JP2022101576A - Timps (tissue inhibitors of metalloproteinase) encapsulating japanese cedar pollen epitopes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide safe and effective means to induce tolerance to cedar pollen antigens (e.g., CRYJ1 and CRYJ2) in subjects that suffer from Japanese cedar pollinosis, or in subjects at risk for developing Japanese cedar pollinosis, taking account of the substantial prevalence of the disease, particularly in Japan and neighboring countries, and the likelihood of antibody cross-reactivity between conifer pollens.

SOLUTION: The present invention provides compositions comprising particles with a negative zeta potential that encapsulate one or more epitopes associated with Japanese cedar pollen. Methods of inducing immunological tolerance to Japanese cedar pollen by administering the particles are also provided.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

Cross-reference to related applications This application claims the priority of US Provisional Application No. 62 / 293,261 filed February 9, 2016, the entire contents of which are incorporated herein by reference.

Description of Text Files Submitted Electronically The contents of text files submitted electronically with this specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename). : COUR_014_02WO_SeqList_ST25.txt, recording date: February 9, 2017, file size: 12 kilobytes).

Sugi pollinosis is a common allergic disease in Japan caused by inhalation of Sugi (Cryptomeria japonica) pollen. The prevalence of Sugi pollinosis in Japanese in non-randomized groups of schools, factories, and communities can be as high as 30%, with up to 20 million people affected by the disease. It is speculated that it can be done. Therefore, this disease is a serious public health problem in Japan, partly due to the severity of symptoms, high morbidity, poor spontaneous cure rates, and high medical costs associated with disease control.

Sugi pollinosis is a severe type I allergic disease. Type I allergic disease is a particularly harmless environment characterized by high systemic levels of allergen-specific immunoglobulin E (IgE), mast cell degranulation, and the release of histamine and other allergic messenger factors. It is mediated by an abnormal Th2-polar immune response to an antigen (eg, cedar pollen). The primary clinical approach to the treatment of allergic diseases is generally targeting antigen (Ag) avoidance (eg, allergen avoidance), and acute effector molecules with drugs such as antihistamines, leukotriene inhibitors, or broadly acting glucocorticoids. It consists of control of symptoms by. However, such treatments are unable to address the development of an abnormal Th2-type immune response that is the underlying cause of promoting allergic inflammation. Alternative techniques such as specific immunotherapy (SIT), in which the patient is exposed to a slowly increasing dose of soluble Ag delivered intramucosally or subcutaneously, are also clinically used. SIT induces both regulatory and Th1 responses that inhibit the established Th2 response. Therefore, SIT can result in a biased immune response to environmental allergens away from pathological Th2 type responses and towards protective or regulatory Th1 / T regulatory responses. However, administration of soluble Ag to sensitized patients poses a great risk of adverse reactions, thereby slow dose escalation of the antigen or co-administration of the antigen with additional drugs such as omalizumab to minimize anaphylaxis. Needs.

Therefore, clinical means for the treatment of allergic diseases in pre-sensitized subjects continue to be sought in improved methods for safely and effectively inducing Ag-specific tolerance. Nanoparticles encapsulating the allergen peptide have traditionally been demonstrated in in vivo models to reduce allergen-induced Th2 responses (see US Patent Application Publication No. 2015/0209293, which is in its entirety by reference. Incorporated in the specification). Therefore, it is possible to enclose pollen derived from various environmental sources in nanoparticles for use in the treatment of allergic diseases such as sugi pollinosis. In addition, the cedar pollen antigen CRYJ1 has high similarity to the mountain cedar Jun a 1 antigen, the hinoki Cha o 1 antigen, and the Cupressus arisonica Cup a 1 antigen, which is a specific antigen derived from cedar pollen. However, it is possible to mediate an allergic antibody response that is cross-reactive across multiple coniferous pollens.

However, attempts to encapsulate Sugi pollen in particles suitable for the treatment of allergic diseases have been only partially successful. This is most likely due to the high stickiness of Sugi pollen, even at low concentrations. Therefore, SIT treatment methods for Sugi pollinosis remain limited. Given the significant prevalence of this disease, especially in Japan and neighboring countries, as well as the potential for cross-reactivity of antibodies between coniferous pollen, there is a risk of developing Sugi pollinosis in subjects suffering from Sugi pollinosis. Safe and effective means for inducing tolerance to Sugi pollen antigens (eg, CRYJ1 and CRYJ2) in a subject are needed in the art.

In some embodiments, the invention comprises carrier particles (eg, PLG particles) that embed or adhere to one or more epitopes derived from Sugi pollen (eg, for the induction of antigen-specific tolerance). ) The composition is provided. In certain embodiments, the carrier particles are poly (lactide-co-glycolide) (PLG) particles with a negative zeta potential.

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

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

Certain embodiments of the invention are directed to compositions comprising biodegradable particles with a negative zeta potential, comprising one or more encapsulated antigen epitopes derived from Sugi pollen. In some embodiments, one or more encapsulated antigen epitopes derived from Sugi 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. Includes LTP and / or Cry j CPA9. In certain embodiments, one or more encapsulated antigen epitopes from Sugi pollen comprises CRYJ1, or a fragment or variant thereof. In some embodiments, CRYJ1 has the following amino acid sequence:
MDNPIDSSWRGDSNWAQNRMKLADSAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPSVFIKRVSNVIIHGLHLYGSSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTISNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNSGQRMPRARYGLVHVANNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGSKTSSSSSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFN（配列番号１）

In some embodiments, the fragment of CRYJ1 comprises at least 10, at least 20, at least 30, at least 40, or at least 50 contiguous amino acids having at least 90% sequence identity to SEQ ID NO: 1. In certain embodiments, the variant of CRYJ1 is 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 relative to SEQ ID NO: 1. Has an amino acid sequence with% sequence identity. 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, one or more encapsulated antigen epitopes derived from Sugi pollen comprise CRYJ2 or a fragment or variant thereof. In some embodiments, CRYJ2 has the following amino acid sequence:
VENGNATPQLTKNAGVLTSSLSKRCRKVEHSRHDAINIFNVEKYGAVGDGKHDSTEAFSTAWQAASKXPSAMLLVPGNKKFVVNNLFFNGPSQPHFTFKVDGIIAAYQNPASWI^NRIWLQFAKITGFTLMGKGVIDGQGKQWWAGQSKXVVNGREISNDRDRPTAIKFDFSTGLIIQGLKMNSPEFHLVFGNSEGVKIIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDSVAIGTGSSNIVIEDLISGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRIKTWQGGSGMASHIIYENVEMINSENPILINQFYSTSASASQNQRSAVQIQDVTYKNTIRGTSATAAAIQLKSSDSMPSKDIKLSDISLKLTSGKIASSL（配列番号２）

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

In certain embodiments, the biodegradable particles described herein contain two or more encapsulated antigenic epitopes derived from Sugi pollen protein. In some embodiments, the two or more encapsulated epitopes are contained in the fusion protein, where 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 within the phagolysosome of the cell and / or a protease located within the cytosol of the cell. In some embodiments, the amino acid sequence of the cleavable linker is cleavable by a protease located within the phagolysosome of the cell and a protease located within the cytosol of the cell. In certain embodiments, the cleavable linker is a furin-sensitive or cathepsin-sensitive linker. In certain embodiments, the cleavable linker is a furin-sensitive linker. In some embodiments, the cleavable linker is a cathepsin-sensitive linker. In certain embodiments, the cathepsin-sensitive linkers are cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, and / or. Sensitive to cleavage by one or more of cathepsins 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 antigen epitopes from Sugi pollen are covalently attached to biodegradable particles. In certain embodiments, one or more encapsulated antigen epitopes from Sugi pollen are covalently attached to biodegradable particles by a conjugate molecule. In some embodiments, the conjugate molecule comprises a carbodiimide compound. In certain embodiments, the carbodiimide compound comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

Certain embodiments are directed 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 are directed to lyophilized compositions comprising the biodegradable particles described herein.

Certain embodiments are directed to methods of inducing antigen-specific tolerance to Sugi pollen in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein. .. Certain embodiments are directed to methods for the treatment of Sugi pollen allergies in subjects in need of treatment, the methods comprising administering the pharmaceutical compositions described herein. Some embodiments are directed to methods for the prevention of Sugi pollen allergy in subjects in need of prevention, the method comprising administering the pharmaceutical compositions described herein.

A particular embodiment is directed to a method of inducing antigen-specific tolerance to Sugi pollen in a subject, wherein the lyophilized particles described herein are returned to obtain the returned pharmaceutical composition. Includes administration of the returned pharmaceutical composition to the subject. Certain embodiments are directed to a method for the treatment of Sugi pollen allergy in a subject in need of treatment, wherein the lyophilized particles described herein are returned and reconstituted pharmaceuticalally. It involves obtaining the composition and administering the returned pharmaceutical composition to the subject. Some embodiments are directed to methods for the prevention of Sugi pollen allergy in subjects in need of prevention, wherein the lyophilized particles described herein are returned and returned pharmaceuticalally. It involves obtaining the composition and administering the returned pharmaceutical composition to the subject.

We show a model of how administration of biodegradable particles with a negative zeta potential encapsulating an antigenic epitope derived from Sugi pollen provides an effective treatment for Sugi pollen allergy. Viscosity of JCP extract (FIG. 2A), exemplary illustration of biodegradable particles (TIMP-JCP) with negative zeta potential encapsulating the antigenic epitope of Sugi pollen (FIG. 2B), and chart of the double emulsion process (FIG. 2B). FIG. 2C) is shown. SDS-PAGE analysis of JCP extract and recombinant JCP protein is shown. The characteristics of the particles related to the TIMP-encapsulated JCP extract are shown. A chart of an acute inflammation mouse model (FIG. 5A) and an antibody response 21 days after sensitization (FIGS. 5B-5D) is shown. It shows changes in body temperature after JCP sensitization, TIMP treatment, and JCP antigen administration. Shows scratching, sneezing, and cough scores after JCP sensitization, TIMP treatment, and JCP antigen administration. Cytokine production from splenocytes of JCP-sensitized and antigen-administered mice treated with TIMP-JCP or TIMP-OVA is shown. The antibody response on the 30th day after sensitization by JCP is shown. The serum levels of histamine (FIG. 10A) and MCPT-1 (FIG. 10B) are shown. Illustrate the effect of the route of administration on the resulting immune response.

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

As used herein, "particle" refers to any composition that is not of tissue origin, which can be a spherical or spherical entity, beads, or liposomes. The terms "particle", term "immunomodified particle", term "carrier particle", and term "bead" may be used interchangeably depending on the context. In addition, the term "particle" can be used to include beads and spheres.

"Negatively charged particles" as used herein refer to particles modified to retain a net surface charge of less than zero.

The "carboxylated particle" or "carboxylated bead" or "carboxylated sphere" includes any particle modified to contain a carboxyl group on its surface. In some embodiments, the addition of a carboxyl group enhances phagocytic / monocyte uptake of particles from the bloodstream, eg, through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be achieved using any compound that adds or incorporates a carboxyl group, including but not limited to poly (ethylene-alt-maleic anhydride) (PEMA).

"Antigen moiety" as used herein refers to any moiety, eg, a peptide recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, enzymes, and / or bacterial or viral proteins, peptides, drugs or components. Without being bound by theory, carboxylated beads can themselves be recognized by the immune system, while carboxylated beads that are not attached to anything are referred to as "antigen moieties" for the purposes of the present invention. Not considered.

"Nude beads" or "naked particles" or "naked spheres" as used herein refer to non-carboxylated beads, particles, or spheres.

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

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

As used herein, the term "inhibitory neutrophil" refers to immunosuppressive cells derived from neutrophils and / or monocytes.

As used herein, the term "Th cells" or "helper T cells" refers to CD4 ⁺ cells. CD4 ⁺ T cells assist other leukocyte cells by immunological processes including B cell maturation to plasma and memory B cells, as well as activation of cytotoxic T cells and macrophages. T cells are activated when presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen presenting cells (APCs).

As used herein, the term "Th1 cells" refers to a subset of Th cells that produce pro-inflammatory mediators. Th1 cells serve as a host defense against pathogens by secreting cytokines, facilitating immune responses and partially mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines, including IFN-γ, IL-2, IL-10, and TNF α / β, to function in defense against intracellular pathogens such as viruses and some bacteria.

As used herein, the term "Th2 cells" refers to a subset of Th cells that mediate the activation and maintenance of antibody-mediated immune responses against extracellular parasites, bacteria, allergens, and toxins. Th2 cells are responsible for antibody production, eosinophil activation, and inhibition of multiple macrophage functions IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25). ), Which mediate these functions and thus provide phagocytic-independent protective responses.

As used herein, the term "Th17 cells" refers to a subset of Th cells. Th17 cells serve as a host defense against pathogens by secreting cytokines, facilitating immune responses and mediating the recruitment 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 function in defense against extracellular pathogens, including fungi and bacteria.

As used herein, "bound" refers to an antigen that is immobilized on the outside of a particle or encapsulated within a particle. Thus, the antigen bound to the particle includes both the surface binding of the particle and the encapsulation within the particle.

The term "IMP" as used herein refers to immunomodifying particles that are not bound to an antigen. The term "TIMP" as used herein refers to tolerant immunomodulatory particles bound to an antigen. In some embodiments, the antigen is attached to the surface of the TIMP. In other embodiments, the antigen is encapsulated within the TIMP.

The particles can have any particle shape or three-dimensional structure. However, in some embodiments, it is preferable to use particles that are less likely to aggregate in vivo. Examples of particles in these embodiments have a spherical shape.

