Compositions and methods for treating autoimmune disorders

Abstract

The present invention provides compositions and methods for treating autoimmune conditions. [Solution] The present invention relates to a composition comprising nanoparticles associated with multiple tolerogenic antigens (e.g., 1 to 30 tolerogenic 5 antigens per nanoparticle) in a manner that promotes potent immune tolerance upon administration to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder (e.g., MS or celiac disease)). The present invention further relates to a method for utilizing the above nanoparticles to treat an autoimmune disorder (e.g., MS or celiac disease).

Description

Detailed description of the invention

[0001] [Sequence Listing] This application includes an electronically submitted sequence listing in ASCII format, the entire listing of which is incorporated herein by reference. The ASCII copy, created on 17 July 2020, is named 37921-601_ST25.txt and is 203,000 bytes in size.

[0002] [Technical Field] The present invention relates to nanoparticles associated with multiple tolerogenic antigens (e.g., 1 to 30 tolerogenic antigens per nanoparticle (e.g., 4 to 30, 5 to 30, 6 to 30, 7 to 30, or 8 to 30 tolerogenic antigens) in a manner that promotes potent immune tolerance upon administration to subjects (e.g., human subjects suffering from or at risk of suffering from autoimmune disorders (e.g., MS or celiac disease)). The present invention further relates to a method for synthesizing such nanoparticles associated with tolerogenic antigens involved in celiac disease, and to a system and method for utilizing such tolerogenic antigen-modified nanoparticles for the treatment of celiac disease.

[0003] [Background technology] Autoimmune disorders are diseases in which the body's immune system attacks its own normal tissues, organs, or other components due to an unexplained abnormality in the immune system. These autoimmune disorders are systemic diseases that can occur in almost any part of the body, including the nervous system, digestive system, endocrine system, skin, skeletal system, and vascular tissue. It is known that approximately 5-8% of the world's population suffers from autoimmune disorders, but due to limitations in our understanding of autoimmune disorders and diagnostic methods, reported prevalence rates are lower than the actual level.

[0004] Improvements in compositions and methods for treating autoimmune conditions are needed.

[0005] Celiac disease is an autoimmune disorder caused by gluten (specifically, proteins classified as gliadin and glutenin found in cereals of the genus *Tetragonia*, including wheat, rye, and barley). Approximately 3 million people in the United States alone suffer from celiac disease, yet 83% of those living with it remain undiagnosed. The number of people with celiac disease has doubled approximately every 20 years since the 1950s, highlighting the growing need for effective diagnostic and treatment methods. Currently, there are no treatments for celiac disease approved by the U.S. Drug Administration. Therefore, individuals with celiac disease must adhere to a strict gluten-free diet. However, given that even a small amount of gluten—50 mg per day (equivalent to 1 / 70th of a slice of bread)—can cause intestinal damage in people with celiac disease, adhering to a strict gluten-free diet can be challenging. Furthermore, some individuals are diagnosed with refractory celiac disease. This means that despite strict adherence to a gluten-free diet, symptoms and damage to the small intestine persist, indicating that the disease is unresponsive.

[0006] Characteristic symptoms associated with celiac disease include diarrhea, abdominal distension, gas, fatigue, weight loss, iron deficiency anemia, constipation, itchy rash, and depression. If left untreated, celiac disease can lead to the development of osteoporosis, anemia, thyroid disorders, and certain types of cancer. While the etiology of celiac disease is complex, it is known to be primarily caused by the presence of a 33-amino acid gliadin polypeptide that is naturally formed during the gastrointestinal digestion of gliadin. In the presence of gliadin, the enzyme tissue transglutaminase 2 (TG2) deamidates a specific glutamine residue of the gliadin peptide to form glutamate. If an individual harbors the human leukocyte antigen (HLA) DQ2 or DQ8 haplotype, antigen-presenting cells expressing HLA-DQ2 or HLA-DQ8 likely have a greater affinity for the deamidated peptide and bind to deamidated gliadin to form the HLA-DQ2 / 8-gliadin complex. Next, this complex stimulates B cells with host-gluten-specific CD4 + It can activate T cells, leading to the production of anti-gliadin and anti-TG2 antibodies. Furthermore, T cell activation triggers cytokine production, causing inflammation and damage to the small intestine. In addition, the HLA-DG2 / 8-gliadin complex can increase IFNγ production, potentially leading to intestinal mucosal lesions. To combat the life-altering symptoms and damaging effects of celiac disease, there remains a need to develop treatments that effectively manage this autoimmune disease.

[0007] [Summary of the Invention] This disclosure describes a composition that, upon administration to a subject (e.g., a human subject with or at risk of developing an autoimmune disorder (e.g., MS or celiac disease)), promotes a potent immune tolerance to antigens associated with the autoimmune disorder, comprising a collection of tolerogenic antigens (e.g., 1 to 30 tolerogenic antigens per nanoparticle (e.g., 8 to 30, e.g., 9 to 15, 12 to 18, 15 to 22, 18 to 25, 20 to 27, 22 to 28, or 25 to 30 tolerogenic antigens) and The present invention provides associated nanoparticles. Autoimmune disorders include, for example, multiple sclerosis (MS), celiac disease, rheumatoid arthritis, diabetes (e.g., type 1 diabetes), thyroid autoimmune diseases (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye disease and dermal disease, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune gastritis, and pernicious anemia. The conditions include celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and its variants, bullous pemphigoid, dermatitis herpetiformis of Duhring, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodbasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease. The present invention further relates to a method for synthesizing nanoparticles associated with toxicogenic antigens involved in celiac disease, and to a system and method for utilizing such toxicogenic antigen-modified nanoparticles for the treatment of celiac disease.

[0008] In a first embodiment, the disclosure provides a composition comprising sHDL nanoparticles associated with a plurality of tolerogenic antigens, wherein the resulting composition can promote potent immune tolerance to antigens associated with autoimmune diseases upon administration to a subject, the sHDL nanoparticles comprising a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. Autoimmune disorders include, for example, MS, celiac disease, rheumatoid arthritis, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye disease and dermal disease, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, and skin herpetiformis of Duhring. These include inflammation, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodbasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease. The composition is not limited to specific nanoparticles. In some embodiments, the average diameter of the nanoparticles is 6-500 nm (e.g., 7-20 nm, 21-50 nm, 51-100 nm, 101-200 nm, 201-300 nm, 301-400 nm, and 401-500 nm). In some embodiments, the average particle size of sHDL nanoparticles is 6–70 nm (e.g., 7–10 nm, 11–20 nm, 21–30 nm, 31–40 nm, 41–50 nm, 51–60 nm, and 61–70 nm).

