Peptide-conjugated particles

Abstract

The present invention provides compositions comprising biodegradable poly(lactide-co-glycolide) (PLG) particles coupled with peptides. Specifically, the PLG particles are surface functionalized to allow peptide molecules to be coupled to the surface of the particles (e.g., for induction of immune tolerance).

Description

[0001] The present invention is a divisional application based on a patent application with an application date of August 13, 2014, application number of "201480046047.X" (international application number of PCT / US2014 / 050962), and invention name of "Peptide-conjugated particles".

[0002] Federal Government Funding

[0003] This invention was made with government support under R01 EB013198 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] Related patent applications

[0005] This application claims priority to U.S. Provisional Patent Application No. 61 / 865,389 filed on August 13, 2013, U.S. Provisional Patent Application No. 61 / 869,279 filed on August 23, 2013, and U.S. Provisional Patent Application No. 61 / 887,112 filed on October 4, 2013. This application is also related to PCT Application No. PCT / US2013 / 047079 filed on June 21, 2013, which claims priority to U.S. Provisional Application No. 61 / 662,687 filed on June 21, 2012. The contents of each of the above patents are hereby incorporated by reference in their entirety.

[0006] Instructions for electronically submitting text documents

[0007] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer readable format copy of the Sequence Listing (file name: COUR-002_01US_Seqlist.txt, record date: August 13, 2013, file size 1.17 megabytes). Background Art

[0008] Inflammatory diseases and disorders are conditions in which an abnormal or otherwise dysregulated inflammatory response contributes to the etiology of the disease or exacerbates the severity of the disease. Examples include autoimmune diseases such as type 1 diabetes and celiac disease.

[0009] Many of these diseases are characterized by mononuclear cell infiltration at sites of tissue injury or other damage. Examples of mononuclear cells that have been observed in these infiltrates include lymphocytes, especially T lymphocytes, and cells of the mononuclear phagocyte system (MPS cells), such as monocytes, macrophages, dendritic cells, microglia, and other cells.

[0010] Many of the cells observed in the mononuclear infiltrate are suspected to play a role in these abnormal inflammatory responses. For example, in diseases such as multiple sclerosis, CD4 + T cells play a central role in pathological autoimmune responses. At early points in T cell activation, dendritic cells and other MPS cells are responsible for CD4 + MPS cells can also promote inflammation through phagocytosis, but in at least some inflammatory diseases, it is unclear how this occurs in the absence of CD4 + Whether such cells can play this role in the case of T cells.

[0011] Peripheral blood mononuclear cells can be divided into one of two groups based on the expression or absence of certain cell surface molecules. Specifically, human "resident monocytes" or "mature monocytes" are understood to be cells with CD14 lo CD16 + Phenotype (corresponding phenotype in mice is CX 3 CR1 hi CCR2 - Gr1 - Another group of cells, "inflammatory monocytes" or "immature monocytes", are understood to have CD14 + CD16 - Phenotype (corresponding phenotype in mice is CX 3 CR1 lo CCR2 + Gr1 + (Geissmann F. et al. 2003 Immunity 19: 71-82 (Geissmann F. et al., 2003, Immunity, Vol. 19, pp. 71-82)

[0012] Importantly, although the latter are understood to be "inflammatory" in the sense that peripheral blood cells of bone marrow origin are observed to migrate into inflamed tissues, these cells have not been shown to cause inflammation directly or through the action of other cells. In addition, the various MPS cells that these cells can form when they differentiate have not been shown to cause inflammation.

[0013] Conventional clinical strategies for general long-term immunosuppression in disorders associated with undesirable immune responses are based on long-term administration of broadly acting immunosuppressive drugs, such as signal 1 blockers such as cyclosporine A (CsA), FK506 (tacrolimus), and corticosteroids. Long-term use of high doses of these drugs can have toxic side effects. In addition, even in patients who can tolerate these drugs, the need for lifelong immunosuppressive drug therapy carries a significant risk of serious side effects, including tumors, serious infections, nephrotoxicity, and metabolic disturbances.

[0014] Methods for inducing antigen-specific tolerance have been developed, which include cell coupling of antigens or peptides. For example, in one method, peptide-induced cell coupling tolerance involves collecting, separating and processing peripheral blood cells under aseptic conditions with disease-specific autoantigens and ethylene carbodiimide (ECDI) coupling agents, and then injecting them into the donor / patient. This process is expensive and must be performed by skilled practitioners under strict monitoring conditions, and is limited by the number of centers that can carry out the procedure. Using red blood cells as donor cell types allows potential sources to be expanded to include allogeneic donors, thereby significantly increasing the supply of source cells and possibly extending the delivery of this therapy to any environment with transfusion permission. These methods have obvious limitations in the supply of source cells and tissue type matching to minimize the necessity of the immune response to donor cells. In addition, there are important quality control issues in local processing cells to couple autoantigens via EDCI. In addition, these methods also require at least some knowledge of pathological antigens for seeking immune tolerance.

[0015] Recently, peptide-coupled particles have been described that eliminate the need for a source cell supply and circumvent the tissue typing requirements of prior methods, see WO 2010 / 085509, which is incorporated herein by reference in its entirety. Nevertheless, the use of antigens coupled to the outside of the particles is still associated with increased allergic reactions and has significant chemical, manufacturing and control problems. Surprisingly, these adverse events are avoided when the antigen is encapsulated within the particles. Even more surprisingly, the size and charge can be varied to enhance tolerance to specific antigens.

[0016] Antigen-specific immune tolerance is generally undesirable because the specific antigen / epitope is generally unknown in human disease. In addition, for antigen-specific methods to be effective, the antigens may differ between subjects, so it will be necessary to determine which antigens each patient will recognize, or a library of possible peptides will need to be coupled to the particles prior to administration. The synthesis and individual coupling of these peptides is both time-consuming and expensive. Therefore, a therapy that solves these two problems is needed, thereby eliminating the need for a tissue-matched cell source. Summary of the invention

[0017] In some embodiments, the present invention provides compositions (e.g., for inducing antigen-specific tolerance) comprising carrier particles (e.g., PLG particles) attached to antigenic peptides. In certain embodiments, the carrier particles are poly(lactide-co-glycolide) (PLG) particles. In other embodiments, the carrier particles are Stabilized polypropylene sulfide particles.

[0018] In some embodiments, the present invention provides a composition comprising: an antigen coupled to a carrier particle having a negative zeta potential. In some embodiments, the zeta potential of the particle is from about -100 mV to about 0 mV. In some embodiments, the zeta potential of the particle is from about -50 mV to about -40 mV. In some embodiments, the particle is a copolymer having a molar ratio of about 50:50, 80:20 to about 100:0. In some embodiments, the copolymer ratio may be, but is not limited to, polystyrene:poly(vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90:10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90:10, polylactic acid:polyglycolic acid / 80:20 or polylactic acid:polyglycolic acid / 90:10 or polylactic acid:polyglycolic acid / 50:50. In other embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, Stabilized polypropylene sulfide particles or poly(lactic-co-glycolic acid) particles. In some embodiments, the particles are poly(lactic-co-glycolic acid) particles.

[0019] In some embodiments, the particles have an average diameter between about 0.1 μm and about 10 μm. In some embodiments, the particles have an average diameter between about 0.2 μm and about 2 μm. In some embodiments, the particles have a diameter between about 0.3 μm and about 5 μm. In some embodiments, the particles have a diameter between about 0.5 μm and about 3 μm. In some embodiments, the particles have a diameter between about 0.5 μm and about 1 μm. In some embodiments, the particles have a diameter of about 0.5 μm.

[0020] In other embodiments, the antigen comprises at least a portion of an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, an enzyme, or an allergen. In some embodiments, the antigen comprises at least a portion of the following substances: myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic β-cell antigen, insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), glutamate decarboxylase (GAD), type II collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, aquaporin 4, desmoglein 1, desmoglein 3, nicotinic acetylcholine receptor, A-gliadin and human tropomyosin subtype 5, bahia grass pollen (BaGP), peach allergen Pru p 3, αs 1-Casein milk allergen, Apig1 celery allergen, Bere1 Brazil nut allergen, B-Lactoglobulin milk allergen, Bovine serum albumin, Cor a1.04 Hazelnut allergen, ovalbumin egg allergen, recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog alpha, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), Ceredase, Alglucerase, Cerezyme, Imiglucerase, Glucocerebrosidase (Elelso), taliglucerase alfa, Fabrazyme, β-galactosidase (Agalsidase beta), laronidase (Aldurazyme), l-iduronidase, α-glucosidase (Myozyme), acid glucosidase, iduronic acid-2-sulfatase (Elaprase), iduronate-2-sulfatase, sulfatase (Naglazyme), arylsulfatase B or N-acetylgalactosamine-4-sulfatase, protein therapeutics for enzyme or coagulation factor replacement therapy such as α-glucosidase (myozyme), alglucerase, imiglucerase, taliglucerase, β-galactosidase (agalsidase beta), l-iduronidase, acid glucosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-4-sulfatase, antihemophilic factor, factor VII, eptacog alfa, factor IX, miglustat, romiplastim, epotetin alpha, protein C, laronidase, lumizyme, or factor VIII.

[0021] In further embodiments, the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, an enzyme, or an allergen. In non-limiting examples, antigens include, for example, myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic β-cell antigen, insulin, glutamate decarboxylase (GAD), type II collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, aquaporin 4, desmoglein 1, desmoglein 3, nicotinic acetylcholine receptor, A-gliadin and human tropomyosin isotype 5, bahia grass pollen (BaGP), peach allergen Pru p 3, αs 1-casein milk allergen, Apig1 celery allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 Hazelnut allergen, insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), ovalbumin egg allergen, protein therapeutics used for enzyme or coagulation factor replacement therapy such as α-glucosidase (myozyme), alglucerase, imiglucerase, taliglucerase, β-galactosidase (agalsidase beta), l-iduronidase, acid glucosidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, antihemophilic factor, factor VII, eptacog alpha, factor IX, miglustat, romiplastin, epotetin alpha, protein C, laronidase, α-glucosidase (lumizyme), factor VIII.

[0022] In further embodiments, the particle is coupled to an antigen comprising one or more epitopes. In yet another embodiment, the epitope is associated with an allergic reaction, an autoimmune disease, an enzyme used in enzyme replacement therapy, a lysosomal storage disease, or an inflammatory disease or disorder. In one embodiment, the epitope is associated with type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, neuromyelitis optica, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, Gaucher's disease, Fabry's disease, Hurler's disease, Hunter's disease, Mar-La disease, hemophilia A, hemophilia B, von Willebrand disease, venous thrombosis, purpura fulminans, mucopolysaccharidosis VI, Pompe's disease, celiac disease, or inflammatory bowel disease (including Crohn's disease or colitis, such as ulcerative colitis). In another embodiment, the epitope is present in a protein therapeutic for enzyme or coagulation factor replacement therapy, such as α-glucosidase (myozyme), alglucerase, imiglucerase, taliglucerase, β-galactosidase (agalsidase beta), l-iduronidase, acid glucosidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, antihemophilic factor, factor VII, eptacog alpha, factor IX, miglustat, romiplastim, epotetin alpha, protein C, laronidase, α-glucosidase (lumizyme), factor VIII. In another embodiment, the epitope is an epitope described in Table 2 or Table 3. In one embodiment, the particle is coupled to an antigen that contains only one epitope associated with a disease and / or disorder. In another embodiment, the antigen comprises more than one epitope associated with the same disease and / or disorder. In another embodiment, the antigen comprises more than one epitope associated with different diseases and / or disorders.

[0023] In some embodiments, the antigen is coupled to the particle via a binding molecule. In some embodiments, the antigen is coupled to the particle via a linker. In some embodiments, the binding molecule is ethylene carbodiimide (ECDI). In certain embodiments, the antigen is connected via a streptavidin-biotin complex. In some embodiments, the linker may include, but is not limited to, a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Specific coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP) to provide disulfide bonds.

[0024] In some embodiments, the antigen is coupled to the exterior of a particle having a negative zeta potential. In some embodiments, the antigen is encapsulated in a particle having a negative surface zeta potential. In some embodiments, the particle is biodegradable. In some embodiments, the particle is surface functionalized. In some embodiments, the particle is carboxylate surface functionalized.

[0025] In some embodiments, the present invention provides a method of inducing antigen-specific tolerance in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential, and wherein the particles and the antigen induce tolerance of the subject to the antigen. In some embodiments, administration is performed to treat or prevent a disease or condition. In some embodiments, administration is performed before or after the occurrence of a disease or condition caused by the antigen. In some embodiments, the disease or condition is selected from: autoimmune diseases, inflammatory diseases, allergies, transplant rejection, lysosomal storage diseases, enzyme deficiencies, inflammatory reactions, and hyperimmune reactions. In some embodiments, the disease or condition is selected from: multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, inflammatory bowel disease including Crohn's disease or ulcerative colitis, and a condition caused by the antigen in the subject that reduces excessive response to the antigen. In some embodiments, the method further comprises repeatedly administering the composition to the subject.

[0026] In another embodiment, administration of the particles results in activation-induced death of effector T cells.

[0027] In another embodiment, administration of the particles results in effector T cell anergy.

[0028] In another embodiment, administration of the particles results in apoptosis of effector T cells.

[0029] In another embodiment, administration of the particles results in conversion of effector T cells into regulatory T cells.

[0030] In another embodiment, the administration of the particles results in the induction and expansion of both antigen-specific and non-specific effector regulatory T cells. In another embodiment, the administration of the particles results in the isolation of effector T cells in lymph nodes and spleen, thereby inhibiting the ability of these cells to traffic to peripheral sites and cause inflammation.

[0031] In another embodiment, administration of the particles results in downregulation of T cell-dependent antibody production.

[0032] In certain embodiments, the present invention provides a method for treating celiac disease in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential. In certain embodiments, the antigen is gliadin or a gliadin epitope. In some embodiments, the antigen is one or more antigens selected from SEQ ID NOs: 1295-1724, SEQ ID NOs: 1726-1766, and SEQ ID NOs: 4986-5140. In some embodiments, the antigen is gliadin and the antigen-coupled particles have an average size after synthesis of about 600-1500 nanometers and an average charge after synthesis of about -30 to about -80 mV. In some embodiments, the particles have an average size after synthesis of about 600-1200 nanometers and an average charge after synthesis of about -40 to about -70 mV. In certain embodiments, the particles have an average size after synthesis of about 600 micrometers and an average charge after synthesis of about -50 mV. In further embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0033] In some embodiments, the present invention provides a method for treating diabetes in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential. In some embodiments, the diabetes is type I diabetes. In some embodiments, the diabetes is type II diabetes.

[0034] In some embodiments, the antigen is insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), or an epitope derived from insulin, proinsulin, or IGRP. In some embodiments, the antigen is one or more antigens selected from SEQ ID NOs: 1767-1840, SEQ ID NOs: 1842-1962, SEQ ID NOs: 1964-2027, SEQ ID NOs: 2029-2073, SEQ ID NOs: 2075-2113, SEQ ID NOs: 2115-2197, SEQ ID NOs: 2199-2248, SEQ ID NOs: 2250-2259, SEQ ID NOs: 2261-2420, SEQ ID NOs: 2422-2486, and SEQ ID NOs: 2489-2505. In some embodiments, the antigen is insulin and the antigen-coupled particles have a post-synthesis average size of about 300-800 nanometers and a post-synthesis average charge of about -30 to about -70 mV. In some embodiments, the particles have an average size after synthesis of about 350-600 nanometers and an average charge after synthesis of about -40 to about -60mV. In some embodiments, the particles have an average size after synthesis of about 500 nanometers and an average charge after synthesis of about -50mV. In some embodiments, the antigen is proinsulin and the antigen-coupled particles have an average size after synthesis of about 300-800 nanometers and an average charge after synthesis of about -30 to about -70mV. In certain embodiments, the particles have an average size after synthesis of about 400-600 nanometers and an average charge after synthesis of about -40 to about -60mV. In some embodiments, the particles have an average size after synthesis of about 570 nanometers and an average charge after synthesis of about -45mV. In some embodiments, the antigen is IGRP and the antigen-coupled particles have an average size after synthesis of about 300-800 nanometers and an average charge after synthesis of about -30 to about -70mV. In some embodiments, the particles have an average size after synthesis of about 400-700 nanometers and an average charge after synthesis of about -40 to about -60 mV. In some embodiments, the particles have an average size after synthesis of about 600 nanometers and an average charge after synthesis of about -40 mV. In certain embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic acid-co-glycolic acid) particles.

[0035] In some embodiments, the present invention provides a method of treating a subject who is receiving enzyme replacement therapy, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential. In some embodiments, the subject is receiving enzyme replacement therapy to treat a disease selected from hemophilia, hemophilia A, hemophilia B, von Willebrand disease, mucopolysaccharidosis, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, Gaucher's disease, Fabry disease, Hurler's disease, Pompe's disease, Hunter's disease, and Mar-La disease. In some embodiments, the antigen-coupled particles comprise a recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog alfa, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), Ceredase, Alglucerase, Cerezyme, Imiglucerase, glucocerebrosidase (Elelso), taliglucerase alfa, Fabrazyme, β-galactosidase (Agalsidase beta), Aldurazyme, l-iduronidase, α-glucosidase (Myozyme), acid glucosidase, Elaprase, iduronate-2-sulfatase, Naglazyme, arylsulfatase B, or N-acetylgalactosamine-4-sulfatase. In some embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, polypropylene sulfide particles stabilized by PLURIONICS, or poly(lactic acid-co-glycolic acid) particles. In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. In certain embodiments, the particles are polystyrene particles, carboxylated polystyrene particles, polypropylene sulfide particles stabilized by PLURIONICS, or poly(lactic acid-co-glycolic acid) particles. In other embodiments, the particles are poly(lactic acid-co-glycolic acid) particles and have a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid. In some embodiments, the particles are poly(lactic-co-glycolic acid) particles and have a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid.

[0036] In another embodiment, administration of the particles of the invention prevents accumulation of neutrophils and other granulocytes in a subject. In another embodiment, the particles of the invention are administered to a subject having cancer.

[0037] In one embodiment, administration of the particles of the invention enhances regeneration of damaged tissue. In another embodiment, the particles enhance regeneration of epithelial cells. In yet another embodiment, the particles enhance remyelination of neurons. In another embodiment, the subject suffers from an autoimmune disease. In yet another embodiment, the subject suffers from inflammatory bowel disease, including ulcerative colitis and / or Crohn's disease. In yet another embodiment, the subject suffers from multiple sclerosis.

[0038] In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered subcutaneously, orally, intramuscularly, intralymphatically, intraportally, or via aerosol. In one embodiment, administration of the negatively charged particles induces antigen-specific tolerance in the subject. In one embodiment, the particles that induce antigen-specific tolerance comprise one or more epitopes associated with allergic reactions, autoimmune diseases, and / or inflammatory diseases. In one embodiment, the epitopes are selected from those described in Table 2 or 3. In one embodiment, the negatively charged particles are polystyrene, diamond, Stable polypropylene sulfide or poly (lactic acid-co-glycolic acid) particles. In one embodiment, the particles are carboxylated. In one embodiment, the particles have a zeta potential of less than about -100 mV. In one embodiment, the particles have a zeta potential between about -75 mV and 0 mV, such as between -50 mV and 0 mV, or between -100 mV and -50 mV, or between -75 mV and -50 mV, or between -50 mV and -40 mV. In one embodiment, the particles have an average diameter of about 0.1 μm to about 10 μm, such as about 0.2 μm to about 2 μm, or about 0.3 μm to about 5 μm, or 0.5 μm to about 3 μm, or about 0.5 μm to about 1 μm.

[0039] In one embodiment, the subject suffers from an autoimmune disease. In one embodiment, the autoimmune disease is multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Raynaud's syndrome, Sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, neuromyelitis optica, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, celiac disease, ulcerative colitis or Crohn's disease. In one embodiment, the particle comprises a full-length polypeptide or a fragment thereof. In one embodiment, the particle comprises one or more myelin basic protein epitopes. In one embodiment, the myelin basic protein epitope is from SEQ ID NO:4975 or SEQID NO:4976. In one embodiment, the particle comprises one or more myelin oligodendrocyte glycoprotein epitopes. In one embodiment, the myelin oligodendrocyte glycoprotein epitope is from SEQ ID NO: 1 or SEQ ID NO: 4978. In one embodiment, the particle comprises one or more insulin epitopes. In one embodiment, the one or more insulin epitopes are from SEQ ID NO: 4981. In one embodiment, the particle comprises one or more glutamate decarboxylase epitopes. In one embodiment, the glutamate decarboxylase epitope is from SEQ ID NO: 4982. In one embodiment, the particle comprises one or more proteolipid protein epitopes. In one embodiment, the proteolipid protein epitope is from SEQ ID NO: 4977. In one embodiment, the particle comprises one or more gliadin epitopes. In one embodiment, the gliadin epitope comprises SEQ ID NO: 4983-4985.

[0040] In some embodiments, the present invention also provides a method for preparing an immune modified particle with a negative zeta potential, the method comprising: contacting an immune modified particle precursor with a buffer solution under conditions effective to form immune modified particles with a negative zeta potential. In some embodiments, the immune modified particle precursor is formed by copolymerization. In some embodiments, the buffer solution has an alkaline pH. In some embodiments, the buffer solution is sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate.

[0041] In some embodiments, the present invention provides a composition comprising an antigen encapsulated in the core of a surface functionalized liposome. In another embodiment, the liposome is composed of phosphatidylcholine:phosphatidylglycerol:cholesterol in a ratio of 30:30:40. In yet another embodiment, the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, or an allergen. In some embodiments, the particle is a poly(lactic-co-glycolic acid) particle and has a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid. In some embodiments, the particle comprises PEMA. In some embodiments, PEMA is present at about 0.1% to about 2.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 (A) Micrograph of poly(lactide-co-glycolide) (PLG) particles. B and C show characterization of surface functionalized poly(lactide-co-glycolide) particles by dynamic light scattering analysis including size distribution, mean size (nm), zeta potential (mV), and OVA 323-339 and PLP 139-151 Peptide coupling efficiency (%) of peptides to PLG-PEMA particles. Peptides were quantified on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at 2.5×10 5 Surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of counts / second. The surface functionalized poly(lactide-co-glycolide) particle population varied by 5-15% from batch to batch, but generally had a Z-average diameter of 567 nm, a peak diameter of 670 nm, and a polydispersity index of 0.209.

[0043] Figure 2 PLG nanoparticles were shown to induce antigen-specific tolerance. Using immunodominant proteolipid protein PLP 139-151 Epitope (PLG-PLP 139-151 ) to induce tolerance to prevent recurrent experimental autoimmune encephalitis (R-EAE). PLP was used on day -7 relative to the immunization time (day 0). 139-151 -PLGA (N=5), OVA 323-339 -PLGA (N=5) or unconjugated PLGA (N=5) treated mice. Peak disease was usually observed around day 12 to 14 and mice were scored for clinical disease. No peptide or control peptide OVA 323-339The modified particles failed to prevent the induction of disease. However, except for the low clinical scores below 1 between days 20 and 30, the treatment with PLP 139-151 The modified PLGA particles produced a clinical score of 0 (no disease).

[0044] Figure 3 shows that the type of particles administered has an effect on the development of EAE in a mouse model. A) shows the mean clinical score, while B) shows the mean cumulative score of EAE animals. 323-339 -PLS (N=5), OVA 323-339 -PLGA PHOSPOREX (N=5), OVA 323-339 -PLGA PEMA (N=5), PLP 139-151 -PLA (N=5), PLP 139-151 -PLGA PHOSPOREX (N=5) or PLP 139-151 -PLG PEMA (N=5) mice were treated. The peak of disease was usually observed around day 12 to day 14, and the mice were scored for clinical disease. 323-339 Modification of any composition of the particles failed to prevent disease induction. However, PLP 139-151 PLG-coupled microbeads were more effective than PLP in down-regulating the induction of R-EAE 139-151 Coupling to commercial (phosphorex) PLG or polystyrene was more effective.

[0045] Figure 4 It is shown that those mice treated with soluble OVA exhibited a decrease in temperature compared to those animals treated with OVA-PLG particles at day 28. No decrease in body temperature was observed within 1 hour of delivery of the particles.

[0046] Figure 5 It was shown that administration of PLP-PLG during remission did not cause any mortality related to anaphylaxis. PLP in CFA was administered subcutaneously to six to eight week old female SJL / J mice. 139-151 EAE was induced, and the development of clinical disease was monitored and recorded. On day 21 relative to disease induction, mice were injected intravenously with soluble PLP 139-151 (hollow square), soluble OVA 323-339 (open circles) or the same peptide coupled to PLG nanoparticles (solid circles).After injection, the temperature of the animals was monitored and recorded every 10 minutes for 1 hour (A).