Another aspect of the invention relates to a composition comprising immunomodifying particles having a negative zeta potential and freed from an antigenic moiety. In a further embodiment, the invention provides a composition comprising immunomodulatory particles having a negative zeta potential bound to an antigen. In a further embodiment, the antigen is bound to the outside of the particle. In a preferred embodiment, the antigen is encapsulated within the particles.

Yet another aspect of the invention relates to a method for the preparation of immunomodifying particles having a negative zeta potential and freed from an antigenic moiety. The method involves contacting the immunomodifying particle precursor with a buffer solution under conditions effective for forming immunomodifying particles with a negative zeta potential. In some embodiments of the invention, the immunomodifying particle precursor is formed via copolymerization. The microstructure of the particles may depend on the method of copolymerization.

In some embodiments, the antigenic peptide molecule is attached to carrier particles (eg, immunomodifying particles) by a conjugate molecule and / or a linker group. In some embodiments, the binding of the antigenic peptide and / or the apoptosis signaling molecule to a carrier (eg, PLG particles) comprises one or more covalent and / or non-covalent interactions. In some embodiments, the antigenic peptide is attached to the surface of carrier particles with a negative zeta potential. In some embodiments, the antigenic peptide is encapsulated in carrier particles with a negative zeta potential.

In one embodiment, the buffer solution in contact with the immunomodifying particles may have a basic pH. Suitable basic pH for basic solutions is 7.1, 7.5, 8.0, 8.5, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0. Includes 1, 12.5, 13.0, and 13.5. Further, the buffer solution may be made of any suitable base and a conjugate thereof. In some embodiments of the invention, the buffer solution may be sodium hydrogen carbonate, potassium hydrogen carbonate, lithium hydrogen carbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate, and a conjugate thereof. Gates may be included, but are not limited to these.

In one embodiment of the invention, the immunomodifying particles contain a copolymer. These copolymers can have a variety of molar ratios. Suitable copolymer ratios of the immunomodifying 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: 0. In another embodiment, the copolymer can be a periodic, statistical, linear, branched (including star, brush, or comb) copolymer. In some embodiments, the copolymer ratios are polystyrene: poly (vinyl carboxylate) / 80:20, polystyrene: poly (vinyl carboxylate) / 90:10, poly (vinyl carboxylate): polystyrene / 80:20, Poly (vinyl carboxylate): polystyrene / 90:10, polylactic acid: polyglycolic acid / 50:50, polylactic acid: polyglycolic acid / 80:20, or polylactic acid: polyglycolic acid / 90:10. , Not limited to these.

In one embodiment, the particles of the invention are made by adding a composition comprising a polymer (eg, PLGA) to a solution of poly (ethylene-maleic anhydride) (PEMA). The concentration of PEMA in the solution can 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 about 0.1% -4%. In another embodiment, the concentration of PEMA in solution is about 0.1% to 2%. In another embodiment, the concentration of PEMA in solution is about 0.5% to 1%. In one embodiment, the proportions of PEMA in the solution are 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0. 8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 6.5% , 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, the proportion of PEMA in solution is about 0.5%. In another embodiment, the proportion of PEMA in solution is about 1.0%. Other compounds that can be used include poly (ethylene-alt-maleic anhydride), poly (isobutylene-co-maleic acid), poly (methyl vinyl ether-alt-maleic acid), poly (methyl vinyl ether-alt-maleic acid). Monoethyl ester), poly (methyl vinyl ether-alt-maleic anhydride), poly (methyl vinyl ether-alt-maleic anhydride) crosslinked with 1,9-decadien powder, poly (styrene-alt-maleic acid) sodium salt , Poly (vinyl alcohol), poly (acrylic acid), and / or sodium deoxycholate, but not limited to these.

In one embodiment, the particles are liposomes. In a further embodiment, the particles are liposomes consisting of the following lipids in the following molar ratios-30: 30: 40 phosphatidylcholine: phosphatidylglycerol: cholesterol. Still in further embodiments, the particles are encapsulated within liposomes.

Each particle does not have to be of uniform size, but the particles are generally sealed within the spleen or liver and phagocytosed by antigen-presenting cells, including endothelial cells, or other MPS cells through receptor or non-receptor-mediated mechanisms. Or it must be large enough to induce uptake. Preferably, the particles are microscale or nanoscale in size to enhance solubility, avoid complications that may be caused by aggregation in vivo, and facilitate pinocytosis. Particle size can be a factor in uptake from the interstitial space into the area of lymphocyte maturation. Particles with a diameter of about 0.1 μm to about 10 μm can induce phagocytosis. Therefore, in one embodiment, the particles have a diameter within these limits. In another embodiment, the particles have an average diameter of about 0.3 μm to about 5 μm. Still in another embodiment, the particles have an average diameter of about 0.5 μm to about 3 μm. In another embodiment, the particles have an average diameter of about 0.2 μm to about 2 μm. In a further embodiment, the particles average about 0.1 μm, or about 0.2 μm, or about 0.3 μm, or about 0.4 μm, or about 0.5 μm, or about 1.0 μm, or about 1.5 μm, Or have a size of about 2.0 μm, or about 2.5 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm. In certain embodiments, the particles have an average size of about 0.5 μm. In some embodiments, the particles have an average diameter of about 0.5 μm to about 0.95 μm. For example, in such embodiments, the particles average 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. It has a diameter of 0.95 μm. In certain embodiments, the particles have an average diameter of about 0.7 μm. In some embodiments, the total weight of the particles is at least about 1000 kDa. In some embodiments, the total weight of the particles is about 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, it. That is all.

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 do not have to be of uniform diameter. As an example, pharmaceutical formulations may contain multiple particles, some of which have a diameter of about 0.5 μm, while others have a diameter of about 1.0 μm. As an additional example, pharmaceutical formulations may contain multiple particles, some of which have a diameter of about 0.7 μm, while others have a diameter of about 0.5 μm to about 0.95 μm. Have. Any mixture of particle sizes within these given ranges is useful.

The particles of the present invention may have a particular zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than about -100 mV (eg, more negative). In one embodiment, the zeta potential is less than about −50 mV (eg, more negative). In certain embodiments, the particles possess a zeta potential of -100 mV to 0 mV. In a further embodiment, the particles possess a zeta potential of −75 mV to 0 mV. In a further embodiment, the particles possess a zeta potential of −60 mV to 0 mV. In a further embodiment, the particles possess a zeta potential of −50 mV to 0 mV. Still in further embodiments, the particles possess a zeta potential of −40 mV to 0 mV. In a further embodiment, the particles possess a zeta potential of −30 mV to 0 mV. In a further embodiment, the particles retain zeta potentials of −20 mV and +0 mV. In a further embodiment, the particles possess zeta potentials of −10 mV and −0 mV. In a further embodiment, the particles possess zeta potentials of -100 mV and -50 mV. In another particular embodiment, the particles possess zeta potentials of −75 mV and −50 mV. In certain embodiments, the particles possess zeta potentials of −50 mV and −40 mV. In another particular embodiment, the particles have a zeta potential of less than about −40 mV (eg, greater than this). In another particular embodiment, the particles possess 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 (eg, greater than this).

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

In some embodiments, the charge of the carrier (eg, positive, negative, neutral) is selected to confer application-specific benefits (eg, physiological compatibility, beneficial surface-peptide interactions, etc.). Will be done. In some embodiments, the carrier has a net neutral or negative charge (eg, to reduce non-specific binding to the generally net negatively charged cell surface). In certain embodiments, the carrier is directed directly to the antigen for which tolerance is desired (also referred to herein as an antigen-specific peptide, antigenic peptide, autoantigen, inducible antigen, or tolerant antigen). Or it can be conjugated either indirectly. In some cases, the carrier has multiple binding sites (eg, for example) to expose on the surface multiple copies of the antigen-specific peptide or multiple different peptides (eg, to increase the likelihood of a tolerant response). , 2, 3, 4, 5, 6, 7, 8, 9, 10 ..., 20 ..., 50 ..., 100, or more). In some embodiments, the carrier presents a single type of antigenic peptide. In some embodiments, the carrier presents a plurality of different antigenic peptides on the surface. In some embodiments, the carrier surface presents a functional group for covalent attachment of selected moieties (eg, antigenic peptides). In some embodiments, the functional groups on the surface of the carrier provide sites for non-covalent interactions with selected moieties (eg, antigenic peptides). In some embodiments, the carrier has a surface on which the conjugate moiety can be adsorbed without forming chemical bonds.

Particle size and particle charge are important in inducing tolerance. The particle size and charge of the particles vary based on the antigens encapsulated therein, but in general, the particles of the invention are from 0 to about -70 mV, they are from about 100 nanometers to about 1500 nanometers. When they have a charge of about -25 mV to -70 mV, they are effective in inducing tolerance, they are 400 to 800 nanometers, and when they have a charge of about -25 mV to -70 mV, they are most effective in inducing tolerance. Moreover, due in part to the concentration of particles in the freeze-drying process, as well as the presence of sucrose and D-mannitol, the average particle size and charge of the particles can vary slightly in the freeze-drying process. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to the particle size and charge of the particles before lyophilization. The terms "size after lyophilization" and "charge after lyophilization" refer to the particle size and charge of particles after lyophilization.

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

The particles of the present invention may also contain additional components. For example, the carrier may have a contrast agent incorporated or conjugated to the carrier. An example of a carrier nanosphere with a contrast agent currently on the market is the Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals known as quantum dots (QDs) have emerged as ideal donors for FRET applications, and their high quantum yields and adjustable size-dependent Stokes shifts have led to a single ultraviolet ray. Allows different sizes from blue to infrared to be emitted when excited at a wavelength. (Brucez, et al., Science, 1998, 281, 2013, Nicemeyer, CM Angle. Chem. Int. Ed. 2003, 42, 5796, Waggoner, A. Methods Enzymol. 1995, 246, 362, Brus, L. EJ Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots, based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the synthesis of conventional inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperature or highly toxic and unstable reagents. (Etienne, et al., Applied. Phys. Lett. 87, 181913, 2005).

Particles can be formed from a wide range of materials. The particles are preferably made of a material suitable for biological use. For example, the particles may consist of glass, silica, polyester of hydroxycarboxylic acid, polyanhydride of dicarboxylic acid, or copolymer of hydroxycarboxylic acid and dicarboxylic acid. More generally, carrier particles are linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, to alkenyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl. Teloaryl, or alkoxyhydroxy acid polyester, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkenyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, It may consist of a heteroaryl or a polyanhydride of an alkoxydicarboxylic acid. In addition, the carrier particles can be quantum dots or can consist of quantum dots such as quantum dot polystyrene particles (Joumaa et al. (2006); Langmuir 22: 1810-6). Carrier particles containing a mixture of esters and anhydride bonds (eg, copolymers of glycolic acid and sebacic acid) can also be used. For example, the carrier particles are polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysevacinic acid polymer (PSA), poly (lactic acid-co-glycol) acid copolymer (PLGA or PLG, these terms are compatible). Includes materials including), [rho] olly (lactic acid-co-sebasin) acid copolymer (PLSA), poly (glycol-co-sebasin) acid copolymer (PGSA), polypropylene sulfide polymer, poly (caprolactone), chitosan, etc. obtain. Other biocompatible, biodegradable polymers useful in the present invention are polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, as well as linear or branched, substituted. Or unsubstituted, alkenyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or these copolymers with aromatic hydroxycarboxylic or dicarboxylic acids. In addition, biologically important amino acids with reactive side chain groups such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or mirror isomers thereof, are antigenic peptides and proteins or conjugates. It may be included in a copolymer with any of the above materials to provide a reactive group for conjugating to a moiety. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used for the carrier particles of the invention. For example, acrylate, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefin, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington). , Del.), And non-biodegradable polymers of nylon can be used.

The particles of the present invention can be produced by any means generally known in the art. Exemplary methods for producing particles include microemulsion polymerization method, interfacial polymerization method, precipitation polymerization method, emulsion evaporation method, emulsion diffusion method, solvent substitution method, and salting out method (Astete and Savliov, J. Biomater. Sci). Polymer Edn., 17: 247-289 (2006)), but is not limited to these. Manipulation of the manufacturing process for PLGA particles can control particle properties such as size, size distribution, zeta potential, morphology, hydrophobicity / hydrophilicity, polypeptide confinement, etc. The particle size is the concentration of PLGA, the solvent used in the production of the particles, the nature of the organic phase, the surfactant used in the production, the viscosity of the continuous and discontinuous phases, the nature of the solvent used, and the nature of the solvent used. It is affected by a number of factors including, but not limited to, water temperature, ultrasonic treatment, evaporation rate, additives, shear stress, sterilization, and any encapsulation antigen or polypeptide.

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

Viscosity of continuous and discontinuous phases is also an important parameter that influences the diffusion process, which is an important step in forming smaller particles. The particle size increases with increasing viscosity of the dispersed phase, while the particle size decreases as the continuous phase is more sticky. In general, the lower the phase ratio of the organic to aqueous solvent, the smaller the particle size.

The homogenizer rate and agitation also affect the particle size, and generally, there is a point that the particle size decreases with higher speed and agitation, but the particle size does not decrease further even if the rate and agitation are further increased. When the emulsion is homogenized with a high pressure homogenizer, it has a beneficial effect on size reduction compared to simply stirring quickly. For example, at a phase ratio of 20% in 5% PVA, the average particle size at stirring is 288 nm and the average particle size at homogenization (high pressure of 300 bars) is 231 nm.