[0009] In some embodiments, the phospholipids are 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-dimiristoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-sn-glycero-3-phosphocholine; 1,2-diarachidoyl-sn-glycero-3-phosphocholine; 1,2-dibehenoyl-sn-glycero-3-phosphocholine; 1,2-dirig Celloyl-sn-glycero-3-phosphocholine; 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine; 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitreoyl-sn- Glycero-3-phosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine); 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine (1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine); 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (1,2-dipetroselenoyl-sn-glycero-3-phosphocholine); 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); 1,2-dielaidoyl-sn-glycero-3-phosphocholine (1,2-dielaidoyl-sn-glycero-3-phosphocholine); 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (1, 2-dieicosenoyl-sn-glycero-3-phosphocholine; 1,2-dinervonoyl-sn-glycero-3-phosphocholine; 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine; 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine;1,2-Dipentadecanoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitreoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dieridoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine; Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3 -(2-pyridyldithio)propionate]; 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanolamine; 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butylamide]; 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butylamide]; 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butylamide] [Cyl(9Z-Octadecenoyl)-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidyl-ethanolamine; N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidyl-ethanolamine; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, distearoyl; N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, Dimyristoy; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, Dioleoyl;N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; phosphatidylcholine; phosphatidylinositol; phosphatidylserine; phosphatidylethanolamine; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, distearoyl; N-(succinimidyloxyglutaryl)-L Selected from the group consisting of -α-phosphatidylethanolamine, dioleoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succinimidyloxyglutaryl)aminopropyl, and polyethylene glycol-carbamyl distearoylphosphatidylethanolamine; and N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoylethanolamine.

[0010] In some embodiments, the components of HDL apolipoprotein are apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein A-II xxx (apoA-II-xxx), apolipoprotein A4 (apoA4), apolipoprotein Cs (apoCs), apolipoprotein E (apoE), apolipoprotein AI milano (ApoA-I-milano), apolipoprotein AI paris (ApoA-I-paris), apolipoprotein M (apoM), HDL apolipoprotein mime, preproapoliprotein, preproApoA-I, proApoA I, preproApoA-II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, preproApoA I milanoproApoA-I milano preproApoA-I paris proApoA-I Paris and a selection from the group consisting of mixtures thereof.

[0011] In some embodiments, the apolipoprotein mimetics are SEQ ID NOs: 1-336, as well as WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 346) 7) PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 3 54), QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASL L (SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),It is described by one of the following codes: PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0012] In some embodiments, the tolerogenic antigens are tolerogenic antigens having lengths of 3 to 50 amino acids (for example, lengths of approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 amino acids).

[0013] In some embodiments, the multiple tolerogenic antigens are tolerogenic antigens comprising polypeptides containing one of the nucleic acid sequences SEQ ID NOs: 375 to 796.

[0014] In some embodiments, the tolerogenic antigens are human allograft antigens. In some embodiments, the human allograft antigens are selected from subunits of various MHC class I and MHC class II haplotype proteins, as well as from single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Duffy, and Ss.

[0015] In some embodiments, multiple tolerogenic antigens are specific to type 1 diabetes. In some embodiments, the tolerogenic antigens for type 1 diabetes are selected from insulin, proinsulin, preproinsulin, glutamate decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2β (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-enteropancreatic / pancreatic-associated protein, S100β, glial fibrillary acidic protein, regenerative gene II, pancreaticoduodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5.

[0016] In some embodiments, the tolerogenic antigen is used in rheumatoid arthritis, multiple sclerosis, diabetes mellitus, autoimmune thyroid diseases, thyroid-associated eye disease, thyroid-associated skin disorders, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and its variants, bullous pemphigoid, dermatitis herpetiformis, acquired epidermolysis bullosa, systemic sclerosis, and other conditions. It is specific to one or more autoimmune disorders among connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodbasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

[0017] In some embodiments, the multiple tolerogenic antigens include thyroglobulin (TG), thyroid peroxidase (TPO), thyroid-stimulating hormone receptor (TSHR), sodium-iodine cotransporter (NIS), megalin, thyroid autoantigens including TSHR, insulin-like growth factor 1 receptor, calcium-sensitive receptor, 21-hydroxylase, 17α-hydroxylase, as well as P450 side-chain cleavage enzyme (P450scc), ACTH receptor, P450c21, P450c17, FSH receptor, α-enolase, pituitary-specific protein factor (PGSF) 1a and 2, as well as type 2 iodothyronine deiodinase, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, collagen II, H + ,K +-ATPase, tissue transglutaminase and gliadin, tyrosinase, tyrosinase-related proteins 1 and 2, acetylcholine receptor, desmocolin 3, 1 and 4, pemphaxin, desmocolin, plakoglobin, perplakin, desmoplakin, acetylcholine receptor, BP180, BP230, plectin, laminin 5, endomysium, tissue transglutaminase, collagen VII, matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, PDGF receptor, Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere protein, topoisomerase I, nucleolar protein, RNA polymerase I, II and III, PM-Slc, fibrillarin, B23, U1snRNP, nuclear antigens SS-A and SS-B, hodrin, poly(ADP-ribose) polymerase, topoisomerase, nucleoproteins including SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins, double-stranded DNA, glomerular basement membrane proteins including collagen IV, cardiac myosin, aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, liver cytochrome P450 P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensitive receptor protein, and type 1 interferon It contains one or more tolerogenic antigens selected from interferon α, β, and ω.

[0018] The above compositions are not limited to specific tolerogenic antigens. In some embodiments, the tolerogenic antigen is an exogenous antigen in which the patient develops an undesirable immune response. In some embodiments, multiple tolerogenic antigens are specific to celiac disease. In some embodiments, the tolerogenic antigen is selected from gliadin, glutenin, and fragments thereof that can induce an immune response. In some embodiments, the tolerogenic antigen is selected from gliadin (e.g., α-, γ-, and ω-gliadin) or fragments thereof. In some embodiments, the tolerogenic antigen is selected from the group consisting of α, γ, and ω-gliadin or fragments thereof. In some embodiments, the immunotolerogenic antigen comprises a polypeptide having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to any one of the polypeptide sequences of SEQ ID NOs. 375-580. In some embodiments, the tolerogenic antigen comprises a polypeptide having at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) sequence identity to any one polypeptide sequence from SEQ ID NOs. 375 to 580. In some embodiments, the tolerogenic antigen comprises a polypeptide having any one polypeptide sequence from SEQ ID NOs. 375 to 580. In some embodiments, the tolerogenic antigen comprises two or more (e.g., two, three, four, five, and six) polypeptide sequences having any one sequence from SEQ ID NOs. 375 to 580.

