5, IL- 17 and IFN [gamma].

Production and / or supply of cedar pollen antigen

The purified cedar pollen extract is derived from the Japan Meteorological Society. Also, recombinant Japanese cedar pollen antigen should be prepared as previously described (Fujimura T, Int. Arch. Allergy Immunol. 2015; 168 (1): 32-43). The amino acid sequence for the immunoprivileged epitope in JCP is CRYJ1 (Accession No. AB081309; SEQ ID NO: 1) and CRYJ2 (Accession No. AB211810; SEQ ID NO: 2).

Research batch production of TIMP-JCP

Recombinant JCP antigens are encapsulated in TIMP as described above. 200 mg of TIMP-CRYJ1 and / or CRYJ2 (TIMP-JCP ^R) and 200 mg TIMP-OVA control group will be used to test the efficacy in a mouse model of allergic JCP. Six groups of 5-10 mice will be treated with either TIMP-OVA or TIMP-JCP ^R once dose (group 1-3) or twice dose (group 3-6). Animals are sensitized to JCP antigens (before and after TIMP treatment). On days 28 and 29, airway infections will be performed and the efficacy of the treatment is determined using the primary readout and success measurements described below. Group 7 should be sensitive to OVA and can be administered with OVA instead of JCP, so it is used as a positive allergic control.

Animal POC: Pre-sensitized TIMP-JCP treatment

Six groups of 5-10 mice are treated with either TIMP-OVA or TIMP-JCP ^R in a single dose (Group 1-3) or two doses (Group 3-6) regimen as shown in Table 5. Animals will be sensitized to JCP antigens. On days 28 and 29, airway infections will be performed and the efficacy of the treatment is determined using the primary readout and success measurements described below. Group 7 should be sensitive to OVA and can be administered with OVA instead of JCP, so it is used as a positive allergic control.

TIMP-JCP treatment after animal POC-sensitization

Six mouse groups were sensitized to JCP and treated as TIMP-JCP ^R or TIMP-OVA as shown in Table 6 below. Two dosing regimens are tested. These include one TIMP dose regimen (group 1-3) and two dosing regimens (group 4-6), divided by 7 day intervals. On days 28 and 29 an air challenge is performed and the efficacy of the treatment is determined using the primary readout and success measurements described below.

Primary reading of animal model and measurement of success

Primary readings and success measurements are measured in mice receiving TIMP-JCP ^R treatment prior to sensitization. The TIMP-JCP ^R treated mice had no temperature change relative to baseline temperature after air challenge. There is no change in the number of blood eosinophils and the number of mast cells compared with the reference value of TIMP-JCP ^R treated mice. TIMP-JCP ^R treated mice significantly reduced JCP-specific IgE compared to JCP-sensitized TIMP-OVA treated mice. TIMP-JCP ^R treated mice also significantly reduced systemic IL-4, IL-5, and IL-13 compared to JCP-sensitized TIMP-OVA treated mice. TIMP-JCP ^R was compared with TIMP-OVA treated mice.

Primary readings and success measurements were measured in mice that received TIMP-JCP ^R treatment after sensitization. The TIMP-JCP ^R treated mice had no temperature change relative to baseline temperature after air challenge. There is no change in the number of blood eosinophils and the number of mast cells compared with the reference value of TIMP-JCP ^R treated mice. TIMP-JCP ^R treated mice significantly reduced JCP-specific IgE compared to JCP-sensitized TIMP-OVA treated mice. In addition, TIMP-JCP ^R treated mice significantly reduced JCP-specific systemic IL-4, IL-5 and IL-13 compared to JCP-sensitized TIMP-OVA treated mice. In addition, TIMP-JCP ^R treated mice reduced specific antigen production compared to TIMP-OVA treated mice.

Example 4; Clinical study on TIMP-JCP

Clinical studies are performed to assess the efficacy and safety of TIMP-JCP in single-center, non-blind, placebo-controlled studies in Japanese seasonal allergic rhinitis (SAR) patients exposed to quantitative high density Japanese cedar pollen .

The primary goal of clinical studies is to analyze and evaluate changes in total nasal congestion scores (TNSS) in Japanese SAR patients treated with oral TIMP-JCP during high-density exposure to Japanese cedar pollen.