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

The properties of the polypeptide encapsulated in the particles also affect the particle size. In general, encapsulation of hydrophobic polypeptides results in the formation of smaller particles compared to encapsulation of more hydrophilic polypeptides. In the double emulsion process, the encapsulation of the more hydrophilic polypeptide is improved by using a high molecular weight PLGA and a high molecular weight first surfactant that causes a higher internal phase viscosity. The interaction between the solvent, the polymer and the polypeptide affects the efficiency of the polypeptide's incorporation into the particles.

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

The solvent used can also affect the particle size, and any solvent that reduces the surface tension of the solution will also reduce the particle size.

The organic solvent is removed by evaporation in vacuum, avoiding damage to the polymer and polypeptide and promoting a reduction in the final particle size. Evaporation of the organic solvent under vacuum is more efficient in forming smaller particles. For example, evaporation in vacuum produces an average particle size approximately 30% smaller than the average particle size produced under normal rate evaporation.

The amplitude of the wavelength of sonication also affects the characteristics of the particles. In order to form a very black miniemulsion with no further change in droplet size, the wavelength amplitude should be greater than 20% with sonication for 600-800 s. However, the main drawback of sonication is the lack of monodisperse of the formed emulsion.

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

Various surfactants can be used in the production of the particles of the present invention. The surfactant can be anionic, cationic, or nonionic. Poloxamers and group of poloaxamine surfactants are commonly used in particle synthesis. Surfactants that can be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, PVA, methylcellulose, lecithin, and DMAB. In addition, a biodegradable and biocompatible surfactant comprising, but not limited to, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate). In certain embodiments, two detergents are required (eg, in a double emulsion evaporation method). These two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

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

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

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

The stability and particle size of the particles of the present invention can be improved by the addition of compounds containing, but not limited to, short chains of fatty acids or carbons. The addition of longer carbon chains of lauric acid is associated with improved particle characteristics. In addition, the addition of hydrophobic additives can improve particle size, incorporation of the polypeptide into the particles, and release profile. Particle preparation can be stabilized by lyophilization. Addition of an antifreeze agent such as trehalose can reduce particle agglomeration during lyophilization.

Suitable beads currently on the market include polystyrene beads such as FluoroSpheres (Molecular Probes, Eugene, Oreg.).

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

Physical properties are also related to the usefulness of nanoparticles after uptake and retention in areas with immature lymphocytes. These include mechanical properties such as rigidity or rubberiness. Some embodiments have recently been developed and characterized for systemic delivery (rather than targeting or immunity), as in the PPS-PEG system, where rubber has an upper layer, eg, a hydrophilic upper layer, such as in PEG. It is based on a shape core, eg, a poly (propylene sulfide) (PPS) core. Rubbery cores are in contrast to substantially rigid cores such as those in polystyrene or metal nanoparticle systems. The term rubbery refers to certain elastic materials other than natural or synthetic rubber, and rubbery is a term well known to those skilled in the field of polymer technology. For example, crosslinked PPS can be used to form a hydrophobic rubbery core. PPS is a polymer that decomposes into polysulfone and finally polysulfone under oxidizing conditions, changing from a hydrophobic rubber to a hydrophilic and water-soluble polymer. Other sulfide polymers may be adapted for use, and the term sulfide polymer refers to polymers that have sulfur in the skeleton of the polymer. Another rubbery polymer that can be used is polyester with a glass transition temperature of less than about 37 ° C. under hydration conditions. Hydrophobic cores can be advantageously used with hydrophilic upper layers because the core and upper layers do not tend to mix and therefore the upper layers tend to expand sterically away from the core. A core refers to a particle having a layer on it. A layer refers to a material that covers at least a portion of the core. The layers can be adsorbed or covalently bonded. The particles or core can be solid or hollow. Rubberic hydrophobic cores are rigid, such as crystalline or glassy (as in polystyrene) cores, in that particles with rubbery hydrophobic cores can be filled with more hydrophobic drugs. It has an advantage over the hydrophobic core.

Another physical property is the hydrophilicity of the surface. Hydrophilic materials can have at least 1 gram of water solubility per liter when uncrosslinked. Three-dimensional stabilization of particles with hydrophilic polymers can improve uptake from the stroma by reducing non-specific interactions, but the high stealth of the particles is phagocytic in areas with immature lymphocytes. It may also reduce internal migration due to. Although the challenge of balancing these competing features has been met, this application demonstrates the creation of nanoparticles for effective lymphatic delivery to DCs and other APCs in the lymph nodes. Some embodiments include a layer of hydrophilic component, eg, a hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers. The molecular weight of the polymer in the layer can be adjusted, for example, from about 1,000 to about 100,000, or even more, to provide a useful degree of steric hindrance in vivo, and those skilled in the art will expressly. It will be immediately appreciated that all ranges and values within the stated ranges, for example 10,000 to 50,000, are intended.

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

Various means well known in the art can be used to conjugate the antigenic peptide and protein to the carrier. These methods do not disrupt or significantly limit the biological activity of the antigenic peptide and protein, and a sufficient number of the antigenic peptide and protein are combined with the antigenic peptide or protein and a T cell receptor homologous to the antigenic peptide or protein. Includes any standard chemical action that can be conjugated to the carrier in an orientation that allows interaction. In general, 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 a carrier is preferred. The exact chemistry will, of course, depend on the nature of the carrier material, the presence or absence of C-terminal fusion to the antigenic peptide or protein, and / or the presence or absence of conjugated moieties.

Functional groups may be located on the particles as needed for availability. One position can be a side group or termination on a core polymer, or a polymer that is a layer on the core, or a polymer that is otherwise tethered to particles. Examples are included herein describing PEGs that stabilize nanoparticles that can be easily functionalized for specific cell targeting or protein and peptide drug delivery.

Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propylene glycol diglycylidyl ether containing two epoxy residues, and epichlorohydrin can be used to secure the peptide or protein to the carrier surface. .. Without being bound by theory, ECDI appears to perform two major functions for inducing tolerance: (a) Catalysis of peptide bond formation between free amino and free carboxyl groups. The protein / peptide is chemically bound to the cell surface via the cell, and (b) the carrier is induced to mimic apoptotic cell death, thereby causing them to host antigen-presenting cells (endothelial cells) in the spleen. Can be included) to induce tolerance. It is the presentation to host T cells in this non-immunogenic mode that results in the direct induction of anergy in self-reactive cells. In addition, ECDI serves as a potent stimulus for inducing specific regulatory T cells.

In a series of embodiments, the antigenic peptide and protein bind to the carrier via a covalent chemical bond. For example, a reactive group or moiety near the C-terminal of an antigen (eg, a C-terminal carboxyl group, or a hydroxyl group, thiol group, or amine group on the amino acid side chain) is a reactive group on the surface of the carrier by direct chemical reaction. Alternatively, it can be directly conjugated to a moiety (eg, 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 a conjugate moiety that covalently conjugates both the antigenic peptide and the protein to the carrier, thereby binding them together.

Reactive carboxyl groups on the surface of the carrier are, for example, by reacting them with 1-ethyl-3- [3,9-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). , Can be attached to a free amine (eg, from a Lys residue) on an antigenic peptide or protein. Similarly, the same chemistry can be used to couple the free amine on the surface of the carrier with the free carboxyl on the antigenic peptide or protein (eg, from the C-terminus, or from the Asp or GIu residue). Alternatively, the free amine group on the surface of the carrier can be found in Arano et al. (1991) Chem. It can be covalently attached to an antigenic peptide and protein, or an antigenic peptide or protein fusion protein, using sulfo-SIAB chemistry as essentially described in 2: 71-6.

In another embodiment, the antigen may be conjugated to the carrier by a non-covalent bond between the ligand bound to the antigen peptide or protein and the anti-ligand attached to the carrier. For example, a biotin ligase recognition sequence tag can be attached to the C-terminus of an antigenic peptide or protein, and this tag can be biotinylated by biotin ligase. Biotin can then serve as a ligand for non-covalently conjugating the antigenic peptide or protein to avidin or streptavidin that is adsorbed or otherwise bound to the surface of the carrier as an antiligand. .. Alternatively, if the antigenic peptide and protein are fused to an immunoglobulin domain carrying the Fc region, the Fc domain can act as a ligand, as described above, and protein A covalently or non-covalently attached to the surface of the carrier. , Can serve as an anti-ligand for non-covalently conjugating an antigenic peptide or protein to a carrier. Non-sharing antigenic peptides and proteins to carriers, including metal ion chelating techniques (eg, using C-terminal poly-His tags of antigenic peptides or proteins or antigenic peptides or protein fusion proteins, and Ni ⁺ coated carriers). Other means that can be used for binding conjugates are well known in the art and these methods can replace the methods described herein.

Conjugation of the nucleic acid moiety to the platform molecule can be achieved in any number of ways, but typically involves one or more cross-linking agents, as well as functional groups on the nucleic acid moiety and platform molecule. Bonding groups are added to the platform using standard synthetic chemistry techniques. Binding groups can be added to nucleic acid moieties using standard synthetic chemical techniques. The practitioner has several options regarding the antigens used in the combinations of the invention. The inducible antigen present in the combination contributes to the specificity of the induced immunotolerant response. It may or may not be the same as the target antigen, is the target of an undesired immunological response, and is the antigen present or given to the subject to whom tolerance is desired.

The inducible antigens of the invention may be polypeptides, polynucleotides, carbohydrates, glycolipids, or other molecules isolated from biological sources, or chemically synthesized small molecules, polymers. Alternatively, it may be a derivative of a biological material, provided that it has the ability to induce tolerance according to the present specification when combined with a mucosal binding component.

In some embodiments, the invention provides a carrier (eg, immunomodifying particles) attached to one or more peptides, polypeptides, and / or proteins. In some embodiments, carriers such as those described herein (eg, PLG carriers) induce antigen-specific tolerance and / or develop immune-related diseases (such as EAE in mouse models). It is effective in preventing and / or reducing the severity of existing immune-related diseases. In some embodiments, the compositions and methods of the invention can cause T cells to initiate early events associated with T cell activation, but not allow T cells to acquire effector function. For example, administration of the compositions of the invention can result in T cells with a quasi-activated phenotype such as CD69 and / or CD44 upregulation, but due to the lack of IFN-γ or IL-17 synthesis. As shown, no effector function is presented. In some embodiments, administration of the compositions of the invention is quasi-without converting naive antigen-specific T cells to a regulatory phenotype, such as those with a CD25 ^{+ /} Foxp3 ⁺ phenotype. It results in T cells with an activated phenotype.

In some embodiments, the surface of the carrier (eg, particles) is a chemical that allows attachment of antigenic peptides and / or other functional elements to the carrier (eg, covalent, non-covalent). Includes moieties and / or functional groups. In some embodiments, the number, orientation, spacing, etc. of chemical moieties and / or functional groups on the carrier (eg, particles) will vary depending on the chemical action of the carrier, desired application, and the like.

In some embodiments, the carrier comprises one or more biological or chemical agents adhered to, adsorbed, encapsulated, and / or contained throughout the carrier. In some embodiments, the chemical or biological agent is encapsulated in the particles and / or contained throughout the particles. 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 different chemical or biological agents (eg, 3, 4, 5, etc.) are included on or in the carrier. In some embodiments, the agent is configured for a particular release rate. In some embodiments, a plurality of different agents are configured for different release rates. For example, the first agent may be released over a period of several hours and the second agent may be released over a longer period (eg, 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, sustained release is biologically active over a period of at least 30 days (eg, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 180 days, etc.). Provides the release of a large amount of drug. In some embodiments, the carrier or portion thereof is configured to be sufficiently porous to allow internal growth into the pores of the cell. The size of the pores can be selected for the particular cell type of interest and / or the amount of internal growth desired. In some embodiments, the particles comprise an antigen of interest without a drug or other non-peptide activator such as an immunomodulator. Moreover, in some embodiments, the particles of the invention do not contain immunostimulatory or immunosuppressive peptides in addition to the antigen of interest. Further, in some embodiments, the particles are other proteins or peptides encapsulated on or within the surface of the particles (eg, costimulatory molecules, MHC molecules, immunostimulatory peptides, or immunosuppressive peptides). Does not contain.

Surprisingly, encapsulation of antigens, biological agents, and / or chemical agents into the particles of the invention has been found to induce immune tolerance and have several advantages. First, the encapsulated particles have a slower cytokine response. Second, when using multiple antigens, biological agents, and / or chemical agents, encapsulation eliminates the possible competition between these various molecules when the agent adheres to the surface of the particles. Third, encapsulation allows more antigens, biological agents, and / or chemical agents to be incorporated into the particles. Fourth, encapsulation makes it easier to use complex protein antigens or organ homogenates (eg, pancreatic homogenates in the case of type 1 diabetes or peanut extracts in peanut allergies). Finally, instead of conjugation to the surface of the particle, an antigen, biological agent, and / or chemical agent is encapsulated within the particle to maintain a net negative charge on the surface of the particle. Encapsulation of antigens, biological agents, and / or chemical agents in the particles of the invention can be performed by any method known in the art. In one embodiment, the polypeptide antigen is encapsulated in particles by a double emulsion process. In a further embodiment, the polypeptide antigen is water soluble.

In another embodiment, the polypeptide antigen is encapsulated in particles by a single emulsion process. In a further embodiment, the polypeptide antigen is more hydrophobic. In some cases, the double emulsion process results in the formation of large particles that can result in leakage of the hydrophilic active ingredient and low confinement efficiency. Fusing and Ostwald ripening are two mechanisms that can destabilize the droplet size of the double emulsion, and diffusion of the hydrophilic active ingredient through the organic phase is the main mechanism responsible for the low levels of trapped active ingredient. Is. In some embodiments, it may be beneficial to reduce the nanoparticle size. One way to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by increasing the viscosity of the internal aqueous phase, increasing the molecular mass of the surfactant, as well as using high polymer concentrations and high polymer molecular mass.