[0019] In some embodiments, a tolerogenic antigen is an autoantigen that a subject (e.g., a human patient) has developed or may develop an autoimmune response to. Examples include proinsulin (e.g., for subjects with or at risk of developing diabetes), collagen (e.g., for subjects with or at risk of developing rheumatoid arthritis), and myelin basic protein (e.g., for subjects with or at risk of developing multiple sclerosis). There are many proteins that are human autoimmune proteins, and the term is used in relation to various autoimmune diseases, and such proteins or the proteins that cause such diseases may be known or established by routine testing. Embodiments include testing a patient to identify an autoimmune protein, creating an antigen for use in molecular fusion, and creating immune tolerance to a protein. Embodiments include including an antigen or selecting an antigen from one or more of the following proteins: In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamate decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2β (IA-2β); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-enteropancreatic / pancreatic-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreaticoduodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5. In autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves' disease, the main antigens include thyroglobulin (TG), thyroid peroxidase (TPO), and thyrotropin receptor (TSHR); other antigens include sodium-iodine cotransporter (NIS) and megalin.In thyroid-associated ophthalmopathy and skin disorders, in addition to thyroid autoantigens including TSHR, the antigen is the insulin-like growth factor 1 receptor. In hypoparathyroidism, the main antigen is the calcium-sensing receptor. In Addison's disease, the main antigens include 21-hydroxylase, 17α-hydroxylase and P450 side-chain cleavage enzyme (P450scc); other antigens include the ACTH receptor, P450c21 and P450c17. In premature ovarian failure, the main antigens include the FSH receptor and α-enolase. In autoimmune hypothyroidism or autoimmune pituitary disease, the main antigens include pituitary-specific protein factors (PGSF) 1a and 2; other antigens are type 2 iodothyronine deiodinase. In multiple sclerosis, the main antigens include myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein. In rheumatoid arthritis, the main antigen is collagen II. In immune gastritis, the main antigen is H. + ,K +-ATPase. In pernicious anemia, the main antigen is intrinsic factor. In celiac disease, the main antigens are tissue transglutaminase and gliadin. In vitiligo, the main antigens are tyrosinase and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the main antigen is acetylcholine receptor. In pemphigus vulgaris and its variants, the main antigens are desmoglein 3, 1 and 4; other antigens include pemfaxine, desmocolin, placoglobin, perplakin, desmoplakin, and acetylcholine receptor. In bullous pemphigoid, the main antigens include BP180 and BP230; other antigens include plectin and laminin 5. In herpetiform dermatitis of Duhring, the main antigens include endomysium and tissue transglutaminase. In acquired epidermolysis bullosa, the main antigen is collagen VII. In systemic sclerosis, the main antigens include matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, and the PDGF receptor; other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere protein, topoisomerase I, nucleolar protein, RNA polymerases I, II, and III, PM-Slc, fibrillarin, and B23. In mixed connective tissue disease, the main antigen is U1snRNP. In Sjögren's syndrome, the main antigens are nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase, and topoisomerase. In systemic lupus erythematosus, the main antigens include nuclear proteins including SS-A, high-mobility group box 1 (HMGB1), nucleosomes, histone proteins, and double-stranded DNA. In Goodpasture syndrome, the main antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the main antigen is cardiac myosin.Other autoantigens identified in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, liver P450 cytochromes P450 1A2 and 2A6, SOX-9, SOX-10, calcium-sensitive receptor proteins, and type 1 interferons interferon α, β, and ω.

[0020] In some cases, a tolerogenic antigen is an exogenous antigen that causes a patient to develop an undesirable autoimmune response. An example is a food antigen. Embodiments include testing a patient to identify an exogenous antigen, creating a molecular fusion containing the antigen, and treating the patient to develop immune tolerance to the antigen or food. Examples of such foods and / or antigens are provided below. Examples include: from peanuts: conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apples: 31kda major allergen / disease-resistant protein homolog (Mal d 2) Lipid transport protein precursors (Mal d 3), major allergens Mal d 1.03D (Mal d 1): From milk: α-lactalbumin (ALA), lactotransferrin; From kiwi: actinidin (Act c 1, Act d 1), phytocystatin, tomatin-like protein (Act d 2), kiuelin (Act d 5); From mustard: 2S globulin (Sin a 1), 11S globulin (Sin a 2) Lipid transport protein (Sin a 3), profilin (Sin a 4); celery-derived: profilin (Api g 4), high molecular weight glycoprotein (Api g 5); shrimp-derived: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform; wheat and / or other cereal-derived: high molecular weight glutenin, low molecular weight glutenin, α- and γ-gliadin, hordein, secarin, avenin; strawberry-derived: major strawberry allergen Fra a 1-E (Fra a 1); banana-derived: profilin (Mus xp 1).

[0021] In some embodiments, the tolerogenic antigen is a multimeric tolerogenic antigen containing the following N-terminal-C-terminal structure: (P4-L4) n4 -(P3-L3) n3 -P2-(L1-P1) n1 In the formula, P1, P2, P3, and P4 are each independently tolerogenic antigens; L1, L3, and L4 are linkers independently of each other; and, n1, n3, and n4 are each independently either 0 or 1, and at least one of n1, n3, and n4 is 1.

[0022] In some embodiments, n1 is 1, n3 is 0, n4 is 0, Tolerogenic antigens include the following N-terminal-C-terminal structures: P2-L1-P1

[0023] In some embodiments, L1 is a peptide linker comprising 2 to 200 amino acids (e.g., 5 to 50 (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150), or 145 to 200 amino acids (e.g., 145 to 160, 155 to 170, 165 to 180, 175 to 190, or 185 to 200). In some embodiments, L1 is a peptide linker comprising glycine (G) and serine (S) residues. In some embodiments, L1 is (GS) x (GGS) x , or (GGGGS) x A peptide linker containing the amino acid sequence, where x is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, P1 and P2 each contain different tolerogenic antigens. In some embodiments, P1 and P2 each contain the same tolerogenic antigen.

[0024] In some embodiments, n1 is 1, n3 is 1, n4 is 0, and the tolerogenic antigen includes the following N-terminal-C-terminal structure: P3-L3-P2-L1-P1

[0025] In some embodiments, L1 and L3 are each independently 2-200 amino acids (e.g., 5-50 (e.g., 5-20, 15-30, 25-40, or 35-50)), 45-100 (e.g., 45-60, 55-70, 65-80, 75-90, or 85-100)), 95-150 (e.g., 95-110, 105-120, 115-130, 125-140, or 135-1) 50), or a peptide linker comprising 145-200 amino acids (e.g., 145-160, 155-170, 165-180, 175-190, or 185-200). In some embodiments, L1 and L3 are peptide linkers comprising glycine (G) and serine (S) residues independently. In some embodiments, L1 and L3 are peptide linkers comprising glycine (G) and serine (S) residues independently. x (GGS) x , or (GGGGS) x A peptide linker containing the amino acid sequence, where x is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, P1, P2, and / or P3 each contain different tolerogenic antigens. In some embodiments, P1, P2, and P3 each contain the same tolerogenic antigen.

[0026] In some embodiments, n1 is 1, n3 is 1, n4 is 1, The aforementioned tolerogenic antigen includes the following N-terminal-C-terminal structure: P4-L4-P3-L3-P2-L1-P1

[0027] In certain embodiments, L1 and L2 are each independently 2 to 200 amino acids (e.g., 5 to 50 (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150), Alternatively, it is a peptide linker containing 145-200 amino acids (e.g., 145-160, 155-170, 165-180, 175-190, or 185-200). In some embodiments, L1, L2, and L3 are each independently peptide linkers containing glycine (G) and serine (S) residues. In some embodiments, L1, L2, and L3 are each independently (GS) x (GGS) x , or (GGGGS(Sequence ID 219)) x A peptide linker containing the amino acid sequence, where x is an integer from 1 to 10. In some embodiments, P1, P2, P3, and / or P4 each contain different tolerogenic antigens. In some embodiments, P1, P2, P3, and P4 each contain the same tolerogenic antigen.