The second goal was to analyze and evaluate the changes in total symptom score (TSS), symptom score, nasal discharge, sneeze number and patient's impression for allergic rhinitis patients, and high density exposure to pure Japanese cedar pollen And to assess the safety of TIMP-JCP administered via the intravenous route of the recipient.

General research design.

Measure the safety and efficacy of TIMP-JCP in patients with allergic reactions to Japanese cedar pollen using environmental exposure units. However, although there are many environmental exposure units (EEU), this study is carried out in cooperation with the Wakayama prefectural Japanese health support network department. Because there are not many limitations of the existing approach, it is possible to design and implement a precise and controlled study through EEU (a similar study design is described in Enomoto, et al., 2009). The diagram below summarizes medication and event schedules.

Object

There are a total of 25 subjects in clinical studies. Five subjects are treated with placebo. Twenty subjects are treated with TIMP-JCP.

Diagnosis and inclusion criteria

Include by:

ㆍ Japanese SAR patients.

ㆍ Patients with a history of Japanese cedar pollen hypersensitivity for at least two years.

Positive IgE for Japanese cedar pollen antigen: Screening is determined by fluorescence-enzyme immunoassay (FEIA) or chemiluminescent enzyme immunoassay (CLEIA) within 1.5 years prior to exposure test day.

Patients with a TNSS greater than 8 and a nasal congestion score greater than or equal to 2 (normal) after the start of screening exposure test (TNSS). (At least one score and nasal congestion score of 90 to 150 minutes after screening exposure has met the criteria If it is, it can be any other evaluation score)

20 years old or older and 65 years old or younger (no sexual preference).

Patients who signed the agreement by notice.

By exclusion:

Patients with symptoms of non-seasonal allergic rhinitis.

Patients with severe asthma, bronchiectasis, severe liver, kidney or heart function abnormalities, blood, endocrine disorders and other serious complications.

• Patients with ocular disease that may interfere with the validity of TIMP-JCP or nasal disease (hypertrophic rhinitis, sinusitis, nasal polyposis, nasal septal deviation, etc.)

Patients with evidence of upper and / or lower respiratory tract inflammation (acute rhinitis, chronic rhinitis, congestive rhinitis, atrophic rhinitis, purulent nasal discharge, sinusitis such as poor circulation, etc.) on the day of treatment exposure.

• Patients taking any of the following medications that may affect the evaluation of TIMP-JCP, including anti-participation agents and immunomodulators.

Additional exclusion criteria:

Within 2 weeks prior to the first administration day:

Anti-allergic drugs, antihistamines (H1 and H2 blockers: oral administration, spot drugs, eye drops, injections and topical use), anticholinergic agents, vasoconstrictor agents, antihistamine-containing cold medicines, antiallergic / antihistamines Other preparations that may be expected to have effects (including herbal medicines and glysirhizin) and allergic symptoms (sneezing, rash, nasal congestion, eye pruritus, etc.).

Histamine containing steroids (oral, inhalation, dentifrice, eye drops or topical use), immunosuppressants (oral, topical or injectable), azole fungicides and gamma globulin preparations.

Formulations containing an azole fungicide, a macrolide antibiotic, and an aluminum hydroxide / magnesium hydroxide.

Within 4 weeks of screening exposure test day:

ㆍ Defoosteroid preparation.

Screening Within six months of exposure test day: Steroid injection.

ㆍ Steroid injection

Within 1 year before screening exposure test day:

Patients undergoing maintenance or nonspecific alternative therapies for specific low-sensitization.

Patients who are participating in other studies or who have previously participated in other studies within 6 months prior to the informed consent.

The investigator / sub-investigator is deemed unsuitable for enrolling in studies for any other criteria.

Patients with hypersensitivity to antihistamines or antihistamine preparations (including fexofenadine HCl) and pseudoephedrine hydrochloride.

Patients participating in other studies or participating in other studies within 6 months prior to screening exposure.

Women who are pregnant, have a chance of becoming pregnant, or are currently breastfeeding.