In certain embodiments, the present invention provides a carrier having cells, or other biological or chemical agents, in (or above). When cells are used, the carrier is not limited to a particular cell type. In some embodiments, the carrier has pancreatic islet cells on it. In some embodiments, the microporous carrier additionally has an ECM protein and / or exendin-4 on it. The carrier is not limited to a particular type. In some embodiments, the carrier has varying porous regions (eg, varying pore sizes, pore depths, and / or pore densities). In some embodiments, the carrier has a drug, DNA, RNA, extracellular matrix protein, exendin-4, etc. on (or in). In certain embodiments, the present invention provides a method for transplanting pancreatic islet cells with such carriers. In certain embodiments of the invention, the inducible antigen is a single isolated molecule or a recombinantly produced molecule. In order to treat a condition in which the target antigen is scattered in various positions of the host, it is generally necessary that the inducible antigen is the same as or immunologically related to the target antigen. be. Most of the examples of such antigens are polynucleotide antigens, and may be carbohydrate antigens (such as blood group antigens).

Any suitable antigen can be found for use within the scope of the invention. In some embodiments, the inducible antigen contributes to the specificity of the induced immunotolerant response. The inducible antigen may or may not be the same as the target antigen, is the target of an undesired immunological response, and is an antigen that is present or given to the subject to whom tolerance is desired.

In certain embodiments of the invention, the inducible antigen is not in the same form as found in pollen, eg, a polypeptide derived from Sugi pollen, but in the same form as a fragment or derivative thereof. The inducible antigens of the invention are based on molecules of appropriate specificity, but are adapted by fusion with peptides having fragmentation, residue substitution, labeling, conjugation, and / or other functional properties. including. Fitting involves eliminating any unwanted properties such as toxicity or immunogenicity; or enhancing any desirable properties such as mucosal binding, mucosal penetration, or stimulation of the immunotolerant arm of an immune response. It can be done for any desired purpose, not limited to these. As used herein, terms such as CRYJ1 or CRYJ2 are not only intact subunits, but at least allotype and synthetic variants, fragments, fusion peptides, conjugates, and analogs of each molecule. Other derivatives containing regions that are homologous to 10 and preferably 20 consecutive amino acids (preferably 70% identical, more preferably 80% identical, and even more preferably 90% identical at the amino acid level). Pointing here, the homologous region of the derivative shares with its respective parent molecule the ability to induce tolerance to the target antigen.

It is recognized that the immunotolerant region of the inducible antigen is often different from the immunodominant epitope for stimulating the antibody response. Immunotolerant regions are generally regions that can be presented in certain cellular interactions involving T cells. Immune-tolerant regions may be present and can induce tolerance when presented with an intact antigen. Some antigens contain latent immunotolerant regions in that the processing and presentation of the native antigen usually does not induce tolerance. Details of the latent antigens and their identification can be found in International Patent Application Publication No. WO94 / 27634.

In certain embodiments of the invention, two, three, or more inducible antigens are used. If there are multiple target antigens, it may be desirable to realize these embodiments or provide multiple bystanders for the target. It may be desirable to provide a cocktail of antigens to cover multiple possible alternative targets. For example, cocktails of CRYJ1 and CRYJ2 fragments, as well as other epitopes derived from Sugi pollen, can be used to tolerate the subject. In another example, a mixture of allergens plays a role in inducing antigens for the treatment of atopy.

Inducible antigens can be prepared by multiple techniques known in the art, depending on the nature of the molecule. Polynucleotides, polypeptides, and carbohydrate antigens can be isolated from cells of the species to be treated that are rich in them. Short peptides are conveniently prepared by amino acid synthesis. Longer proteins of known sequence are to synthesize the coding sequence or PCR amplify the coding sequence from a natural source or vector and then express the coding sequence in a suitable bacterial or eukaryotic host cell. Can be prepared by

In certain embodiments of the invention, the combination comprises a complex mixture of antigens obtained from pollen grains, one or more of which serves as an inducible antigen. The antigen can be in the form of whole cells, either intact or treated with a fixed solution such as formaldehyde, glutaraldehyde, or alcohol. The antigen can be in the form of a solubilized product created by solubilization of the cell or tissue with a detergent or clarification after mechanical rupture. The antigen can also be obtained by concentrating the plasma membrane by a technique such as subcellular fractionation, in particular fraction centrifugation, and then optionally solubilizing with a detergent and dialysis. Other separation techniques such as affinity chromatography or ion exchange chromatography of solubilized membrane proteins are also suitable.

Allergens are other antigens for which tolerance of the immune response to them is also desirable. In one embodiment, the antigen is a polypeptide associated with Sugi pollen. In some embodiments, the antigen is CRYJ1. In certain embodiments, the antigen is CRYJ2.

In one embodiment, the particles of the invention are bound to an antigen comprising one or more epitopes associated with allergies, autoimmune diseases, and / or inflammatory diseases or disorders. The antigen may contain one or more copies of the epitope. In one embodiment, the antigen comprises a single epitope associated with one disease or disorder. In a further embodiment, the antigen comprises more than one epitope associated with the same disease or disorder. Still in further embodiments, 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.

Further non-limiting examples of epitopes associated with allergies to Sugi pollen, Table 1.

Any suitable antigen associated with allergies can be found for use within the scope of the invention. In some embodiments, the particles described herein are encapsulated with one or more antigens or epitopes associated with allergies. In certain embodiments, the antigen or epitope is a dust, pet allergen, tree pollen, grass pollen, mold, vegetable allergen, fruit allergen, nut allergen, seed allergen, spice allergen, grain allergen, seafood allergen, meat allergen, nematode. Related to allergens, latex allergens, and / or toxic allergens.

In some embodiments, the antigenic epitopes derived from Sugi 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. Includes Cry j CPA9, or fragments or variants thereof. In certain embodiments, the antigenic epitope derived from Sugi pollen comprises a CRYJ1 polypeptide, or a fragment or variant thereof. In some embodiments, the CRYJ1 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the antigenic epitope derived from Sugi pollen is a fragment of CRYJ1. In some embodiments, the fragment of CRYJ1 has 10, 11, 12, 13, 14, 15 having at least 90%, at least 95%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 1. , 16, 17, 18, 19, 20, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive amino acids. In certain embodiments, the antigen epitope is a variant of CRYJ1. In certain embodiments, the variants are at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91% relative to SEQ ID NO: 1. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or contains amino acid sequences having more than 99% sequence identity.

In some embodiments, the antigenic epitope derived from Sugi 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. Yes, these are Sone et al. , J. Immunol, vol. 161 (1) 448-457 (1998), which is incorporated herein by reference in its entirety.

In some embodiments, the antigenic epitope derived from Sugi 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 derived from Sugi pollen is a fragment of CRYJ2. In some embodiments, the CRYJ2 fragment has 10, 11, 12, 13, 14, 15 having at least 90%, at least 95%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 2. , 16, 17, 18, 19, 20, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive amino acids. In certain embodiments, the antigen epitope is a variant of CRYJ2. In certain embodiments, the variants are at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91% relative to SEQ ID NO: 2. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or contains amino acid sequences having more than 99% sequence identity.

In some embodiments, the antigenic epitopes derived from Sugi pollen consist of p66-80, p81-95, p141-155, p186-200, p236-250, p346-360, p351-365, and p336-350. Fragments of CRYJ2 selected from, these are described in Sone et al (1998).

In certain embodiments, the particles described herein are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 of which are derived from Sugi pollen. Encapsulate 10 or more, 10 or more, 15 or more, or 20 or more antigen epitopes. In certain embodiments, two or more antigenic epitopes were contained in the fusion protein. In some embodiments, the antigenic epitopes contained in the fusion protein are linked by a cleavable linker. Cleavable linker, as used herein, refers to an amino acid sequence containing a particular cleavage site.

In some embodiments, the cleavable linker has a length of 5, 6, 7, 8, 9, 10, more than 10, more than 15, more than 20, more than 25 amino acids. Is. In some embodiments, the cleavable linker is cleaved by a protease at a particular site. In certain embodiments, the cleavable linker contains more than one particular site that is cleaved by a protease. In some embodiments, the cleavable linker contains more than one specific site, where the specific site is cleaved by a different protease. In some embodiments, the cleavable linker contains more than one specific site, where the specific site is cleaved by the same protease.

In some embodiments, the cleavable linker is a furin-sensitive linker and / or a cathepsin-sensitive linker. In certain embodiments, the cleavable linkers are furin, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, and. / Or is cleaved at a specific site on the linker by one or more of cathepsin Z. In some embodiments, the cleavable liner 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 from CRYJ1. In certain embodiments, the fusion protein comprises two or more epitopes from CRYJ2. In some embodiments, the fusion protein comprises epitopes from CRYJ1 and CRYJ2.

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

In some embodiments, the tolerance-inducing compositions of the invention contain an apoptotic signaling molecule (eg, in addition to an antigenic peptide or other antigenic molecule). In some embodiments, the apoptotic signaling molecule binds and / or associates with the surface of the carrier. In some embodiments, the apoptotic signaling molecule allows the carrier to be recognized as an apoptotic body by the host's antigen presenting cells, eg, cells of the host reticuloendothelial system, thereby the relevant peptide. It is possible to present the epitope in a manner that induces tolerance. Without being bound by theory, it is presumed to prevent upregulation of molecules involved in immune cell stimulation, such as MHC class I / II, and co-stimulatory molecules. These apoptotic signaling molecules can also serve as phagocytotic markers. For example, apoptotic signaling molecules suitable for the present invention are described in US Patent Application No. 2005/0113297, which is incorporated by reference in its entirety. Molecules suitable for the present invention include molecules that target phagocytic cells, including macrophages, dendritic cells, monocytes, granulocytes, and neutrophils.

In some embodiments, a molecule suitable as an apoptotic signaling molecule acts to enhance the tolerance of the associated peptide. 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, in its entirety herein by reference. Will be incorporated into). For example, molecules that may be useful as apoptotic signaling molecules include phosphatidylserine, anexin-1, anexin-5, milk fat globules-EGF-factor 8 (MFG-E8), or the thrombospondin family (eg, thrombospondin). (TSP-1)) is included. A variety of molecules suitable for use as apoptotic signaling molecules with the present invention are discussed, for example, in US Patent Application Publication No. 2012/0076831 and are incorporated herein by reference in their entirety).

In some embodiments, the apoptotic signaling molecule can be conjugated to an antigen-specific peptide. In some examples, apoptotic signaling molecules and antigen-specific peptides are conjugated by the creation of fusion proteins. For example, a fusion protein may comprise at least one antigen-specific peptide (or fragment or variant thereof) attached to at least one molecule of an apoptotic signaling molecule (or fragment or variant thereof). With respect to the creation of fusion proteins, the terms "fusion protein", "fusion peptide", "fusion polypeptide", and "chimeric peptide" are used interchangeably. Suitable fragments of the antigen-specific peptide include any fragment of the full-length peptide that retains the ability to produce the desired antigen-specific tolerance function of the invention. Fusion proteins can be created by a variety of means understood in the art (eg, gene fusion, chemical binding, etc.). The two proteins can be fused either directly or via an amino acid linker. Polypeptides that form fusion proteins typically bind at the C-terminus to the N-terminus, but they can also be attached at the C-terminus to the C-terminus, the N-terminus to the N-terminus, or the N-terminus to the C-terminus. The polypeptides of the fusion protein can be in any order. The peptide linker sequence is used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structure. obtain. Amino acid sequences that can be usefully used as linkers include Maratea et al. , Gene 40: 39-46 (1985), Murphy et al. , Proc. Natl. Acad. Sci. USA 83: 8258-8262 (1986), US Pat. No. 4,935,233, and US Pat. No. 4,751,180 include those disclosed in their entirety, which are incorporated herein by reference in their entirety. Is done. The linker sequence can generally be 1 to about 50 amino acids in length. In some embodiments, the linker sequence is not required, for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric hindrance. Not and / or not used.

An alternative to immunotolerant activity is the ability of an intact antigen or fragment to stimulate the production of appropriate cytokines at the target site. The immunoregulatory cytokine released by T immunosuppressive cells at the target site is thought to be TGF-β (Miller et al., Proc. Natl. Acad. Sci. USA 89: 421, 1992). Other factors that can be produced during tolerance are the cytokines IL4 and IL-10, as well as the transduction factor PGE. In contrast, lymphocytes in tissues causing active immune disruption secrete cytokines such as IL-1, IL-2, IL-6, and IFN-γ. Therefore, the efficacy of a candidate to induce an antigen can be assessed by measuring its ability to stimulate the appropriate type of cytokine.

With this in mind, rapid screening tests for immunotolerant epitopes of inducible antigens, effective mucosal binding components, effective combinations, or effective modes and schedules of mucosal administration are donors for in vitro cell assays. Can be performed using identical gene animals. Animals may be treated with the test composition on the mucosal surface and administered with parenteral administration of the target antigen in a complete Freund's adjuvant. Spleen cells are isolated and cultured in vitro in the presence of a target antigen at a concentration of approximately 50 μg / mL. The target antigen can be replaced with a candidate protein or fragment and the location of the immunotolerant epitope can be mapped. Cytokine secretion into the medium can be quantified by standard immune assays.