[0028] In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle includes a population of 1 to 30 (e.g., 1 to 10, 9 to 15, 12 to 18, 15 to 22, 18 to 25, 20 to 27, 22 to 28, or 25 to 30) tolerogenic antigens per nanoparticle. In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle includes a population of 6 tolerogenic antigens per particle. In other embodiments, the number of tolerogenic antigens associated with a particular nanoparticle includes a population of 8 tolerogenic antigens per particle. In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle is the same tolerogenic antigen. In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle includes 1 to 5 (e.g., 2, 3, 4, and 5) different tolerogenic antigens. In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle includes 3 to 4 different tolerogenic antigens. In some embodiments, the population of tolerogenic antigens is specific to one to three different diseases. In certain embodiments, the population of tolerogenic antigens is specific to the same disease.

[0029] In some embodiments, the population of tolerogenic antigens associated with specific nanoparticles includes (i) a first polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-588 or a biologically active fragment or variant thereof, (ii) a second polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-588 or a biologically active fragment or variant thereof, and (iii) a third polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-588 or a biologically active fragment or variant thereof.

[0030] In some embodiments, (ii) a first polypeptide population comprises the amino acid sequence of SEQ ID NO: 474 or a biologically active fragment or variant thereof; (ii) a second polypeptide population comprises any one of the amino acid sequences of SEQ ID NOs. 406 to 588 or a biologically active fragment or variant thereof; and (iii) the third polypeptide population comprises any one of the amino acid sequences of SEQ ID NOs. 406 to 588 or a biologically active fragment or variant thereof.

[0031] In some embodiments, the population of tolerogenic antigens associated with specific nanoparticles includes (i) a first polypeptide population comprising the amino acid sequence of SEQ ID NO: 474 or a biologically active fragment or variant thereof, (ii) a second polypeptide population comprising the amino acid sequence of SEQ ID NO: 475 or a biologically active fragment or variant thereof, and (iii) a third polypeptide population comprising any one of the amino acid sequences of SEQ ID NOs. 406 to 588 or a biologically active fragment or variant thereof. In some embodiments, the third polypeptide population comprises the amino acid sequence of SEQ ID NO: 476 or a biologically active fragment or variant thereof. In some embodiments, the second polypeptide population comprises the amino acid sequence of SEQ ID NO: 477 or a biologically active fragment or variant thereof, and / or the third polypeptide population comprises the amino acid sequence of SEQ ID NO: 478 or a biologically active fragment or variant thereof.

[0032] In some embodiments, the tolerogenic antigen comprises a polypeptide having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the polypeptide sequence of SEQ ID NO: 374. In some embodiments, the tolerogenic antigen comprises the polypeptide sequence of SEQ ID NO: 374. In some embodiments, the tolerogenic antigen comprises a fragment of SEQ ID NO: 373 having a length of 6 to 12 amino acid residues.

[0033] In some embodiments, the tolerogenic antigen contains an amide group at the C-terminus. In some embodiments, the tolerogenic antigen contains a pyroglutamic acid residue at the N-terminus. In some embodiments, the tolerogenic antigen contains an acetyl group at the N-terminus. In some embodiments, the tolerogenic antigen contains a pyroglutamic acid residue at the N-terminus and an amide group at the C-terminus. In some embodiments, the tolerogenic antigen contains an acetyl group at the N-terminus and an amide group at the C-terminus. In certain embodiments, the tolerogenic antigen contains an N-terminus or C-terminus modified with a linker-bound cysteine ​​residue. In some embodiments, the tolerogenic antigen contains an N-terminus and a C-terminus modified with a linker-bound cysteine ​​residue.

[0034] In some embodiments of any one of the compositions described herein, a population of tolerogenic antigens is conjugated to nanoparticle phospholipids in a manner that promotes potent immune tolerance upon administration to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder). Autoimmune disorders include, for example, MS, celiac disease, rheumatoid arthritis, diabetes (e.g., type 1 diabetes), autoimmune thyroid disorders (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye disease and dermal disease, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, and herpetiform dermatosis. These include dermatitis, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodbasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, or inflammatory bowel disease.

[0035] In some embodiments, multiple tolerogenic antigens are bound to nanoparticle phospholipids via thiol-reactive and reduction-insensitive binding between each tolerogenic antigen and the nanoparticle phospholipid. Indeed, the thiol-reactive and reduction-insensitive binding between the tolerogenic antigen and the nanoparticle phospholipid promotes potent immunotolerance. In some embodiments, the phospholipid is N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine.

[0036] In some embodiments, tolerogenic antigens are bound to nanoparticle phospholipids via amine-mediated interactions (e.g., N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl (DOPE-NHS)). In some embodiments, the amine-mediated interaction is via amine-reactive phospholipids having self-immolative linkages. Self-immolative linkages include linkers containing o-dithiobenzyl, p-dithiobenzyl, β-dithiobenzylcarbamate moieties, 2,2-dimethyl-4-mercaptobutyric acid, or disulfide-carbonate-based traceless linkers.

[0037] In some embodiments, the composition further comprises at least one therapeutic agent (e.g., at least one immunomodulator or immunosuppressant). Examples of at least one immunomodulator or immunosuppressant include fingolimod; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or related ligands; trichostatin A; suberoylanilide hydroxamic acid (SAHA); statins; mTOR inhibitors; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors; corticosteroids; mitochondrial function inhibitors; NF-κβ inhibitors; adenosine receptor agonists Prostaglandin E2 agonists (PGE2); phosphodiesterase inhibitors; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB inhibitors; autophagy inhibitors; aromatic hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); oxidized ATP IDO; Vitamin D3; Cyclosporine; Aromatic hydrocarbon receptor inhibitors; Resveratrol; Azathioprine (Aza); 6-Mercaptopurine (6-MP); 6-Thiogunine (6-TG); FK506; Sangrifferin A; Salmeterol; Mycophenolate mofetil (MMF); Aspirin and other COX inhibitors; Diflumic acid; Estriol; Triptolide; OPN-305; OPN-401; Eristran (E5664); TAK-242; Cpn10; NI -0101;1A6;AV411;IRS-954(DIV-1079);IMO-3100;CPG-52363;CPG-52364;OPN-305;ATNC05;NI-0101;IMO-8400;Hydroxychloroquine;CU-CPT22;C29;Ortho-vanillin;SSL3 protein;OPN-305;SsnB;Byzantine();(+)-N-phenethylnoroxymorphone;VB3323;Monosaccharide 3;(+)-naltrexone and (+)-naloxone;HT52;HTB2;Compound 4a;CNTO2424;TH1020;INH-ODN;E6446;AT791;CpG ODN 2088;ODN TTAGG;COV08-0064;2R9;GpG oligonucleotide;2-aminopurine;Anlexanox;Bay11-7082;BX795;CH-223191;Chloroquine;CLI-095;CU-CPT9a;Cyclosporine A;CTY387;Gefinitib;Glibenclamide;H-89;H-131;Isoliquitigenin;MCC950;MRT67307;OxPAPC;Parthenolide;Pepinh-MYD;Pepinh-TRIF;Polymyxin B;R406;RU.521;VX-765;YM201636;Z-VAD-FMK;and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD);Tryptamine (TA);and 6-formylindrol[3,2 b) AHR-specific ligands including but not limited to carbazole (FICZ). In certain embodiments, at least one therapeutic agent is fingolimod, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or related ligand; trichostatin A;and / or suberoylanilide hydroxamic acid (SAHA). In some embodiments, at least one therapeutic agent is contained within sHDL nanoparticles. In some embodiments, the sHDL nanoparticles are further mixed with an adjuvant. Examples of adjuvants include CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, reciquimod, gardicomod, 3M-052, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, badimezan, AsA404 (DMXAA), and any derivatives of adjuvants.