[0047] Figure 6 PLP administered intravenously seven days before disease induction is shown139-151 -The optimal dose of PLG. Compared with OVA 323-339 -PLG-treated SJL / J mice, and the development of clinical disease was measured (A). Six to eight-week-old female SJL / J mice were injected intravenously with PLP 139-151 (Square) or OVA 323-339 (Circles) PLG nanoparticles coupled to the PD-L1 complex. PLG nanoparticles were injected subcutaneously in CFA after 7 (B), 25 (C), or 50 (D) days. 139-151 To induce EAE. Animals in panel B were followed for clinical disease for 100 days. Delayed-type hypersensitivity (DTH) reactions (E) were performed in the subgroups of mice shown in panel B on day 8 relative to disease induction. PLP from panel B was used to induce EAE. 139-151 / CFA priming group (OVA 323-339 -PLG and PLP 139-151 -PLG) selected representative animals were primed with PLP 139-151 Epitopes and OVA 323-339 Control peptides were used for ear challenge. Ear swelling as a measure of DTH was determined 24 hours later and the response before challenge was subtracted. Six to eight week old female SJL / J mice were injected intravenously with PLP 178-191 (Triangle), OVA 323-339 (Circular) or PLP 139-151 (squares) conjugated PLG nanoparticles or unconjugated particles alone (open circles) (F). PLG nanoparticles were injected subcutaneously in CFA 7 days later. 178-191 EAE was induced, and the disease was monitored at the indicated time points.

[0048] Figure 7 AD shows that when PLG-PLP is administered intravenously or intraperitoneally 139-151 PLP is most effective in preventing tolerance when administered intravenously. 139-151 -PLG treated animals did not develop disease and had a mean clinical score of 0 at most time points.

[0049] Figure 8 AF shows the administration of OVA 323-339 -PLG particles suppressed Th1 and Th17 responses in treated animals.

[0050] Fig. 9 AC shows the PLP 139-151 - PLG-treated animals have reduced immune cell infiltration in the spinal cord and are more similar to native tissue than OVA 323-339 - Tissues from animals treated with PLG.OVA 323-339 -PLG treated animals had positive staining for CD45, CD4, and CD11b; whereas PLP139-151 -PLG treated animals had minimal staining for these factors.

[0051] Fig.10 AC shows the administration of PLP 139-151 -PLG particles inhibited blood-brain barrier (BBB) ​​disruption and macrophage activation in the spinal cord of treated mice. Animals were treated with complete Freund's adjuvant (CFA), OVA 323-339 PLG particles or PLP 139-151 -PLG particle treatment. The clinical scores and incidence percentage of EAE were determined (B) and the spinal cord was observed via in vivo imaging (A and C).

[0052] Fig.11 A and B show the spinal cord of treated mice demonstrated via in vivo imaging. CF are graphs showing quantification of the image data.

[0053] Fig.12 The administration of PLP encapsulated 139-151 PLG particles of inhibited the induction of R-EAE in mice. The ability to encapsulate self-antigens allows the use of complex mixtures of proteins or even organ homogenates that are not possible in the case of surface binding, thus allowing higher antigen coverage and thus more effective response to epitope expansion.

[0054] Fig.13 Shown using PLP 139-151 - Animals treated with PLG particles and anti-CD25 antibodies sometimes showed more 139-151 - Higher mean clinical scores in animals treated with PLG particles and control IgG antibody.

[0055] Fig.14 Shown by PLP 139-151 -PLG particles induce therapeutic tolerance in active and adoptive EAE. 2.5 × 10 6 PLP 139-151 Activation of blasts to induce adoptive EAE. Mice were injected intravenously with PLP coupled to 500 nm PLG nanoparticles 2 days (A), 14 days (C), 18 days (E), or 21 days (F) after disease induction. 139-151 (Square) or OVA 323-339 (Circles) Peptides. Clinical disease scores were compared with those after treatment with antigen-coupled splenocytes (A). 139-151 or OVA 323-339Brain and spinal cord were collected from tolerized mice for histological analysis. Sections from mice in panel A were stained for PLP protein and CD45. Spinal cord sections from mice in panel C were stained with Luxol Fast Blue (D). Areas of demyelination and cellular infiltration are indicated by arrows.

[0056] Fig.15 The figure shows the conjugation to OVA 323-339 or PLP 139-151 Figure 3 is a graph of the mean clinical scores of mice with active EAE and adoptive EAE after treatment with SP or PLG particles. Mice were injected intravenously with PLG at 10 days (A) or 2 days (B) after disease induction. 139-151 -SP, PLP 139-151 -PLG or OVA 323-339 -SP or OVA coupled to 500 nm nanoparticles 323-339 -PLG peptide, and the mean clinical score was determined. 139-151 -PLG particles all induced tolerance in mice.

[0057] Fig.16 It was shown that the infiltration of central nervous system immune cells was also significantly reduced in PLP-PLG-tolerized mice. SJL / J mice were intravenously injected with PLP-PLG 2 days after EAE induction by adoptive transfer. 139-151 (Square) or OVA 323-339 (Circle) coupled 500nm PLG nanoparticles. At the peak of the disease (day 14), the brain and spinal cord were removed and the number of lymphocytes (B), APCs (C), microglia (D), peripheral dendritic cells (E), myeloid dendritic cells (F) and macrophages (G) were calculated by flow cytometry. The gating strategy of these colonies is depicted in (A). CNS cell preparations were stimulated with PM and ionomycin for 5 hours, and then intracellular staining (H) was performed for IL-17A and IFN-γ.

[0058] Fig.17 Administration of PLP encapsulated in PLG particles is shown 139-151 The peptide induced tolerance when the particles were administered together with PBS. However, administration of anti-PD-1 antibody reduced this tolerance.

[0059] Fig.18 Administration of PLP encapsulated in PLG particles is shown 139-151 The peptide induced tolerance when the particles were administered together with PBS. Administration of anti-CD40 antibody reduced this tolerance, but this reduction in tolerance was reversed by the addition of anti-IL-12 antibody.

[0060] Fig.19It was shown that prophylactic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and decreased the levels of serum OVA IgE and eosinophils in the lung.

[0061] Fig. 20 The OVA specific in vitro recall reaction of mediastinal lymph node that OVA encapsulated in PLG granules preventively inhibits is shown. Lymph node proliferation observed after restimulation with 25 μ g OVA reduces in those animals processed with OVA-PLG (A). In addition, the release of cytokines after restimulation with OVA is reduced by processing with OVA-PLG. The level of IL-4, IL-5, IL-13 and IL-10 is reduced in the mice processed with OVA-PLG (B).

[0062] Fig.21 A and B show that therapeutic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and decreased the levels of serum OVA IgE and eosinophils in the lung.

[0063] Fig. 22 It is shown that OVA encapsulated in PLG particles therapeutically down-regulates OVA-specific Th2 cytokines in bronchoalveolar lavage fluid better than OVA-coupled PLG particles. Mice were treated intraperitoneally with OVA / alum at a dose of 10 μg / mouse on days 0 and 14. OVA coupled to PLG particles or OVA encapsulated in PLG particles was administered intravenously to mice on days 28 and 42. Between days 56 and 58, mice were treated three times with aerosolized OVA. These graphs depict cytokine secretion when animals were treated with OVA (A) coupled to PLG particles or OVA (B) encapsulated in PLG particles.

[0064] Fig.23 Figure 2 shows blood glucose levels in type 1 diabetic animals after treatment with p31-PLG particles. Administration of p31 peptide-coupled PLG particles produced similar results to those observed after administration of MOG 35-55 In mice treated with p31-PLG, compared with MOG 35-55 The percentage of IFNγ-secreting cells observed in peptide-PLG treated mice was also reduced compared to animals (C).

[0065] Fig.24 AB shows that p31-PLG-induced tolerance requires Tregs. Type 1 diabetes was induced in mice by adoptive transfer. Two hours after the activated cells were transferred into NOD.SCID mice, the mice were treated with p31-PLG or MOG 35-55PLG particles tolerize. Depletion of Tregs abolished tolerance induced by administration of p31-PLG particles.

[0066] Fig.25 It was shown that administration of insulin-coupled PLG particles significantly increased the percentage of mice that did not develop diabetes within 300 days (69.6% vs. 22.7%; p=0.0027). NOD mice were treated with BSA (N=22) or insulin (N=23)-coupled PLG particles via intravenous administration at 6, 8, and 10 weeks of age. The mice were then analyzed for the development of diabetes.

[0067] Fig.26 The percentage of CD45.1 donor cells observed in recipient mice is shown. Female CD45.2 mice were tolerized with OVA-PLG or Dby-PLG on day -7. Mice were irradiated with 200 rads on day -1 and then transplanted with 1×10 PD-1 cells from male CD45.1 mice on day 0. 6 , 5×10 6 or 1×10 7 Recipient mice were then tolerized with OVA-PLG, Dby-SP or Dby-PLG on day 1 and blood was harvested for FACS analysis of chimerism.

[0068] Fig. 27 The percentage of donor CD45.1 cells in recipient mice after tolerization with OVA-PLG, Dby-SP or Dby-PLG on day 1 is shown. One positive control mouse did not show significant implantation (about 10%). All negative control mice were not implanted with donor cells. One Dby-SP mouse did not show significant implantation (about 10%). Two OVA-PLG mice were implanted with donor cells (about 10%): one completely rejected by week 16. One Dby-PLG mouse began to reject at week 12 and was 10% by week 16. The Dby-PLG group was within the implantation range of 10%-56% by week 16. OVA-PLG mice showed: 1) spontaneous implantation, 2) sequence homology between OVA323 and Dby, or 3) tolerogenicity of the particles. Dby-PLG allowed more implantation than Dby-SP and OVA-PLG.

[0069] Fig.28 It is shown that timed tolerance has an effect on the percentage of CD45.1 cells in the recipient mice. The positive control showed less implantation (about 4%) than expected (about 10%). One negative control mouse had 5% implantation. In all 3 OVA-PLG groups, one mouse in the -7 day, +1 day group showed implantation (12%). Tolerance on day 1 is more clinically relevant than tolerance on day -7.

[0070] Fig.29 Shown that coumarin-6 PLGA particles coupled to antigen or free of antigen were detectable 3 hours after administration, but undetectable 24 hours after administration. Particles were detectable 3 hours after administration, but undetectable 24 hours after administration. Uninjected mice (top row) that were not tested for the first time were compared with spleen (left column), liver (middle column) and lung (left column) sections of mice injected with intravenous fluorescent PLGA / PEMA microparticles (3 hours after injection (middle row) and 24 hours after injection (bottom row), counterstained with DAPI).

[0071] Fig.30 F4 / 80 in the liver after 6 and 15 hours is shown. + Cell co-localization with PLGA particles.

[0072] Fig.31 Figure 2 shows that TAMRA-labeled PLP was predominantly taken up by marginal zone macrophages 24 hours after intravenous infusion. 139-151 Coupled particles. Highest percentage of PLP 139-151 + cells are marginal zone macrophages.

[0073] Fig.32 The daily mean clinical scores relative to the day of PLP139-151 / CFA priming are depicted.Induction of immune tolerance in SJL / J mice by using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core suppressed PLP139-151 / CFA-induced R-EAE.

[0074] Fig.33 It was shown that mice treated with encapsulated OVA-PLG showed the greatest reduction in eosinophil accumulation.

[0075] Fig.34 It was shown that mice treated with encapsulated OVA-PLG showed the greatest reduction in serum IgE levels compared to untreated or control treated animals.

[0076] Fig.35Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core by dynamic light scattering analysis is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of 1.792×105 counts / second on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water. The population of surface functionalized poly(lactide-co-glycolide) particles had a Z-average diameter of 584 nm, a peak diameter of 679 nm, and a polydispersity index of 0.162. These results represent the synthesis of 6 batches following the above protocol.

[0077] Fig.36 Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core by zeta potential measurement is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of 6.67×104 counts / second on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water. The population of surface functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -48.9 mV and a zeta deviation of 5.14 mV. These results are representative of the synthesis of 6 batches following the above protocol.

[0078] Fig.37 Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core by dynamic light scattering analysis is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.822×105 counts / second. The population of surface functionalized poly(lactide-co-glycolide) particles had a Z-average diameter of 569.7 nm, a peak diameter of 700.3 nm, and a polydispersity index of 0.230. These results are representative of the synthesis of 3 batches following the protocol described above.

[0079] Fig.38Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core by zeta potential measurement is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.67×104 counts / second. The population of surface functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -52.2 mV and a zeta deviation of 5.38 mV. These results are representative of the synthesis of 3 batches following the protocol described above.

[0080] Fig.39 This shows that the core contains soluble PLP 139-151 Figure 1. Surface-functionalized peptide liposomes induce immune tolerance in a mouse model of multiple sclerosis. Animals were treated with liposomes containing soluble PLP in their core. 139-151 Surface functionalized liposomes with peptides (round) or containing soluble OVA 323-339 The surface of the liposomes functionalized with peptides (squares) was treated. Those receiving PLP 139-151 The mean clinical scores of animals receiving peptide liposomes were lower than those receiving OVA 323-339 Peptide liposomes in animals.

[0081] Fig.40 The charge of the administered particles has been shown to have an effect on the development of EAE in a mouse model. Panel (A) shows the mean clinical scores, while panel (B) shows the mean cumulative scores of EAE animals. Mice received TIMPs (tolerogenic immune modifying particles) with a charge of -60 mv or -25 mv bound to the antigen. Mice were treated with OVA 323-339 -TIMP -60mv 、OVA 323-339 -PLGA -25mv 、PLP 139-151 -TIMP -60mv or PLP 139-151 -PLGA -25mv Treatment and clinical disease scoring. More negatively charged particles TIMP -60mv PLGA -25mv The particles induced tolerance more effectively.

[0082] Figure 41 shows that the charge of immune modified particles is important for targeting immune modified particles to antigen presenting cells. Wild type or MARCO+ / - animals were treated with PS-IMP or vehicle. The results show that particles with reduced negative charge are less efficient because they interact less with the scavenger receptor MARCO (A). Anti-MARCO antibodies alone do not provide similar efficiency as PLGA IMP (B).

[0083] Fig.42 The key particle parameters required for tolerance in the EAE mouse model are shown. Figure (A) shows that the most effective average particle size is 500nm. Mice were treated with 500nm OVA 323-339 -PSB, 100nm PLP 139-151 -PSB, 500nmPLP 139-151 -PSB, 1.75μm PLP 139-151 -PSB or 4.5μm PLP 139-151 -PSB treatment and clinical disease scoring. Panel (B) shows that fluorescently labeled particles with a 50:50 lactide:glycolide ratio were significantly cleared from the spleen, liver and lungs 24 hours after intravenous infusion.

[0084] Fig.43 Demonstrated superiority of TIMP with encapsulated antigen over peptide-coupled particles. In a murine allergy model, animals were exposed to OVA as an allergen and then given the following treatments: sham-PLG, no treatment, PLGA particles with OVA coupled to the outside of the particles (A), or PLGA particles with OVA encapsulated within the particles (TIMP) (B). Panel (A) shows that OVA-PLG surface-coupled particles failed to reduce TH2 responses. Panel (B) shows that TIMP PEMA-60mv (OVA encapsulated in the particles) inhibits TH2 responses. Figure (C) shows that TIMP PEMA-60mv (OVA encapsulated within the particles) inhibits the recall response. DETAILED DESCRIPTION

[0085] The inventors have found that nanoparticles coupled to antigens can induce tolerance to autoimmune diseases and reduce immune responses. These particles can induce tolerance regardless of whether the antigen is bound to the surface of the particle or encapsulated within the particle. Therefore, these particles can be used to treat any disease or condition characterized by an excessive inflammatory immune response, such as an autoimmune disease or an allergic reaction.

[0086] As used herein, "particle" refers to any non-tissue derived material composition, which can be a microsphere or microspheroidal entity, a microbead or a liposome. The term "particle", the term "immune modified particle", the term "carrier particle" and the term "microbead" can be used interchangeably depending on the context. In addition, the term "particle" can be used to cover microbeads and microspheres.

[0087] As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge of less than zero.

[0088] "Carboxylated particles" or "carboxylated microbeads" or "carboxylated microspheres" include any particle modified to include carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups enhances the uptake of particles from the circulation by phagocytes / monocytes, for example, by interacting with scavenger receptors such as MARCO. Carboxylation of the particles can be achieved using any compound that can add carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA).

[0089] As used herein, "antigenic portion" refers to any portion, such as a peptide, that is recognized by the host's immune system. Examples of antigenic portions include, but are not limited to, autoantigens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components. Without being bound by theory, although the carboxylated microbeads themselves may be recognized by the immune system, for the purposes of the present invention, carboxylated microbeads that do not have any other substance attached thereto are not considered "antigenic portions."

[0090] As used herein, "naked microbead" or "naked particle" or "naked microsphere" refers to a microbead, particle or microsphere that has not been carboxylated.

[0091] As used herein, "proinflammatory mediator" or "proinflammatory polypeptide" refers to a polypeptide or fragment thereof that induces, maintains or prolongs inflammation in a subject. Examples of proinflammatory mediators include, but are not limited to, cytokines and chemokines.

[0092] As used herein, the term "inflammatory monocyte" refers to any myeloid cell expressing any combination of CD14 / CD26 and CCR2.

[0093] As used herein, the term "suppressor neutrophils" refers to neutrophils, and / or monocyte-derived suppressor cells.

[0094] As used herein, the term "Th cell" or "helper T cell" refers to a CD4 + Cells. CD4 + T cells contribute to the immune processes of other white blood cells, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. T cells are activated when peptide antigens are presented by MHC class II molecules, which are expressed on the surface of antigen presenting cells (APCs).

[0095] As used herein, the term "Th1 cells" refers to a subpopulation of Th cells that produce proinflammatory mediators. Th1 cells secrete cytokines to promote immune responses and play a role in host defense against pathogens, in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines including IFN-γ, IL2, IL-10, and TNFα / β to coordinate defense against intracellular pathogens such as viruses and certain bacteria.

[0096] As used herein, the term "Th2 cell" refers to a Th cell subpopulation that mediates the activation and maintenance of antibody-mediated immune responses against extracellular parasites, bacteria, allergens and toxins. Th2 cells mediate these functions by producing various cytokines such as IL-4, IL-5, IL-6, IL-9, IL-13 and IL-17E (IL-25), which are responsible for the production of antibodies, the activation of eosinophils and the inhibition of various macrophage functions, thereby providing a non-phagocyte-dependent protective response.

[0097] As used herein, the term "Th17 cells" refers to a subpopulation of Th cells. Th17 cells secrete cytokines to promote immune responses and play a role in host defense against pathogens by mediating the recruitment of neutrophils and macrophages to infected tissues. TH17 cells secrete cytokines such as IL17, IL21, IL22, IL24, IL26, and TNFα to coordinate defense against extracellular pathogens including fungi and bacteria.

[0098] As used herein, "coupled" means that the antigen is fixed to the outside of the particle or encapsulated within the particle. Therefore, antigens coupled to particles include both surface coupling and encapsulation within the particle.

[0099] As used herein, the term "IMP" refers to an immune modified particle that is not coupled to an antigen. As used herein, the term "TIMP" refers to a tolerized immune modified particle that is coupled 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.

[0100] The particles may have any particle shape or conformation. However, in some embodiments, it is preferred to use particles that are less likely to clump in vivo. Examples of particles within these embodiments are those that have a spherical shape.

[0101] Another aspect of the invention relates to a composition comprising immune modified particles having a negative zeta potential and not containing an antigenic portion. In another embodiment, the invention provides a composition comprising immune modified particles having a negative zeta potential coupled to an antigen. In another embodiment, the antigen is coupled to the exterior of the particle. In a preferred embodiment, the antigen is encapsulated within the particle.

[0102] Another aspect of the invention relates to a method for preparing an immune modified particle having a negative zeta potential and not containing an antigenic portion. The method involves contacting an immune modified particle precursor with a buffer solution under conditions effective to form an immune modified particle having a negative zeta potential. In some embodiments of the invention, the immune modified particle precursor is formed via copolymerization. The particle microstructure may depend on the copolymerization method.

[0103] In certain embodiments, the antigen peptide molecule is coupled to carrier particles (e.g., immune modified particles) by binding molecules and / or linking groups. In certain embodiments, the antigen peptide and / or apoptosis signal transduction molecules are coupled to carriers (e.g., PLG particles) and include one or more covalent and / or non-covalent interactions. In certain embodiments, the antigen peptide is attached to the surface of the carrier particles with negative zeta potential. In certain embodiments, the antigen peptide is encapsulated in the carrier particles with negative zeta potential.

[0104] In one embodiment, the buffer solution contacted with the immune modified particles may have an alkaline pH. Suitable alkaline pHs for alkaline solutions include 7.1, 7.5, 8.0, 8.5, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5. The buffer solution may also be made of any suitable alkali and a combination thereof. In some embodiments of the invention, the buffer solution may include, but is not limited to, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate and a combination thereof.

[0105] In one embodiment of the invention, the immune modified particles comprise copolymers. These copolymers may have different molar ratios. Suitable copolymer ratios of the immune modified particles of the invention may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1 or 100:0. In another embodiment, the copolymer may be a periodic, statistical, linear, branched (including star, brush or comb copolymers) copolymer. In some embodiments, the copolymer ratio may be, but is not limited to, polystyrene:poly(vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90:10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90:10, polylactic acid:polyglycolic acid / 50:50, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90:10.

[0106] In one embodiment, the particles of the present invention are prepared by adding a composition comprising a polymer (e.g., PLGA) to a solution of poly(ethylene-maleic anhydride) (PEMA). The concentration of PEMA in the solution may be between about 0.1% and about 10%. In one embodiment, the concentration of PEMA in the solution is between about 0.2% and about 5%. In another embodiment, the concentration of PEMA in the solution is between about 0.1% and 4%. In another embodiment, the concentration of PEMA in the solution is between about 0.1% and 2%. In another embodiment, the concentration of PEMA in the solution is between about 0.5% and 1%. In one embodiment, the percentage of PEMA in the solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, the percentage of PEMA in the solution is about 0.5%. In another embodiment, the percentage of PEMA in the solution is about 1.0%. Other compounds that may be used include, but are not limited to, poly(ethylene-alt-maleic anhydride), poly(isobutylene-co-maleic acid), poly(methyl vinyl ether-alt-maleic acid), poly(methyl vinyl ether-alt-maleic acid monoethyl ester), poly(methyl vinyl ether-alt-maleic anhydride), poly(methyl vinyl ether-alt-maleic anhydride) powder crosslinked with 1,9-decadiene, and / or poly(styrene-alt-maleic acid) sodium salt.

[0107] In one embodiment, the particle is a liposome. In another embodiment, the particle is a liposome composed of the following lipids in the following molar ratio: 30:30:40 phosphatidylcholine:phosphatidylglycerol:cholesterol. In yet another embodiment, the particle is encapsulated in a liposome.

[0108] The size of each particle need not be uniform, but the particles must generally be of a size sufficient to achieve the following effects: sequestration in the spleen or liver and triggering phagocytosis or uptake by antigen presenting cells (including endothelial cells or other MPS cells) through receptor or non-receptor mediated mechanisms. Preferably, the particle size is micrometer or nanometer in order to enhance solubility, avoid possible complications caused by aggregation in the body, and facilitate pinocytosis. Particle size can be a factor in uptake from interstitial spaces into areas of lymphocyte maturation. Particles with a diameter of about 0.1 μm to about 10 μm can trigger 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. In yet 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 another embodiment, the particles have an average size of about 0.1 μm, or about 0.2 μm, or about 0.3 μm, or about 0.4 μm, or about 0.5 μm, or about 1.0 μm, or about 1.5 μm, or about 2.0 μm, or about 2.5 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm. In a specific embodiment, the particles have a size of about 0.5 μm. In some embodiments, the overall 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 need to have a uniform diameter. For example, a pharmaceutical preparation may include a plurality of particles, some of which are about 0.5 μm and others are about 1.0 μm. Any mixture having particle sizes within these given ranges will be useful.