Safety and Stopping Criteria

If one of the following parameters is met, the study can be suspended at the discretion of the data safety monitoring board:

a) one or more unexpected drug-related serious adverse events (SAEs) are reported to the data safety monitoring board.

b) Excessive and / or unexpected harmful events not clearly associated with the study drug.

c) Unexpected patient death

Dosage, duration and mode of administration:

Drug: TIMP-JCP, poly (lactic acid-co-glycolic acid) immunogenic immunodeficiency

Dosage and Form: TIMP-JCP is provided as a lyophilized white powder sterilized in vials. Each vial contains 500 mg of TIMP-JCP. TIMP-JCP is provided at a dose of [10 mg / kg initially adjusted for outcome per step].

Duration of therapy / study: Patient registration occurs after patient screening and notification by notice. When the study is mobilized, dosing begins. Dosage will be provided on day 0 and will be provided more than once on day 14. The first high density Japanese cedar pollen exposure occurs on days 18-21 (the patient is exposed to 8,000 particles / m ³ of JCP for 5 hours in environmental exposure units). The second high density Japanese cedar pollen exposure occurs in 32 to 35 days (the patient is exposed to 8000 grains / m ³ of JCP for 5 hours to environmental exposure unit). Follow-up visits occur on days 42, 49, and 56. Final follow-up visits occur on the 63rd day, four weeks after the second exposure.

Dosage Modes: The reconstituted TIMP-JCP should be diluted with normal saline (NS; 0.9% NaCl, NaCl) by slow IV infusion over 30 minutes. The TIMP-JCP may be discontinued at any time if the investigator determines that alternative therapies should be instituted.

Evaluation standard

safety:

The incidence and severity of AE, plus the specificity of plus AE, including standard reporting criteria for hypersensitivity and immune-mediated responses.

ㆍ Vital signs (armpit temperature, blood pressure in a sitting position, pulse rate in sitting position)

ㆍ Standard laboratory evaluation

ㆍ 12-lead ECG laboratory test (hematology, biochemistry and urinalysis)

ㆍ Prick test for Japanese cedar pollen

Parameters developed by antibody and histamine release tests.

Primary endpoint:

TNSS: Total score of sneezing, rash, nasal congestion and itchy nose. Each symptom is assessed by the following five categories of patients: 1 = none (no symptoms); 2 = mild (symptomatic but easily tolerated); 3 = moderate (awareness of symptoms; inconvenience, but tolerable); 4 = Severe (a clear recognition of symptoms; difficult to tolerate but not interfering with activities of daily living); 5 = severe (difficult to tolerate and interfering with daily activities)

Secondary efficacy endpoint:

Immunity monitoring:

(Anti-JCP, anti-CRYJ1 and anti-CRYJ2), specific IgG4 antibody (anti-JCP), IgE antibody, specific IgE antibody (anti-JCP)

(IL-4, IL-5, IL-10, IL-12 and IL-13)

Immunocytochemistry (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's impression.

ㆍ Amount of nasal secretion: Measure the weight of tissue paper after rinsing patient's nose every hour. The difference from the weight before uncoiling is calculated as the amount of nasal secretion.

Number of sneezes: The patient records the number of sneezes themselves every hour.

Pharmacokinetics (PK):

Serum concentrations of TIMP-JCP measured daily in rare sample designs during study drug administration.

conclusion:

Subjects treated with TIMP-JCP showed less occurrence and severity of allergic reactions to Japanese cedar pollen exposure. This indicates that TNSS of subjects treated with TIMP-JCP was reduced compared to subjects treated with placebo. JCP-treated subjects also exhibit 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 Japanese cedar pollen; Have a lower TSS score, symptom score, nasal discharge and sneeze frequency compared to placebo groups.

Although specific embodiments of the invention have been described and illustrated, it should be understood that these embodiments are to be considered merely exemplary of the invention and are not to be construed as limiting the invention as interpreted in accordance with the appended claims.