[0038] In some embodiments, the composition does not contain an adjuvant.

[0039] In another embodiment, the Disclosure provides a method for treating a subject having or at risk of having one or more autoimmune disorders, comprising administering an effective amount of any one of the compositions of the embodiments described herein. Autoimmune disorders include, for example, MS, celiac disease, rheumatoid arthritis, diabetes (e.g., type 1 diabetes), autoimmune thyroid disorders (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye disease and dermal disease, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and its variants, bullous pemphigoid, and herpetiform dermatosis of Duhring. These include dermatitis, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodbasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, or inflammatory bowel disease. In some embodiments, one or more autoimmune disorders are a single autoimmune disorder. In some embodiments, the single autoimmune disorder is celiac disease. In some embodiments, the subject is a human subject. In some embodiments, the method includes administering one or more further therapeutic agents to the subject. In some embodiments, one or more further therapeutic agents are administered to the subject simultaneously with an effective amount of any one composition of the embodiments described herein. In some embodiments, one or more further therapeutic agents are administered at a different time than an effective amount of any one composition of the embodiments described herein.

[0040] In some embodiments, one or more additional therapeutic agents are selected from the group consisting of corticosteroids (e.g., prednisone, betamethasone, clobetazone butyrate, and butesonide), immunosuppressants (e.g., etanercept, adalimumab, azathioprine, infliximab, cyclosporine, alemtuzumab, and cladribine), and anti-inflammatory agents (e.g., dapsone and sulfonamides). In some embodiments, one or more additional therapeutic agents are selected from infliximab, adalimumab, etanercept, dapsone, or clobetazone butyrate. In some embodiments, the subject strictly adheres to a gluten-free diet.

[0041] Additional embodiments will be apparent to those skilled in the art based on the teachings contained herein.

[0042] [Brief explanation of the drawing] Figure 1 is an image showing T cell activation.

[0043] Figure 2 is a graph showing the pathological scores for EAE induced by free MOG (100 μg / administered) and HDL-MOG nanodiscs (100 μg / administered).

[0044] Figures 3A-3C show that HDL-MOG nanodiscs, administered from day 2, exhibit potent efficacy against EAEs. Figure 3A is a schematic diagram of the treatment plan. Figure 3B is a graph showing the pathological scores for EAEs induced by MOG35-55. Figure 3C is a graph showing the pathological scores for EAEs induced by MOG1-125.

[0045] Figures 4A-4C show that HDL-MOG nanodiscs, administered starting on day 15, exhibit potent efficacy against EAEs. Figure 4A is a schematic diagram of the treatment plan. Figure 4B is a graph showing the pathological scores for EAEs induced by MOG35-55. Figure 4C is a graph showing the pathological scores for EAEs induced by MOG1-125.

[0046] Figures 5A-5B are graphs showing that HDL-MOG nanodiscs exhibit stronger efficacy than FTY720. Pathological scores of EAE mice treated with HDL-MOG versus EAE mice treated with FTY720, when treatment was initiated on day 15 (Figure 5A) or day 30 (Figure 5B).

[0047] Figures 6A-6B show HDL-MOG nanodiscs that reduce the secretion of inflammatory cytokines in the CNS of EAE mice. Figure 6A is a schematic diagram showing the treatment plan. Figure 6B is a graph showing the amounts of GM-CSF, IFN-γ, and IL-17 released after ex vivo treatment of CNS tissue with MOG35-55 peptides.

[0048] Figure 7 shows images of BMDCs and microglia after incubation with HDL-MOG-FITC or MOG-FITC peptide and visualization of antigen uptake. Actin filaments were stained with AlexaFluor 647-Phalloidin, and nuclei were stained with DAPI.

[0049] Figures 8A–8B are images illustrating the experimental design and results for naive mice (Figure 8A) or EAE-induced mice (Figure 8B). EAE-induced mice were subcutaneously administered PBS, free MOG-TMR, or HDL-MOG-TMR. At the indicated time points, DCs, macrophages, and B cells were isolated from influx region lymph nodes or spinal cord and analyzed for TMR fluorescence signals.

[0050] Figure 9A shows HDL-MOG-NOTA- for in vivo distribution studies. 64 Cu or MOG-NOTA- 64 This is a schematic diagram showing the treatment plan for EAE-induced mice administered with Cu.

[0051] Figure 9B is a photograph showing 24-hour positron emission tomography (PET) images of EAE-induced mice.

[0052] Figure 9C shows the major organs 24 hours after injection. 64 This graph shows the quantitative analysis of the Cu signal.

[0053] Figure 10A is a schematic diagram of EAE-induced mice treated with PBS, free MOG, HDL-M30, or HDL-MOG, as shown.

[0054] Figure 10B is a graph showing the results from ELISA from the CNS, where cells were collected on day 40, processed individually, restimulated ex vivo with MOG peptide, and quantified for levels of IL-17, IFN-gamma, and GM-CFS.

[0055] Figures 10C-10D are graphs showing the results of intracellular cytokine staining for CNS (Figure 10C) and spleen cells (Figure 10D) in exovivotive restimulation with or without MOG peptide, examining the frequency of CD4 T cells secreting IL-17, IFN-gamma, and GM-CFS.

[0056] Figure 11 shows the experimental design and results for EAE-induced mice treated with PBS, free MOG, HDL-M30, or HDL-MOG. CNS tissue collected on day 40 was examined for levels of IL-17, IFN-gamma, GM-CFS, and IL-10.

[0057] Figure 12A is a schematic diagram of EAE-induced mice treated as shown with PBS, free MOG, HDL-M30, or HDL-MOG. The frequency of Treg cells was examined in CNS tissue collected on day 40.

[0058] Figures 12B-12C are graphs showing the frequencies of CD25+Foxp3+Tregs (Figure 12B) and MOG-tetramer+Foxp3+Tregs in the CNS.

[0059] Figure 13 shows the experimental design for EAE-inducing mice treated with PBS or HDL-MOG as indicated. Red arrows indicate the treatment day. In addition, some mice were administered anti-CD25 IgG at the indicated time points. Mice were monitored over time for EAE scores.

[0060] Figure 14 is an HPLC chromatogram that quantifies the amount of HDL-FTY720 loaded compared to a free drug standard.

[0061] Figure 15 is an HPLC chromatogram that quantifies the amount of ITE loaded into HDL (HDL-ITE) compared to a free drug standard.

[0062] Figure 16 is an HPLC chromatogram that quantifies the amount of TSA loaded into HDL (HDL-TSA) compared to a free drug standard.

[0063] Figure 17 is an HPLC chromatogram that quantifies the amount of SAHA loaded into HDL (HDL-SAHA) compared to a free drug standard.

[0064] Figure 18 is a table showing the drug loading efficiencies for FTY72-, ITE, TSA, and SAHA in HDL nanodiscs, quantified by HPLC / MS. The size of the nanodiscs was measured by dynamic light scattering (DLS).