[0109] The particles of the present invention may have a specific zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than about -100 mV. In one embodiment, the zeta potential is less than about -50 mV. In certain embodiments, the particles have a zeta potential between -100 mV and 0 mV. In another embodiment, the particles have a zeta potential between -75 mV and 0 mV. In another embodiment, the particles have a zeta potential between -60 mV and 0 mV. In another embodiment, the particles have a zeta potential between -50 mV and 0 mV. In yet another embodiment, the particles have a zeta potential between -40 mV and 0 mV. In another embodiment, the particles have a zeta potential between -30 mV and 0 mV. In another embodiment, the particles have a zeta potential between -20 mV and +0 mV. In another embodiment, the particles have a zeta potential between -10 mV and -0 mV. In another embodiment, the particles have a zeta potential between -100 mV and -50 mV. In another specific embodiment, the particles have a zeta potential between -75 mV and -50 mV. In a particular embodiment, the particles have a zeta potential between -50 mV and -40 mV.

[0110] In some embodiments, the charge of the carrier (e.g., positive, negative, neutral) is selected to impart a beneficial effect for a specific application (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier has a net neutral charge or negative charge (e.g., to reduce nonspecific binding to the cell surface that generally has a net negative charge). In certain embodiments, the carrier is capable of directly or indirectly binding to an antigen (also referred to herein as an antigen-specific peptide, antigenic peptide, self-antigen, induced antigen, or tolerant antigen) to which tolerance is required. In some cases, the carrier has multiple binding sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ... 20 ... 50 ... 100 or more) so as to have multiple antigen-specific peptide copies or multiple different antigens exposed on the surface (e.g., to increase the possibility of a tolerance reaction). In some embodiments, the carrier displays a single type of antigenic peptide. In some embodiments, the carrier displays a variety of different antigenic peptides on the surface. In some embodiments, the carrier surface displays functional groups for covalent attachment of selected parts (e.g., antigenic peptides). In some embodiments, the carrier surface functional groups provide sites for non-covalent interactions with selected moieties (eg, antigenic peptides). In some embodiments, the carrier has a surface such that a binding moiety can be adsorbed thereon without forming a chemical bond.

[0111] The size and charge of the particles are crucial for tolerance induction. Although the particles vary in size and charge based on the antigens encapsulated therein (see Table 1 for examples of specific particles), generally speaking, the particles of the present invention are effective in inducing tolerance when they are between about 100 nanometers and about 1500 nanometers and have a charge of 0 to about -70 mV, and are most effective in inducing tolerance when they are 400-800 microns and have a charge between about -25 mV and -70 mV. In addition, as shown in Table 1, partly due to the concentration of the particles and the presence of sucrose and D-mannitol during the freeze-drying process, the average particle size and charge of the particles may change slightly during the freeze-drying process, so both the average value after synthesis and the average value after freeze-drying are given below. As used herein, the terms "size after synthesis" and "charge after synthesis" refer to the size and charge of the particles before freeze-drying. The terms "size after freeze-drying" and "charge after freeze-drying" refer to the size and charge of the particles after freeze-drying.

[0112] Table 1 Representative particle analysis

[0113]

[0114] In certain embodiments, particle is nonmetallic. In these embodiments, particle can be formed by polymer. In a preferred embodiment, particle is biodegradable in individuality. In this embodiment, particle can provide in individuality with multiple dosages, and particle can not accumulate in individuality. The example of suitable particle comprises polystyrene particle, PLGA particle, PLURIONICS stable polypropylene sulfide particle and diamond particle.

[0115] Preferably, the particle surface is composed of materials that minimize nonspecific or unwanted biological interactions. Interactions between the particle surface and the stroma can be a factor that plays a role in lymphatic absorption. The particle surface can be coated with materials that prevent or reduce nonspecific interactions. This can be achieved by using a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers such as Coating the particles (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) provides steric stabilization and can reduce nonspecific interactions with interstitial proteins, as demonstrated by improved lymphatic absorption after subcutaneous injection. All of these facts indicate the importance of the physical properties of the particles in lymphatic absorption. Biodegradable polymers can be used to prepare all or some of the polymers and / or particles and / or layers. Biodegradable polymers can degrade, for example, by reaction of functional groups with water in solution. The term "degrade" as used herein refers to becoming soluble by reducing the molecular weight or by converting hydrophobic groups into hydrophilic groups. Polymers with ester groups generally undergo spontaneous hydrolysis, for example, polylactide and polyglycolide.

[0116] The particles of the present invention may also contain additional components. For example, the carrier may have an imaging agent incorporated into or bound to the carrier. An example of a currently commercially available carrier nanosphere with an imaging agent is the Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals (called quantum dots (QDs)) have become ideal donors in FRET applications: when excited at a single ultraviolet wavelength, their high quantum yield and tunable size-dependent Stokes shift allow different sizes to emit blue light to infrared light. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LE J Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers called dendrimers, can be used for bio-labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the synthesis of traditional inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0117] The particle can be formed by various materials. The particle is preferably made of materials suitable for biological use. For example, the particle can be made of glass, silicon dioxide, polyester of hydroxycarboxylic acid, polyanhydride of dicarboxylic acid or copolymer of hydroxycarboxylic acid and dicarboxylic acid. More generally, the carrier particle can be made of the following materials: polyester of straight or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy hydroxy acid, or polyanhydride of straight or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy dicarboxylic acid. In addition, the carrier particles can be quantum dots, or consist of quantum dots such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6 (Joumaa et al., 2006, "Langmuir", Vol. 22, pp. 1810-1816)). Carrier particles comprising a mixture of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) can also be used. For example, the carrier particles can include materials including the following substances: polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysebacic acid polymer (PSA), poly (lactic acid-co-glycolic acid) copolymer (PLGA or PLG; these terms are interchangeable), poly (lactic acid-co-sebacic acid) copolymer (PLSA), poly (glycolic acid-co-sebacic acid) copolymer (PGSA), polysulfide propylene polymer, poly (caprolactone), chitosan, etc. Other biocompatibility, biodegradable polymers that can be used for the present invention include polymers or copolymers of caprolactone, carbonate, amides, amino acid, orthoester, acetal, cyanoacrylate and degradable polyurethane, and copolymers of them and straight or branched, substituted or unsubstituted alkyl, haloalkyl, sulfanyl, aminoalkyl, alkenyl or aromatic hydroxy acid or dicarboxylic acid. 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 their enantiomers, can be included in the copolymer with any of the above-mentioned materials, so that reactive groups are provided for the combination with antigenic peptides and protein or binding moiety. Biodegradable materials that are suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in carrier particles of the present invention. For example, acrylates, ethylene vinyl acetate, acyl-substituted cellulose acetate, non-degradable polyurethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, (DuPont, Wilmington, Del.) and non-biodegradable polymers of nylon.

[0118] The particles of the present invention can be manufactured by any means generally known in the art. Exemplary methods for manufacturing particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, precipitation polymerization, emulsification evaporation, emulsification diffusion, solvent replacement and salting out (Astete and Sabliov, J.Biomater.Sci.Polymer Edn., 17:247-289 (2006) (Astete and Sabliov, Journal of Biomaterials Science-Polymer Edition, Vol. 17, pp. 247-289, 2006)). The manipulation of the manufacturing process of PLGA particles can control particle properties (e.g., size, size distribution, zeta potential, morphology, hydrophobicity / hydrophilicity, polypeptide retention, etc.). The size of the particle is affected by a variety of factors, including but not limited to the concentration of PLGA, the solvent used to make the particles, the properties of the organic phase, the surfactant used to make, the viscosity of the continuous and discontinuous phases, the properties of the solvent used, the temperature of the water used, ultrasonic treatment, evaporation rate, additives, shear stress, sterilization, and the properties of any encapsulated antigen or polypeptide.

[0119] Particle size is affected by polymer concentration; larger particles are formed from higher polymer concentrations. For example, when using the solvent propylene carbonate, increasing the PLGA concentration from 1% to 4% (w / v) increases the average particle size from about 205 nm to about 290 nm. Alternatively, in ethyl acetate and 5% Pluronic F-127, increasing the PLGA concentration from 1% to 5% (w / v) increases the average particle size from 120 nm to 230 nm.

[0120] The viscosity of the continuous and discontinuous phases is also an important parameter that affects the diffusion process, a key step in the formation of smaller particles. The size of the particles increases with increasing viscosity of the dispersed phase, while the size of the particles decreases with a more viscous continuous phase. In general, the lower the ratio of organic solvent to aqueous solvent, the smaller the particle size.

[0121] Homogenizer speed and agitation also affect particle size; generally speaking, higher speeds and agitation will reduce particle size, but there is a point where further increases in speed and agitation do not reduce particle size. Size reduction is favorably affected when the emulsion is homogenized with a high pressure homogenizer compared to high speed agitation alone. For example, at a 20% phase ratio in 5% PVA, the average particle size with agitation was 288 nm, while the average particle size with homogenization (high pressure of 300 bar) was 231 nm.

[0122] Significant particle size reduction can be achieved by varying the temperature of the added water to improve diffusion of the solvent. The average particle size decreases as the water temperature increases.

[0123] The properties of the polypeptide encapsulated in the particle also affect the particle size. Generally speaking, the encapsulation of hydrophobic polypeptides leads to the formation of smaller particles compared to the encapsulation of more hydrophilic polypeptides. In the double emulsification process, the retention of more hydrophilic polypeptides is improved by using high molecular weight PLGA and a high molecular weight first surfactant that causes a higher internal phase viscosity. The interaction between the solvent, polymer and polypeptide affects the efficiency of the polypeptide incorporation into the particle.

[0124] PLGA molecular weight affects the final average particle size. Generally speaking, the higher the molecular weight, the larger the average particle size. For example, when the composition and molecular weight of PLGA vary (e.g., 12 to 48 kDa for 50:50 PLGA; 12 to 98 kDa for 75:25 PLGA), the average particle size also varies (about 102 nm-154 nm, respectively; about 132 nm to 152 nm). Even when the particles have the same molecular weight, their composition can also affect the average particle size; for example, particles with a 50:50 ratio generally form particles that are smaller than particles with a 75:25 ratio. The end groups on the polymer also affect the particle size. For example, particles prepared with ester end groups form particles with an average size of 740 nm (PI=0.394), compared to 240 nm (PI=0.225) for acidic PLGA end groups.

[0125] The solvent used can also affect particle size; solvents that reduce the surface tension of the solution also reduce particle size.

[0126] The organic solvent is removed by vacuum evaporation to avoid damage to the polymer and polypeptide and to promote final particle size reduction. Vacuum evaporation of the organic solvent is more efficient in forming smaller particles. For example, the average particle size produced by vacuum evaporation is about 30% smaller than the average particle size produced by normal evaporation rate.

[0127] The amplitude of the ultrasonic treatment wavelength also affects the particle properties. The amplitude of the wavelength should exceed 20% and the ultrasonic treatment should be 600 to 800 s to form a stable fine emulsion without any change in droplet size. However, the main disadvantage of ultrasonic treatment is that the emulsion formed is not monodisperse.

[0128] Organic phases that can be used to produce particles of the present 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, surfactants such as poloxamer 188.

[0129] A variety of surfactants can be used to make the particles of the present invention. The surfactant can be anionic, cationic or nonionic. Surfactants in the poloxamer and poloxamine families are commonly used for particle synthesis. Available surfactants include but are not limited to PEG, Tween-80, gelatin, dextran, pluronic L-63, PVA, methylcellulose, lecithin and DMAB. In addition, biodegradable and biocompatible surfactants include but are not limited to vitamin E TPGS (D-α-tocopherol polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are required (for example, in a multiple emulsion volatilization method). The two surfactants can include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

[0130] Solvents that can be used to produce particles of the present invention include, but are not limited to, acetone, tetrahydrofuran (THF), chloroform, and a member of the chlorine family, methyl chloride. The choice of organic solvent requires two selection criteria: the polymer must be soluble in the solvent, and the solvent must be completely miscible with the aqueous phase.

[0131] Salts that can be used to produce the particles of the present invention include, but are not limited to, magnesium chloride hexahydrate, magnesium acetate tetrahydrate.

[0132] Common salting-out agents include, but are not limited to, electrolytes (eg, sodium chloride, magnesium acetate, magnesium chloride) or non-electrolytes (eg, sucrose).

[0133] The stability and size of the particles of the present invention can be improved by adding compounds, including but not limited to fatty acids or short carbon chains. The addition of lauric acid with a longer carbon chain is associated with improved particle properties. In addition, the addition of hydrophobic additives can improve particle size, incorporation of polypeptides into the particles, and release characteristics. The formulation of the particles can be stabilized by freeze drying. The addition of cryoprotectants such as trehalose can reduce the aggregation of particles during freeze drying.

[0134] Suitable microbeads currently available commercially include polystyrene microbeads, such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[0135] In some embodiments, the system provided by the present invention comprises: (a) a delivery scaffold configured to deliver a chemical agent and / or a biological agent to a subject; and (b) antigen-coupled poly(lactide-co-glycolide) particles for inducing antigen-specific tolerance. In some embodiments, at least a portion of the delivery scaffold is microporous. In some embodiments, the antigen-coupled poly(lactide-co-glycolide) particles are encapsulated within the scaffold. In some embodiments, the chemical agent and / or biological agent is selected from: proteins, peptides, small molecules, nucleic acids, cells and particles. In some embodiments, the chemical agent and / or biological agent comprises cells, and the cells comprise pancreatic islet cells.

[0136] Physical properties are also related to the suitability of nanoparticles after uptake and retention in areas with immature lymphocytes. They include mechanical properties, such as rigidity or elasticity. Some embodiments are based on an elastic core, such as a poly(propylene sulfide) (PPS) core with a covering layer, such as a hydrophilic covering layer, as in PEG, such as in a PPS-PEG system recently developed and characterized for systemic (but not targeted or immune) delivery. The elastic core is opposite to a substantially rigid core, such as in a polystyrene or metal nanoparticle system. The term elastic refers to certain elastic materials other than natural or synthetic rubber, and elasticity is a term familiar to technicians in the field of polymers. For example, cross-linked PPS can be used to form a hydrophobic elastic core. PPS is a polymer that degrades to polysulfoxide under oxidative conditions and ultimately forms polysulfone, converting from a hydrophobic rubber to a hydrophilic, water-soluble polymer. Other sulfide polymers may be suitable for use, wherein the term sulfide polymer refers to a polymer having sulfur in the main chain of the matrix. Other elastic polymers that can be used are polyesters with a glass transition temperature of less than about 37° C. under hydrated conditions. A hydrophobic core can be advantageously used with a hydrophilic covering because the core and covering will tend not to mix and thus the covering will tend to spatially expand away from the core. A core refers to a particle having a layer thereon. A layer refers to a material covering at least a portion of a core. A layer can be adsorbed or covalently bonded. A particle or core can be solid or hollow. An elastic hydrophobic core is preferred over a rigid hydrophobic core, such as a crystalline or glass (as in the case of polystyrene) core, because particles having an elastic hydrophobic core can carry a higher hydrophobic drug load.

[0137] Another physical property is the hydrophilicity of the surface. The hydrophilic material may have a solubility of at least 1 gram per liter in water when it is not cross-linked. The steric stabilization of particles with hydrophilic polymers can improve uptake from the interstitium by reducing nonspecific interactions; however, the increased stealth properties of the particles can also reduce internalization by phagocytes in areas with immature lymphocytes. However, the challenge of balancing these conflicting features has been encountered, and the present application describes the production of nanoparticles for effective lymphatic delivery to DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic component, such as a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of the following: polyalkylene oxide, polyethylene oxide, polysaccharides, polyacrylic acid, and polyethers. The molecular weight of the polymer in the layer can be adjusted to provide a useful degree of steric hindrance in vivo, such as from about 1,000 to about 100,000 or even more; the technician will immediately recognize that all ranges and values ​​within the explicitly stated range are expected, such as between 10,000 and 50,000.

[0138] Nanoparticles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles; they facilitate reaction with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

[0139] Various means well known in the art can be used for antigen peptides and proteins are attached to carriers.These methods comprise any standard chemical method, these methods do not destroy or seriously limit the biological activity of antigen peptides and proteins, and allow sufficient number of antigen peptides and proteins to be attached to carriers with the interactional orientation of allowing antigen peptides or proteins and homologous T cell receptors.Usually, the method that the C-terminal region of antigen peptides or proteins or the C-terminal region of antigen peptides or protein fusion proteins are attached to carriers is preferred.Certainly, accurate chemical method will depend on the existence or not of the character of carrier material, the existence or not of antigen peptides or protein C-terminal fusion and / or the existence or not of the binding part.

[0140] Functional groups can be located on the particle as needed for utilization. One location can be as a side group or end on the following polymer: a core polymer, a polymer as a layer on the core, or a polymer otherwise tethered to the particle. For example, examples included herein describe PEG stabilization of the following nanoparticles, which can be easily functionalized for targeting specific cells or delivering protein and peptide drugs.

[0141] Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, polypropylene glycol diglycidyl ether containing 2 epoxy residues, and epichlorohydrin can be used to fix peptides or proteins to the surface of the carrier. Without being bound by theory, ECDI is suspected to perform the following two functions to induce tolerance: (a) ECDI chemically couples proteins / peptides to the cell surface by catalyzing the formation of peptide bonds between free amino groups and free carboxyl groups; and (b) ECDI induces the carrier to simulate apoptotic cell death, so that they are taken up by host antigen presenting cells in the spleen (which may include endothelial cells) and induce tolerance. It is this non-immune presentation to host T cells that leads to the direct induction of anergy in autoreactive cells. In addition, ECDI acts as a potent stimulator for the induction of specific regulatory T cells.

[0142] In a series of embodiments, antigen peptides and proteins are bound to carriers via covalent chemical bonds. For example, reactive groups or moieties near the antigen C-terminal (e.g., C-terminal carboxyl or hydroxyl, thiol or amine groups from amino acid side chains) can be directly bound to reactive groups or moieties on the carrier surface (e.g., hydroxyl or carboxyl of PLA or PGA polymers, terminal amine or carboxyl of dendritic polymers, or hydroxyl, carboxyl or phosphate groups of phospholipids) by direct chemical reactions. Alternatively, there may be a binding moiety that is covalently bound to both antigen peptides and proteins and is also covalently bound to the carrier, thereby linking them together.

[0143] Reactive carboxyl groups on the surface of the carrier can be connected to free amines (e.g., from Lys residues) on the antigenic peptide or protein by reacting these carboxyl groups with, for example, 1-ethyl-3-[3,9-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). Similarly, the same chemical method can be used to bind free amines on the surface of the carrier to free carboxyl groups (e.g., from the C-terminus, or from Asp or Glu residues) on the antigenic peptide or protein. Alternatively, free amine groups on the surface of the carrier can be covalently bound to antigenic peptides and proteins, or antigenic peptide or protein fusion proteins using sulfo-SIAB chemical methods, generally as described in Arano et al. (1991) Chem. 2: 71-6 (Arano et al., 1991, "Chemistry", Vol. 2, pp. 71-76).

[0144] In another embodiment, the non-covalent bond between the ligand that binds to the antigenic peptide or protein and the anti-ligand attached to the carrier can bind the antigen to the carrier. For example, a biotin ligase recognition sequence tag can be connected to the C-terminus of the antigenic peptide or protein, and this tag can be biotinylated by a biotin ligase. Biotin can then serve as a ligand to non-covalently bind the antigenic peptide or protein to avidin or streptavidin that is adsorbed as an anti-ligand or otherwise bound to the surface of the carrier. Alternatively, if the antigenic peptide and protein are fused to an immunoglobulin domain with an Fc region as described above, the Fc domain can serve as a ligand, and protein A that is covalently or non-covalently bound to the surface of the carrier can serve as an anti-ligand to non-covalently bind the antigenic peptide or protein to the carrier. Other means that can be used for non-covalently binding antigenic peptides and proteins to the carrier are well known in the art, and these means include metal ion chelation technology (e.g., using a poly-His tag at the C-terminus of the antigenic peptide or protein or the antigenic peptide or protein fusion protein, and Ni + coated carrier), and these methods may be substituted for the methods described here.

[0145] The binding of the nucleic acid moiety to the platform molecule can be achieved in many ways, which generally involve one or more cross-linking agents and functional groups on the nucleic acid moiety and the platform molecule. The linking group is added to the platform using standard synthetic chemistry techniques. The linking group can be added to the nucleic acid moiety using standard synthetic techniques. The practitioner has many choices regarding the antigens used in the combination of the present invention. The inducing antigen present in the combination contributes to the specificity of the induced tolerogenic response. The inducing antigen may be the same as or different from the target antigen, which is an antigen present in or to be placed in the body of the subject being treated, which is the target of an undesirable immune response and against which tolerance is desired.

[0146] The inducing antigen of the present invention can be a polypeptide, polynucleotide, carbohydrate, glycolipid or other molecule isolated from a biological source, or the inducing antigen can be a chemically synthesized small molecule, a polymer or 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.

[0147] In some embodiments, the present invention provides a carrier (e.g., immune modified particles) coupled to one or more peptides, polypeptides, and / or proteins. In some embodiments, carriers such as those described herein (e.g., PLG carriers) effectively induce antigen-specific tolerance and / or prevent the onset of immune-related diseases (e.g., EAE in mouse models) and / or reduce the severity of existing immune-related diseases. In some embodiments, the compositions and methods of the present invention may cause T cells to undergo early events associated with T cell activation, but do not allow T cells to acquire effector functions. For example, administration of the compositions of the present invention may produce T cells with a quasi-activated phenotype, such as upregulation of CD69 and / or CD44, but without displaying effector functions, as indicated by lack of IFN-γ or IL-17 synthesis. In some embodiments, administration of the compositions of the present invention produces T cells with a quasi-activated phenotype without converting natural antigen-specific T cells into a regulatory phenotype, such as having CD25 + / Foxp3 + Those of the phenotype.

[0148] In some embodiments, the surface of the carrier (e.g., particle) comprises chemical moieties and / or functional groups that allow antigenic peptides and / or other functional elements to attach (e.g., covalently, non-covalently) to the carrier. In some embodiments, the number, orientation, spacing, etc. of chemical moieties and / or functional groups on the carrier (e.g., particle) vary according to carrier chemistry, desired application, etc.

[0149] In some embodiments, the carrier comprises one or more biological agents or chemical agents, which are adhered to the carrier, adsorbed on the carrier, encapsulated in the carrier and / or contained in the entire carrier. In some embodiments, the chemical agent or biological agent is encapsulated in the particle and / or contained in the entire particle. The present invention is not limited by the nature of the chemical agent or biological agent. Such agents include but are not limited to proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cell components, etc. In some embodiments, two or more (e.g., 3, 4, 5, etc.) different chemical agents or biological agents are included on or in the carrier. In some embodiments, the agent is configured to have a specific release rate. In some embodiments, a variety of different agents are configured to have different release rates. For example, the first agent can release for a few hours, while the second agent releases for a longer time (e.g., days, weeks, months, etc.). In some embodiments, the carrier or a portion thereof is configured to slowly release the biological agent or chemical agent. In some embodiments, the slow release allows the release of a biologically active amount of the agent for at least 30 days (e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 180 days, etc.). In some embodiments, the carrier or a portion thereof is configured to have a porosity sufficient to allow cells to grow into the pores. The size of the pores can be selected for the specific cell type of interest and / or the amount of ingrowth required. In some embodiments, the particles include the antigen of interest, but do not include other non-peptide active agents, such as drugs or immunomodulators. In addition, in some embodiments, the particles of the present invention do not contain immunostimulatory or immunosuppressive peptides except the antigen of interest. In addition, in some embodiments, the particles do not contain other proteins or peptides (e.g., co-stimulatory molecules, MHC molecules, immunostimulatory peptides or immunosuppressive peptides) on its surface or encapsulated therein.

[0150] It has been surprisingly found that encapsulating antigens, biological agents and / or chemicals in the particles of the present invention induces immune tolerance and has several advantages. First, the encapsulated particles have a slower cytokine response. Second, when using multiple antigens, biological agents and / or chemicals, encapsulation eliminates competition between these different molecules, which may occur if the agent is attached to the surface of the particle. Third, encapsulation allows more antigens, biological agents and / or chemicals to be incorporated into the particles. Fourth, encapsulation allows for easier use of complex protein antigens or organ homogenates (e.g., pancreatic homogenates for type 1 diabetes or peanut extracts in peanut allergies). Finally, encapsulating antigens, biological agents and / or chemicals in particles rather than being bound to the surface of particles maintains the net negative charge on the particle surface. Encapsulating antigens, biological agents and / or chemicals in the particles of the present invention can be performed by any method known in the art. In one embodiment, polypeptide antigens are encapsulated in particles by a double emulsion method. In another embodiment, the polypeptide antigens are water-soluble.