The ability of cells to suppress the activity of other cells can be determined by using cells isolated from animals immunized with the target antigen or by creating 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 variant of this experiment, immunosuppressive cell populations were slightly irradiated (about 1000-1250 rads) to prevent proliferation, immunosuppressive cells were co-cultured with killer cells, and then tritium-labeled thymidine. Integration (or MTT) is used to quantify the proliferative activity of killer cells. In another variant, immunosuppressive and killer cell populations are cultured in higher and lower level dual chamber transwell culture systems (Costar, Cambridge Mass.), whereby the groups are cultured with each other in a polycarbonate membrane. Allows coincubate within 1 mm separated by (WO93 / 16724). Irradiation of immunosuppressive cell populations is not required in this procedure, as the proliferative activity of killer cells can be measured separately.

In embodiments of the invention in which the target antigen is already present in the individual, it is not necessary to isolate the antigen or pre-combine it with a mucosal binding component. For example, the antigen can be expressed in an individual in a particular manner as a result of pathological conditions (such as inflammatory bowel disease or celiac disease) or by digestion of food allergens. The test is performed by giving one or more doses or mucosal binding components in the formulation and determining its ability to promote tolerance to the antigen with insights.

The efficacy and mode of administration of the composition for the treatment of a particular disease may also be detailed in the corresponding animal disease model. The ability of treatment to reduce or delay the overall symptoms of the disease is the cyclical biochemical and immunological characteristics of the disease of the model used, the immunohistology of the affected tissue, and optionally. Monitored at the level of overall clinical features. Non-limiting examples of animal models that can be used in the test are included in the following sections. The present invention contemplates the regulation of tolerance by regulating the TH1 response, TH2 response, TH17 response, or a combination of these responses. Modulating the TH1 response involves, for example, altering the expression of interferon-gamma. Modulating the TH2 response involves, for example, altering the expression of any combination of IL-4, IL-5, IL-10, and IL-13. Typically, an increase (decrease) in TH2 response involves an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10, or IL-13, more typically. , TH2 response increase (decrease) includes an increase in expression of at least two of IL-4, IL-5, IL-10, or EL-13, most typically an increase (decrease) in TH2 response. ) Contains at least three increases in IL-4, IL-5, IL-10, or IL-13, but ideally an increase (decrease) in TH2 response is IL-4, IL-. 5. Includes increased (decreased) expression of all IL-10, and IL-13. Modulating TH17 includes, for example, altering the expression of TGF-beta, IL-6, IL-21, and IL23, as well as the effect levels of IL-17, IL-21, and IL-22. In some embodiments, the invention contemplates the regulation of tolerance by promoting one type of immune response that surpasses another type of immune response (eg, immune switching or immune bias). In such embodiments, the established immune response of one phenotype is suppressed or reduced, and the immune response of different phenotypes is increased or enhanced. For example, immune switching from Th2 to Th1 response in an allergic immune response promotes the generation of IgG2a antibody response and the production of IFNγ and / or IL-12, thereby reducing allergen-specific IgE response and Th2 cells. It results in a decrease in T cell polarization to. Other suitable for assessing the effectiveness of the compositions and methods of the invention, for example, as discussed in US Patent Application Publication No. 2012/0076831 (incorporated herein by reference in its entirety). Methods are understood in the art.

Certain embodiments of the invention relate to pre-stimulation of immune tolerance in individuals that have not been previously tolerated by therapeutic intervention. These embodiments generally involve multiple doses of the combination of antigen and mucosal binding component. Typically, subjects receive at least 3 doses, often at least 4 doses, and sometimes at least 6 doses during prestimulation to achieve long-term results, but the subject is treated. It may show signs of tolerance early in the process. In most cases, each dose is given as a bolus dose, but long-acting formulations capable of mucosal release are also suitable. When multiple doses are given, the time between doses is generally 1 day to 3 weeks, and typically about 3 days to 2 weeks. Generally, the same antigen and mucosal binding components are present at the same concentration and administration is given to the same mucosal surface, but variations of any of these variables may be indicated during the course of treatment. Other embodiments of the invention relate to boosting or prolonging the persistence of previously established immune tolerance. These embodiments generally involve a single dose or short-term treatment when established tolerance diminishes or is at risk of diminishing. Boosting is generally done 1 month to 1 year, and typically 2 to 6 months after pre-stimulation or previous boost. The invention also includes embodiments involving regular maintenance of tolerance in a schedule of dosing that occurs twice weekly, once weekly, biweekly, or on any other periodic schedule.

The particles of the invention may be given at any dose effective to diminish the inflammatory immune response in a subject in need of medication or to treat a bacterial or viral infection in a subject in need of medication. ^In certain embodiments, about ¹⁰² to about 1020 particles are provided to an individual. ^In a further embodiment, about ¹⁰³ to about 1015 particles are provided. Still in a further embodiment, about ¹⁰⁶ to about ¹⁰¹² particles are provided. Still in further embodiments, about ¹⁰⁸ to about 10 ¹⁰ particles are provided. In a preferred embodiment, the preferred dose is 0.1% solid / ml. Thus, for 0.5 μm beads, the preferred dose is approximately 4 × 10 ⁹ beads, and for 0.05 μm beads, the preferred dose is approximately 4 × 10 ¹² beads, for 3 μm beads. The preferred dose is 2 × ¹⁰⁷ beads. However, any dose effective to treat a particular condition to be treated is included by the invention.

INDUSTRIAL APPLICABILITY The present invention is useful for the treatment of immune-related disorders such as autoimmune diseases, transplant rejection, enzyme deficiency, and allergic reactions. Substitution of synthetic biocompatible particle systems to induce immune tolerance, ease of manufacture, wide availability of therapeutic agents, increased uniformity between samples, increased number of possible therapeutic sites, against carrier cells It can result in a dramatic reduction in the likelihood of an allergic response.

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 response), and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes, eg, B cells and T cells (CD4 ⁺ , CD8 ⁺ , Th1 cells, and Th2 cells); antigen presenting cells (eg, professional antigen presenting cells, eg, dendritic). Cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, stellate glue cells, fibroblasts, oligodendrogliol cells); natural killer cells; bone marrow cells such as macrophages. Includes eosinocytes, mast cells, basal spheres, and granulocytes. In some embodiments, the modified particles of the invention are effective in reducing the transport of inflammatory cells to the site of inflammation.

As used herein, the terms "anergy," "tolerance," or "antigen-specific tolerance" refer to T cell insensitivity to T cell receptor-mediated stimuli. Such insensitivity is generally antigen-specific and persists after the end of exposure to the antigenic peptide. For example, anergy in T cells is characterized by a lack of cytokine production, eg IL-2. T-cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (co-stimulation signal). Under these conditions, re-exposure of cells to the same antigen can prevent cytokines from being produced (even when re-exposed in the presence of co-stimulatory molecules) and then proliferate. become unable. Therefore, the inability to produce cytokines prevents proliferation. However, anergy T cells can proliferate when cultured with cytokines (eg IL-2). For example, T cell anergy can also be observed by lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, a reporter gene construct may be used. For example, anergy T cells are unable to initiate transcription of the DL-2 gene induced by a heterologous promoter under the control of a 5'IL-2 gene enhancer or by a multimer of API sequences that can be found within the enhancer. (Kang et al. 1992 Science. 257: 1134).

As used herein, a) reduced levels of specific immunological responses are believed to be at least partially mediated by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or their equivalents. The term "immunological tolerance" in the case of), b) initiation or delay of progression of a specific immunological response, or c) reduction of the risk of initiation or progression of a specific immunological response is being treated. Refers to a method that is performed based on the proportion of treatment targets compared to non-subjects. "Specific" immunological tolerance occurs when immunological tolerance is preferentially evoked for a particular antigen compared to others. "Non-specific" immunological tolerance occurs when immunological tolerance is indiscriminately evoked against an antigen that provides an inflammatory immune response. "Semi-specific" immunological tolerance occurs when immunological tolerance is elicited semi-discriminatively against an antigen that provides a pathogenic immune response rather than another antigen that provides a protective immune response.

Tolerance to autoantigens and autoimmune diseases includes a variety of mechanisms of peripheral tolerance to autoreactive T cells found in the periphery, avoiding negative selection of thymic autoreactive T cells and thymic deletion. Achieved by the mechanism. Examples of mechanisms that provide peripheral T cell tolerance include "ignore" of self-antigens, anergy or non-responsiveness to self-antigens, cytokine immune bias, and activation-induced cell death of self-reactive T cells. .. Furthermore, regulatory T cells have been shown to be involved in mediating peripheral tolerance. For example, Walker et al. (2002) Nat. Rev. Immunol. 2: 11-19, Shevac et al. (2001) Immunol. Rev. See 182: 58-67. In certain situations, peripheral tolerance to autoantigens is lost (or destroyed), resulting in an autoimmune response. For example, in an animal model of EAE, activation of antigen-presenting cells (APCs) by TLR congenital immune receptors has been shown to disrupt self-tolerance and lead to the induction of EAE (Waldner et al. (2004)). J. Clin. Invest. 113: 990-997).

Accordingly, in some embodiments, the invention provides a method for increasing antigen presentation while suppressing or reducing TLR7 / 8, TLR9, and / or TLR7 / 8/9 dependent cell stimulation. .. As described herein, administration of specific modified particles suppresses the TLR7 / 8, TLR9, and / or TLR7 / 8/9 dependent cellular responses associated with immunostimulatory polynucleotides, while still suppressing. Antigen presentation by DC or APC is brought about. Such inhibition may include reduced levels of one or more TLR-related cytokines.

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

Accordingly, in another aspect of the invention, a pharmaceutical composition comprising immunomodifying particles and optionally a pharmaceutically acceptable carrier is provided. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the modified particles of the invention may be administered to a patient in need of the modified particles in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for co-administration with the compounds of the invention or for inclusion in pharmaceutical compositions comprising the compounds of the invention may be approved anti-inflammatory agents or are uncontrolled. With any one of several drugs in the Food and Drug Administration stage of approval that will ultimately be approved for the treatment of an inflammatory immune response or any disorder characterized by a bacterial or viral infection. could be. It will also be appreciated that certain modified particles of the invention may be present in free form for treatment or, if desired, as pharmaceutically acceptable derivatives thereof. ..

In addition, the pharmaceutical compositions of the invention, when used herein, are any and all solvents, diluents, or other liquid vehicles, dispersion aids, suitable for the particular dosage form desired. Includes pharmaceutically acceptable carriers including agents or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.I. W. Martin (Mac Publishing Co., Easton, Pa., 1980) discloses a variety of carriers used to formulate pharmaceutical compositions and known techniques for preparing them. The compounds of the invention are such that any conventional carrier medium produces any undesired biological effect, or interacts with any other component (s) of the pharmaceutical composition in a detrimental manner. Its use is intended to be within the scope of the invention, unless it is incompatible with. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, such as. Sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; starch; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, benivana oil, sesame oil, olive oil, corn oil. , And soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic physiological saline. Water; Ringer solution; ethyl alcohol and phosphate buffer, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, and in accordance with the discretion of the formulator. , Colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives, and antioxidants may also be present in the composition.

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, liquid dosage forms include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsions, such as ethyl alcohol, isopropyl alcohol, carbonic acid. Ethyl, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), It may contain fatty acid esters of glycerol, tetrahydrofurfuryl alcohols, polyethylene glycols, and sorbitans, as well as mixtures thereof. In addition to the Inactive Diluent, the oral composition may include an adjuvant, such as a wetting agent, an emulsion and a suspending agent, a sweetening agent, a flavoring agent, and a fragrance.

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

Effective amounts and methods of administration of the invention in regulating an immune response can vary based on the individual, the condition to be treated, and other factors apparent to those of skill in the art. Factors to consider include the route of administration and the number of doses administered. Such factors are known in the art and can be determined by those skilled in the art without undue experimentation. A suitable dose range is one that provides the desired immune control. A useful dose range of carriers, as indicated by the amount of carrier delivered, is, for example, about 0.5-10 mg / kg, 1-9 mg / kg, 2-8 mg / kg, 3-7 mg / kg, 4- It can be either 6 mg / kg, 5 mg / kg, 1-10 mg / kg, or 5-10 mg / kg. Alternatively, the dose may be administered based on the number of particles. For example, a useful dose of carrier indicated by the carrier to be delivered can be, for example, the number of particles per dose of about ¹⁰⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ or more. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, clearance rate, and route of administration. Details of pharmaceutically acceptable carriers, diluents, and excipients, as well as methods of preparing pharmaceutical compositions and formulations, are described in ^Remmingtons Pharmaceutical Sciences 18th Edition, 1990, Mac Publishing Co., Ltd. , Easton, Pa. , Provided to USA and incorporated in its entirety by reference.

Effective amounts and methods of administration of a particular carrier formulation may vary based on the individual patient, desired outcome and / or type of disorder, stage of disease, and other factors apparent to those of skill in the art. The route of administration (s) useful for a particular application will be apparent to those of skill in the art. Routes of administration include topical, cutaneous, percutaneous, transmucosal, epidermal, parenteral, gastrointestinal, and intranasopharyngeal and intrapulmonary, eg, transbronchial and transalveolar bone administration. Included, but not limited to. A suitable dose range is a dose that provides sufficient IRP-containing composition to achieve a tissue concentration of about 1-50 μΜ as measured by blood levels. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, clearance rate, and route of administration.