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

                         SEQUENCE LISTING <110> Cour Pharmaceuticals Development Company        Getts, Daniel R.   <120> TIMPs Encapsulating Japanese Cedar Pollen Epitopes <130> COUR-014 / 02WO <150> US 62 / 293,261 <151> 2016-02-09 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 329 <212> PRT <213> Cryptomeria japonica <400> 1 Met Asp Asn Pro Ile Asp Ser Ser Trp Arg Gly Asp Ser Asn Trp Ala 1 5 10 15 Gln Asn Arg Met Lys Leu Ala Asp Ser Ala Val Gly Phe Gly Ser Ser             20 25 30 Thr Met Gly Gly Lys Gly Gly Asp Leu Tyr Thr Val Thr Asn Ser Asp         35 40 45 Asp Asp Pro Val Asn Pro Ala Pro Gly Thr Leu Arg Tyr Gly Ala Thr     50 55 60 Arg Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys 65 70 75 80 Leu Lys Met Pro Met Tyr Ile Ala Gly Tyr Lys Thr Phe Asp Gly Arg                 85 90 95 Gly Ala Gln Val Tyr Ile Gly Asn Gly Gly Pro Ser Val Phe Ile Lys             100 105 110 Arg Val Ser Asn Val Ile Ile His Gly Leu His Leu Tyr Gly Ser Ser         115 120 125 Thr Ser Val Leu Gly Asn Val Leu Ile Asn Glu Ser Phe Gly Val Glu     130 135 140 Pro Val His Pro Gln Asp Gly Asp Ala Leu Thr Leu Arg Thr Ala Thr 145 150 155 160 Asn Ile Trp Ile Asp His Asn Ser Phe Ser Asn Ser Ser Asp Gly Leu                 165 170 175 Val Asp Val Thr Leu Ser Ser Thr Gly Val Thr Ile Ser Asn Asn Leu             180 185 190 Phe Phe Asn His His Lys Val Met Leu Leu Gly His Asp Asp Ala Tyr         195 200 205 Ser Asp Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly     210 215 220 Pro Asn Ser Gly Gln Arg Met Met Arg Ala Arg Tyr Gly Leu Val His 225 230 235 240 Val Ala Asn Asn Asn Tyr Asp Pro Trp Thr Ile Tyr Ala Ile Gly Gly                 245 250 255 Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser Phe Thr Ala Pro             260 265 270 Asn Glu Ser Tyr Lys Lys Gln Val Thr Ile Arg Ile Gly Ser Lys Thr         275 280 285 Ser Ser Ser Ser Asn Trp Val Trp Gln Ser Thr Gln Asp Val Phe     290 295 300 Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn 305 310 315 320 Ile Tyr Thr Lys Lys Glu Ala Phe Asn                 325 <210> 2 <211> 391 <212> PRT <213> Cryptomeria japonica <400> 2 Val Glu Asn Gly Asn Ala Thr Pro Gln Leu Thr Lys Asn Ala Gly Val 1 5 10 15 Leu Thr Ser Ser Leu Ser Lys Arg Cys Arg Lys Val Glu His Ser Arg             20 25 30 His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala Val Gly         35 40 45 Asp Gly Lys His Asp Ser Thr Glu Ala Phe Ser Thr Ala Trp Gln Ala     50 55 60 Ala Ser Lys Lys Pro Ser Ala Met Leu Leu Val Pro Gly Asn Lys Lys 65 70 75 80 Phe Val Val Asn Asn Leu Phe Phe Asn Gly Pro Ser Gln Pro His Phe                 85 90 95 Thr Phe Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser             100 105 110 Trp Lys Asn Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe         115 120 125 Thr Leu Met Gly Lys Gly Val Ile Asp Gly Gln Gly Lys Gln Trp Trp     130 135 140 Ala Gly Gln Ser Lys Trp Val Asn Gly Arg Glu Ile Ser Asn Asp Arg 145 150 155 160 Asp Arg Pro Thr Ala Ile Lys Phe Asp Phe Ser Thr Gly Leu Ile Ile                 165 170 175 Gln Gly Leu Lys Leu Met Asn Ser Pro Glu Phe His Leu Val Phe Gly             180 185 190 Asn Ser Glu Gly Val Lys Ile Ile Gly Ile Ser Ile Thr Ala Pro Arg         195 200 205 Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Ala Ser Lys Asn Phe     210 215 220 His Leu Gln Lys Asn Thr Ile Gly Thr Gly Asp Asp Ser Val Ala Ile 225 230 235 240 Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu Ile Ser Gly Pro                 245 250 255 Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Glu Asn Ser Arg Ala             260 265 270 Glu Val Ser Tyr Val His Val Asn Gly Ala Lys Phe Ile Asp Thr Gln         275 280 285 Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser Gly Met Ala Ser     290 295 300 His Ile Ile Tyr Glu Asn Val Glu Met Ile Asn Ser Glu Asn Pro Ile 305 310 315 320 Leu Ile Asn Gln Phe Tyr Ser Thr Ser Ala Ser Ala Ser Gln Asn Gln                 325 330 335 Arg Ser Ala Val Gln Ile Gln Asp Val Thr Tyr Lys Asn Ile Arg Gly             340 345 350 Thr Ser Ala Thr Ala Ala Ile Gln Leu Lys Ser Ser Asp Ser Met         355 360 365 Pro Ser Lys Asp Ile Lys Leu Ser Asp Ile Ser Leu Lys Leu Thr Ser     370 375 380 Gly Lys Ile Ala Ser Ser Leu 385 390 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide linker <400> 3 Gly Ala Val Val Gly Ala 1 5