[0151] In another embodiment, polypeptide antigen is encapsulated in particle by single emulsification.In another embodiment, the hydrophobicity of polypeptide antigen is higher.Sometimes, double emulsification causes the formation of large particles, which can cause the hydrophilic active component to leak and the interception efficiency is low. Coagulation and Ostwald ripening are two mechanisms that can make the double emulsion droplet instability, and the diffusion of the organic phase by the hydrophilic active component is the main mechanism that causes the low interception level of active component.In certain embodiments, it may be useful to reduce the nanoparticle size.A strategy to achieve this is to apply the second strong shear rate.The leakage effect can be reduced by using high polymer concentration and high polymer molar mass, which is accompanied by the rise of internal water phase viscosity and the increase of surfactant molar mass.

[0152] In certain embodiments, the present invention provides a carrier having cells or other biological or chemical agents thereon (or inside). In the case of using cells, the carrier is not limited to a specific type of cell. In some embodiments, the carrier has pancreatic islet cells thereon. In some embodiments, the microporous carrier additionally has ECM proteins and / or exendin-4 thereon. The carrier is not limited to a specific type. In some embodiments, the carrier has regions of different porosity (e.g., different pore sizes, pore depths, and / or pore densities). In some embodiments, the carrier has agents, DNA, RNA, extracellular matrix proteins, exendin-4, etc. thereon (or inside). In certain embodiments, the present invention provides a method for transplanting pancreatic islet cells using such carriers. In certain embodiments of the present invention, the inducing antigen is a single isolated or recombinantly produced molecule. To treat diseases in which the target antigen is distributed in multiple locations in the host, it is generally necessary that the inducing antigen is the same as the target antigen or is immunologically related to the target antigen. Examples of such antigens are most polynucleotide antigens and some sugar antigens (such as blood group antigens).

[0153] Any suitable antigen may be used within the scope of the present invention. In some embodiments, the inducing antigen contributes to the specificity of the induced tolerogenic response. The inducing antigen may be the same as or different from the target antigen, which is an antigen present in or to be placed in the body of the subject being treated that is the target of an undesirable immune response and against which tolerance is desired.

[0154] In the case where the target antigen is preferentially expressed on a specific organ, cell or tissue type, the practitioner may again choose to use an inducing antigen that is the same as the target antigen or immunologically related to the target antigen. However, there is also another option, namely, to use an antigen that is a bystander for the target. This is an antigen that may be immunologically unrelated to the target antigen, but it is preferentially expressed in tissues that express the target antigen. The working principle of the effectiveness of bystander suppression is that suppression is an active cell-mediated process that downregulates the effector arm of the immune response at the target cell. Suppressor cells are specifically stimulated by the inducing antigen on the mucosal surface and return to the tissue site where the bystander antigen is preferentially expressed. Through an interaction or cytokine-mediated mechanism, the local suppressor cells then downregulate nearby effector cells (or inducers of effector cells), regardless of what they are reactive to. If the effector cell is specific for a target different from the inducing antigen, the result is a bystander effect. For a further detailed description of the bystander response and a list of tolerogenic peptides with this effect, the reader is referred to International Patent Publication WO 93 / 16724. The implication of the bystander theory is that to practice the present invention, the skilled artisan need not identify or isolate a specific target antigen for which tolerance is desired. The practitioner need only have access to at least one molecule that is preferentially expressed at the target site to use as an inducing antigen.

[0155] In certain embodiments of the present invention, the form of the inducing antigen is different from the form expressed in the individual being treated, but is a fragment or derivative thereof. The inducing antigens of the present invention include peptides that are based on molecules with appropriate specificity but are modified by fragmentation, residue substitution, labeling, conjugation and / or fusion with peptides having other functional properties. Such modifications can be made for any desired purpose, including but not limited to eliminating any unwanted properties, such as toxicity or immunogenicity; or enhancing any desired properties, such as mucosal binding, mucosal penetration or stimulation of the tolerogenic arm of the immune response. Terms such as insulin peptides, collagen peptides and myelin basic protein peptides as used herein refer not only to complete subunits, but also to heterotypic and synthetic variants, fragments, fusion peptides, conjugates and other derivatives, which contain at least 10 and preferably 20 consecutive amino acids homologous to the corresponding molecule as an analog of the derivative (preferably 70% identity at the amino acid level, more preferably 80% identity and even more preferably 90% identity), wherein the homologous region of the derivative shares the ability to induce tolerance to the target antigen with the respective parent molecule.

[0156] It is recognized that the tolerogenic region of an inducing antigen is often different from the immunodominant epitope used to stimulate an antibody response. Tolerogenic regions are generally regions that can participate in specific cell interactions involving T cells. Tolerogenic regions can participate in, and are able to induce tolerance when the intact antigen is presented. Some antigens contain hidden tolerogenic regions because the processing and presentation of native antigens generally do not trigger tolerance. A detailed description of hidden antigens and their identification is found in International Patent Publication WO94 / 27634.

[0157] In certain embodiments of the present invention, two, three or more induction antigens are used. These embodiments may need to be implemented when there are multiple target antigens, or they may need to be implemented to provide multiple bystanders for the target. For example, in the treatment of diabetes, both insulin and glucagon can be mixed with mucosal binding components. It may also be necessary to provide a mixture of antigens to cover several possible alternative targets. For example, a mixture of histocompatibility antigen fragments can be used to tolerize a subject who is expected to transplant an allogeneic transplant of an unknown phenotype in the future. The allogeneic variant region of human leukocyte antigens is known in the art: for example, Immunogenetics 29:231, 1989 ("Immunogenetics", Vol. 29, p. 231, 1989). In another example, a mixture of allergens can serve as an induction antigen for the treatment of atopy.

[0158] Depend on molecular properties, inducing antigens can be prepared by many techniques known in the art. Polynucleotides, polypeptides and carbohydrate antigens can be separated from cells of species to be treated that are rich in them. Short peptides are easily prepared by amino acid synthesis. Longer proteins with known sequences can be prepared by synthesizing coding sequences or by amplifying coding sequences from natural sources or vector PCR, then expressing the coding sequences in suitable bacteria or eukaryotic host cells.

[0159] In certain embodiments of the present invention, the combination comprises a complex mixture of antigens obtained from cells or tissues, one or more of which act as inducing antigens. Antigens can be in the form of whole cells, which are complete or treated with fixatives such as formaldehyde, glutaraldehyde or alcohol. Antigens can be in the form of cell lysates, which are produced by making cells or tissues undergo detergent solubilization or mechanical disruption followed by purification. Antigens can also be obtained by subcellular fractionation, particularly by plasma membrane enrichment through techniques such as differential centrifugation, optionally followed by detergent solubilization and dialysis. Other separation techniques are also suitable, such as affinity or ion exchange chromatography of dissolved membrane proteins.

[0160] In one embodiment, the antigenic peptide or protein is an autoantigen, an alloantigen or a transplant antigen. In another specific embodiment, the autoantigen is selected from myelin basic protein, collagen or fragments thereof, DNA, nuclear and nucleolar proteins, mitochondrial proteins and pancreatic β-cell proteins.

[0161] The present invention induces tolerance to self-antigens for the treatment of autoimmune diseases by administering antigens to which tolerance is desired. For example, autoantibodies against myelin basic protein (MBP) are observed in patients with multiple sclerosis, and therefore, MBP antigen peptides or proteins can be used in the present invention for delivery using the composition of the present invention, thereby treating and preventing multiple sclerosis.

[0162] As another non-limiting example, an individual who is a candidate for a transplant from a fraternal twin may experience rejection of the implanted cells, tissues or organs because the implanted antigens are foreign to the recipient. The recipient individual's prior tolerance to the target transplant eliminates or reduces subsequent rejection. Long-term anti-rejection therapy can be reduced or eliminated by practicing the present invention. In another example, many autoimmune diseases are characterized by cellular immune responses to endogenous or self-antigens. Tolerance of the immune system to endogenous antigens is required for controlling the disease.

[0163] In another example, sensitization of an individual to industrial pollutants or chemicals such as may be encountered at work poses a risk of immune response. Prior tolerance of the individual's immune system to the chemical (especially a form of the chemical that reacts with the individual's endogenous proteins) may be necessary to prevent the later occupational occurrence of an immune response.

[0164] Allergens are other antigens to which an immune response tolerance is also required. In one embodiment, the antigen is gluten. In another embodiment, the antigen is A-gluten.

[0165] It is noteworthy that even in diseases where the pathogenic autoantigen is unknown, antigens present near the anatomical location can be used to induce bystander inhibition. For example, autoantibodies to collagen are observed in rheumatoid arthritis, and therefore, genes encoding collagen can be used as gene modules expressing antigens to treat rheumatoid arthritis (see, for example, Choy (2000) Curr Opin Investig Drugs 1: 58-62 (Choy, 2000, "Modern Research Drug Perspectives", Vol. 1, pp. 58-62)). In addition, tolerance to β cell autoantigens can be used to prevent the occurrence of type 1 diabetes (see, for example, Bach and Chatenoud (2001) Ann Rev Immunol 19: 131-161 (Bach and Chatenoud, 2001, "Annual Review of Immunology", Vol. 19, pp. 131-161)).

[0166] As another example, autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have been observed in autoimmune encephalomyelitis and many other CNS diseases as well as multiple sclerosis (see, e.g., Iglesias et al. (2001) Glia 36:22-34). Thus, use of MOG antigen expression constructs in the present invention can treat multiple sclerosis and related autoimmune diseases of the central nervous system.

[0167] Other examples of candidate autoantigens for the treatment of autoimmune diseases include: pancreatic β-cell antigens, insulin, and GAD for the treatment of insulin-dependent diabetes mellitus; type II collagen, human cartilage gp39 (HCgp39), and gp130-RAPS for the treatment of rheumatoid arthritis; myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG, see above) for the treatment of multiple sclerosis; fibrillarin and small nucleolar protein (snoRNP) for the treatment of scleroderma; thyroid stimulating factor receptor (TSH-R) for the treatment of Graves' disease; nuclear antigens, histones, glycoprotein gp70, and ribosomal proteins for the treatment of systemic lupus erythematosus; pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2) for the treatment of primary biliary cirrhosis; hair follicle antigens for the treatment of alopecia areata; and human tropomyosin isoform 5 (hTM5) for the treatment of ulcerative colitis.

[0168] In one embodiment, the particles of the invention are coupled to an antigen comprising one or more epitopes associated with an allergic reaction, an autoimmune disease, and / or an inflammatory disease or disorder. The antigen may comprise one or more copies of an epitope. In one embodiment, the antigen comprises a single epitope associated with one disease or disorder. In another embodiment, the antigen comprises more than one epitope associated with the same disease or disorder. In yet another embodiment, the antigen comprises more than one epitope associated with different diseases or disorders. In another embodiment, the antigen comprises one or more epitopes associated with one or more allergic reactions. In another embodiment, the antigen comprises one or more epitopes associated with multiple sclerosis, type 1 diabetes, celiac disease, and / or inflammatory bowel disease (including Crohn's disease or ulcerative colitis). In one embodiment, the epitope is from myelin basic protein (e.g., SEQ ID NO: 4975 and 4976), proteolipid protein (e.g., SEQ ID NO: 4977), myelin oligodendrocyte glycoprotein (e.g., SEQ ID NO: 1 and 4978), aquaporin (e.g., SEQ ID NO: 4979), myelin-associated glycoprotein (e.g., SEQ ID NO: 4980), insulin (e.g., SEQ ID NO: 4981), glutamate decarboxylase (e.g., SEQ ID NO: 4982), gliadin (e.g., SEQ ID NO: 4983-4985 or 5136-5140), type IV collagen α3 chain (e.g., SEQ ID NO: 5017) or fragments, homologs or isoforms thereof. In another embodiment, the epitope is from gluten, including from gliadin and / or glutenin. In one embodiment, the epitope is from an insulin homolog, such as those described in U.S. Patent No. 8,476,228, which is hereby incorporated in its entirety for all purposes. In one embodiment, the gliadin epitope is SEQ ID NO: 13, 14, 16, 320 or 321 in U.S. Patent Application No. 20110293644, which is hereby incorporated in its entirety for all purposes.

[0169] Additional non-limiting examples of epitopes associated with various autoimmune and / or inflammatory diseases or disorders contemplated by the present invention are described in Tables 2 and 3.

[0170] Table 2 - Representative linear epitopes

[0171]

[0172] Not all epitopes are linear epitopes; epitopes may also be discontinuous conformational epitopes. A variety of discontinuous epitopes associated with autoimmune or inflammatory diseases and / or disorders are known. Non-limiting examples of discontinuous epitopes are described in Table 3.

[0173] Table 3 - Representative discontinuous epitopes

[0174]

[0175]

[0176] The ability of combinations of antigens and / or epitopes to promote tolerance can be tested using isolated cells or by conducting experiments in animal models.

[0177] In some embodiments, the tolerance inducing composition of the present invention comprises apoptosis signal transduction molecules (e.g., in addition to antigenic peptides or other antigenic molecules). In some embodiments, apoptosis signal transduction molecules are coupled and / or associated with the surface of the carrier. In some embodiments, apoptosis signal transduction molecules allow the carrier to be perceived as apoptotic bodies by antigen presenting cells of the host (e.g., cells of the host reticuloendothelial system); this allows the relevant peptide epitopes to be presented in a tolerance inducing manner. Without being bound by theory, it is speculated that this prevents the upregulation of molecules (e.g., MHC class I / II) and co-stimulatory molecules involved in immune cell stimulation. These apoptosis signal transduction molecules can also serve as phagocyte markers. For example, apoptosis signal transduction molecules suitable for use in the present invention have been described in U.S. Patent Application No. 20050113297, which is hereby incorporated by reference in its entirety. Molecules suitable for use in the present invention include molecules targeting phagocytes, and the phagocytes include macrophages, dendritic cells, monocytes, and neutrophils.

[0178] In some embodiments, molecules suitable for use as apoptosis signal transduction molecules play a role in enhancing the tolerance of related peptides. In addition, carriers bound to apoptosis signal transduction molecules can be bound by C1q in apoptotic cell recognition (Paidassi et al., (2008) J. Immunol. 180: 2329-2338 (Paidassi et al., 2008, Journal of Immunology, Vol. 180, pp. 2329-2338); the entire text is incorporated herein by reference). For example, molecules that can be used as apoptosis signal transduction molecules include phosphatidylserine, annexin-1, annexin-5, milk fat globule-EGF-factor 8 (MFG-E8) or thrombospondin family (e.g., thrombospondin-1 (TSP-1)). Various molecules suitable for use as apoptosis signal transduction molecules in the context of the present invention are discussed in, for example, U.S. Patent Application 2012 / 0076831; the entire text is incorporated herein by reference).

[0179] In some embodiments, apoptosis signal transduction molecules can be bound to antigen-specific peptides. In some cases, apoptosis signal transduction molecules and antigen-specific peptides are combined by forming a fusion protein. For example, a fusion protein may include at least one antigen-specific peptide (or a fragment or variant thereof), which is coupled to at least one molecule (or a fragment or variant thereof) of apoptosis signal transduction molecules. Regarding the formation of fusion proteins, the terms "fusion protein", "fusion peptide", "fusion polypeptide" and "chimeric peptide" are used interchangeably. Suitable fragments of antigen-specific peptides include any fragments that maintain the following functions in the full-length peptide: antigen-specific tolerance function required for the present invention can be produced. Fusion proteins can be formed by various means known in the art (such as gene fusion, chemical coupling, etc.). Two proteins can be fused directly or via an amino acid linker. The polypeptides forming the fusion protein usually connect the C-terminus to the N-terminus, but they can also connect 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 can be used to space the first and second polypeptide components at a distance sufficient to ensure that each polypeptide is folded into its secondary and tertiary structure. Amino acid sequences that can be effectively used as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180; the entire texts of which are incorporated herein by reference. The length of the linker sequence can generally be 1 to about 50 amino acids. In some embodiments, for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent spatial interference, a linker sequence is not required and / or is not utilized.

[0180] One representative of tolerogenic activity is the ability of the complete antigen or fragment to stimulate the generation of appropriate cytokines at the target site. The immunomodulatory cytokine released by T suppressor cells at the target site is considered to be TGF-β (Miller et al., Proc. Natl. Acad. Sci. USA 89: 421, 1992 (Miller et al., "Proceedings of the National Academy of Sciences of the United States", Vol. 89, p. 421, 1992)). Other factors that can be produced during tolerance are cytokines IL4 and IL-10, and mediator PGE. In contrast, lymphocytes in tissues that undergo active immune destruction secrete cytokines such as IL-I, IL-2, IL-6, and γ-IFN. Therefore, the efficacy of candidate inducing antigens can be evaluated by measuring their ability to stimulate cytokines of the appropriate type.

[0181] With this in mind, rapid screening tests for tolerogenic epitopes of inducing antigens, effective mucosal binding components, effective combinations, or effective mucosal administration modes and schedules can be performed using isogenic animals as donors for in vitro cell assays. The mucosal surfaces of the animals are treated with the test composition and sometimes challenged with the target antigen in complete Freund's adjuvant, parenterally. Splenocytes are isolated and cultured in vitro in the presence of the target antigen at a concentration of about 50 μg / mL. Candidate proteins or subfragments can be used in place of the target antigen to map the location of tolerogenic epitopes. Cytokine secretion into the culture medium can be quantified by standard immunoassays.

[0182] The ability of cells to suppress the activity of other cells can be determined using cells isolated from animals immunized with the target antigen or by forming a cell line responsive to the target antigen (Ben-Nun et al., Eur. J. Immunol. 11: 195, 1981, incorporated herein by reference in its entirety). In one variation of this assay, the suppressor cell population is mildly irradiated (about 1000 to 1250 rads) to prevent proliferation, the suppressor cells are cultured with responder cells, and the proliferative activity of the responder cells is quantified using the tritiated thymidine incorporation method (or MTT). In another variation, the suppressor cell population and the responder cell population are cultured in the upper and lower layers of a two-chamber transwell culture system (Costar, Cambridge Mass.), which allows the cell populations to be co-cultured within 1 mm of each other separated by a polycarbonate membrane (WO 200). 93 / 16724). In this method, irradiation of the suppressor cell population is unnecessary because the proliferative activity of the responding cells can be measured alone.

[0183] In embodiments of the invention where the target antigen is already present in the individual, there is no need to isolate the antigen or pre-combine it with a mucosal binding component. For example, the antigen may be expressed in the individual in some manner due to a pathological state such as inflammatory bowel disease or celiac disease or through digestion of a food allergen. Testing is performed by administering the mucosal binding component in one or more doses or formulations and determining its ability to promote tolerance to the antigen in situ.

[0184] The effectiveness of the compositions and modes of administration for treating a particular disease can also be demonstrated in the corresponding animal disease models. The ability of the treatment to attenuate or delay disease symptoms is monitored at the level of circulating biochemical and immunological markers of the disease, immunohistology of the affected tissues, and overall clinical features appropriate to the model employed. Non-limiting examples of animal models that can be used for testing are included in the following sections.

[0185] The present invention contemplates regulating tolerance by regulating TH1 response, TH2 response, TH17 response, or a combination of these responses. Regulating TH1 response encompasses changing the expression of, for example, interferon-γ. Regulating TH2 response encompasses changing the expression of any combination of, for example, IL-4, IL-5, IL-10, and IL-13. Usually, an increase (decrease) in TH2 response will include an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10, or IL-13; more usually, an increase (decrease) in TH2 response will include an increase in the expression of at least two of IL-4, IL-5, IL-10, or IL-13, and most usually, an increase (decrease) in TH2 response will include an increase in at least three of IL-4, IL-5, IL-10, or IL-13, and ideally, an increase (decrease) in TH2 response will include an increase (decrease) in the expression of all of IL-4, IL-5, IL-10, and IL-13. Modulating TH 17 encompasses altering the expression of, for example, TGF-β, IL-6, IL-21 and IL23, and the effector levels of IL-17, IL-21 and IL-22.

[0186] Other methods suitable for assessing the effectiveness of the compositions and methods of the invention are known in the art, as described, for example, in US Patent Application 2012 / 0076831 (incorporated herein by reference in its entirety).

[0187] Certain embodiments of the present invention relate to inducing immune tolerance in individuals who have not been previously tolerized by therapeutic intervention. These embodiments generally relate to a combination of multiple administrations of antigens and mucosal binding components. Typically, at least three administrations, often at least four administrations and sometimes at least six administrations are performed in the primary immunization process to achieve long-lasting results, but the subject may show tolerance in the early stages of the treatment process. Most often, each dose is given in a one-time large dose administration (bolus administration), but a continuous formulation that can be released through the mucosa is also suitable. In the case of multiple administrations, the time between each administration is generally between 1 day and 3 weeks, and generally between about 3 days and 2 weeks. Typically, the same antigen and mucosal binding component are present at the same concentration and are administered to the same mucosal surface, but changes in any of these variables during the treatment process are allowed.

[0188] Other embodiments of the present invention relate to strengthening or extending the persistence of previously established immune tolerance. These embodiments generally relate to a single administration or short-term treatment course when the established tolerance declines or is at risk of decline. Strengthening is generally performed 1 month to 1 year after the initial immunization or prior boost, and generally 2 to 6 months. The present invention also includes embodiments that relate to regular maintenance of tolerance according to an administration schedule that occurs once every half week, every week, every two weeks, or according to any other regular schedule.

[0189] The particles of the present invention may be administered at any dose effective to suppress an inflammatory immune response in a subject in need thereof, or to treat a bacterial or viral infection in a subject in need thereof. 2 to about 10 20 In another embodiment, about 10 3 to about 10 15 In yet another embodiment, about 10 6 to about 10 12 In yet another embodiment, about 10 8 to about 10 10 In a preferred embodiment, the preferred dose is 0.1% solid content / ml. Thus, for 0.5 μm microbeads, the preferred dose is approximately 4×10 9 For 0.05 μm beads, the preferred dose is about 4 × 10 12 For 3 μm beads, the preferred dose is 2×10 7 However, the present invention contemplates any dosage amount that is effective in treating the particular condition to be treated.

[0190] The present invention can be used to treat immune-related disorders such as autoimmune diseases, transplant rejection, enzyme deficiencies and allergic reactions. The use of synthetic biocompatible particle systems as an alternative to induce immune tolerance can lead to easier manufacturing, expanded availability of therapeutic agents, increased uniformity between samples, an increased number of potential treatment sites, and a significantly reduced likelihood of allergic reactions to carrier cells.

[0191] 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 cellular cytotoxicity. In addition, the term immune response includes immune responses 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 an immune response include lymphocytes, such as B cells and T cells (CD4 + 、CD8 + , Th1 and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; bone marrow cells, such as macrophages, eosinophils, mast cells, basophils and granulocytes. In some embodiments, the modified particles of the present invention effectively reduce inflammatory cells transported to the site of inflammation.

[0192] As used herein, the term "anergy", "tolerance", or "antigen-specific tolerance" refers to the insensitivity of T cells to T cell receptor-mediated stimulation. This insensitivity is usually antigen-specific and continues after exposure to antigenic peptides has ceased. For example, T cell anergy is characterized by lack of cytokine production, such as IL-2. T cell anergy can occur when T cells are exposed to antigens and receive a first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, cells are re-exposed to the same antigen (even if the re-exposure occurs in the presence of co-stimulatory molecules) resulting in the inability to produce cytokines and subsequently unable to proliferate. Therefore, the inability to produce cytokines can prevent proliferation. However, if cultured with cytokines (such as IL-2), anergic T cells can proliferate. For example, it can also be observed that T cells are anergic due to lack of IL-2 production of T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, a reporter gene construct can be used. For example, anergic T cells are unable to initiate transcription of the DL-2 gene induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of the API sequence found in the enhancer (Kang et al. 1992 Science. 257: 1134).

[0193] As used herein, the term "immune tolerance" refers to a method performed on a subset of treated subjects, compared to untreated subjects, in which: a) the level of a specific immune response (thought to be mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or their equivalents) is reduced; b) the onset and development of a specific immune response is delayed; or c) the risk of onset or development of a specific immune response is reduced. "Specific" immune tolerance occurs when immune tolerance is preferentially elicited against certain antigens compared to other antigens. "Non-specific" immune tolerance occurs when immune tolerance is indiscriminately elicited against antigens that cause inflammatory immune responses. "Quasi-specific" immune tolerance occurs when immune tolerance is semi-discriminately elicited against antigens that cause pathogenic immune responses without semi-discriminatory elicited against other antigens that cause protective immune responses.