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

Transdermal administration is achieved by application of creams, rinses, gels, etc. that allow the carrier to penetrate the skin and enter the bloodstream. Compositions suitable for transdermal administration include pharmaceutically acceptable suspending agents, oils, which are applied directly to the skin or incorporated into a protective carrier such as a transdermal device (so-called "patch"). , Creams, and ointments, but not limited to these. Examples of suitable creams, ointments, etc. can be found, for example, in the medical package insert. Percutaneous permeation can also be achieved, for example, by ion migration using commercially available patches that continuously deliver their products through intact skin over a period of several days or longer. The use of this method allows controlled permeation of pharmaceutical compositions at relatively high concentrations, allows infusion of combined drugs, and allows simultaneous use of absorption enhancers.

Parenteral routes of administration include electric injection (ion migration) or direct injection into a central venous catheter, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Not limited to. Formulations of carriers suitable for parenteral administration are generally formulated in USP water or water for injection, with additional pH buffers, salt enhancers, preservatives, and other pharmaceutically acceptable excipients. Can include. Immunomodulatory polynucleotides for parenteral injection can be formulated in pharmaceutically acceptable sterile isotonic solutions such as saline for injection and phosphate buffered saline.

Gastrointestinal routes of administration include, but are not limited to, oral and rectal routes, such as the use of pharmaceutically acceptable powders, pills, or liquids for oral ingestion, and suppositories for rectal administration. May be included.

Intranasopharyngeal and intrapulmonary administration is included, achieved by inhalation, and includes delivery routes such as the nasal cavity, transbronchial, and transalveolar bone pathways. The present invention includes liquid suspending agents for forming aerosols, as well as powder forms for dry powder inhalation delivery systems, including, but not limited to, formulations of carriers suitable for administration by inhalation. Devices suitable for administration by inhalation of the carrier formulation include, but are not limited to, atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.

Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension, or emulsion in a non-toxic parenteral acceptable diluent or solvent, eg, as a solution in 1,3-butanediol. .. Among other acceptable vehicles and solvents in particular are water, Ringer's solution, U.S.A. S. P. And isotonic sodium chloride solution. In addition, sterile fixed oils have traditionally been used as solvents or suspension media. Any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used for this purpose. In addition, fatty acids such as oleic acid are used in injectable preparations.

Injectable formulations are sterile, for example, prior to use by filtration through a bacterial retention filter or by incorporating a sterile agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium. Can be done.

It may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be achieved by the use of liquid suspending agents with poor water solubility, or crystalline or amorphous materials. The rate of absorption of the drug then depends on its rate of dissolution, which may depend on crystal size and crystalline morphology. Alternatively, delayed absorption of the drug form administered parenterally is achieved by dissolving or suspending the drug in an oil vehicle. The injectable depot form is made by forming a microcapsule matrix of the drug in a biodegradable polymer 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 (orthoester) and poly (anhydride). Depot injectable formulations can also be prepared by enclosing the drug in liposomes or microemulsions that are compatible with body tissues.

In some embodiments, the synthetic biodegradable particles of the invention provide ease of manufacture, wide availability of therapeutic agents, and increased therapeutic sites. In certain embodiments, the surface-functionalized biodegradable poly (lactide-co-glycolide) particles with a high density surface carboxylate group synthesized using the surfactant poly (ethylene-alt-maleic anhydride) are. , Other carrier particles and / or carriers that provide a number of advantages over surfaces. Experiments performed during the development of embodiments of the invention have demonstrated conjugation of peptides to these particles (eg, PLP _139-151 peptide). Such peptide-bonded particles have been shown to be effective in preventing disease onset and inducing immunological tolerance (eg, SJL / J PLP _139-151 / CFA-induced in multiple sclerosis). EAE mouse model). The peptide binding carrier of the present invention offers many advantages over other tolerance-inducing structures. In some embodiments, the particles are biodegradable and therefore do not last long in the body. The time for complete decomposition can be controlled. In some embodiments, the particles are functionalized to facilitate internal migration without activating cells (eg, phosphatidylserine packed in PLG microparticles). In some embodiments, the particles incorporate a target ligand for a particular cell population. In some embodiments, to limit the activation of cell types that translocate particles and facilitate the induction of tolerance through energy and / or regulatory T cell deletion and activation. Anti-inflammatory cytokines such as IL-10 and TGF-β are included on or in the particles. The composition of the particles has been found to affect the length of time it takes for the particles to persist in the body and forgiveness for rapid particle uptake and clearance / degradation. The particles of the present invention have a lactide: glycolide ratio of about 50:50 or less because the ratio of 50:50 lactide: glycolide slows the rate of degradation. In one embodiment, the particles of the invention have a D, L-lactide: glycolide ratio of about 50:50.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate and dicalcium phosphate, and / or a) filler or extender. For example, starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) moisturizers such as glycerol, d). Disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders such as paraffin, f) absorption accelerators such as four. Grade ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene. It is mixed with glycol, sodium lauryl sulfate, and a mixture thereof. For capsules, tablets, and pills, the dosage form may also include a buffer.

Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using such excipients as lactose or lactose, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules with coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutical formulation can be prepared. They may optionally contain opaque agents, which optionally release only the active ingredient (s) or preferentially the active ingredient (s) in a particular portion of the intestinal tract, in a delayed manner. It can also have a composition. Examples of embedded compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using such excipients as lactose or lactose, as well as high molecular weight polyethylene glycols and the like.

The modified particles can also be in microencapsulated form with one or more excipients as described above. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules with coatings and shells such as enteric coatings, release control coatings, and other coatings well known in the art of pharmaceutical formulation can be prepared. In such solid dosage forms, the active compound may be miscible with at least one inert diluent such as sucrose, lactose, and starch. Such dosage forms may also include, in normal practice, additional substances other than the inert diluent, such as tablet lubricants, as well as other tablet aids, such as magnesium stearate and microcrystalline cellulose. For capsules, tablets, and pills, the dosage form may also include a buffer. They may optionally contain opaque agents, which may optionally have a composition that, in a delayed manner, releases only the modified particles or preferentially the modified particles in a particular portion of the intestinal tract. .. Examples of embedded compositions that can be used include polymeric substances and waxes.

The present invention includes pharmaceutically acceptable topical formulations of the modified particles of the present invention. The term "pharmaceutically acceptable topical formulation" as used herein is any pharmaceutically acceptable formulation for intradermal administration of the modified microparticles of the invention by application of the formulation to the epidermis. Means. In certain embodiments of the invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include solvents (eg, alcohol, polyalcohol, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffer solutions (eg, hypopenetration). Pressurized or buffered physiological saline), or any other carrier known in the art for pharmaceuticals for topical administration, but not limited to these. A more complete list of carriers known in the art is provided by reference texts standard in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985. Both are described in Mack Publishing Company, Easton, Pa. Published by, these disclosures are incorporated herein by reference in their entirety. In certain other embodiments, the topical formulations of the invention may comprise excipients. Any pharmaceutically acceptable excipient known in the art can be used to prepare a pharmaceutically acceptable topical formulation of the invention. Examples of excipients that may be included in the topical formulations of the present invention include preservatives, antioxidants, moisturizers, softeners, buffers, solubilizers, other penetrants, skin protectants, surfactants. , And additional therapeutic agents used in combination with, but not limited to, sprays and / or modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, or organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylhydroxytoluene, butylhydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable moisturizers include, but are not limited to, glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers for use with the present invention include, but are not limited to, citric acid, hydrochloric acid, and lactic acid buffers. Suitable solubilizers include, but are not limited to, quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin, and polysorbate. Suitable skin protective agents that can be used in the topical formulations of the present invention include, but are not limited to, vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topical formulation of the invention comprises at least the modified particles of the invention and a penetration enhancer. The choice of topical formulation depends on or is treated, the physicochemical characteristics presented by the compounds and other excipients of the invention, their stability in the formulation, the equipment available, and the cost. There are multiple factors, including constraints. As used herein, the term "penetration enhancer" is used to transport a pharmacologically active compound through the stratum corneum, preferably into the epidermis or dermis with little or no systemic absorption. It means a drug that can be used. A wide variety of compounds have been evaluated for their effectiveness in increasing the rate of penetration of drugs through the skin. For example, Percutaneous Penetration Enhancers, Maibach H. et al., Who are investigating and testing the use of various skin penetration enhancers. I. and Smith H. E. (Eds.), CRC Press, Inc. , Boca Raton, Fla. (1995), and Buyuktimkin et al. , Chemical Mens of Transfer Permeation Enhancement in Transdermal and Basic Drug Delivery Systems, Gosh T. et al. K. , Pfister W. R. , Yum S. I. (Eds.), Interpharm Press Inc. , Buffalo Grove, Ill. (1997). In certain exemplary embodiments, penetrants for use with the present invention include triglycerides (eg, soybean oil), aloe compositions (eg, aloe veragel), ethyl alcohol, isopropyl alcohol, octeryphenyl polyethylene. (Octolyphenylpolyethylene) Glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (eg, isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N- Includes, but is not limited to, methylpyrrolidone.

In certain embodiments, the composition may be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant, or patch. In certain exemplary embodiments, the formulation of the composition according to the invention is a cream, which is a saturated or unsaturated fatty acid such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl alcohol. , Or may further contain oleic alcohol, with stearic acid being particularly preferred. The creams of the invention may also contain nonionic surfactants such as polyoxy-40-stearate. In certain embodiments, the active ingredient is miscible under sterile conditions with a pharmaceutically acceptable carrier and, if required, any required preservative or buffer. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. In addition, the invention contemplates the use of transdermal patches with the additional advantage of providing controlled delivery of compounds into the body. Such dosage forms are made by dissolving or dispensing the compound in a suitable medium. As discussed above, penetration enhancers can also be used to increase the flow of the compound over the skin. The rate can be controlled either by providing a rate control membrane or by dispersing the compound in a polymer matrix or gel.

The modified particles can be administered by aerosol. This is achieved by preparing aqueous aerosols, liposome preparations, or solid particles containing the modified particles. Non-aqueous (eg, fluorocarbon spray) suspensions can be used.

Aqueous aerosols are usually made by formulating an aqueous solution or suspension of the drug with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of the particular compound, but are typically nonionic surfactants (Tweens, Pluronics®, or polyethylene glycol), harmless proteins such as serum albumin, sorbitan. Includes amino acids such as esters, oleic acid, lecithin, glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

Also, the modified particles and pharmaceutical compositions of the present invention can be formulated and used in combination therapy, i.e., the compound and pharmaceutical composition are one or more other desired therapeutic agents or medical. It will also be appreciated that it can be formulated using the procedure, or at the same time, administered before or after. The particular combination of therapies (eg, therapies or procedures) for use in the combination regimen takes into account the desired therapeutic agent and / or procedure, as well as the suitability of the desired therapeutic effect achieved. Also, the treatments used may achieve the desired effect on the same disorder (eg, the compounds of the invention may be administered at the same time as another anti-inflammatory agent), or they may have different effects. It will also be understood that it can be achieved (eg, control of any adverse effects).

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

The present invention provides a method of controlling an immune response in an individual, preferably a mammal, more preferably a human, the method comprising administering to the individual the modified particles described herein. The methods of immunoregulation provided by the present invention include, but are not limited to, suppress and suppress innate or adaptive immune responses stimulated by immunostimulatory polypeptides, or viral or bacterial components. / Or includes methods of inhibiting.

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

In some embodiments, the compositions described herein are with implants (eg, devices) and / or implants (eg, tissues, cells, organs) (eg, simultaneously, before or after). When administered, it mediates, nullifies, controls, and / or reduces the immune responses associated with them.

In certain embodiments, the individual suffers from an allergic disease or condition thereof, allergies, and disorders associated with unwanted immune activation such as asthma. An individual with an allergic disease or asthma is an individual with a recognizable symptom of an existing allergic disease or asthma. Tolerance can be induced in such individuals, for example, by a particle complex inhaler (eg, Sugi pollen protein) that induces an allergic reaction.

In some embodiments, the invention relates to the use of the compositions of the invention prior to the onset of allergies, eg, allergies to sugi pollen. In certain embodiments, the present invention relates to the use of the compositions of the present invention to inhibit ongoing or existing allergies. In some embodiments, the invention relates to alleviating an allergic or allergic response in a subject. Relieving it means treating, preventing, or suppressing an allergic or allergic response in a subject.

In some embodiments, the compositions of the invention (eg, PLG carriers bound to antigen molecules associated with Sugi pollen allergy) find use with one or more scaffolds, matrices, and / or delivery systems. (For example, US Patent Application Publication No. 2009/02388879, US Patent No. 7,846,466, No. 7,427,602, No. 7,029,697, No. 6,890,556, Please refer to No. 6,797,738 and No. 6,281,256, which are incorporated herein by reference in their entirety). In some embodiments, the particles (eg, antigen-bound PLG particles) are directed to the subject of a scaffold, matrix, and / or delivery system (eg, chemical / biological material, cell, tissue, and / or organ). For delivery), associate, adsorb, implant and conjugate. In some embodiments, scaffolds, matrices, and / or delivery systems (eg, for delivery of chemical / biological materials, cells, tissues, and / or organs to a subject) are described herein. Containing and / or making from them (eg, PLG conjugated to one or more antigenic peptides).

In some embodiments, a microporous scaffold (eg, for transplanting a biological material (eg, cell, tissue, etc.) into a subject) is provided. In some embodiments, microporous scaffolds having agents (eg, extracellular matrix proteins, exendin-4) and biological materials (eg, pancreatic islet cells) on top are provided. In some embodiments, the scaffold is used in the treatment of a disease (eg, type 1 diabetes) and related methods (eg, diagnostic methods, research methods, drug screening). In some embodiments, a scaffold having a carrier conjugated to an antigen described herein is provided on and / or in the scaffold. In some embodiments, the scaffold is produced from an antigen-conjugated material (eg, an antigen-conjugated PLG).