[0065] Figure 19 is a gel permeation chromatograph showing the HDL-FTY720 compared to an empty HDL nanodisk.

[0066] Figure 20 shows graphs of HDL loaded with rapamycin (Rapa), analyzed by DLS and gel permeation chromatography. HDL-Rapa showed a uniform particle size distribution with an average hydrodynamic size of ~10 nm.

[0067] Figure 21 is a graph showing EAE-induced mice on day 0, and mice that received intraperitoneal administration of HDL-FTY720 (1 mg / kg FTY720) on days 14, 21, and 28. The mice were monitored for EAE scores.

[0068] Figure 22 shows HPLC chromatograms of HDL nanodiscs loaded with EA peptides, empty HDL (blank HDL), lipid-EA conjugates, and MPB lipids. The amount of EA peptide loaded into the HDL was quantified by HPLC-MS.

[0069] Figure 23 shows HPLC chromatograms of HDL nanodiscs loaded with OVA-II peptide, empty HDL, lipid-OVA-II conjugates, and MPB lipids. The amount of OVA-II peptide loaded into the HDL was quantified by HPLC-MS.

[0070] Figure 24 shows HPLC chromatograms of HDL nanodiscs loaded with CIA peptide, empty HDL, lipid-CIA conjugates, and MPB lipids. The amount of CIA peptide loaded into the HDL was quantified by HPLC-MS.

[0071] Figure 25 is a table showing the antigen binding efficiency for forming antigen-lipid conjugates (EA, OVA-II, and CIA), the antigen-lipid loading efficiency in HDL, and the hydrodynamic size of antigen-loaded HDL.

[0072] [Definition] As used herein, the term "about" means a value that is ±10% of the listed value.

[0073] As used herein, the term “absorbed” refers to a biopolymer (e.g., an antigen, adjuvant, etc.) that is incorporated into the interior (i.e., the interior of the outer surface) of nanoparticles and / or microparticles and stably retained therein.

[0074] As used herein, “administering” means a method of administering a dose of the composition described herein (e.g., nanoparticles or nanoparticles associated with an antigen) to a subject. The composition used in the methods described herein may be administered by any preferred route, including, for example, inhalation, spraying, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, or oral. The composition used in the methods described herein may also be administered topically or systemically. The preferred method of administration may vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).

[0075] As used herein, the term “admixed” refers to a biopolymer (e.g., an antigen, an adjuvant, etc.) dissolved, dispersed, or suspended in nanoparticles and / or microparticles. In some cases, the biopolymer may be homogeneously mixed in the nanoparticles and / or microparticles.

[0076] As used herein, the term “adsorbed” refers to the attachment of a biopolymer (e.g., antigen, adjuvant, etc.) to the outer surface of nanoparticles and / or fine particles. Such adsorption is preferably caused by electrostatic attraction, which is an attractive force or bond generated between two or more oppositely charged or ionic chemical groups. Generally, adsorption is typically reversible.

[0077] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a conformational arrangement consisting of a continuous stretch of amino acids or different regions of discontinuous amino acids). Useful antigenic determinants may be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, in the free in serum, and / or in the extracellular matrix (ECM). Proteins referred to herein as antigens include full-length 33-mer polypeptides derived from α-gliadin (SEQ ID NO: 374) or any fragment thereof, or CD4 + The epitope recognized by T cells may be any of the polypeptides disclosed in Table 3 (SEQ ID NOs: 375-405). When referring to a particular protein as used herein, the term encompasses both the “full-length” unprocessed protein and any form of protein resulting from processing in cells. The term also encompasses naturally occurring protein variants (e.g., splicing variants or allele variants).

[0078] As used herein, the terms “autoimmune disorder” and “autoimmune disease,” as used interchangeably herein, refer to a medical condition in which the immune system of a subject mistakenly attacks the body's own tissues.

[0079] As used herein, “combination therapy” or “administered in combination” means administering two or more different drugs or treatments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a subject as part of a defined treatment regimen for a particular disease or condition (e.g., autoimmune disorder (e.g., MS or celiac disease)). The treatment regimen defines the dosage and periodicity of administration of each drug so that the effects of the separate drugs on the subject overlap. In some embodiments, the delivery of two or more drugs is simultaneous or concurrent, and the drugs may be co-formulated. In some embodiments, the two or more drugs are not combined but administered sequentially as part of a prescribed regimen. In some embodiments, the administration of two or more drugs or a combination treatment results in a greater reduction in other parameters related to the symptom or disorder than that observed with one drug, or with a treatment delivered alone or in the absence of the other drugs. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be influenced by any suitable route, including, but not limited to, inhalation, spray, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, oral, or direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, the first therapeutic agent of the combination may be administered by intravenous injection, while the second therapeutic agent of the combination may be administered orally.

[0080] As used herein, the term “complexed” refers to a non-covalent interaction between a biomolecular agent (e.g., an antigen, an adjuvant, etc.) and nanoparticles and / or microparticles.

[0081] As used herein, the term “conjugated” refers to the covalent association of a biomolecular agent (e.g., an antigen, an adjuvant, etc.) with nanoparticles and / or microparticles.

[0082] As used herein, the terms “drug” or “therapeutic agent” include any molecule, molecular complex, or substance administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraception, cosmetic, nutritional supplement, pharmaceutical, and preventive uses. The term “drug” further includes any such molecule, molecular complex, or substance that is chemically modified and / or functionally bound to a biological or biocompatible structure.

[0083] As used herein, the term “fragment” refers to less than 100% of the amino acid sequence of a full-length reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but includes, for example, 5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350 or more amino acids. A fragment may be long enough to maintain the desired function of the full-length protein. For example, a fragment such as factor H maintains the regulation of alternative complement pathways in the liquid phase. Such fragments are “biologically active fragments.”

[0084] As used herein, the terms “gluten-free” and “gluten-free diet” refer to a diet in which grains of the genus *Tetris*, such as wheat, barley, and rye, are not consumed directly or in any food. A gluten-free diet is one in which gluten is consumed at a rate of 10 to 50 mg or less per day. Gluten-free diets are often adopted to minimize or eliminate the effects of celiac disease.