[0194] Tolerance to self-antigens and autoimmune diseases is achieved through a variety of mechanisms, including mechanisms of negative selection of autoreactive T cells in the thymus, and mechanisms of peripheral tolerance of autoreactive T cells that escape thymic clearance and are found in the periphery. Examples of mechanisms that provide peripheral T cell tolerance include "ignorance" of self-antigens, no reaction or non-response to self-antigens, cytokine immune deviation, and activation-induced cell death of autoreactive T cells. In addition, regulatory T cells have been shown to be involved in mediating peripheral tolerance. See, for example, Walker et al. (2002) Nat. Rev. Immunol. 2: 11-19 (Walker et al., 2002, "Nature Review: Immunology", Vol. 2, pp. 11-19); Shevach et al. (2001) Immunol. Rev. 182: 58-67 (Shevach et al., 2001, "Review of Immunology", Vol. 182, pp. 58-67). In some cases, peripheral tolerance to self-antigens is lost (or damaged) and thus an autoimmune reaction is generated. For example, in an animal model of EAE, it has been demonstrated that activation of antigen presenting cells (APCs) by TLR innate immune receptors destroys self-tolerance and leads to the induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113: 990-997 (Waldner et al., 2004, Journal of Clinical Research, Vol. 113, pp. 990-997)).

[0195] Therefore, in some embodiments, the present invention provides a method for increasing antigen presentation while inhibiting or reducing TLR7 / 8, TLR9 and / or TLR 7 / 8 / 9-dependent cell stimulation. As described herein, administration of specific modified particles results in antigen presentation of DC or APC while inhibiting TLR 7 / 8, TLR9 and / or TLR7 / 8 / 9-dependent cell responses associated with immunostimulatory polynucleotides. This inhibition may include a reduction in the level of one or more TLR-related cytokines.

[0196] As discussed above, the present invention provides novel compounds having biological properties useful for treating Mac-1 and LFA-1 mediated disorders.

[0197] Therefore, in another aspect of the present invention, a pharmaceutical composition is provided, which comprises immune modified particles and optionally comprises a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally also comprise one or more additional therapeutic agents. Alternatively, the modified particles of the present invention may be administered to patients in need in combination with one or more other therapeutic agents. For example, the additional therapeutic agent used for co-administration or contained in the pharmaceutical composition with the compound of the present invention may be an approved anti-inflammatory agent, or it may be any of a variety of agents being approved by the Food and Drug Administration, which are ultimately approved for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or bacterial or viral infection. It should also be understood that some modified particles of the present invention may exist in free form for treatment, or in the form of a pharmaceutically acceptable derivative thereof when appropriate.

[0198] The pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable carrier, which as used herein includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effect, or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated to be within the scope of the present invention. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower 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 saline; Ringer's solution; ethanol, and phosphate buffered solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0199] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may also include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0200] The particles of the invention can be administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, intraperitoneally or subcutaneously. In one embodiment, the particles of the invention are administered intravenously.

[0201] The effective amount and method of administration of the present invention for regulating immune response can vary based on the individual, the condition to be treated, and other factors obvious to those skilled in the art. Factors to be considered include the route of administration and the number of doses administered. Such factors are known in the art, and those skilled in the art can make such decisions well without undue experimentation. A suitable dosage range is a dosage range that provides the desired immunomodulation. The applicable dosage range of the vector given in the amount of the delivered vector can be, for example, approximately any of the following: 0.5 to 10 mg / kg, 1 to 9 mg / kg, 2 to 8 mg / kg, 3 to 7 mg / kg, 4 to 6 mg / kg, 5 mg / kg, 1 to 10 mg / kg, 5 to 10 mg / kg. Alternatively, the dosage can be administered based on the number of particles. For example, the applicable dosage of the vector given in the amount of the delivered vector can be, for example, about 10 per dose. 6 , 10 7 , 10 8 , 10 9 , 10 10 or a greater number of particles. The absolute amount administered to each patient depends on pharmacological properties such as bioavailability, clearance and route of administration. Details of pharmaceutically acceptable carriers, diluents and excipients and methods of preparing pharmaceutical compositions and formulations are described in Remmingtons Pharmaceutical Sciences 18 th The invention is provided in Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co., Easton, Pa., USA. (which is hereby incorporated by reference in its entirety).

[0202] The effective amount of a particular vector formulation and the method of administration may vary based on the individual patient, the desired outcome and / or the type of disease, the stage of the disease, and other factors apparent to one skilled in the art. The routes of administration useful for a particular application will be apparent to one skilled in the art. The routes of administration include, but are not limited to, topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal, and nasopharyngeal and pulmonary administration, including transbronchial and transalveolar administration. A suitable dosage range is one that provides sufficient IRP-containing composition to obtain a tissue concentration of about 1-50 μM, as measured by blood levels. The absolute amount administered to each patient depends on pharmacological properties, such as bioavailability, clearance, and route of administration.

[0203] The present invention provides carrier formulations suitable for topical application, including but not limited to physiologically acceptable implants, ointments, creams, lotions and gels.Exemplary skin administration routes are the least invasive skin administration routes, such as transdermal delivery, epidermal administration and subcutaneous injection.

[0204] Transdermal administration is accomplished by applying creams, lotions, gels, etc. that enable the carrier to penetrate the skin and enter the bloodstream. Compositions suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments that are applied directly to the skin or incorporated into a protective carrier such as a transdermal device (so-called "patch"). Examples of suitable creams, ointments, etc. can be found in, for example, the Physician's Desk Reference. Transdermal delivery can also be accomplished by iontophoresis, for example, using commercially available patches that continuously deliver their products across unbroken skin for several days or longer. Use of this method allows controlled delivery of pharmaceutical compositions at relatively high concentrations, allows combined drug infusions, and allows the simultaneous use of absorption enhancers.

[0205] Parenteral administration routes include, but are not limited to, electricity (iontophoresis) or direct injection, such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. Carrier formulations suitable for parenteral administration are typically prepared in USP water or water for injection, and may also include pH buffers, salt expanders, preservatives and other pharmaceutically acceptable excipients. Immunomodulatory polynucleotides for parenteral injection may be formulated into pharmaceutically acceptable sterile isotonic solutions, such as saline for injection and phosphate buffered saline.

[0206] Gastrointestinal routes of administration include, but are not limited to, swallowing and rectal routes, and may include the use of, for example, pharmaceutically acceptable powders, pills or liquids for swallowing and suppositories for rectal administration.

[0207] Nasopharyngeal and pulmonary administration include administration by inhalation and include delivery routes such as intranasal, transbronchial and transalveolar routes. The present invention includes carrier formulations suitable for administration by inhalation, including but not limited to liquid suspensions for forming aerosols and powder forms for dry powder inhalation delivery systems. Devices suitable for administration by inhalation of carrier formulations include but are not limited to nebulizers, vaporizers, sprayers and dry powder inhalation delivery devices.

[0208] Injectable preparations, such as sterile injection aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any non-irritating fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectables.

[0209] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0210] In order to prolong the effect of the drug, it is usually necessary to slow down the absorption of the drug injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension or crystalline or amorphous material with poor water solubility. The absorption rate of the drug therefore depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form. Alternatively, the delayed absorption of the drug form administered parenterally is achieved by dissolving or suspending the drug in an oily solvent. The injectable long-acting form is prepared by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. According to the ratio of the drug to the polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Long-acting injectable preparations can also be prepared by trapping the drug in a liposome or microemulsion compatible with body tissues.

[0211] In some embodiments, the synthetic biodegradable particles of the present invention provide ease of manufacture, broad availability of therapeutic agents, and increased treatment sites. In a specific embodiment, surface functionalized biodegradable poly(lactide-co-glycolide) particles with a high density of surface carboxylate groups synthesized using the surfactant poly(ethylene-alt-maleic anhydride) provide a carrier that offers a number of advantages over other carrier particles and / or surfaces. Experiments performed during the development of embodiments of the present invention demonstrated that peptides (e.g., PLP 139-151 Such peptide-coupled particles have been shown to be effective in preventing disease progression and inducing immune tolerance (e.g., in SJL / J PLP 139-151 / CFA-induced R-EAE multiple sclerosis mouse model). The peptide-coupled carriers of the present invention provide many advantages over other tolerance-inducing structures. In some embodiments, the particles are biodegradable and therefore will not exist for a long time in the body. The time for complete degradation can be controlled. In some embodiments, the particles are functionalized to promote internalization without cell activation (e.g., phosphatidylserine loaded into PLG microspheres). In some embodiments, the particles contain targeting ligands for specific cell populations. In some embodiments, anti-inflammatory cytokines such as IL-10 and TGF-β are included on or within the particles to limit the activation of cell types that are internalizing the particles and promote the induction of tolerance via energy and / or loss and activation of regulatory T cells. It has been found that the composition of the particles affects the length of time that the particles remain in the body, and tolerance requires rapid particle uptake and clearance / degradation. Since a ratio of more than 50:50 lactide: glycolide slows the degradation rate, the particles of the present invention have a lactide: glycolide ratio of about 50:50 or less. In one embodiment, the particles of the present invention have a D,L-lactide:glycolide ratio of about 50:50.

[0212] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and gum arabic, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retardants, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glyceryl monostearate, h) adsorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer.

[0213] Solid compositions of similar types can also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose (lactose) or milk sugar (milk sugar) and high molecular weight polyethylene glycol, etc. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared to have coatings and shells, such as other coatings well-known in the field of enteric coatings and pharmaceutical formulations. They can optionally include opacifiers and can also have a composition that allows them to release active ingredients only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. The example of an embedding composition that can be used includes polymeric substances and waxes. Solid compositions of similar types can also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose (lactose) or milk sugar (milk sugar) and high molecular weight polyethylene glycol, etc.

[0214] Modified particles can also be in the form of microencapsulation with one or more excipients described above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, release-controlled coatings and other coatings well-known in the field of pharmaceutical formulation. In such solid dosage forms, the active compound can be mixed with at least one inert diluent (such as sucrose, lactose and starch). Such dosage forms can also include other substances except inert diluents in normal practice, such as tableting lubricants and other tableting aids (such as magnesium stearate and microcrystalline cellulose). In the case of capsules, tablets and pills, the dosage form can also include a buffer. They can optionally include an emulsifier and can also have a composition that allows them to release the modified particles only in or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. The example of an embedding composition that can be used includes polymeric substances and waxes.

[0215] The present invention encompasses pharmaceutically acceptable topical formulations of modified particles of the present invention. As used herein, the term "pharmaceutically acceptable topical formulation" means any formulation that is pharmaceutically acceptable for intradermal administration of modified microparticles of the present invention by applying the formulation to the epidermis. In certain embodiments of the present invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (such as alcohols, polyols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (such as hypotonic or buffered saline) or any other carrier known in the art for topical administration of drugs. A more complete list of known carriers in the industry is provided by standard reference works in the art, for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both versions are published by Mack Publishing Company, Easton, Pa., Pennsylvania, the disclosure of which is incorporated herein by reference in its entirety. In certain other embodiments, the topical formulations of the present invention may include excipients. Any pharmaceutically acceptable excipient known in the art can be used to prepare the pharmaceutically acceptable topical formulations of the present invention. Examples of excipients that may be included in the topical formulations of the present invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, other penetrants, skin protectants, surfactants and propellants and / or additional therapeutic agents used in combination with modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable moisturizers include, but are not limited to, glycerol, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers for the present invention include, but are not limited to, citric acid buffer, hydrochloric acid buffer, and lactic acid buffer. Suitable solubilizers include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbate. Suitable skin protectants that may be used in the topical formulations of the present invention include, but are not limited to, vitamin E oil, allantoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0216] In certain embodiments, the pharmaceutically acceptable topical formulations of the present invention comprise at least the modified particles of the present invention and a penetration enhancer. The choice of topical formulation will depend on several factors, including the condition to be treated, the physicochemical characteristics of the compounds of the present invention and other excipients used, their stability in the formulation, available manufacturing equipment, and cost constraints. As used herein, the term "penetration enhancer" means an agent that is capable of transporting a pharmacologically active compound through the stratum corneum and into the epidermis or dermis (preferably with little or no systemic absorption). The effectiveness of a variety of compounds in enhancing the permeability of drugs through the skin has been evaluated. See, for example, Percutaneous Penetration Enhancers, Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the use and testing of various skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum SI (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplary embodiments, penetrants for use in the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe gel), ethanol, isopropyl alcohol, octylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decyl methyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glyceryl monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0217] In certain embodiments, the composition can be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant or patch. In certain exemplary embodiments, the preparation of the composition according to the present invention is an ointment, which may further include saturated or unsaturated fatty acids, such as stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl alcohol or oleyl alcohol, and stearic acid is particularly preferred. The ointment of the present invention may also include a nonionic surfactant, such as polyoxy-40-stearate. In certain embodiments, when it may be necessary, the active component is mixed with a pharmaceutically acceptable carrier and any required preservative or buffer under aseptic conditions. It can also be imagined that ophthalmic preparations, ear drops and eye drops are also included in the scope of the present invention. In addition, the present invention can envision the use of a transdermal patch, which has the additional advantage of providing controlled compound delivery to the body. Such dosage forms are prepared by dissolving or dispersing the compound in an appropriate medium. As discussed above, penetration enhancers can also be used to increase the flow of the compound through the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0218] The modified particles can be administered by aerosol. This is achieved by preparing an aqueous aerosol, liposomal formulation or solid particles containing the modified particles. Non-aqueous (e.g., fluorocarbon propellant) suspensions can be used.

[0219] Typically, aqueous aerosols are prepared by combining an aqueous solution or suspension of the agent with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the specific compound, but typically include nonionic surfactants (Tween, or polyethylene glycol), non-toxic proteins (such as serum albumin), sorbitan esters, oleic acid, lecithin, amino acids (such as glycine), buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0220] It will also be appreciated that the modified particles and pharmaceutical compositions of the invention can be formulated and used in combination therapies, i.e., the compounds or pharmaceutical compositions can be formulated with or administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. The specific combination of therapies (therapeutics or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies used may achieve the desired effect for the same disorder (e.g., a compound of the invention may be administered simultaneously with another anti-inflammatory agent), or they may achieve different effects (e.g., control of any adverse effects).

[0221] In certain embodiments, the pharmaceutical composition comprising the modified particles of the present invention further comprises one or more additional therapeutically active ingredients (e.g., anti-inflammatory agents and / or palliative agents). For the purposes of the present invention, the term "palliative agent" refers to a treatment that focuses on alleviating disease symptoms and / or side effects of a treatment regimen but is not a cure. For example, palliative treatments encompass analgesics, anti-sickness drugs, and antiemetics.

[0222] The present invention provides a method for regulating an immune response in an individual (preferably a mammal, more preferably a human), the method comprising administering to the individual a modified particle as described herein. The immunomodulatory methods provided by the present invention include methods for suppressing and / or inhibiting an innate immune response or an adaptive immune response, including but not limited to an immune response stimulated by an immunostimulatory polypeptide or a viral or bacterial component.

[0223] The modified particles are administered in an amount sufficient to modulate the immune response. Modulation of the immune response can be humoral and / or cellular, as described herein, and is measured using standard techniques in the art and as described herein.

[0224] In some embodiments, the compositions described herein are administered with (e.g., simultaneously with, before, or after) implants (e.g., devices) and / or transplants (e.g., tissues, cells, organs) to mediate, abrogate, modulate, and / or reduce immune responses associated therewith.

[0225] In certain embodiments, the individual suffers from a disorder associated with undesirable immune activation, such as an allergic disease or condition, anaphylaxis, and asthma. An individual suffering from an allergic disease or asthma is an individual with recognizable symptoms of an existing allergic disease or asthma. In such individuals, tolerance can be induced, for example, by particles complexed with specific foods that cause allergic reactions (e.g., peanut protein, etc.), injected substances (e.g., bee venom, etc.), or inhaled substances (e.g., ragweed pollen protein, pet dander protein, etc.).

[0226] In certain embodiments, the individual suffers from a disorder associated with undesirable immune activation, such as autoimmune diseases and inflammatory diseases. An individual suffering from an autoimmune disease or inflammatory disease is an individual with recognizable symptoms of an existing autoimmune disease or inflammatory disease. In such an individual, tolerance can be induced, for example, by particles complexed with relevant autoantigens that drive the particular autoimmune disease.

[0227] In certain embodiments, the individual suffers from a disorder associated with enzyme replacement therapy. In such an individual, tolerance can be induced, for example, by particles complexed with an enzyme that a genetically defective patient cannot produce, thereby preventing the patient from developing a neutralizing antibody response to a recombinantly produced enzyme administered to treat their specific defect, for example, tolerance to human factor VIII in a patient with hemophilia due to a genetic defect in the ability to produce factor VIII. Another example can include enzyme replacement for conditions such as mucopolysaccharidoses, gangliosidosis, hypoalkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, or lysosomal storage diseases, including Fabry disease, Gaucher disease, Hurler disease, Hunter disease, Mar-La disease, Niemann-Pick disease, Tay-Sachs disease, and Pompe disease.

[0228] In certain embodiments, the individual produces a strong or adverse immune response to the medicament used for treatment, which may actually or may endanger the patient's health or treatment. In addition, the novel compound provided by the invention can be provided to individuals who do not show an immune response to the medicament but may show an immune response in the future. In certain embodiments, the individual is receiving enzyme replacement therapy. In certain embodiments, therapeutic agents include but are not limited to peptides or protein-based medicaments, DNA vaccines, siRNA, splice site conversion oligomers and RNA-based nanoparticles. In some embodiments, the therapeutic agent includes, but is not limited to, recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog alfa, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), Ceredase, Alglucerase, Cerezyme, Imiglucerase, glucocerebrosidase (Elelso), taliglucerase alfa, Fabrazyme, β-galactosidase (Agalsidase beta), laronidase (Aldurazyme), l-iduronidase, alpha-glucosidase (Myozyme), acid glucosidase, iduronic acid-2-sulfatase (Elaprase), iduronate-2-sulfatase, sulfatase (Naglazyme), arylsulfatase B, or N-acetylgalactosamine-4-sulfatase. In some embodiments, the subject is administered a therapeutic agent used to treat a disease including, but not limited to, hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Hunter disease, mucopolysaccharidosis, gangliosidosis, hypoalkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, and Mar-La disease.

[0229] In certain embodiments, the subject suffers from a rare autoimmune disorder. Such disorders include, but are not limited to, idiopathic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, and myasthenia gravis.

[0230] In certain embodiments, the individual suffers from a condition associated with the treatment of the disease. In the case of recombinant antibodies, tolerance is induced, for example, to humanized antibodies used in therapeutic situations to prevent patients from forming neutralizing antibodies to the antibody therapeutic, such as tolerance to humanized immune subgroup depleting antibodies or anti-cytokine antibodies used as therapeutics for autoimmune diseases.

[0231] Autoimmune diseases can be divided into two major categories: organ-specific and systemic. Autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes, type II diabetes, multiple sclerosis (MS), immune-mediated infertility (such as premature ovarian failure), scleroderma, Sjögren's disease, vitiligo, alopecia (baldness), polyglandular failure, Graves' disease, hypothyroidism, polymyositis, pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), autoimmune hepatitis (including hepatitis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV)), hypopituitarism, graft-versus-host disease (GvHD), myocarditis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, celiac disease, hypoparathyroidism, neuromyelitis optica, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis.

[0232] Autoimmune diseases may also include, but are not limited to, Hashimoto's thyroiditis, autoimmune polyglandular syndrome type I and II, paraneoplastic pemphigus, bullous pemphigoid, dermatitis herpetiformis, linear IgA disease, acquired epidermolysis bullosa, erythema nodosum, pemphigoid gestationis, cicatricial pemphigoid, primary mixed cryoglobulinemia, chronic bullous disease of childhood, hemolytic anemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmune neutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff-man syndrome, acute disseminated encephalomyelitis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal with conduction block motor neuropathy, chronic neuropathy with monoclonal gammopathy, opsoclonus-myoclonus syndrome, cerebellar degeneration, encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing cholangitis, gluten-sensitive enteropathy, ankylosing spondylitis, reactive arthritis, polymyositis / dermatomyositis, mixed connective tissue disease, Behcet's syndrome, psoriasis, polyarteritis nodosa, allergic vasculitis and granulomatosis (Churg-Strauss disease), polyangiitis overlap syndrome, hypersensitivity vasculitis, Wegener's granulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki disease, isolated vasculitis of the central nervous system, thromboangiitis obliterans, sarcoidosis, glomerulonephritis, and cold sickness. These conditions are well known in the medical art and are described, for example, in Harrison's Principles of Internal Medicine, 14th ed., Fauci AS et al., eds., New York: McGraw-Hill, 1998.

[0233] Animal models for studying autoimmune diseases are known in the art. For example, animal models whose behavior most closely resembles human autoimmune diseases include strains of animals that spontaneously develop high morbidity for a particular disease. Examples of such models include, but are not limited to, non-obese diabetic (NOD) mice that develop a disease similar to type 1 diabetes, and animals that are susceptible to lupus-like disease, such as New Zealand hybrids, MRL-Fas lprAnd BXSB mice. Animal models of induced autoimmune diseases include, but are not limited to: experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis; collagen-induced arthritis (CIA), which is a model of rheumatoid arthritis; desmoglein 3 transgenic T cell mice, which can be used as an experimental model of pemphigus vulgaris; and experimental autoimmune uveitis (EAU), which is a model of uveitis. Animal models of autoimmune diseases have also been constructed by genetic manipulation, and include, for example, IL-2 / IL-10 knockout mice for inflammatory bowel disease, Fas or Fas ligand knockout for SLE, and IL-I receptor antagonist knockout for rheumatoid arthritis.

[0234] In certain embodiments, the subject suffers from a bacterial or viral infection. A subject suffering from a bacterial or viral infection is a subject with recognizable symptoms of an existing bacterial or viral infection.

[0235] A non-limiting list of viral infections that can be treated with the modified particles of the present invention includes herpes virus infections, hepatitis virus infections, West Nile virus infections, flavivirus infections, influenza virus infections, rhinovirus infections, papillomavirus infections, paramyxovirus infections, parainfluenza virus infections, and retrovirus infections. Preferred viruses are those that infect the central nervous system of a subject. Most preferred viruses are those that cause hemorrhagic fever, encephalitis, or meningitis.

[0236] A non-limiting list of bacterial infections that can be treated with the modified particles of the present invention include Staphylococcus infections, Streptococcus infections, Mycobacterium infections, Bacillus infections, Salmonella infections, Vibrio infections, Spirochete infections, and Neisseria infections. Preferred are bacteria that infect the central nervous system of a subject. Most preferred are bacteria that cause encephalitis or meningitis.

[0237] In some embodiments, the present invention relates to using the compositions of the present invention before the onset of a disease. In other embodiments, the present invention relates to using the compositions of the present invention to inhibit a developing disease. In some embodiments, the present invention relates to improving a subject's disease. The so-called improving a subject's disease is intended to include treating, preventing, or inhibiting a subject's disease.

[0238] In some embodiments, the present invention relates to preventing the recurrence of a disease. For example, an undesirable immune response may occur in one region of a peptide (such as an antigenic determinant). The recurrence of a disease associated with an undesirable immune response may occur due to the presence of an immune response attack in different regions of the peptide. Since the immune modified particles of the present invention do not contain attached peptides or antigenic portions, the particles will be effective for multiple epitopes. T cell responses in some immune response disorders including MS and other ThI / 17-mediated autoimmune diseases can be dynamic and evolve during relapsing-remitting and / or chronic progressive diseases. The dynamic nature of the T cell repertoire suggests that it can be used to treat certain diseases because the target can change as the disease develops. Prior to this, it is necessary to understand the reaction pattern in advance in order to predict the development of the disease. The present invention provides a composition that can prevent the effects of dynamically changing diseases (i.e., "epitope expansion" function). As a model of multiple sclerosis (MS), a known model for relapse is an immune response to proteolipid protein (PLP). The initial immune response can occur through a response to PLP139-15. Subsequent disease attacks can occur through a recurrent immune response to PLP[pi]s-iβi.

[0239] Other embodiments of the invention relate to transplantation. This refers to the transfer of a tissue sample or graft from a donor individual to a recipient individual, and is often performed on a human recipient who needs the tissue in order to restore the physiological function provided by the tissue. Transplanted tissues include (but are not limited to) whole organs such as kidney, liver, heart, lung; organ components such as skin grafts and cornea; and cell suspensions such as bone marrow cells and cell cultures selected and expanded from bone marrow or circulating blood, as well as whole blood transfusions.