Claims (55)

A composition comprising biodegradable particles comprising at least one encapsulated antigenic epitope from Japanese cedar pollen, wherein said biodegradable particles have a negative zeta potential. The composition of claim 1, wherein the biodegradable particles comprise poly (lactide-co-glycolide) (PLG). The composition of claim 1 or 2, wherein the biodegradable particles comprise PLG having a copolymer ratio of polylactic acid: polyglycolic acid of about 50:50. The composition according to any one of claims 1 to 3, wherein the surface of the biodegradable particles is carboxylated. 5. The composition of claim 4, wherein the carboxylation is achieved using poly (ethylene-alt-maleic anhydride), (PEMA) poly (acrylic acid) or sodium deoxycholate. The composition according to any one of claims 1-5, wherein the biodegradable particles have a zeta potential of about -100 mV to about 0 mV. The composition of any one of claims 1-6, wherein the biodegradable particles have a zeta potential of about -50 mV to about -40 mV. The composition of any one of claims 1 to 7, wherein the biodegradable particles have a zeta potential of about -75 mV to about -50 mV. The composition according to any one of claims 1 to 8, wherein the biodegradable particles have a zeta potential of about -50 mV. The composition according to any one of claims 1 to 9, wherein the biodegradable particles have a zeta potential of about-30 mV. The composition according to any one of claims 1 to 10, wherein the biodegradable particles have a zeta potential of about -40 mV. The composition according to any one of claims 1 to 11, wherein the biodegradable particles have a diameter of from about 0.1 μm to about 10 μm. The composition according to any one of claims 1 to 12, wherein the biodegradable particles have a diameter of about 0.3 microns to about 5 microns. The composition of any one of claims 1-13, wherein the biodegradable particles have a diameter of about 0.5 μm to about 3 μm. The composition according to any one of claims 1 to 14, wherein the biodegradable particles have a diameter of from about 0.5 μm to about 1 μm. The composition according to any one of claims 1 to 15, wherein the biodegradable particles have a diameter of from about 0.2 μm to about 0.7 μm. 17. The composition of claim 16, wherein the biodegradable particles have a diameter of about 0.7 m. 17. The composition of claim 16, wherein the biodegradable particles have a diameter of about 0.5 microns. The composition of any one of claims 1 to 18, wherein the at least one encapsulated antigenic epitope from Japanese cedar pollen comprises CRYJ1 or a fragment or variant thereof. 20. The composition of claim 19, wherein CRYJ1 has the amino acid sequence of SEQ ID NO: 1. 20. The composition of claim 20, wherein the fragment of CRYJ1 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids having at least 90% sequence identity to SEQ ID NO: 20. The method of claim 20, wherein the variant of CRYJ1 comprises a sequence identity of 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% &Lt; / RTI &gt; 21. The composition of claim 20, wherein the fragment of CRYJ1 is selected from the group consisting of p16-30, p81-95, p106-120, p111-125, p211-225 and p301-315. The composition of any one of claims 1-23, wherein the at least one encapsulated antigenic epitope from Japanese cedar pollen comprises CRYJ2 or a fragment or variant thereof. 25. The composition of claim 24, wherein CRYJ2 has the amino acid sequence of SEQ ID NO: 2. 26. The composition of claim 25, wherein the fragment of CRYJ2 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids having at least 90% sequence identity to SEQ ID NO: 26. The variant of claim 25 wherein the variant of CRYJ2 has 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: &Lt; / RTI &gt; 26. The composition of claim 25, wherein the 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. The composition of any one of claims 1 to 28, wherein the biodegradable particles comprise a ratio of antigen to polymer from 1 μg / mg to 15 μg / mg. 31. The composition of claim 29, wherein the biodegradable particles comprise a ratio of antigen to polymer from 5 占 퐂 / mg to 15 占 퐂 / mg. The composition of any one of claims 1 to 30, wherein the biodegradable particles comprise a ratio of antigen to a polymer of at least about 5 μg / mg. The composition of any one of claims 1 to 31, wherein the biodegradable particles comprise at least two encapsulated antigenic epitopes from Japanese cedar pollen protein. 