[0085] As used herein, the term "HDL" or "high-density lipoprotein" refers to high-density lipoprotein. HDL is a complex of nearly equal amounts of lipids and proteins that functions as a transporter for cholesterol in the blood. HDL is primarily synthesized and secreted by epithelial cells of the liver and small intestine. Immediately after secretion, HDL is in the form of disc-shaped particles containing apolipoprotein AI (also called apoA-I) and phospholipids as its main components; this is also called nascent HDL. In the blood, this nascent HDL receives free cholesterol from the cell membranes of peripheral cells or is produced by the hydrolysis process of other lipoproteins, and forms mature spherical HDL, holding cholesterol esters converted from the aforementioned cholesterol at its hydrophobic center by the action of LCAT (lecithin cholesterol acyltransferase). HDL plays a crucial role in a lipid metabolic process called "reverse cholesterol transport," which takes cholesterol from peripheral tissues into the blood and transports it to the liver. Since reverse cholesterol transport is considered one of the main mechanisms by which HDL has a protective effect against atherosclerosis, high levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0086] As used herein, the term “immunomodulatory agent” refers to a compound that stimulates or suppresses the immune system. As used herein, the term “immunosuppressant” refers to a compound that causes an APC to have an immunosuppressive effect (e.g., a tolerogenic effect). An immunosuppressive effect generally refers to the production or expression of cytokines or other factors by an APC that reduce, inhibit, or prevent an undesirable immune response, or promote a desired immune response. When an APC exerts an immunosuppressive effect on immune cells that recognize an antigen presented by the APC, that immunosuppressive effect is said to be specific to the presented antigen. Such an effect is also referred to herein as a tolerogenic effect. While not bound by any particular theory, an immunosuppressive or tolerogenic effect is considered to be a result of an immunosuppressant delivered to an APC, preferably in the presence of an antigen (e.g., an administered antigen or an antigen already present in vivo). Thus, immunosuppressants include compounds that provide a tolerogenic immune response to an antigen that may or may not be provided in the same or different compositions. In one embodiment, the immunosuppressant promotes a regulatory phenotype in one or more immune effector cells in the APC. For example, the regulatory phenotype may be characterized by inhibition of the production, induction, stimulation, or recruitment of antigen-specific CD8+ T cells, the production, induction, stimulation, or recruitment of Treg cells, or the production, induction, stimulation, or recruitment of Treg cells. This may result in the conversion of CD8+ T cells or B cells into a regulatory phenotype. This may also result in the induction of FoxP3 in other immune cells (e.g., CD4+ T cells, macrophages, and iNKT cells). In one embodiment, the immunosuppressant affects the APC response after antigen processing. In another embodiment, the immunosuppressant does not interfere with antigen processing. In a further embodiment, the immunosuppressant is not an apoptosis signaling molecule. In yet another embodiment, the immunosuppressant is not a phospholipid.

[0087] Immunomodulators include statins; mTOR inhibitors (e.g., rapamycin or rapamycin analogs); TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors (e.g., trichostatin A); corticosteroids; mitochondrial function inhibitors (e.g., rotenone); P38 inhibitors; NF-κβ inhibitors (e.g., 6Bio, dexamethasone, TCPA-1, IKK). VII); adenosine receptor agonists; prostaglandin E2 agonists (PGE2) (e.g., misoprostol); phosphodiesterase inhibitors (e.g., phosphodiesterase 4 inhibitors (PDE4) such as rolipram); proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB inhibitors (e.g., TGX-221); autophagy inhibitors (e.g., 3-methyladenine); aromatic hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATP (e.g., P2X receptor blockers), which are included, but not limited to these.Immunosuppressants include IDO, vitamin D3, cyclosporine (e.g., cyclosporine A), aromatic hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol; triptolide; OPN-305, OPN-401; Eritran (E5564); TAK-242; Cpn10; NI-0101; 1A6; AV411; IRS-954 (DV-1079); IMO-3100; CPG-52363; CPG-52364; OPN-305; ATNC05; NI-0101; IMO-8400; Hydroxychloroquinone; CU-CPT22; C29; Orthovanillin; SSL3 protein; OPN-305; 5 SsnB; Byzantine; (+)-N-phenethylnoroxymorphone; VB3323; Monosaccharide 3; (+)-naltrexone and (+)-naloxone; HT52; HTB2; Compound 4a; CNTO2424; TH1020; INH-ODN; E6446; AT791; CpG ODN 2088; ODN TTAGGG;COV08-0064;2R9;GpG oligonucleotide;2-aminopurine;Anlexanox;Bay11-7082;BX795;CH-223191;Chloroquine;CLI-095;CU-CPT9a;Chlorosporin A;CTY387;Gefitinib;Glybenclamide;H-89;H-131;Isoliquitiche The AHR-specific ligands include, but are not limited to, nin; MCC950; MRT67307; OxPAPC; parthenolide; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU.521; VX-765; YM201636; Z-VAD-FMK; and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); tryptamine (TA); and 6-formylindoro[3,2b]carbazole (FICZ).In certain embodiments, the immunosuppressant is FTY720 (also known as fingolimod) (Chung and Harung, Clin. Neuropharmacol 33: 91-101, 2010), AhR activation by 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or related ligands (Yeste A, et al. Proc. Natl. Acad. Sci. USA 109: 11270-11275, 2012; Quintana FJ, et al Proc. Natl. Acad. Sci. USA 107: 20768-20773, 2010), or trichostatin (TSA) (Reilly CM et al. J. Autoimmun 31: 123-130. 2008). Suberoylanilide hydroxamic acid (SAHA), histone deacetylase inhibitors (Lucas JL, et al. Cell Immunol 257: 97-104, 2009), and / or rapamycin (Rapa) (Maldonado, RA, et al. Proc. Natl. Acad. Sci. USA 112:E156-165, 2015). In embodiments, the immunosuppressant may include any of the agents provided herein.

[0088] Immunosuppressants may be compounds that directly provide an immunosuppressive (e.g., tolerogenic) effect on APCs, or compounds that provide an immunosuppressive (e.g., tolerogenic) effect indirectly (i.e., after some treatment following administration). Therefore, immunosuppressants include any prodrug form of the compounds provided herein.

[0089] The immunosuppressant also includes nucleic acids encoding peptides, polypeptides, or proteins provided herein that produce an immunosuppressive (e.g., tolerogenic) immune response. Thus, in embodiments, the immunosuppressant is a nucleic acid encoding a peptide, polypeptide, or protein that produces an immunosuppressive (e.g., tolerogenic) immune response, and it is the nucleic acid that is bound to the sHDL nanoparticles.

[0090] The nucleic acid may be DNA or RNA (e.g., mRNA). In embodiments, the composition comprises a complement, such as a full-length complement, or a degenerate (due to degeneracy of the genetic code) of any of the nucleic acids provided herein. In embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may comprise, among other things, a plasmid, a retrovirus, or an adenovirus. The nucleic acid may be isolated or synthesized using standard molecular biological approaches. For example, the nucleic acid may be isolated or synthesized by producing nucleic acid fragments using polymerase chain reaction, which are then cloned into an expression vector. Further techniques useful for carrying out the present invention are found in Current Protocols in Molecular Biology 2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of You can also find it at Texas Southwestern Medical Center, Dallas, Cold Spring Harbor.

[0091] Other exemplary immunosuppressants include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, and CD4), biologic-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506), etc. Further immunosuppressants are known to those skilled in the art, and the present invention is not limited in this respect.

[0092] As used herein, the term "in vitro" refers to an artificial environment and the processes or reactions that occur within it. An in vitro environment may, but is not limited to, test tubes and cell cultures.

[0093] The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and the processes or reactions that occur within that natural environment.