[0240] Serious potential complications of any transplant will occur due to antigenic differences between the host receptor and the transplanted tissue. Depending on the nature and extent of the differences, there may be a risk of host-to-graft or graft-to-host immune attack, or both. The extent of the risk is determined by tracking the response pattern in a population of subjects with similar phenotypes who have received similar treatments and linking various possible contributing factors according to recognized clinical procedures. The immune attack may be the result of a pre-existing immune response (such as pre-formed antibodies) or the result of an immune response initiated approximately at the time of transplantation (such as the production of Th cells). Antibodies, Th cells, or Tc cells may participate in any combination with each other and with various effector molecules and cells. However, the antigens involved in the immune response are usually unknown, thus causing difficulties in designing antigen-specific therapies or inducing antigen-specific tolerance.

[0241] Certain embodiments of the present invention are directed to reducing the risk of rejection of a tissue graft by a recipient due to host-versus-graft disease. Treatment may be administered to prevent or reduce the effects of hyperacute rejection, acute rejection, or chronic rejection. Treatment is preferably initiated long enough before transplantation so that tolerance is in place when the graft is placed; however, when this is not feasible, treatment may be initiated simultaneously with or after the transplant. Regardless of the time of initiation, treatment will generally continue at regular intervals for at least the first month after transplantation. Subsequent doses may not be required if the graft is adequately adapted, but may be restarted if there are any signs of graft rejection or inflammation. Of course, the tolerance program of the present invention may be combined with other forms of immunosuppression to achieve even lower levels of risk.

[0242] Certain embodiments of the invention relate to reducing or otherwise ameliorating an inflammatory response induced as a response to surgery. In one embodiment of the invention, the immune-modifying particles are administered prior to surgery. In another embodiment of the invention, the immune-modifying particles are administered concurrently with or during surgery. In yet another embodiment of the invention, the immune-modifying particles are administered after surgery.

[0243] The particles of the invention may also be used to treat abscesses or empyema to reduce the inflammatory response produced in a subject after exposure to an infectious pathogen such as bacteria or parasites. In one embodiment of the invention, the immune-modifying particles are administered in conjunction with antibacterial and / or antiparasitic treatments known in the art.

[0244] The particles of the invention may also be used to reduce or otherwise ameliorate an inflammatory response induced in response to physical trauma or injury, including, but not limited to, sports injuries, wounds, spinal cord injuries, brain injuries, and / or soft tissue injuries. In one embodiment of the invention, the immune-modifying particles are administered after a subject has experienced trauma or injury.

[0245] The particles of the present invention can also be used to reduce the inflammatory response associated with the development and / or growth of cancer cells. Treatable cancers include, but are not limited to, central nervous system cancer, basal cell carcinoma, cancerous brain tumors, Burkitt's lymphoma, lymphoma, cervical cancer, ovarian cancer, testicular cancer, liver cancer, non-small cell and small cell lung cancer, melanoma, bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney (renal cell) cancer, leukemia, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, melanoma and thyroid cancer. In one embodiment, subcutaneous injection of the particles of the present invention prevents the accumulation of inhibitory neutrophils, thereby reducing inflammation in cancer patients.

[0246] The particles of the present invention can also be used to regenerate damaged tissue. In one embodiment, administering the particles to a patient can enhance the regeneration of damaged epithelial cells in the digestive tract. In another embodiment, the patient suffers from colitis, Crohn's disease, or inflammatory bowel disease. In another embodiment, administering the particles of the present invention to a patient can enhance the remyelination of neurons. In another embodiment, the patient suffers from multiple sclerosis.

[0247] In some embodiments, the compositions of the invention (e.g., PLG carriers coupled to antigen molecules) can be used with one or more scaffolds, matrices, and / or delivery systems (see, e.g., U.S. Patent Application 2009 / 0238879, U.S. Patent No. 7,846,466, U.S. Patent No. 7,427,602, U.S. Patent No. 7,029,697, U.S. Patent No. 6,890,556, U.S. Patent No. 6,797,738, U.S. Patent No. 6,281,256; the entireties of which are incorporated herein by reference). In some embodiments, the particles (e.g., antigen-coupled PLG particles) are associated with, adsorbed to, embedded in, bound to, or the like of a scaffold, matrix, and / or delivery system (e.g., for delivering chemical / biological materials, cells, tissues, and / or organs to a subject). In some embodiments, a scaffold, matrix and / or delivery system (e.g., for delivering chemical / biological materials, cells, tissues and / or organs to a subject) comprises and / or is made of materials described herein (e.g., PLG conjugated to one or more antigenic peptides).

[0248] In some embodiments, a microporous scaffold is provided (e.g., for transplanting biomaterials (e.g., cells, tissues, etc.) into a subject). In some embodiments, a microporous scaffold having a pharmaceutical agent (e.g., extracellular matrix protein, exenatide-4) and a biomaterial (e.g., pancreatic islet cells) thereon is provided. In some embodiments, the scaffold is used to treat a disease (e.g., type 1 diabetes) and related methods (e.g., diagnostic methods, research methods, drug screening). In some embodiments, a scaffold having an antigen-binding carrier described herein thereon and / or therein is provided. In some embodiments, the scaffold is made of an antigen-binding material (e.g., antigen-bound PLG).

[0249] In some embodiments, the scaffold and / or delivery system comprises one or more layers and / or has one or more chemical and / or biological entities / agents (e.g., proteins, peptide-coupled particles, small molecules, cells, tissues, etc.), see, e.g., U.S. Patent Application 2009 / 0238879; the entire text of which is incorporated herein by reference. In some embodiments, antigen-coupled particles are co-administered with a scaffold delivery system to induce induction of immune tolerance to scaffolds and related materials. In some embodiments, a microporous scaffold having particles described herein thereon or therein is administered to a subject. In some embodiments, the antigen-coupled particles are coupled to a scaffold delivery system. In some embodiments, the scaffold delivery system comprises antigen-coupled particles.

[0250] Various modifications, reconstructions, and variations of the features described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes and embodiments that are apparent to those skilled in the relevant art are also intended to fall within the scope of the following claims. For example, U.S. patent applications 2012 / 0076831, 2002 / 0045672, 2005 / 0090008, 2006 / 0002978, and 2009 / 0238879, each of which is incorporated herein by reference in its entirety, and U.S. Patent Nos. 7,846,466, 7,427,602, 7,029,697, 6,890,556, 6,797,738, and 6,281,256, each of which is incorporated herein by reference in its entirety, provide details, modifications, and variations that may be used in the various embodiments described herein.

[0251] All publications and patents mentioned in this application and / or listed below are incorporated herein by reference in their entirety.

[0252] Example

[0253] The following examples are provided to further illustrate the advantages and features of the present invention but are not intended to limit the scope of the present disclosure.

[0254] Materials and Methods

[0255] Generation of chimeric mice

[0256] Six to eight-week-old B6.SJL-Ptprc a Pep3 b / BoyJ (CD45.1) mice were irradiated once with a dose of 950 rads. Twelve hours later, 10 7Mice were reconstituted with bone marrow cells from a C57BL / 6-7.2fms-EGFP donor. After irradiation, mice were given drinking water containing sulfamethoxazole (Sigma Aldrich) and trimethoprim (Sigma Aldrich) for 10 days. Six weeks after irradiation as described above, mice were infected with WNV. Chimerism was examined using flow cytometry, and as previously demonstrated, 96-99% chimerism was always donor-derived (Getts et al., J Neurochem. 103: 1019, 2007).

[0257] Immunohistology

[0258] Mice were anesthetized and perfused with 50 mL of sterile PBS. All organs were isolated and snap-frozen in Optimum Cutting Temperature Compound (OCT; Tissue-Tek, Tokyo, Japan) except the heart, which was processed into paraffin blocks (Getts et al., J. Neurochem 103:10919-1030, 2007). Eight micron tissue sections were cut in a cryostat, air-dried overnight, and then stored at -80°C until needed. Frozen sections were thawed and subjected to histological (standard hematoxylin and eosin staining) or immunohistochemical analysis (Getts et al., J. Exp Med 205:2319-2337, 2008 (Getts et al., Journal of Experimental Medicine, Vol. 205, pp. 2319-2337, 2008). Antibodies to MARCO, SIGN-R1 and SIGLEC-1 (R&D Systems, MN, USA), CD68 (Abcam, MA, USA) and Ki67 (Abcam) were used as indicated. Images were acquired on an Olympus BX-51 microscope (Olympus, Tokyo, Japan) using a DP-70 camera and DP manager 2.2.1 software.

[0259] Microscopy and image acquisition

[0260] Images were acquired on an Olympus BX-51 microscope (Olympus, Japan) using a DP-70 camera and DP manager 2.2.1 software (Olympus).

[0261] Isolation of leukocytes from brain and liver

[0262] Leukocytes were obtained from brains of PBS-perfused mice by digestion of the brains with DNase (0.005 g / ml; Sigma-Aldrich) and collagenase IV (0.05 g / ml; Sigma-Aldrich) in PBS for 60 min at 37°C as described previously (Getts et al, J Exp Med. 29:2319, 2007). Digestion was stopped with 10% FCS and the homogenate was passed through a 70 μm nylon cell strainer (Becton, NJ, USA). Dickinson, NJ, USA). The pellet obtained after centrifugation at 340 x g for 10 minutes was resuspended in 30% Percoll (Amersham, Norway) and layered on 80% Percoll. Leukocytes were collected from the 30% / 80% interface after centrifugation at 1140 x g for 25 minutes at room temperature. Leukocytes can also be obtained from the liver using the same protocol, and the tissue is weighed before processing.

[0263] Isolation of leukocytes from spleen, blood and bone marrow

[0264] For flow cytometry analysis, right femurs were dissected and bone marrow cells were flushed out using a syringe filled with PBS. For bone marrow precursor isolation, femurs and tibiae from at least 4 mice were used. The cell suspension obtained after flushing was filtered through a 70 μm cell strainer and centrifuged at 340 g for 5 minutes. The red blood cells in the resulting pellet were precipitated in NH based 4 Red blood cell lysis buffer (BD Pharm Lyse TM ; BD Pharmingen) and then centrifuged at 340xg for 5 minutes. For peripheral blood, blood was collected via cardiac puncture and immediately transferred to citrate buffer (mMol, Sigma Aldrich). The resulting suspension was layered on 70% Percoll and centrifuged at 1140×g for 20 minutes at room temperature with the brake off. The junction was collected and the cells were washed once in PBS and centrifuged at 340×g. For the isolation of spleen leukocytes, the spleen was passed through a 7070 μm cell strainer and centrifuged at 340g for 5 minutes. The red blood cells in the resulting pellet were separated in a NH based 4Red blood cell lysis buffer (BD Pharm Lyse TM ; BD Pharmingen) and then centrifuged at 340×g for 5 min.

[0265] Flow cytometry

[0266] Cells collected from brain, liver, blood and bone marrow (as described above) were washed in PBS and blocked with anti-CD16 / CD32 antibody (Biolegend). Live cells were counted using trypan blue exclusion and typically showed >95% cell viability.

[0267] Cell surface molecule expression is measured, and cell sorting is performed on a FACS ARIA (Beidi) equipped with an argon ion and HeNe laser. The living cell population is gated by forward scatter and side scatter, and the fluorescent cell population of the marker is subsequently determined by forward gate determination. Sorting is performed using the specific fluorescence and scattering parameters of the cell population of interest identified. For the bone marrow cell population, the sorting severity according to purity is set to achieve a purity of >98%.

[0268] The acquired FACS data files were analyzed using the flow cytometry program Flow Jo (Tree Star, Inc. OR, USA). Quantification of the cell population of interest was calculated based on the flow cytometric percentage and the absolute cell count from each organ at the time of analysis.

[0269] Adoptive transfer

[0270] During the development of the embodiments of the present invention, experiments were performed to study a second model of active disease known as adoptive transfer. Rather than immunizing animals with peptides, lymphocytes from the spleen of mice with active disease were transferred into recipients, which would subsequently develop disease. During the development of the embodiments of the present invention, experiments were performed to characterize the ability of PLG nanoparticles to inactivate adoptively transferred activated effector cells. On day 4, clinical scores began to increase in mice treated with control peptide-coupled particles or spleen cells. On day 2, mice treated with PLG-PLP nanoparticles showed an increase in clinical scores. 139-151 The mean clinical score of the particle-treated mice was 0 at all but two time points during the first 40 days, when the mean clinical score was 0.25.

[0271] Multiple ELISA

[0272] Multiplex plate ELISA was performed according to the manufacturer's instructions (Quansys Biosciences, Logan, Utah, USA). Briefly, brain, spleen, and liver tissues were homogenized in PBS, clarified by spinning at 1000 × g, and stored at -20°C until assayed. Serum samples were also used. Thawed samples and standards were diluted in the provided buffer and 30 μl was spread in each well, each well containing 16 spots, each containing capture antibodies for specific soluble proteins. The plate was then incubated on an orbital shaker at 120 rpm for 1 hour. The plate was washed 3 times, and 30 μl of detection antibody was added to each well and incubated for another 1 hour. After washing 3 times, streptavidin-HRP was added and incubated for another 15 minutes. The plate was then washed 6 times and a substrate mixture was added. The plates were immediately read on a CCD imager (Kodak, Rochester NY, USA). Plate images were analyzed using Quansys Q-view software (Quansys Biosciences). Induction and evaluation of experimental autoimmune encephalitis (EAE)

[0273] Mice were injected subcutaneously with an emulsion containing 0.1 mg of MOG peptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 1); Osbi, Parkville, Victoria, Australia; >95% purified by HPLC) and 2 mg / mL of Mycobacterium tuberculosis (Sigma-Aldrich) in complete Freund's adjuvant. Two days later, 500 μl of pertussis toxin (Sigma-Aldrich) were administered intraperitoneally to the mice. Disease progression of mice was monitored and graded according to the following scale: 1, tail weakness and / or weakness in one hind limb; 2, weakness in more than one limb, gait disturbance; 3, paralysis of one limb; 4, paralysis of more than one limb, incontinence; 5, imminent death.

[0274] Statistical analysis

[0275] Graphs were drawn and computerized statistical analysis was performed in GraphPad Prism and InStat (both software were obtained from GraphPad software, San Diego, CA, USA). Unpaired two-tailed Student t-test or one-way ANOVA plus Tukey-Kramer post test were performed according to the data, with P < 0.05 considered significant.

[0276] For correlation analysis between parameters such as weight loss, infiltration and viral titer, nonlinear regression (curve fitting) with a second order polynomial (Y = A + B*X + C*X^2) was used.

[0277] Example 1

[0278] Preparation of Negatively Charged Immunomodified Particles (IMPs)

[0279] Towards D 2 A solution of poly(lactide-co-glycolic acid) (PLG) in dichloromethane (DCM) (2 mL, 20% w / v) was added dropwise to a D O solution (4 mL, 1% w / v). The mixture was sonicated on ice at 16 watts for 30 seconds using a VC30 Ultrasonic Processor. The resulting homogenized crude product was then poured into D 2 The homogenized slurry was stirred overnight using a Bellstir Multi-stir 9 magnetic stirrer (10 W for 10 s, 16 W for 10 s, 16 W for 30 s) from Bellco Glass, Inc. at a speed setting of 3.5.

[0280] result

[0281] After three hours of stirring, particle size analysis was performed using dynamic light scattering in disposable polystyrene cuvettes.

[0282] a. 10 W, 10 s – Z-average = 499.9 nm – PdI = 0.23, peak = 634.5 nm

[0283] b. 16 W, 10 s – Z-average = 528.9 nm – PdI = 0.227, peak = 657.5 nm

[0284] c. 16W, 30s – Z-average = 471.6nm – PdI = 0.228, peak = 580.5nm

[0285] d. 16W, 60s – Z-average = 491.1nm – PdI = 0.275, peak = 600.8nm

[0286] After the reaction is complete, the resulting crude suspension is then purified.

[0287] purification

[0288] The fresh D 2D O and 10× sodium bicarbonate buffer cooled to 4°C overnight. Using a 40 μm cell strainer, filter 36 mL of the particle suspension from each batch into an appropriately labeled 50 mL centrifuge tube filled with 4 mL of cooled 10× sodium bicarbonate buffer. Make approximately 6 such tubes per beaker. Centrifuge all tubes at 7000 g for approximately 15 minutes at 4°C and aspirate the supernatant. Repeat the above procedure to suspend as much of the particle pellet as possible in 1 mL of cooled D O and 10× sodium bicarbonate buffer cooled to 4°C overnight. 2 In O.

[0289] Transfer the suspended pellet to a new tube containing 4 mL of chilled 10x sodium bicarbonate buffer. (Step 1)

[0290] Repeat the resuspension of the pellet until all pellets are successfully resuspended. (Step 2)

[0291] The six centrifuge tubes were then combined into one centrifuge tube (50 mL tube), and the remaining volume in the tube was replaced by cooled D 2 O was filled up to 40 mL (wash 1).

[0292] The tubes were centrifuged at 7000 g for 20 min at 4°C and the supernatant was aspirated.

[0293] Steps 1 and 2 and washing 1 of the obtained particles were repeated at least twice each time. Finally, the obtained particle pellet was snap-frozen in liquid nitrogen and lyophilized to anhydrous in a manifold to obtain negatively charged IMPs.

[0294] Figure 1 Characterization of surface functionalized poly(lactide-co-glycolide) particles by dynamic light scattering analysis is shown. The particles were analyzed in 18.2 MΩ water at 2.5×10 5 The surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of counts / second. The population of surface functionalized poly(lactide-co-glycolide) particles had a Z-average diameter of 567 nm, a peak diameter of 670 nm, and a polydispersity index of 0.209.

[0295] Table 4 shows the measured values ​​of surface functionalized PLG-PEMA particles. The data in the table are representative because each batch is slightly different. However, the numbers in the table are based on the merging of several batches of particles. The measured values ​​of the re-emulsified particles are similar to those in Table 2.

[0296] Table 4 - Measurements of surface functionalized PLG-PEMA particles

[0297]

[0298] Example 2

[0299] Administration of antigen-coupled PLGA microbeads to prevent recurrent experimental autoimmune encephalitis

[0300] Immunodominant proteolipid protein (PLP) 139-151 Epitope (PLG-PLP 139-151 ) PLG nanoparticles were studied to induce tolerance to prevent recurrent experimental autoimmune encephalitis (R-EAE). R-EAE mice were generated as described above.

[0301] The peptides administered to the animals were coupled to particles with an average diameter of 500 nm. 139-151 -PLGA (N=5), OVA 323-339 -PLGA (N=5) or unconjugated PLGA (N=5) treated mice. Peak disease was usually observed around day 12 to 14 and mice were scored for clinical disease. No peptide or control peptide OVA 323-339 The modified particles failed to prevent the induction of disease. However, except for the low clinical scores below 1 between days 20 and 30, the treatment with PLP 139-151 The modified PLGA particles produced a clinical score of 0 (no disease) ( Figure 2 Previous studies using unmodified PLG or using polystyrene particles, where the polystyrene-bound particles often trigger allergic reactions, did not produce such effective disease reduction.

[0302] In addition, the lack of two immune PLP 139-151 Myelin-specific CD4 + Specific inactivation of T cells. In summary, PLG-PLP 139-151 Prophylactic treatment specifically prevented the development of EAE and demonstrated an improvement in the ability of the particles to prevent disease. Scores generated using the particles were as good as, and possibly better than, those generated using antigen-coupled splenocytes.

[0303] The type of particles administered also has an effect on the development of EAE in the mouse model. 323-339 -PLS (N=5), OVA 323-339 -PLGA PHOSPOREX (N=5), OVA 323-339 -PLGA PEMA (N=5), PLP 139-151-PLA (N=5), PLP 139-151 -PLGA PHOSPOREX (N=5) or PLP 139-151 -PLG PEMA (N=5) mice were treated. The peak of disease was usually observed around day 12 to day 14, and the mice were scored for clinical disease. 323-339 Modification of any composition of the particles failed to prevent disease induction. However, PLP 139-151 PLG-coupled microbeads were more effective than PLP in downregulating the induction of R-EAE. 139-151 Coupling with commercial (Phosphorex) pLG or polystyrene is more effective ( Figure 3A and Figure 3B ).

[0304] Example 3

[0305] Intravenous infusion of antigen-coupled PLG particles does not induce anaphylaxis-induced hypothermia in OVA / alum presensitized animals

[0306] Due to the presence of active disease, allergic reactions to antigens are worrying, which may lead to immediate death and have been described in the case of polystyrene-bound particles. Allergic reactions are associated with a significant drop in body temperature. To test whether the intravenous administration of OVA-PLG can cause a drop in temperature induced by allergic reactions in pre-sensitized animals, mice were immunized via intraperitoneal injection with 10 μg OVA / alum on day 0. On the 14th day, mice were re-immunized via intraperitoneal injection with 10 μg OVA / alum, and then tolerated with intravenously administered OVA-PLG on the 21st day. Then, on the 28th day, mice were tolerated via intravenous administration of OVA-PLG particles or OVA.

[0307] like Figure 4 As shown in Figure 2, those mice treated with soluble OVA at day 28 showed a decrease in temperature compared to those animals treated with OVA-PLG particles. No decrease in body temperature was observed within 1 hour of particle delivery.

[0308] Figure 5 It was shown that administration of PLP-PLG during remission did not cause any mortality related to anaphylaxis. PLP in CFA was administered subcutaneously to six to eight week old female SJL / J mice. 139-151 EAE was induced, and the occurrence of clinical disease was monitored and recorded ( Figure 5 B) On day 21 relative to disease induction, mice were injected intravenously with soluble PLP 139-151 (hollow square), soluble OVA 323-339(open circles) or the same peptide coupled to PLG nanoparticles (solid circles). The temperature of the animals was monitored and recorded every 10 minutes for 1 hour after injection ( Figure 5 A).

[0309] Example 4

[0310] Prophylactic treatment with PLP-PLG particles to induce long-term antigen-specific tolerance

[0311] Seven days before disease induction, increasing concentrations of PLP were administered intravenously. 139-151 -PLG to determine the optimal dose and monitor the effect of OVA 323-339 - Development of clinical disease in PLG-treated SJL / J mice ( Figure 6 A). Six to eight week old female SJL / J mice were injected intravenously with PLP. 139-151 (Square) or OVA 323-339 (Circle) coupled PLG nanoparticles. Figure 6 B) 25 days Figure 6 C) or 50 days ( Figure 6 D) PLP in CFA after subcutaneous injection 139-151 Animals in panel B were followed for clinical disease for 100 days. Figure 6 E shows delayed-type hypersensitivity (DTH) in a subset of mice shown in panel B at day 8 relative to disease induction. 139-151 / CFA priming group (OVA 323-339 -PLG and PLP 139-151 -PLG) selected representative animals were primed with PLP 139-151 Epitopes and OVA 323-339 Control peptides were used for ear challenge. After 24 hours, ear swelling was determined as a measure of DTH and the response before challenge was subtracted. Figure 6 F shows intravenous injection of PLP into six- to eight-week-old female SJL / J mice. 178-191 (Triangle), OVA 323-339 (Circular) or PLP 139-151 (squares) conjugated PLG nanoparticles or unconjugated particles alone (open circles). After 7 days, PLG nanoparticles were injected subcutaneously in CFA. 178-191 EAE was induced, and the disease was monitored at the indicated time points.

[0312] Example 5

[0313] Treatment of recurrent experimental autoimmune encephalitis with antigen-coupled particles

[0314] During the development of embodiments of the present invention, experiments were conducted to study the139-151 Particles treat The ability of PLP to induce disease rather than prevent disease and to determine whether the route of administration affects disease development. 139-151 and adjuvant to immunize mice. Mice usually have maximum clinical scores on days 12-14. In this model, mice are treated with PLG-PLP via intravenous (iv), intraperitoneal (ip), subcutaneous (sc) or oral administration on day 10. 139-151 PLG-OVA 323-339 Particle processing. Figure 7 As shown in 139-151 PLP administered intravenously is most effective in preventing tolerance. 139-151 -PLG treated animals did not develop disease and had a mean clinical score of 0 at most time points. 139-151 This was in contrast to animals treated with polystyrene particles, in which >70% of the observed deaths were due to anaphylaxis.

[0315] Example 6

[0316] Tolerance to antigen-coupled particles suppresses the induction of antigen-specific Th1 and Th17 responses in active recurrent experimental autoimmune encephalitis

[0317] To determine whether administration of antigen-coupled particles inhibits the induction of T helper cells, MOG was administered intravenously to BALB / c mice on day -7. 35-55- PLG or OVA 323-339 -PLG particles. On day 0, OVA was administered subcutaneously to mice 323-339 -PLG particles and complete Freund's adjuvant (CFA). On day 10, the animals were treated with MOG 35-55- PLG or OVA 323-339 -PLG particles were restimulated and draining lymph node cells were isolated. CPM and levels of IL-17, GM-CSF, IFN-γ, IL-10, and IL-4 were measured on day 10. Figure 8 As shown in 323-339 -PLG particles suppressed Th1 and Th17 responses in treated animals.