In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or is conjugated to one or more chemical and / or biological entities / agents (eg, proteins, peptides). (Particles, small molecules, cells, tissues, etc.), eg, US Patent Application Publication No. 2009/02388879, which is incorporated herein by reference in its entirety. In some embodiments, the antigen-bound particles are co-administered with the scaffold delivery system to induce induction of immunological tolerance to the scaffold and associated materials. In some embodiments, the microporous scaffold is administered to the subject with the particles described herein on or in the scaffold. In some embodiments, the antigen-bound particles are attached to the scaffold delivery system. In some embodiments, the scaffold delivery system comprises antigen-bound particles.

Various modifications, reconstructions, and variations of the features and embodiments described will be apparent to those skilled in the art without departing from the scope and gist of the invention. Although the specific embodiments have been described, it should be understood that the claimed invention should not be overly limited to such specific embodiments. In fact, various modifications of the forms and embodiments that are apparent to those skilled in the art are intended to be within the scope of the following claims. For example, US Application Nos. 62 / 221,504, PCT Application Nos. PCT / US2016 / 068423, and PCT / US2017 / 0121173 (each of which is incorporated herein by reference in its entirety). , US Patent Application Publication Nos. 2012/0076831, 2002/0045672, 2005/0090008, 2006/0002978, 2009/0238879, 2012/0076831 and 2015/ 0209293, 2015/0283218 (each of which is incorporated herein by reference in its entirety), and US Pat. Nos. 7,846,466, 7,427,602, ibid. No. 7,029,697, No. 6,890,556, No. 6,797,738, and No. 6,281,256 (each of which, by reference in its entirety, is described herein in its entirety. (Incorporated in) provides details, modifications, and variations found for use in the various embodiments described herein.

All publications and patents described in this application and / or listed below are incorporated herein by reference in their entirety.

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

Example 1: Encapsulation of recombinant Sugi pollin protein in TIMP As described above, due to the problem of encapsulating JCP in which the stickiness of the solution remains high even at low concentrations, Sugi pollen in the treatment of Sugi pollinosis. There is a limit to the use of particles that encapsulate (JCP) extract. An experiment was conducted to prepare TIMP particles (TIMP-JCP ^P ) encapsulating JCP using the double emulsion-solvent evaporation method (FIG. 2C). In summary, depending on the batch of JCP extract being tested, 200 μL of 5 mg / mL JCP solution may be added to 0.5 mL of 20% w / v PLGA in DCM, or 400 μL of 5 mg / mL. The JCP solution was added to 1.0 mL of 20% w / v PLGA in DCM. Each solution was emulsified by sonication to produce a primary emulsion. A PEMA solution (10 mL of 1% w / v aqueous Pema) was then added to the emulsion and re-emulsified by sonication to produce a secondary emulsion. The emulsion was poured into 200 mL of 0.5% w / v aqueous Pema with stirring. The particles were purified, lyophilized and stored at -20 ° C for later use. CBQCA analysis was performed after determining the antigen dose of the particles as described above by dissolving the particles in DMSO. The nanoparticle size and zeta potential in water were measured using a dynamic light scattering method on the Zetasizer Nano ZSP (Malvern Instruments, Westborough, MA). Lot release criteria require a TIMP-JCP ^P diameter of 700 nm ± 250 nm, are charged at at least -30 mV, and contain at least 5 μg of antigen / 1 mg of PLGA. The conditions for particle production and the results of JCP encapsulation are shown in FIG.

The first attempt to encapsulate all JCP extracts in TIMP was only partially successful because the maximum antigen filling achieved was approximately 1 μg / 1 mg TIMP or less protein (FIG. 4). This may be due to the high viscosity of the JCP even at low concentrations (FIG. 2A). Therefore, an experiment was conducted to determine the optimum conditions for encapsulating the recombinant JCP proteins (CRY1, CRY2, Lifeome) in TIMP (TIMP-JCP®, chart shown in FIG. 2B).

Generate TIMP-JCP® particles using low molecular weight PLGA (0.17 dL / g), Sugi basic protein (CRYJ1, 55,180 Da), and polygalacturonase (CRYJ2, 59,070 Da). did. The purity of the recombinant protein was assessed by SDS-PAGE (FIG. 3C) and compared with the results previously determined (Y. Mitobe et al., Toxicology and pharmacology: RTP 2015 71). Table 2 shows the conditions for producing TIMP-JCP (registered trademark) particles.

CRYJ1 or CRYJ2 was encapsulated for particle formulation and using the dual emulsion protocol described above (the chart shown in FIG. 2C). For each sample, protein concentration and burst release were determined by CBQCA. The results of these analyzes are shown in Table 3 below. The particle size and particle charge were determined by dynamic light scattering (DLS) using Malvern Zethasizer Nano ZS or ZSP, and the results are shown in Table 4 below.

Example 2: Mouse model of JCP allergy A mouse model with an acute allergic response to an inhaled allergen is used to elucidate the underlying mechanisms of the immunological and inflammatory response in asthma and to control allergic inflammation. Widely used for the identification and investigation of new targets for.

The characteristics of the acute inflammation model can be influenced by mouse strains, allergens, and selection of sensitization and antigen administration protocols (FIG. 5A). In summary, Sugi pollen extract (Sugi pollen extract-Cj, LSL-LG5280, Lo # 153101, Cosmo Bio USA) was resuspended in MilliQ water at 1 mg / mL. The JCP solution was diluted 1: 1 in Imject Alum (Thermo Scientific, Cat. # 77161) so that the final concentration of alum was 20 mg / mL. On day 0, Balb / c mice were injected intraperitoneally with 100 μL of JCP + alum solution (ie, 50 μg of JCP adsorbed on 2 mg / mL alum, n = 10). Control mice were injected with alum diluted in PBS (n = 10). On day 14, mice received a second equivalent dose. On day 21, sera were collected by bleeding the posterior orbit of mice for antibody analysis (FIGS. 5B-5D). On day 22, some mice in each county were given a single dose of 2.5 mg of either TIMP-OVA (n = 5) or TIMP-JCP (n = 4) intravenously via the tail vein. It was tolerated by oral administration. In addition, mice were tolerated with TIMP-OVA or TIMP-JCP and then sensitized with JCP.

On days 28 and 29, mice were administered intranasal antigen with 100 μg of JCP extract in 20 μL MilliQ water. On day 29, mice were monitored for scratches (FIG. 7) and temperature changes (FIG. 6) 1 hour after antigen administration. After 1 hour, blood was collected and the serum levels of MCPT-1 (FIG. 10B) and histamine (FIG. 10A) were assessed.

On day 30, mice were rebleeded for analysis of antibodies in serum (FIGS. 9A-9C). Mice were then sacrificed and spleens were collected from each mouse and homogenized for Exvivocytokine analysis and proliferation (FIG. 8). The spleen, and collected, treated into a single cell suspension free of red blood cells, and incubated at 37 ° C. for 48 hours in 1, 25, or 50 μg / mL JCP complete RPMI culture medium. 50 μL of supernatant was removed for cytokine analysis, followed by instantaneous application of 1 μCi / well [3H] TdR to cells for the second 24 hours of culture. Growth was determined by incorporation with [3H] TdR, detected by the Topcount Microplate Scintillation Counter (PerkinElmer, Waltham, MA).

These data indicate that treatment with JCP-TIMP after JCP sensitization sufficiently reduces the production of IFNγ, IL-17, and IL-5 in Exvivo analysis. Similar results were observed for IL-4, IL-10, and IL-13 levels when splenocytes were stimulated with higher doses of JCP. After TIMP treatment, antibody levels did not change.

Example 3: A mouse model of JCP allergy, and a treated allergic airway inflammation model mouse using CRYJ1 and CRYJ2TIM, a large amount of cedar pollen allergen in alum (3 mg), 10 μg of CRYJ1 and CRYJ2 in two doses. , Or intraperitoneal (ip) immunization using only alum and PBS. Mice were then tissue collected after antigen administration with aerosolized CRYJ1 and CRYJ2 (10 mg / ml) for 20 minutes for 3 consecutive days.

Analysis of eosinophils in bronchoalveolar lavage fluid Lungs are flushed with 1 mL of bronchoalveolar lavage fluid (BALF, 1 mM EDTA and 10% FBS in PBS). The total number of cells is determined, the sample is site-spun on the slide, and the number of fractionated cells is stained with DiffQuik (Siemens, Newark, DE).

Airway histological examination Lungs are collected, fixed in formalin and processed into paraffin. Paraffin sections are stained with hematoxylin and eosin (H & E) or periodic acid shiff (PAS).

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

Cytokine Quantification For IL-4, IL-5, IL-13, IL-10, IL-17, and IFNγ, BAL fluid and reresponsive cultures by magnetic Milliporex MAP multiplex assay (Millipore, Billilica, MA). The supernatant from (collected at 48 hours) is assayed.

Production and / or source of Sugi pollen antigen The purified Sugi pollen extract is sourced from the Japan Meteorological Agency (Japane Aerology Society). Further, as described above, recombinant Sugi pollen antigen is produced (Fujimura T, Int. Arch. Allergy Immunol. 2015; 168 (1): 32-43). The amino acid sequences of the immunodominant epitopes in the JCP are CRYJ1 (accession number AB081309, SEQ ID NO: 1) and CRYJ2 (accession number AB21810, SEQ ID NO: 2).

Production of TIMP-JCP research batch As described above, the recombinant JCP antigen is encapsulated in TIMP. For efficacy studies in a mouse model of JCP allergy, 200 mg TIMP-CRYJ1 and / or CRYJ2 (TIMP-JCP®) and 200 mg TIMP-OVA controls are used. Six groups of 5-10 mice were given either a single dose (groups 1-3) or two doses (groups 3-6) regimens to TIMP-OVA or TIMP-JCP (groups 3-6). Treat with registered trademark). Sensitize animals to JCP antigen (either before or after TIMP treatment). On days 28 and 29, airway antigens are administered and the efficacy of treatment is determined using the primary readout and success measurement scales described below. Note that group 7 mice are antigenized with OVA instead of JCP to sensitize these mice to OVA and use as a positive allergy control.

Animal POC: TIMP-JCP Treatment Before Sensitization As shown in Table 5, 6 groups of 5-10 mice were divided into single doses (groups 1-3) or double doses (groups 4 to 4). Give any of the regimens of 6) and treat with TIMP-OVA or TIMP-JCP®. The animal is then sensitized to the JCP antigen. On days 28 and 29, airway antigens are administered and the efficacy of treatment is determined using the primary readout and success measurement scales described below. Note that group 7 mice are antigenized with OVA instead of JCP to sensitize these mice to OVA and use as a positive allergy control.

Post-sensitized animal POC-TIMP-JCP treatment As shown in Table 6 below, six groups of mice are treated with TIMP-JCP® or TIMP-OVA after being sensitized to JCP. Test two dose regimens. This includes a single TIMP dose regimen (Groups 1-3) and two dose regimens (Groups 4-6) and is divided for 7 days. On days 28 and 29, air antigens are administered and the efficacy of treatment is determined using the primary readout and success measures described below.

Primary readout and success measurement scale in animal models Measure the primary readout and success measurement scale in mice treated with TIMP-JCP® prior to sensitization. Mice treated with TIMP-JCP® do not have a temperature change compared to baseline temperature after administration of air antigen. There is no change in the number of blood eosinophils and mast cells compared to the baseline measurement scale in mice treated with TIMP-JCP®. Mice treated with TIMP-JCP® significantly reduced JCP-specific IgE compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® also significantly reduced systemic IL-4, IL-5, and IL-13 compared to mice treated with TIMP-OVA sensitized to JCP. Mice treated with TIMP-JCP® are compared to mice treated with TIMP-OVA.

The primary readout and success measure scales are measured in mice treated with TIMP-JCP® after sensitization. Mice treated with TIMP-JCP® do not have a temperature change compared to baseline temperature after administration of air antigen. There is no change in the number of blood eosinophils and mast cells compared to the baseline measurement scale in mice treated with TIMP-JCP®. Mice treated with TIMP-JCP® significantly reduced JCP-specific IgE compared to mice sensitized to JCP and treated with TIMP-OVA. Mice treated with TIMP-JCP® also significantly reduced JCP-specific systemic IL-4, IL-5, and IL-13 compared to mice treated with JCP-sensitized TIMP-OVA. did. Mice treated with TIMP-JCP® also had reduced specific antigen production compared to mice treated with TIMP-OVA.

Example 4: Clinical study on TIMP-JCP Efficacy and safety of TIMP-JCP in a single-center, open-label, placebo-controlled study of Japanese seasonal allergic rhinitis (SAR) patients exposed to quantitative high-density cedar pollen. Conduct clinical studies to evaluate.

The primary purpose of the clinical study is to analyze and evaluate changes in total nasal symptoms score (TNSS) in Japanese SAR patients treated with oral TIMP-JCP during high-density exposure to Sugi pollen.

The second purpose is to analyze and evaluate changes in total symptom score (TSS), symptom score, nasal discharge, number of sneezes, and patient impressions in patents with allergic rhinitis, and pure cedar pollen. To assess the safety of TIMP-JCP administered to the venous route of patients exposed to high-density exposure to.