33. The composition of claim 32, wherein the two or more encapsulated epitopes are contained in a fusion protein, wherein the two or more encapsulated epitopes in the fusion protein are separated by a cleavable linker. 34. The composition of claim 33, wherein the cleavable linker amino acid sequence is cleavable by a protease located in the phagocytose of the cell and / or a protease located in the cytosol of the cell. 35. The composition of claim 34, wherein the amino acid sequence of the cleavable linker is cleavable by a protease located in the phagocytose of the cell and a protease located in the cytosol of the cell. 35. The composition of claim 34 or 35 wherein the cleavable linker is a purine-susceptible linker or a cathepsin-sensitive linker. 34. The composition of any one of claims 34-36, wherein the cleavable linker is a purine susceptible linker. 34. The composition of any one of claims 34 to 36, wherein the cleavable linker is a cathepsin-sensitive linker. 39. The method of claim 38, wherein the cathepsin-sensitive linker is selected from the group consisting of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, , Cathepsin O, cathepsin W, and / or cathepsin Z. 35. The composition of claim 34, wherein the amino acid sequence of the linker is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 3). 41. The composition of any one of claims 1-40, wherein the at least one encapsulated antigenic epitope from Japanese cedar pollen is covalently bound to biodegradable particles. 43. The composition of claim 41, wherein the at least one encapsulated antigenic epitope from Japanese cedar pollen is covalently bound to the biodegradable particle by a conjugate molecule. 43. The composition of claim 42, wherein the conjugate molecule comprises a carbodiimide compound. 43. The composition of claim 43, wherein the carbodiimide compound comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). A composition comprising biodegradable particles comprising at least one encapsulated antigenic epitope from Japanese cedar pollen, wherein the biodegradable particles have a negative zeta potential of at least about -30 mV, a diameter of about 0.7 탆, a diameter of at least about 5 ug / mg of the antigen to the polymer. A pharmaceutical composition comprising the biodegradable particles of any one of claims 1 to 45. 47. The pharmaceutical composition of claim 46, further comprising a pharmaceutically acceptable carrier. 48. The pharmaceutical composition of claim 47, further comprising a pharmaceutically acceptable excipient. A lyophilized composition comprising the biodegradable particles of any one of claims 1 to 45. 46. A method of inducing antigen-specific resistance to Japanese cedar pollen in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of any one of claims 46-49. A method for treating Japanese cedar pollen allergy in a subject in need of treatment, comprising administering the pharmaceutical composition of any one of claims 46 to 49. A method for preventing Japanese cedar pollen allergy in a subject in need of treatment comprising administering the pharmaceutical composition of any one of claims 46 to 49. 49. A method of inducing antigen-specific resistance to Japanese cedar pollen in a subject, comprising reconstituting the lyophilized particles of claim 49 to obtain a reconstituted pharmaceutical composition and administering the reconstituted pharmaceutical composition to the subject How to. 49. A method of treating Japanese cedar pollen allergy in a subject in need of treatment comprising reconstituting the lyophilized particles of claim 49 to obtain a reconstituted pharmaceutical composition and administering the reconstituted pharmaceutical composition to a subject Way. Comprising reconstituting the lyophilized particles of claim 49 to obtain a reconstituted pharmaceutical composition and administering the reconstituted pharmaceutical composition to a subject, wherein the method comprises the steps of: Way.

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