[0318] Example 7

[0319] By PLP- 139-151 Conjugated PLGA particles induce tolerance

[0320] By delivering PLP 139-151 -PLG or OVA 323-339PLG was introduced into mice to implement another therapeutic tolerance strategy. Histological analysis showed that administration of PLG 139-151 -PLG particles inhibit cervical spinal cord inflammation and demyelination. Mice were treated with PLP-PLG or OVA 323-339 -PLG treatment and tissue retrieval on day 40. Cervical spinal cords were isolated and sectioned to study immune responses within the CNS, which underlie the pathological basis of R-EAE and multiple sclerosis. Fig. 9 Shows the PLP in use 139-151 - PLG-treated animals have reduced immune cell infiltration in the spinal cord and are more similar to native tissue than OVA 323-339 - Tissues from animals treated with PLG.OVA 323-339 -PLG treated animals had positive staining for CD45, CD4, and CD11b; whereas PLP 139-151 -PLG treated animals had minimal staining for these factors.

[0321] Administration of PLP 139-151 -PLG particles also inhibited blood-brain barrier (BBB) ​​disruption and macrophage activation in the spinal cord of treated mice. Animals were treated with complete Freund's adjuvant (CFA), OVA 323-339 PLG particles or PLP 139-151 -PLG particle treatment. The clinical scores and incidence rate of EAE were determined ( Fig.10 B) and observed the spinal cord through in vivo imaging ( Fig.10 A and Fig.11 ). Angiosense measures vascular leakage in the CNS and prosense reports activated macrophages (activation of cathepsins cleaves the reporter protein, revealing a fluorescent signal). The bar graph gives numerical values ​​for the signal intensity shown in the brain and SC scans.

[0322] Tolerance can also be induced by particles in which antigens are encapsulated. Fig.12 The administration of PLP encapsulated 139-151 PLG particles of inhibited the induction of R-EAE in mice. The ability to encapsulate self-antigens allows the use of complex mixtures of proteins or even organ homogenates to achieve higher antigen coverage and thus more effectively address epitope expansion.

[0323] Example 8

[0324] By LP- 139-151 Tolerance induced by coupled PLGA particles is partially dependent on the expansion / activation of regulatory T cells

[0325] SJL / J mice were treated with anti-CD25 antibody, a common marker for regulatory T cells (Treg), on day -9 and then with OVA on day -7.323-339 PLG particles, anti-CD25 antibody, OVA 323-339 PLG particles and control IgG antibody, PLP 139-151 -PLG particles and anti-CD25 antibody or PLP 139-151 -PLG particles and control IgG antibody treatment. Fig.13 As shown in 139-151 - Animals treated with PLG particles and anti-CD25 antibody sometimes showed worse 139-151 -PLG particles and control IgG antibody treated animals showed greater mean clinical scores. This confirms that Tregs or at least CD25 expressing T cells play a role in inducing tolerance.

[0326] Example 9

[0327] PLP in active and adoptive EAE 139-151 -PLG particles induce therapeutic tolerance

[0328] Comparison of PLP in active and adoptive EAE 139-151 -PLG particles induced therapeutic tolerance. 2.5 × 10 6 PLP 139-151 Activate mother cells to induce adoptive EAE. 2 days after disease induction ( Fig.14 A) 14 days ( Fig.14 C) 18 days Fig.14 E) or 21 days ( Fig.14 F), mice were intravenously injected with PLP coupled to 500 nm PLG nanoparticles 139-151 (Square) or OVA 323-339 (Circle) Peptide. Clinical disease scores were compared with those after treatment with antigen-coupled splenocytes ( Fig.14 A) On day 42, from PLP 139-151 or OVA 323-339 Brains and spinal cords were collected from tolerized mice for histological analysis. Sections from mice in panel A were stained for PLP protein and CD45 ( Fig.14 B). Spinal cord sections from mice in panel C were stained with Luxol fast blue ( Fig.14 D). Areas of demyelination and cell infiltration are indicated by arrows. Results show that PLP 139-151 -PLG particles induced tolerance.

[0329] Fig.15 The figure shows the binding of OVA 323-339 or PLP 139-151Graph of the mean clinical scores of mice with active EAE and adoptive EAE after treatment with SP or PLG particles of Fig.15 A) or 2 days ( Fig.15 B), mice were injected intravenously with PLP 139-151 -SP, PLP 139-151 -PLG or OVA 323-339 -SP or OVA coupled to 500 nm nanoparticles 323-339 -PLG peptide, and the mean clinical score was determined. 139-151 -PLG particles alleviated the disease, implying the induction of tolerance.

[0330] In PLP-PLG-tolerized mice, the infiltration of central nervous system immune cells was also significantly reduced. SJL / J mice were intravenously injected with PLP-PLG-tolerized mice 2 days after EAE induction by adoptive transfer. 139-151 (Square) or OVA 323-339 (Circles) 500 nm PLG nanoparticles conjugated. At the peak of disease (day 14), brain and spinal cord were removed and lymphocytes were counted by flow cytometry ( Fig.16 B) APC( Fig.16 C) microglia ( Fig.16 D) peripheral dendritic cells ( Fig.16 E), myeloid dendritic cells ( Fig.16 F) and macrophages ( Fig.16 G). The gating strategy for these cell populations is depicted in ( Fig.16 CNS cell preparations were stimulated with PM and ionomycin for 5 h and then intracellularly stained for IL-17A and IFN-γ ( Fig.16 H).

[0331] Example 10

[0332] Treatment with anti-PD-1 mAb eliminates PLP encapsulation in adoptively transferred EAE 139-151 Tolerance induction achieved by PLG nanoparticles

[0333] To test the effect of anti-PD-1 antibody treatment on PLP in mice with adoptive EAE, 139-151 To induce tolerance, on day 0, mice received 3 × 10 6 PLP 139-151 On day 2, they received PLP encapsulated in PLG particles along with PBS or anti-PD-1 antibody via intravenous administration. 139-151 or OVA 323-339On days 4, 6, 8, 10, and 12, all animals received 250 μg of anti-PD-1 antibody or PBS.

[0334] like Fig.17 As shown in FIG, administration of PLP encapsulated in PLG particles when the particles were administered with PBS 139-151 The peptide induced tolerance. However, administration of anti-PD-1 antibodies reduced this tolerance.

[0335] Embodiment 11

[0336] Treatment with antagonist anti-CD40 mAb abolishes PLP-encapsulated mitochondria in adoptively transferred EAE in an IL-12-dependent manner 139-151 Tolerance induction achieved by PLG nanoparticles

[0337] To test the effect of antagonist anti-CD40 antibody treatment on PLP in mice with adoptive EAE, 139-151 To induce tolerance, on day 0, mice received 3 × 10 6 PLP 139-151 On day 2, mice received PLP encapsulated in PLG particles via intravenous administration. 139-151 or OVA 323-339 On day 3, animals received control IgG2a antibody, anti-CD40 antibody, or anti-CD40 antibody and anti-Il-12 antibody.

[0338] like Fig.18 As shown in FIG, administration of PLP encapsulated in PLG particles when the particles were administered with PBS 139-151 The peptide induced tolerance. Administration of antagonist CD40 antibodies reduced this tolerance, but this reduction in tolerance was reversed by the addition of anti-IL-12 antibodies.

[0339] Example 12

[0340] OVA encapsulated in PLG particles prevents allergic airway inflammation and OVA-specific Th2 responses in vivo

[0341] To test the preventive effect of OVA encapsulated in PLG particles on airway inflammation, mice were treated intravenously with OVA-PLG on day -7. On day 0, mice received an intraperitoneal injection of OVA / alum at a dose of 10 μg / mouse. On day 7, mice were treated intravenously with OVA-PLG again, and on day 14 mice received another intraperitoneal injection of OVA / alum at 10 μg / mouse. Between days 28 and 30, mice were treated three times with aerosolized OVA.

[0342] like Fig.19As shown in , prophylactic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and decreased the levels of serum OVA IgE and eosinophils in the lung.

[0343] OVA encapsulated in PLG particles preventively inhibited the OVA-specific in vitro recall response of mediastinal lymph nodes. Fig. 20 Lymph node proliferation observed after restimulation with 25 μg OVA was reduced in those animals treated with OVA-PLG, as shown in A. In addition, treatment with OVA-PLG reduced the release of cytokines after restimulation with OVA. Fig. 20 B shows that the levels of IL-4, IL-5, IL-13 and IL-10 were reduced in mice treated with OVA-PLG.

[0344] Example 13

[0345] OVA encapsulated in PLG particles therapeutically inhibits allergic airway inflammation and OVA-specific Th2 responses in vivo

[0346] To test the therapeutic effect of OVA encapsulated in PLG particles on airway inflammation, mice were treated intraperitoneally with OVA / alum at a dose of 10 μg / mouse on days 0 and 14. OVA-PLG was administered intravenously to mice on days 28 and 42. Between days 56 and 58, mice were treated three times with aerosolized OVA.

[0347] like Fig.21 As shown in , therapeutic administration of OVA-PLG reduced the secretion of IL-4, IL-5, IL-13, and IL-10, and decreased the levels of serum OVA IgE and eosinophils in the lung.

[0348] Fig. 22 It was shown that OVA encapsulated in PLG particles therapeutically downregulated OVA-specific Th2 cytokines in bronchoalveolar lavage fluid better than OVA-coupled PLG particles. Animals were treated as described above, but on days 28 and 42, mice were treated with OVA encapsulated in PLG particles or OVA coupled to PLG particles. Surprisingly, encapsulated OVA inhibited the secretion of Th2 cytokines more than OVA peptide coupled to the surface of PLG particles.

[0349] Embodiment 14

[0350] Tolerance induced by chromogranin A p31 peptide-PLG particles suppresses type 1 diabetes

[0351] Type 1 diabetes was induced in BDC2.5 mice at 3 weeks by isolating spleen, axillary, brachial, inguinal, and pancreatic lymph node cells from mice. The isolated cells were cultured and 2 × 10 6 5×10 cells / mL were incubated with 0.5 μM p31 peptide for 96 hours for in vitro activation. 6 10 cells were transferred into NOD.SCID mice (6-8 weeks). p31 or MOG coupled to SP or PLG was administered intravenously 2 hours to 3 days later. 35-55 Mice were tolerized.

[0352] Fig.23 A and Fig.23 B shows the blood glucose level of the treated animals. Administration of p31 peptide-coupled PLG resulted in the same level of blood glucose as that of MOG. 35-55 The blood glucose levels seen after coupling the particles were lower than those seen after coupling. Fig.23 C shows the MOG 35-55 The percentage of IFNγ-secreting cells observed in animals was also reduced in P31-PLG-treated mice compared to P31-PLG-treated mice.

[0353] p31-PLG-induced tolerance requires Tregs. Type 1 diabetes was induced in mice as described above, and 2 hours later, activated cells were transferred to NOD.SCID mice and treated with p31-PLG or MOG 35-55 PLG particles tolerant mice. Fig.24 As shown in , depletion of Tregs abolished tolerance induced by administration of p31-PLG particles.

[0354] Embodiment 15

[0355] Tolerance induced by insulin-coupled PLG particles suppresses the development of spontaneous type 1 diabetes in NOD mice

[0356] NOD mice were treated with BSA (N=22) or insulin (N=23)-coupled PLG particles via intravenous administration at 6, 8, and 10 weeks of age. The mice were then analyzed for the development of diabetes, which was defined as blood glucose >250 mg / dL. Fig.25 As shown in , administration of insulin-coupled PLG particles significantly increased the percentage of mice that did not develop diabetes over 300 days (69.6% vs. 22.7%; p=0.0027).

[0357] Example 16

[0358] Implantation dynamics

[0359] Female CD45.2 mice were tolerized with OVA-PLG or control peptide Dby-PLG (the major HY antigen expressed by male C57BL / 6 mice) on day -7. Mice were irradiated at 200 rads on day -1 and then transplanted with 1×10 100 μg / mL of 10 μg / mL from male CD45.1 mice on day 0. 6 , 5×10 6 or 1×10 7 Recipient mice were then tolerized with OVA-PLG, Dby-SP or Dby-PLG on day 1 and blood was harvested for FACS analysis of chimerism. Fig.26 The percentage of CD45.1 donor cells observed in recipient mice is shown.

[0360] Fig. 27 The percentage of donor CD45.1 cells in recipient mice after tolerization with OVA-PLG, Dby-SP or Dby-PLG on day 1 is shown. One positive control mouse did not show significant implantation (about 10%). All negative control mice were not implanted with donor cells. One Dby-SP mouse did not show significant implantation (about 10%). Two OVA-PLG mice were implanted with donor cells (about 10%): one of them was completely rejected by week 16. One Dby-PLG mouse began to reject at week 12 and was 10% by week 16. By week 16, the Dby-PLG group was within the implantation range of 10%-56%. OVA-PLG mice showed: 1) spontaneous implantation, 2) sequence homology between OVA323 and Dby, or 3) tolerogenicity of the particles. Dby-PLG allowed more implantation than Dby-SP and OVA-PLG.

[0361] Fig.28 It is shown that timed tolerance has an effect on the percentage of CD45.1 cells in the recipient mice. The positive control showed less implantation (about 4%) than expected (about 10%). One negative control mouse had 5% implantation. In all 3 OVA-PLG groups, one mouse in the -7 day, +1 day group showed implantation (12%). Tolerance on day 1 is more clinically relevant than tolerance on day -7.

[0362] Embodiment 17

[0363] Coumarin-6 PLGA particles were undetectable 24 hours after administration

[0364] Mice were treated with coumarin-6 PLGA particles conjugated to antigen or without antigen. Fig.29As shown in , particles were detectable 3 hours after administration, but not 24 hours after administration. Spleen (left column), liver (middle column), and lung (left column) sections of naive, uninjected mice (top row) were compared with those of mice injected with intravenous fluorescent PLGA / PEMA microparticles 3 hours after injection (middle row) and 24 hours after injection (bottom row), counterstained with DAPI.

[0365] Embodiment 18

[0366] Nanoparticles associate with macrophages in vivo

[0367] Liver analysis at 6 and 15 hours after administration showed that PLGA particles were bound to F4 / 80 in the liver. + Cell colocalization ( Fig.30 ).

[0368] 24 hours after intravenous infusion, marginal zone macrophages mainly took up TAMRA-labeled PLP 139-151 Coupled particles. Fig.31 As shown in 139-151 + cells are marginal zone macrophages.

[0369] Embodiment 19

[0370] Suppression of R-EAE in SJL / J mice using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core

[0371] Groups of SJL / J mice were injected intravenously with 2.5 mg of 500 nm-700 nm surface functionalized poly(lactide-co-glycolide) particles with soluble PLP139-151 peptide in the core on days -7 and -1 relative to priming with PLP139-151 / CFA on day 0. Control mice were primed on day 0 but did not receive particle treatment on days -7 or -1. Mice were observed for clinical signs of R-EAE for an additional 20 days.

[0372] Depicted on Fig.32 The results in depict the daily mean clinical scores relative to the day of PLP139-151 / CFA priming.Induction of immune tolerance in SJL / J mice by using surface-functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core inhibited PLP139-151 / CFA-induced R-EAE.

[0373] Embodiment 20

[0374] Inhibition of allergic airway inflammation by surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin

[0375] Allergic airway inflammation (AIA) was induced in mice. Groups of Balb / c mice were injected intravenously with 2.5 mg of 500nm-700nm surface functionalized poly(lactide-co-glycolide) particles with soluble ovalbumin or soluble bovine serum albumin (control) in the core on days -7 and +7 prior to priming with ovalbumin / alum on days 0 and +14. On days +28-30, mice were challenged with aerosolized ovalbumin. Mice were then sacrificed and bronchoalveolar lavage fluid was obtained. Serum levels of ovalbumin-specific IgE were also measured.

[0376] Eosinophil counts in bronchoalveolar lavage fluid indicate the severity of AAI - higher counts indicate worse disease. Serum levels of IgE indicate the severity of AAI - higher levels indicate worse disease.

[0377] Fig.33 It was shown that mice treated with encapsulated OVA-PLG showed the greatest reduction in eosinophil accumulation. Fig.34 It was shown that mice treated with encapsulated OVA-PLG showed the greatest reduction in serum IgE levels compared to untreated or control treated animals.

[0378] Induction of immune tolerance by using surface-functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core suppressed ovalbumin / alum-induced allergic airway inflammation in Balb / c mice.

[0379] Embodiment 21

[0380] Synthesis of surface functionalized poly(lactide-co-glycolide) particles encapsulating antigens

[0381] This example details the formulation and partial characterization of biodegradable poly(lactide-co-glycolide) particles that have been surface functionalized with a high density of carboxylate groups and contain soluble antigens within their core surrounded by a poly(lactide-co-glycolide) shell for tolerance induction in autoimmune diseases and for the treatment of allergies.

[0382] The high density of carboxylate groups was achieved by using poly(ethylene-alt-maleic anhydride (PEMA)), a polymer with carboxylate groups incorporated into the backbone, as a surfactant for the emulsification process.

[0383] As described above, biodegradable poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core and functionalized with high density of carboxylate groups on the surface effectively induced immune tolerance in the SJL / J PLP139-151 / CFA-induced R-EAE multiple sclerosis mouse model. In addition, biodegradable poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core and functionalized with high density of carboxylate groups on the surface effectively induced immune tolerance in the Balb / c ovalbumin / alum-induced AAI allergic asthma mouse model.

[0384] Poly(lactide-co-glycolide) particles containing soluble ovalbumin or bovine serum albumin in the core and surface functionalized with a high density of carboxylate groups were synthesized using the double emulsion-solvent evaporation method as follows:

[0385] 1. Add 150 μL of endotoxin-free water containing 200 mg / mL ovalbumin or bovine serum albumin dropwise to 2 mL of 20% w / v poly(lactide-co-glycolide) in dichloromethane in a 20 mL scintillation vial.

[0386] 2. The resulting mixture was placed on ice and sonicated using a probe sonicator at 10 watts for 30 seconds.

[0387] 3. Add 10 mL of 1% w / v poly(ethylene-alt-maleic anhydride) in water.

[0388] 4. The resulting mixture was placed on ice and sonicated using a probe sonicator at 16 watts for 30 seconds.

[0389] 5. The resulting emulsion was poured into 200 mL of 0.5% w / v poly(ethylene-alt-maleic anhydride) in a 600 mL beaker and stirred overnight to allow the particles to harden.

[0390] 6. The hardened particles were then purified by centrifugation and washed 3 times with bicarbonate buffer (pH 9.6).

[0391] 7. purification The pellet was resuspended in water containing 4% w / v sucrose and 3% w / v D-mannitol, flash frozen in liquid nitrogen and lyophilized to dryness.

[0392] Fig.35Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core by dynamic light scattering analysis is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of 1.792×105 counts / second on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water. The population of surface functionalized poly(lactide-co-glycolide) particles had a Z-average diameter of 584 nm, a peak diameter of 679 nm, and a polydispersity index of 0.162. These results represent the synthesis of 6 batches following the above protocol.

[0393] Fig.36 Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble PLP139-151 in the core by zeta potential measurement is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed at a count rate of 6.67×104 counts / second on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water. The population of surface functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -48.9 mV and a zeta deviation of 5.14 mV. These results are representative of the synthesis of 6 batches following the above protocol.

[0394] Fig.37 Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core by dynamic light scattering analysis is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 1.822×105 counts / second. The population of surface functionalized poly(lactide-co-glycolide) particles had a Z-average diameter of 569.7 nm, a peak diameter of 700.3 nm, and a polydispersity index of 0.230. These results are representative of the synthesis of 3 batches following the protocol described above.

[0395] Fig.38Characterization of surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin in the core by zeta potential measurement is shown. The surface functionalized poly(lactide-co-glycolide) particles were analyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) in 18.2 MΩ water at a count rate of 2.67×104 counts / second. The population of surface functionalized poly(lactide-co-glycolide) particles had a peak zeta potential of -52.2 mV and a zeta deviation of 5.38 mV. These results are representative of the synthesis of 3 batches following the protocol described above.

[0396] Embodiment 22

[0397] The core contains soluble PLP 139-151 Surface-functionalized liposomes induce immune tolerance in the murine R-EAE multiple sclerosis model

[0398] The present inventors have also discovered that biodegradable liposomal delivery vehicles that have been surface functionalized with a high density of negatively charged groups and contain a soluble antigen within the core induce immune tolerance in the murine R-EAE multiple sclerosis model.

[0399] The liposomes used in this study were composed of the following lipids in the following molar ratios: 30:30:40 phosphatidylcholine:phosphatidylglycerol:cholesterol. 139-151 On day -7 of CFA priming, each group of SJL / J mice was intravenously injected with soluble PLP 139-151 200 nm surface functionalized liposomes (10 μmol total lipid / mouse) containing peptide. Control mice were primed on day 0 and received soluble OVA in the core on day -7. 323-339 500nm-700nm surface functionalized liposomes of peptide (10 μmol total lipid / mouse). Mice were observed for clinical signs of R-EAE for another 17 days.

[0400] The results depict relative to PLP 139-151 / The average clinical score of the day of CFA priming. Fig.39 As shown in 323-339 Animals treated with peptide-functionalized liposomes had soluble PLP in the core compared to animals treated with peptide-functionalized liposomes. 139-151 Animals treated with peptide surface functionalized liposomes had lower clinical scores.

[0401] The results of this study confirmed that the core contains soluble PLP 139-151And the biodegradable liposomes functionalized with high density of negatively charged groups on the surface are in SJL / J PLP 139-151 / CFA-induced R-EAE mouse multiple sclerosis model effectively induced immune tolerance.

[0402] Tolerance induced by antigen-coupled or antigen-encapsulated particles is antigen-specific, dose-dependent, and long-lasting (>150 days). Tolerance is best induced by intravenous administration of coupled particles with diameters between 500 nm and 1 μm and zeta potentials <-5 mV. The induction of tolerance depends on the uptake of particles by MARCO scavenger receptors found on the surface of polyanions (e.g., carboxylated PS / PLG particles). Tolerance is induced and maintained by a combination of anergy (partially reversed by anti-PD-1 and antagonist CD40 antibodies) and iTregs (partially reversed by anti-CD25 antibodies). The particles of the present invention accumulate primarily in marginal zone macrophages (CD11b hi CD11c lo MARCO + Sign-R1 + Siglec-1 - )middle.

[0403] Compared with the use of antigen-pulsed or antigen-directed immature tolerogenic dendritic cells or engineered antigen-specific Tregs, the use of antigen-coupled particles for the treatment of autoimmune diseases has many advantages. Tolerogen preparation and induction using GMP-manufacturable off-the-shelf universal tolerogenic carriers is rapid and simple; there is no need to isolate and expand immature dendritic cells or Tregs ex vivo; there is no need to worry about immature dendritic cells being activated and becoming stimulatory rather than tolerogenic when manipulated ex vivo or about Tregs converting to Th1 / 17 after transfer; because host immature marginal zone APCs process and present antigens in a tolerogenic manner, host APCs can select relevant immunodominant self-epitopes from PLG particles or tissue extracts encapsulating intact self-antigens (e.g., OVA-encapsulated PLG particles prevent OVA / alum-induced AAD); and the regimen is antigen-specific without bystander suppression, safe, highly effective, and can induce unresponsiveness in both effector T cells (Th1, Th2, Th17, and CD8) involved in epitope expansion and naive T cells.

[0404] Synthetic biodegradable particles and liposomes can achieve ease of manufacturing, widespread availability of therapeutic agents, and increase the number of potential treatment sites. To this end, we have engineered surface-functionalized biodegradable poly(lactide-co-glycolide) particles with a high density of surface carboxylate groups using the surfactant poly(ethylene-alt-maleic anhydride).

[0405] We have also developed surface functionalized liposomes using a 30:30:40 ratio of phosphatidylcholine:phosphatidylglycerol:cholesterol.

[0406] We have further engineered these particles to contain soluble ovalbumin within the core, thereby circumventing the chemical contamination and purity issues that plague surface binding of peptides or proteins. These surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within the core effectively prevent disease onset and thus induce immune tolerance in the Balb / c ovalbumin / alum-induced AAI mouse allergic asthma model. Peptides or proteins conjugated to carboxylate functionalized poly(lactide-co-glycolide) particles using EDC are attached in an indiscriminate manner, resulting in antigen aggregates and particle-antigen-particle aggregates that are difficult to characterize and purify into a homogeneous population.