General Research Program Environmental exposure units will be used to determine the safety and efficacy of TIMP-JCP in patients allergic to Sugi pollen. There are many environmental exposure units (EEUs), but this study will be conducted in collaboration with the Japan Health Support Network Unit in Wakayama Prefecture. Without being bound by many of the limitations of conventional methods, the EEU allows precise controlled studies to be planned and performed (similar studies are described by Enomoto, et al., 2009). The following diagram outlines dosing and event schedules.

Subjects There are a total of 25 subjects in clinical studies. Treat 5 subjects with placebo. Twenty subjects will be treated with TIMP-JCP.

Diagnosis and incorporation criteria Incorporation criteria:
-Japanese patients with SAR.
-Patients with a history of Sugi pollinosis symptoms for at least 2 years.
-Positive IgE (against cedar pollen antigen): Determined by fluorescent enzyme immunoassay (FEIA) or chemiluminescent enzyme immunoassay (CLEIA) within 1.5 years prior to the day of the screening exposure test.
Patients with a TNSS of 8 or higher and a nasal congestion score of 2 (moderate) or higher at at least one rating point 90-150 minutes after the start of screening exposure. (TNSS) and nasal congestion score) may be at the time of any other rating point.・ Age ≧ 20 and ≦ 65 years old (gender does not matter).
-Patients who have signed a consent form based on the notice.

Exclusion criteria ・ Patients with symptoms of perennial allergic rhinitis.
-Patients with severe asthma, bronchiectasis, severe hepatic dysfunction, renal dysfunction, or cardiac dysfunction, blood disease, endocrine disease, and other serious complications.
-Patients with nasal disorders (such as hypertrophic rhinitis, sinusitis, nasal polyps, septal curvature) or eye disorders that can interfere with the determination of TIMP-JCP efficacy.
-Patients with evidence of upper respiratory tract inflammation and / or lower respiratory tract inflammation (acute rhinitis in the presence of a cold, chronic rhinitis, congestive rhinitis, atrophic rhinitis, purulent rhinitis, sinusitis) on the day of treatment exposure.
-Patients taking any of the following drugs that may affect the evaluation of TIMP-JCP, including anti-competitants and immunomodulators.

Additional exclusion criteria:

Within 2 weeks before the day of the first dose:
-Anti-allergic drugs, antihistamines (H1 and H2 blockers: oral administration, nasal drops, eye drops, injections, and topical use), anticholinergic agents, vasoconstrictor nasal drops, antihistamine-containing cold drugs, antiallergic effects / Drugs that may be expected to have antihistamine effects (including Chinese herbs and glycyrrhizin), and other drugs that are indicated for allergic symptoms (squeezing, nasal leakage, nasal congestion, and itchy eyes).
Gamma globulin preparations containing steroids (oral, inhalation, nasal drops, eye drops, or topical use), immunosuppressants (oral, topical, or injectable), azole fungicides, and histamine.
-Preparations containing azole fungicides, macrolide antibiotics, and aluminum hydroxide / magnesium hydroxide.

Within 4 weeks before the screening exposure test day:
-Depot steroid preparation.

Within 6 months before the day of the screening exposure test: Steroid injection.
・ Steroid injection

Within one year before the screening exposure test day:
-Patients who have received maintenance therapy for specific desensitization or who have received non-specific alternative therapy.
-Patients participating in another study or who have previously participated in another study within 6 months prior to the consent based on the announcement.
Patients who are deemed inappropriate for participation in this study by the investigator / deputy investigator for any other criteria.
-Patients with a history of hypersensitivity to antihistamines or antihistamines (including fexofenadine HCI) and pseudoephedrine hydrochloride.
-Patients participating in another study or who have previously participated in another study within 6 months prior to the day of the screening exposure study.
-Women who are pregnant, may become pregnant, or are currently breastfeeding.

Safety and stop criteria

This study may be withheld at the discretion of the Data Safety Monitoring Committee if any of the following parameters are met:
a) When a severe adverse event (SAE) associated with one or more unexpected drugs is reported to the Data Safety Monitoring Committee.
b) Excess and / or unexpected adverse events not explicitly related to the study drug.
c) Unexpected patient death (s).

Dose, duration, and mode of administration:
Drugs: TIMP-JCP, poly (lactic acid-co-glycolic acid) immunotolerant immunomodulation

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

Duration of treatment / study: Patient participation is made after patient screening and informed consent. Dosing will begin after recruitment and entry into the study. Dosing is given on days 0 and 14. The first high density Sugi pollen exposure takes place on days 18-21 (patients are exposed to 8,000 grains / m ³ JCP in an environmental exposure unit for 5 hours). The second high-density Sugi pollen exposure takes place on days 32-35 (patients are exposed to 8,000 grains / m3 of JCP in an environmental exposure unit for 5 hours). Patients are visited and followed up on days 42, 49, and 56. Visit on day 63, four weeks after the second exposure, for final follow-up.

Dosing Mode: The returned TIMP-JCP is diluted by slow IV infusion in normal saline (NS, 0.9% sodium chloride, NaCl) for 30 minutes. TIMP-JCP can be stopped at any time if the investigator decides that an alternative therapy should be given.

Evaluation Criteria Safety:
-Onset and severity of AEs and special purpose AEs (including standard reporting criteria for hypersensitivity and immune-mediated responses).
Vital signs (axillary temperature, sitting blood pressure, and sitting pulse rate)
・ Standard laboratory evaluation ・ 12-lead ECG laboratory test (hematology, biochemical action, and urine analysis)
-Prick test on Sugi pollen-Parameters generated by antibody and histamine release test.

Key endpoint:

TNSS: Total score for sneezing, rhinorrhea, nasal congestion, and itching of the nose Each symptom is rated by the patient in the following five categories: 1 = none (no symptom), 2 = mild (symptoms but easily) Tolerable), 3 = moderate (symptom recognition, annoying but tolerable), 4 = severe (clear symptom recognition, intolerable but not disturbing daily activities), and 5 = very serious (tolerable) Sneeze, hinders daily activities)

Secondary efficacy endpoint:

Immune surveillance:
-Antibodies: IgG antibody, specific IgG antibody (anti-JCP, anti-CRYJ1, and anti-CRYJ2), specific IgG4 antibody (anti-JCP), IgE antibody, specific IgE antibody (anti-JCP).
Cytokines (IFN-gamma, IL-4, IL-5, IL-10, IL-12, and IL-13)
-Immune cell phenotype (T-leg, effector T-cell, and monocyte)
-Peripheral B and T cell response to Sugi pollen.
-Changes in TSS, changes in each symptom score, amount of runny nose, number of sneezes, and patient's impression.
-Amount of nasal discharge: Weigh the tissue paper after the patient blows the nose every hour. The difference from the weight before blowing the nose is calculated as the amount of nasal discharge.
-Number of sneezes: Record the number of sneezes per hour by the patient himself.

Pharmacokinetics (PK):
-Serum concentration of TIMP-JCP measured daily in a small collection program during study drug administration.

result:

Subjects treated with TIMP-JCP show an onset and reduced severity of allergic reactions to Sugi pollen exposure. This is manifested by a reduction in TNSS in subjects treated with TIMP-JCP compared to subjects treated with placebo. Subjects treated with JCP also had reduced levels of specific IgG antibodies (anti-JCP, anti-CRYJ1, and anti-CRYJ2), specific IgG4 antibodies (anti-JCP), IgE antibodies, and specific IgE compared to the placebo group. It had an antibody (anti-JCP), a reduced peripheral B and T cell response to cedar pollen, and a lower TSS score, symptom score, nasal discharge volume, and number of squeaks.

Although certain embodiments of the invention have been described and exemplified, such embodiments should be considered merely exemplary of the invention, as may be construed in accordance with the appended claims. It should not be seen as limiting.

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

Claims (55)

A composition comprising biodegradable particles comprising one or more encapsulated antigen epitopes derived from Sugi pollen, wherein the biodegradable particles have a negative zeta potential. The composition according to claim 1, wherein the biodegradable particles contain poly (lactide-co-glycolide) (PLG). The composition according to claim 1 or 2, wherein the biodegradable particles contain a PLG having a polylactic acid: polyglycolic acid copolymer ratio of about 50:50. The composition according to any one of claims 1 to 3, wherein the surface of the biodegradable particles is carboxylated. The composition according to claim 4, wherein the carboxylation is achieved by using poly (ethylene-alt-maleic anhydride) (PEMA), poly (acrylic acid), or sodium deoxycholate. The composition according to any one of claims 1 to 5, wherein the biodegradable particles have a zeta potential of about -100 mV to about 0 mV. The composition according to any one of claims 1 to 6, wherein the biodegradable particles have a zeta potential of about -50 mV to about -40 mV. The composition according to 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 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 μm to about 5 μm. The composition according to any one of claims 1 to 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 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 about 0.2 μm to about 0.7 μm. 16. The composition of claim 16, wherein the biodegradable particles have a diameter of about 0.7 μm. 16. The composition of claim 16, wherein the biodegradable particles have a diameter of about 0.5 μm. The composition according to any one of claims 1 to 18, wherein the one or more encapsulated antigen epitopes derived from Sugi pollen contain CRYJ1 or a fragment or variant thereof. 19. 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 contiguous amino acids having at least 90% sequence identity to SEQ ID NO: 1. thing. The CRYJ1 variant 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: 1. The composition according to claim 20, which has an amino acid sequence having. 20. 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 according to any one of claims 1 to 23, wherein the one or more encapsulated antigen epitopes derived from Sugi pollen contain CRYJ2, or a fragment or variant thereof. 24. The composition of claim 24, wherein CRYJ2 has the amino acid sequence of SEQ ID NO: 2. 25. 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 contiguous amino acids having at least 90% sequence identity to SEQ ID NO: 2. thing. The CRYJ2 variant 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: 2. 25. The composition according to claim 25, which has an amino acid sequence having. 25. 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 described. The composition according to any one of claims 1 to 28, wherein the biodegradable particles contain an antigen-to-polymer ratio of 1 μg / mg to 15 μg / mg. 29. The composition of claim 29, wherein the biodegradable particles contain an antigen-to-polymer ratio of 5 μg / mg to 15 μg / mg. The composition according to any one of claims 1 to 30, wherein the biodegradable particles contain an antigen-to-polymer ratio of at least about 5 μg / mg. The composition according to any one of claims 1 to 31, wherein the biodegradable particles contain two or more encapsulated antigen epitopes derived from Sugi pollen protein. 32. The composition of claim 32, wherein the two or more encapsulated epitopes are contained in the fusion protein and the two or more encapsulated epitopes in the fusion protein are separated by a cleavable linker. 33. The composition of claim 33, wherein the amino acid sequence of the cleavable linker is cleavable by a protease located within the phagolysosome of the cell and / or a protease located within the cytosol of the cell. 34. The composition of claim 34, wherein the amino acid sequence of the cleavable linker is cleavable by a protease located within the phagolysosome of the cell and a protease located within the cytosol of the cell. The composition according to claim 34 or 35, wherein the cleavable linker is a furin-sensitive linker or a cathepsin-sensitive linker. The composition according to any one of claims 34 to 36, wherein the cleavable linker is a furin-sensitive linker. The composition according to any one of claims 34 to 36, wherein the cleavable linker is a cathepsin-sensitive linker. The cathepsin-sensitive linker is one of cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin W, and / or cathepsin Z. 38. The composition of claim 38, which is sensitive to cleavage by one or more. The composition according to claim 34, wherein the amino acid sequence of the linker is Gly-Ala-Val-Val-Arg-Gly-Ala (SEQ ID NO: 3). The composition according to any one of claims 1 to 40, wherein the one or more encapsulated antigen epitopes derived from Sugi pollen are covalently bound to the biodegradable particles. The composition according to claim 41, wherein the one or more encapsulated antigen epitopes derived from Sugi pollen are covalently bound to the biodegradable particles by a conjugate molecule. 42. The composition of claim 42, wherein the conjugate molecule comprises a carbodiimide compound. The composition of claim 43, wherein the carbodiimide compound comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). A composition comprising biodegradable particles comprising one or more encapsulated antigen epitopes derived from cedar pollen, wherein the biodegradable particles have a negative zeta potential of at least about -30 mV, a diameter of about 0.7 μm, and. A composition having an antigen-to-polymer ratio of at least about 5 μg / mg. A pharmaceutical composition comprising the biodegradable particles according to any one of claims 1 to 45. 46. The pharmaceutical composition of claim 46, further comprising a pharmaceutically acceptable carrier. 47. The pharmaceutical composition of claim 47, further comprising a pharmaceutically acceptable excipient. A lyophilized composition comprising the biodegradable particles according to any one of claims 1 to 45. A method for inducing antigen-specific tolerance to Sugi pollen in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition according to any one of claims 46-49. A method for the treatment of Sugi pollen allergy in a subject in need of treatment, comprising administering the pharmaceutical composition according to any of claims 46-49. A method for the prevention of Sugi pollen allergy in a subject in need of prevention, comprising administering the pharmaceutical composition according to any one of claims 46-49. A method for inducing antigen-specific tolerance to Sugi pollen in a subject, wherein the lyophilized particles according to claim 49 are returned to obtain a returned pharmaceutical composition, and the returned pharmaceutical composition is used as described above. A method comprising administering to a subject. A method for the treatment of Sugi pollen allergy in a subject in need of treatment, wherein the lyophilized particles according to claim 49 are returned to obtain a returned pharmaceutical composition, wherein the returned pharmaceutical composition is obtained. A method comprising administering a substance to said subject. A method for the prevention of Sugi pollen allergy in a subject in need of prevention, wherein the lyophilized particles according to claim 49 are returned to obtain a returned pharmaceutical composition, and the returned pharmaceutical composition is obtained. A method comprising administering a substance to said subject.

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