[0407] We have generated a homogeneous population of surface functionalized poly(lactide-co-glycolide) particles containing soluble ovalbumin within the core that does not require surface binding of antigen.

[0408] We have further demonstrated that the core contains soluble PLP 139-151 And biodegradable liposomes functionalized with high density of negatively charged groups on the surface of SJL / J PLP 139-151 / CFA-induced R-EAE mouse multiple sclerosis model effectively induced immune tolerance.

[0409] The liposomes and poly(lactide-co-glycolide) particles of the present invention provide many advantages. These advantages include:

[0410] 1) Biodegradable particles will not last long in the body, and the time to complete degradation can be controlled.

[0411] 2) Particles and liposomes can be functionalized to facilitate internalization without cell activation. To this end, we have loaded phosphatidylserine into PLG microspheres.

[0412] 3) Particles and liposomes can also be designed to contain targeting ligands for specific cell populations.

[0413] 4) Anti-inflammatory cytokines such as IL-10 and TGF-β may also be included to limit the activation of cell types that internalize the particles and promote the induction of tolerance via anergy and / or deletion and activation of regulatory T cells.

[0414] From multiple perspectives, this combination of functionality of particles or liposomes can be targeted for tolerance induction, thus providing a significant advance for designer particles relative to polystyrene particles. Potential clinical applications of this tolerance induction technology include:

[0415] (1) T cell and antibody mediated autoimmune diseases (e.g. multiple sclerosis, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.) - Tolerance will be induced using particles complexed with relevant autoantigens driving the specific autoimmune disease

[0416] (2) Food and lung allergies, skin allergies, and asthma - Tolerance will be induced using particles complexed with specific foods that cause allergic reactions (e.g., peanut protein, etc.), injected substances (e.g., bee venom protein, etc.), or inhaled substances (e.g., ragweed pollen protein, pet dander protein, etc.)

[0417] (3) Transplant rejection - Tolerance will be induced against transplant antigens on the donor organ or cells before organ transplantation to prevent recipient rejection

[0418] (4) Enzyme replacement therapy - Tolerance is induced against the enzyme that the patient with the genetic defect cannot produce, thereby preventing the patient from developing a neutralizing antibody response to the recombinantly produced enzyme administered to treat his or her specific defect.

[0419] Embodiment 23

[0420] The most effective particles for inducing tolerance were negatively charged and had an average diameter of 500 nm.

[0421] The key particle parameters for inducing tolerance are the size and charge of the composition. Fig.40 As shown in A and B, the charge of the particles affects the efficiency of tolerance induction. Comparison of EAE mice treated with OVA conjugated particles with a charge of -25mv or -60mv found that the composition containing particles with a charge of -60mv was more effective in inducing tolerance than those with a charge of -25mv. Mice were treated with TIMPs (tolerogenic immune modifying particles) with a charge of -60mv or -25mv. 323-339 -TIMP -60mv 、OVA 323-339 -PLGA -25mv 、PLP 139-151 -TIMP -60mv or PLP 139-151 -PLGA -25mv (all antigens were encapsulated) were treated and scored for clinical disease. Panel (A) shows the mean clinical score, while panel (B) shows the mean cumulative score for EAE animals.

[0422] The negative charge on the particles affects the ability of the particles to interact with the MARCO scavenger receptor. Figure 41 shows that the charge of the immune modified particles is important for targeting the immune modified particles to antigen presenting cells. Wild type or MARCO+ / - animals were treated with PS-IMP or vehicle. The results show that particles with reduced negative charge are less effective because they interact less with scavenger receptors (such as MARCO) with positively charged collagen-like domains.

[0423] In addition to charge, the size and composition of biodegradable TIMPs also influence the induction of tolerance. Fig.42 As shown in A, the most effective particles for inducing tolerance in the EAE model are those with an average diameter of approximately 500 nm. 323-339 -PSB, 100nm PLP 139-151 -PSB, 500nm PLP 139-151 -PSB, 1.75μm PLP 139-151 -PSB or 4.5μm PLP 139-151 -PSB treatment, and clinical disease scoring. PLGA carriers have slow release kinetics over 1 month, and changing the polymer ratio can affect the release of particles. Tolerance requires rapid particle uptake and clearance / degradation. Since a lactide:glycolide ratio of more than 50:50 slows the degradation rate, the particles of the present invention are 50:50 lactide:glycolide in one embodiment. Fig.42 B shows that the particles are rapidly destroyed.

[0424] In addition to the charge and average diameter of the TIMPs, antigens encapsulated within the particles outperformed particles coupled to the antigen in allergy models. In allergy models, coupled nanoparticles have a tendency to cause allergic reactions and are therefore not effective therapies. Fig.43 As shown in Figure 1, TIMP with a charge of -60 mV was effective in a mouse allergy model. Animals were exposed to OVA as an allergen and then treated with sham-PLG or TIMP particles, PLG with OVA or TIMP particles or no treatment. Figure (A) shows that OVA-PLG particles failed to reduce TH2 responses in allergy. Figure (B) shows that TIMP PEMA-60mv Figure (C) shows that TIMP PEMA-60mv Inhibit recall response.

[0425] Embodiment 24

[0426] Single emulsion synthesis of surface functionalized poly(lactide-co-glycolide) particles encapsulating antigens

[0427] The polypeptide antigen can be incorporated into the poly(lactide-co-glycolide) particles using a double emulsion method (see Example 21), however, the inventors have found that when incorporating more hydrophobic polypeptides such as gliadin, it is best to incorporate the antigen into the particles by a single emulsion method using a solvent.

[0428] Poly(lactide-co-glycolide) particles with carboxylate end groups, a 50:50 D,L-lactide:glycolide ratio, and an intrinsic viscosity of 0.18 dl / g in hexafluoro-2-propanol were used to form particles containing gliadin. Poly(lactide-co-glycolide) particles containing gliadin in the core and surface functionalized with a high density of carboxylate groups were synthesized using a single emulsion-solvent evaporation method as follows:

[0429] 1. Dissolve five milliliters of gliadin and 200 mg of PLG in 50 μL of trifluoroacetic acid (TFA), 700 μL of dimethyl sulfoxide, and 1250 μL of dichloromethane (DCM).

[0430] 2. The resulting mixture was added dropwise to 4 mL of 1% w / v PEMA aqueous solution and sonicated at 100% amplitude for 30 seconds.

[0431] 3. The resulting emulsion was poured into 200 mL of 0.5% w / v PEMA aqueous solution under stirring and stirred for 12 hours to allow DCM to evaporate completely.

[0432] 4. The particles were then washed three times in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). Alternatively, ddHO can be used. 2 O Wash the pellet.

[0433] 5. The purified particles were resuspended in water containing 4% w / v sucrose and 3% w / v D-mannitol, gradually frozen to -80°C and lyophilized to dryness.

[0434] While specific embodiments of the present invention have been described and illustrated, these embodiments should be considered merely illustrative of the invention and not limiting of the invention, which should be construed in accordance with the appended claims.

[0435] All patents, applications, and other references cited herein are incorporated by reference in their entirety.

[0436] The present invention relates to the following technical solutions:

[0437] 1. A composition comprising: an antigen coupled to a carrier particle having a negative zeta potential.

[0438] 2. The composition of embodiment 1, wherein the particles have a zeta potential of about -100 mV to about 0 mV.

[0439] 3. The composition of embodiment 2, wherein the particles have a zeta potential of about -50 mV to about -40 mV.

[0440] 4. The composition of embodiment 1, wherein the particles are copolymers having a molar ratio of about 80:20 to about 100:0.

[0441] 5. The composition of embodiment 1, wherein the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0442] 6. The composition of embodiment 1, wherein the particles have a diameter between about 0.1 μm and about 10 μm.

[0443] 7. The composition of embodiment 6, wherein the particles have a diameter between about 0.3 μm and about 5 μm.

[0444] 8. The composition of embodiment 7, wherein the particles have a diameter between about 0.5 μm and about 3 μm.

[0445] 9. The composition of embodiment 8, wherein the particles have a diameter between about 0.5 μm and about 1 μm.

[0446] 10. The composition of embodiment 9, wherein the particles have a diameter of about 0.5 μm.

[0447] 11. The composition of embodiment 1, wherein the antigen is coupled to the surface of the carrier particle.

[0448] 12. The composition of embodiment 1, wherein the antigen is encapsulated within the carrier particle.

[0449] 13. A composition according to embodiment 1 or 12, wherein the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, an enzyme for enzyme replacement therapy, or an allergen.

[0450] 14. The composition of embodiment 13, wherein the antigen comprises at least a portion of a protein selected from the group consisting of myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic β-cell antigen, insulin, glutamate decarboxylase (GAD), type II collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, gliadin, insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), human tropomyosin isotype 5, bahia grass pollen (BaGP), peach allergen Pru p 3, αs 1-casein milk allergen, Apig1 celery allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 Hazelnut allergen, myelin-associated glycoprotein, aquaporin, type IV collagen α3 chain, ovalbumin egg allergen, recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog α, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), ceredase, arabinocerebrosidase (Alglucerase), imiglucerase (Cerezyme), imiglucerase (Imiglucerase), glucocerebrosidase (Elelso), taliglucerase α (taliglucerase alfa), Fabrazyme, Agalsidase beta, Aldurazyme, L-iduronidase, α-glucosidase (Myozyme), acid glucosidase, Elaprase, iduronate-2-sulfatase, Naglazyme, arylsulfatase B, or N-acetylgalactosamine-4-sulfatase.

[0451] 15. The composition according to embodiment 13, wherein the antigens include myelin basic protein, acetylcholine receptor, endogenous antigen, myelin oligodendrocyte glycoprotein, pancreatic β-cell antigen, insulin, proinsulin, IGRP, glutamate decarboxylase (GAD), type II collagen, human cartilage gp39, fp130-RAPS, proteolipid protein, fibrin, small nucleolar protein, thyroid stimulating factor receptor, histone, glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, A-gliadin, gliadin, human tropomyosin isotype 5, bahia grass pollen (BaGP), peach allergen Pru p 3, αs 1-casein milk allergen, Apig1 celery allergen, Bere1 Brazil nut allergen, B-lactoglobulin milk allergen, bovine serum albumin, Cor a 1.04 Hazelnut allergen, myelin-associated glycoprotein, aquaporin, type IV collagen α3 chain, ovalbumin egg allergen, recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog α, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), ceredase, arabinocerebrosidase (Alglucerase), imiglucerase (Cerezyme), imiglucerase (Imiglucerase), glucocerebrosidase (Elelso), taliglucerase α (taliglucerase alfa), Fabrazyme, Agalsidase beta, Aldurazyme, L-iduronidase, α-glucosidase (Myozyme), acid glucosidase, Elaprase, iduronate-2-sulfatase, Naglazyme, arylsulfatase B, or N-acetylgalactosamine-4-sulfatase.

[0452] 16. A composition according to embodiment 1, wherein the antigen is coupled to the particle via a conjugated molecule.

[0453] 17. A composition according to embodiment 16, wherein the conjugated molecule is ethylene carbodiimide (ECDI).

[0454] 18. The composition of embodiment 1, wherein the particles are biodegradable.

[0455] 19. The composition of embodiment 1, wherein the particles are surface functionalized.

[0456] 20. The composition of embodiment 19, wherein the particles are carboxylate surface functionalized.

[0457] 21. The composition according to embodiment 1, further comprising a pharmaceutically acceptable carrier.

[0458] 22. A method of inducing antigen-specific tolerance in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0459] 23. The method of embodiment 22, wherein the administering is performed to treat or prevent a disease or disorder.

[0460] 24. The method of embodiment 23, wherein the disease or condition is selected from the group consisting of: autoimmune disease, lysosomal storage disease, enzyme deficiency, inflammatory disease, allergy, transplant rejection and hyperimmune response.

[0461] 25. The method of embodiment 24, wherein the disease or condition is selected from: multiple sclerosis, type 1 diabetes, asthma, food allergies, environmental allergies, celiac disease, inflammatory bowel disease including Crohn's disease and ulcerative colitis, mucopolysaccharidoses, gangliosidosis, low alkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher's disease, Fabry disease, Hurler's disease, Pompe's disease, Hunter's disease, Mar-La disease, and a condition caused by the antigen reducing an overreaction to the antigen in the subject.

[0462] 26. The method of embodiment 22, wherein the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0463] 27. The method of embodiment 26, wherein the particles are poly(lactic-co-glycolic acid) particles.

[0464] 28. The method of embodiment 27, wherein administration of the poly(lactic-co-glycolic acid) particles results in less allergic reaction than administration of polystyrene particles.

[0465] 29. The method of embodiment 22, wherein the composition is administered intravenously.

[0466] 30. A method for preparing immune modified particles having a negative zeta potential, the method comprising: contacting an immune modified particle precursor with a buffer solution under conditions effective to form the immune modified particles having a negative zeta potential.

[0467] 31. A method according to embodiment 30, wherein the immune modified particle precursor is formed by copolymerization.

[0468] 32. A method according to embodiment 30, wherein the buffer solution has an alkaline pH.

[0469] 33. The method of embodiment 30, wherein the buffer solution is sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate or lithium dihydrogen phosphate.

[0470] 34. A composition comprising an antigen encapsulated within the core of a surface functionalized liposome.

[0471] 35. A composition according to embodiment 34, wherein the liposomes are composed of phosphatidylcholine:phosphatidylglycerol:cholesterol in a ratio of 30:30:40.

[0472] 36. A composition according to embodiment 34, wherein the antigen comprises an autoimmune antigen, an antigen expressed on a tissue to be transplanted into a subject, or an allergen.

[0473] 37. A composition according to embodiment 13, wherein the antigen comprises one or more epitopes selected from those in Table 2 or Table 3.

[0474] 38. A method of reducing suppressive neutrophil accumulation in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0475] 39. A method according to embodiment 38, wherein the subject has cancer.

[0476] 40. The method of embodiment 38, wherein the particles are administered subcutaneously.

[0477] 41. A method of enhancing tissue regeneration in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0478] 42. A method according to embodiment 41, wherein the particles enhance epithelial cell regeneration in patients with colitis.

[0479] 43. A method according to embodiment 41, wherein the particles enhance myelin regeneration in patients with multiple sclerosis.

[0480] 44. The composition of embodiment 14, wherein the antigen comprises at least a portion of myelin basic protein.

[0481] 45. A composition according to embodiment 44, wherein the antigen comprises at least a portion of SEQ ID NO:4975 or SEQ ID NO:4976.

[0482] 46. ​​A composition according to embodiment 14, wherein the antigen comprises at least a portion of myelin oligodendrocyte glycoprotein.

[0483] 47. A composition according to embodiment 46, wherein the antigen comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:4978.

[0484] 48. A composition according to embodiment 14, wherein the antigen comprises at least a portion of insulin.

[0485] 49. A composition according to embodiment 48, wherein the antigen comprises at least a portion of SEQ ID NO:4981.

[0486] 50. The composition of embodiment 14, wherein the antigen comprises at least a portion of glutamate decarboxylase (GAD).

[0487] 51. A composition according to embodiment 50, wherein the antigen comprises at least a portion of SEQ ID NO:4982.

[0488] 52. A composition according to embodiment 14, wherein the antigen comprises at least a portion of a proteolipid protein.

[0489] 53. A composition according to embodiment 52, wherein the antigen comprises at least a portion of SEQ ID NO:4977.

[0490] 54. The composition of embodiment 14, wherein the antigen comprises at least a portion of gliadin.

[0491] 55. The composition of embodiment 54, wherein the antigen comprises at least a portion of SEQ ID NO: 4983-4985 or 5136-5140.

[0492] 56. The composition of embodiment 14, wherein the antigen comprises at least a portion of a water channel protein.

[0493] 57. A composition according to embodiment 56, wherein the antigen comprises at least a portion of SEQ ID NO:4979.

[0494] 58. A composition according to embodiment 14, wherein the antigen comprises at least a portion of myelin-associated glycoprotein.

[0495] 59. A composition according to embodiment 58, wherein the antigen comprises at least a portion of SEQ ID NO:4980.

[0496] 60. A composition according to embodiment 14, wherein the antigen comprises at least a portion of the type IV collagen α3 chain.

[0497] 61. A composition according to embodiment 60, wherein the antigen comprises at least a portion of SEQ ID NO:5017.

[0498] 62. A method of treating celiac disease in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0499] 63. The method of embodiment 62, wherein the antigen is gliadin or a gliadin epitope.

[0500] 64. A method according to embodiment 63, wherein the antigen is one or more antigens selected from SEQ ID NO: 1295-1724, SEQ ID NO: 1726-1766 and SEQ ID NO: 4986-5140.

[0501] 65. The method of embodiment 63, wherein the antigen is gliadin and the antigen-coupled particles have a post-synthesis average size of about 600-1500 nanometers and a post-synthesis average charge of about -30 to about -80 mV.

[0502] 66. The method of embodiment 65, wherein the particles have a post-synthesis average size of about 600-1200 nanometers and a post-synthesis average charge of about -40 to about -70 mV.

[0503] 67. A method according to embodiment 66, wherein the particles have a post-synthesis average size of about 600 microns and a post-synthesis average charge of about -50 mV.

[0504] 68. The method of any one of embodiments 62-67, wherein the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0505] 69. A method of treating diabetes in a subject, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0506] 70. The method of embodiment 69, wherein the antigen is insulin, proinsulin, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), or an epitope derived from insulin, proinsulin, or IGRP.

[0507] 71. The method of embodiment 69, wherein the antigen is one or more antigens selected from SEQ ID NO: 1767-1840, SEQ ID NO: 1842-1962, SEQ ID NO: 1964-2027, SEQ ID NO: 2029-2073, SEQ ID NO: 2075-2113, SEQ ID NO: 2115-2197, SEQ ID NO: 2199-2248, SEQ ID NO: 2250-2259, SEQ ID NO: 2261-2420, SEQ ID NO: 2422-2486 and SEQ ID NO: 2489-2505.

[0508] 72. The method of embodiment 70, wherein the antigen is insulin and the antigen-coupled particles have a post-synthesis average size of about 300-800 nanometers and a post-synthesis average charge of about -30 to about -70 mV.

[0509] 73. The method of embodiment 72, wherein the particles have a post-synthesis average size of about 350-600 nanometers and a post-synthesis average charge of about -40 to about -60 mV.

[0510] 74. A method according to embodiment 73, wherein the particles have a post-synthesis average size of about 500 nanometers and a post-synthesis average charge of about -50 mV.

[0511] 75. The method of embodiment 70, wherein the antigen is proinsulin and the antigen-coupled particles have a post-synthesis average size of about 300-800 nanometers and a post-synthesis average charge of about -30 to about -70 mV.

[0512] 76. The method of embodiment 75, wherein the particles have a post-synthesis average size of about 400-600 nanometers and a post-synthesis average charge of about -40 to about -60 mV.

[0513] 77. The method of embodiment 76, wherein the particles have a post-synthesis average size of about 570 nanometers and a post-synthesis average charge of about -45 mV.

[0514] 78. The method of embodiment 70, wherein the antigen is IGRP and the antigen-coupled particles have a post-synthesis average size of about 300-800 nanometers and a post-synthesis average charge of about -30 to about -70 mV.

[0515] 79. The method of embodiment 78, wherein the particles have a post-synthesis average size of about 400-700 nanometers and a post-synthesis average charge of about -40 to about -60 mV.

[0516] 80. The method of embodiment 79, wherein the particles have a post-synthesis average size of about 600 nanometers and a post-synthesis average charge of about -40 mV.

[0517] 81. The method of any one of embodiments 69-80, wherein the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0518] 82. A method of treating a subject receiving enzyme replacement therapy, the method comprising: administering to the subject an effective amount of a composition comprising antigen-coupled particles, wherein the particles have a negative zeta potential.

[0519] 83. The method of embodiment 82, wherein the subject is receiving enzyme replacement therapy to treat a disease selected from the group consisting of hemophilia, hemophilia A, hemophilia B, von Willebrand disease, Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Hunter disease, mucopolysaccharidoses, gangliosidosis, hypoalkaline phosphatase, cholesterol ester storage disease, hyperuricemia, growth hormone deficiency, renal anemia, and Mar-La disease.

[0520] 84. A method according to embodiment 82, wherein the antigen-coupled particles comprise one or more enzymes selected from the group consisting of recombinant human coagulation factor VIII (Advate), antihemophilic factor, human gene recombinant antihemophilic factor (Kogenate), recombinant antihemophilic factor (Eloctate), recombinant factor VIII Fc fusion protein, recombinant antihemophilic factor VIII (Refacto), Novo VIIa, recombinant factor VII, eptacog alfa, human gene recombinant antihemophilic (FVIII) factor (Helixate), Monanine, coagulation factor IX, von Willebrand factor and factor VIII concentrate (Wilate), Ceredase, Alglucerase, Cerezyme, Imiglucerase, glucocerebrosidase (Elelso), taliglucerase alfa, Fabrazyme, β-galactosidase (Agalsidase beta), laronidase (Aldurazyme), l-iduronidase, alpha-glucosidase (Myozyme), acid glucosidase, iduronic acid sulfatase (Elaprase), iduronate-2-sulfatase, sulfatase (Naglazyme), arylsulfatase B, and N-acetylgalactosamine-4-sulfatase.

[0521] 85. The method of any one of embodiments 82-84, wherein the particles are polystyrene particles, carboxylated polystyrene particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) particles.

[0522] 86. The composition of embodiment 4 or 5, wherein the particles are poly(lactic-co-glycolic acid) particles and have a polylactic acid:polyglycolic acid copolymer ratio of about 50:50.

[0523] 87. The method of any one of embodiments 26, 27, 68, 81 or 85, wherein the particles are poly(lactic-co-glycolic acid) particles and have a polylactic acid:polyglycolic acid copolymer ratio of about 50:50.

[0524] 88. The composition of embodiment 86, further comprising PEMA.

[0525] 89. The composition of embodiment 88, wherein the PEMA is present at about 0.1% to about 2.0%.

[0526] 90. The method of embodiment 87, wherein the particles further comprise PEMA.

[0527] 91. The method of embodiment 90, wherein the PEMA is present at about 0.1% to about 2.0%.

Claims

1. Negatively charged carrier particles comprising one or more encapsulated antigens or epitope peptides thereof, wherein the particles are poly(lactic-co-glycolic acid) (PLGA) particles having a negative zeta potential of -100 mV to -30 mV and a diameter of 400 to 800 nm, and wherein the antigen is pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2).

2. The particle according to claim 1, wherein the negative zeta potential of the particle is obtained by surface functionalization.

3. The particle of claim 2, wherein the surface functionalization comprises carboxylation.

4. The particle of claim 1, wherein the particle has a zeta potential of -80 mV to -30 mV. The particle according to claim 1 , further comprising a pharmaceutically acceptable carrier or excipient.

6. A composition for inducing antigen-specific tolerance in a subject with primary biliary cirrhosis, comprising an effective amount of negatively charged poly(lactic-co-glycolic acid) (PLGA) particles having a negative zeta potential of -100 mV to -30 mV and a diameter of 0.2 μm to 2 μm, wherein the particles comprise one or more encapsulated antigens or epitope peptides thereof, wherein the particles have a negative zeta potential, wherein the antigen is pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2).

7. The composition of claim 6, wherein the composition is administered intravenously.

8. The composition of claim 6, wherein the particles have a negative zeta potential of -80 mV to -30 mV.

9. The composition of claim 6, wherein the particles have a diameter of 400 to 800 nm.

10. The composition of claim 6, wherein the composition is administered intravenously.

11. The composition of claim 6, wherein the composition is lyophilized.

12. The composition according to claim 11, wherein the particles are lyophilized and reconstituted to obtain a reconstituted pharmaceutical composition comprising the particles.

13. Use of negatively charged carrier particles comprising one or more encapsulated antigens or epitope peptides thereof in the preparation of a medicament for treating primary biliary cirrhosis, wherein the particles are poly(lactic-co-glycolic acid) (PLGA) particles having a negative zeta potential of -100 mV to -30 mV and a diameter of 400 to 800 nm, and wherein the antigen is pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2).

14. The use according to claim 13, wherein the antigen is pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2).

15. Use according to claim 13, wherein the negative zeta potential of the particles is obtained by surface functionalization.

16. Use according to claim 15, wherein the surface functionalization comprises carboxylation.

17. The use according to claim 13, wherein the particles have a zeta potential of -80 mV to -30 mV.

18. The use according to claim 13, wherein the particles further comprise a pharmaceutically acceptable carrier or excipient.

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