[0094] As used herein, the terms “lipid” or “lipid molecule” refer to fatty substances that are insoluble in water and include fats, oils, waxes, and related compounds. They may be produced in the blood (endogenous) or ingested through diet (exogenous). Lipids are essential for normal bodily functions and, whether produced from exogenous or endogenous sources, must be transported for use by cells and then released for use by cells. The production, transport, and release of lipids for use by cells is called lipid metabolism. There are several classes of lipids, but the two main classes are cholesterol and triglycerides. Cholesterol may be ingested through diet and may be produced by cells in most organs and tissues of the body, primarily the liver. Cholesterol may be found in free form, or more frequently, as cholesterol esters bound to fatty acids. As used herein, “lipid” or “lipid molecule” refers to any lipid-soluble compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogues, which are typically classified into at least three classes: (1) "simple lipids" including fats and oils, as well as waxes; (2) "lipid compounds" including phospholipids and glycolipids; and (3) "lipid derivatives" such as steroids. Lipids or lipid molecules suitable for use in the present invention include both membrane-forming lipids and non-membrane-forming lipids.

[0095] As used herein, the term “lipoprotein” refers to a spherical compound structured such that water-insoluble lipids are partially contained within a water-soluble outer shell. Depending on the type of lipoprotein, the contents may include varying amounts of free and esterified cholesterol, triglycerides, and apoproteins, or apoproteins. There are five main types of lipoproteins, classified according to their function and lipid content, as well as their apoprotein content, and increasing concentration: (i) chylomicrons and chylomicron remnants, (ii) very low-density lipoproteins ("VLDL"), (iii) intermediate-density lipoproteins ("IDL"), (iv) low-density lipoproteins ("LDL"), and (v) high-density lipoproteins ("HDL"). Cholesterol circulates in the bloodstream as particles in conjunction with lipoproteins.

[0096] As used herein, the term “non-naturally occurring amino acid” means an α-amino acid that is not produced or found naturally in mammals. Examples of non-natural amino acids include D-amino acids; amino acids having an acetylaminomethyl group bonded to the sulfur atom of cysteine; pegylated amino acids; omega amino acids of the formula NH2(CH2)nCOOH (where n is 2-6), neutral nonpolar amino acids (e.g., sarcosine), t-butylalanine, t-butylglycine, n-methylisoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteine ​​acid; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxypiperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutanoic acid; 3-(2-naphthyl)alanine, and hydroxyproline. Other amino acids include α-aminobutyric acid, α-amino-α-methylbutyric acid, aminocyclopropane-carboxylate, aminoisobutyric acid, aminonorbonyl-carboxylate, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylnorvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-norleucine, α-methylaminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, D -α-methylarginine, D-α-methylasparagine, D-α-methylaspartic acid, D-α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine, D-α-methylisoleucine, D-α-methylleucine, D-α-methyllysine, D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-methylserine, DN-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, DN-methylalanine, DN-methylarginine,DN-methylasparagine, DN-methylaspartic acid, DN-methylcysteine, DN-methylglutamine, DN-methylglutamic acid, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, DN-methylcyclohexylalanine, DN-methylornithine, N-methylglycine, N-methylaminoisobutyric acid, N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyltyrosine, DN-methylvaline, γ-aminobutyric acid, Lt -Butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α-methylaspartic acid, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-methylleucine, L-α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine, L-α-methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-diphenylethyl)carbamylmethylglycine, 1-carboxy- 1-(2,2-diphenyl-ethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidinedione-4-carboxylic acid, L-homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan- Phosphorus, meta-tyrosine, nor-tyrosine, LN,N',N''-trimethyllysine, homolysine, norlysine, N-glycan asparagine, 7-hydroxy-1,2,3,4-tetrahydro-4-fluorophenylalanine, 4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid,1-amino-cyclohexaneacetic acid, D / L-allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutanecarboxylic acid, 2 or 3 or 4-aminocyclohexanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetralin-2-carboxylic acid, azetidine-3-carboxylic acid, 4-benzyl-pyrrolidine-2-carboxylic acid, tert-butylglycine, β-(benzothiazolyl-2-yl)-alanine, β-cyclopropylalanine, 5 ,5-dimethyl-1,3-thiazolidined-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S) and (2S,4R)-4-(2-naphthylmethoxy)-pyrrolidine-2-carboxylic acid, (2S,4S) and (2S,4R)-4-phenoxypyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-1-benzoylpyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenylpyrrolidine LN-2-carboxylic acid, 1-aminomethylcyclohexaneacetic acid, 3,5-bis-(2-amino)ethoxybenzoic acid, 3,5-diaminobenzoic acid, 2-methylaminobenzoic acid, N-methylantranilic acid, LN-methylalanine, LN-methylarginine, LN-methylasparagine, LN-methylaspartic acid, LN-methylcysteine, LN-methylglutamine, LN-methylglutamic acid, LN-methylhistidine, LN-methylisoleucine, LN-methyllysine, LN-methylnorleucine, LN-methylornithine, LN-methyl Tilthreonine, LN-methyltyrosine, LN-methylvaline, LN-methyl-t-butylglycine, L-norvaline, α-methyl-γ-aminobutyric acid, 4,4'-biphenylalanine, α-methylcylcopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine,N-(2-carbamylethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cyclododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine, N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N-(hydro N-(Imidazolylethyl)glycine, N-(3-indolylyethyl)glycine, N-methyl-γ-aminobutyric acid, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN-methylthreonine, N-(1-methylethyl)glycine, N-methylnaphthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methyl L-Alanine, L-α-Methylasparagine, L-α-Methyl-t-Butylglycine, L-Methylethylglycine, L-α-Methylglutamic acid, L-α-Methylhomophenylalanine, N-(2-Methylthioethyl)glycine, L-α-Methyllysine, L-α-Methylnorleucine, L-α-Methylornithine, L-α-Methylproline, L-α-Methylthreonine, L-α-Methyltyrosine, LN-Methylhomophenylalanine, N-(N-(3,3-Diphenylpropyl)carbamylmethylglycine, L-Pyroglutamic acid, D-Pyroglutamic acid Calcium phosphate, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamic acid, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine, L-dimethyldopa or L-dimethoxyphenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cyclopeptylglycine, L-diphenylalanine, O-methyl-L-homoterosine, L-β-homolisine, O-glycan-threonine, ortho-tyrosine,LN,N'-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantylalanine, symmetric dimethylarginine, 3-carboxythiomorpholine, D-1,2,3,4-tetrahydronorharman-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethylpyridine-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-amino-tetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylic acid, b-(benzothiazole-2-I (2S,4R)-alanine, neopentylglycine, 2-carboxymethylpiperidine, β-cyclobutylalanine, allylglycine, diaminopropionic acid, homo-cyclohexylalanine, (2S,4R)4-hydroxypiperidine-2-carboxylic acid, octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, nicopetic acid, (2S,4R) and (2 These include S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercaptopyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and selenocysteine. In some embodiments, the amino acid residues may be charged or polar. Charged amino acids include alanine, lysine, aspartic acid or glutamic acid, or analogs thereof that do not exist in nature. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine or tryptophan, or analogs thereof that do not exist in nature. In some embodiments, the terminal amino group in the amino acid isIt is specifically intended that the group may be an amide group or a carbamate group.

[0097] The "percent sequence identity (%)" of a candidate sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after sequence alignment and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining the percentage of sequence identity of nucleic acids or amino acids can be achieved in various ways within the capabilities of a person skilled in the art, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. For example, the value of the percentage of sequence identity may be generated using the sequence comparison computer program BLAST. As an example, the percentage of sequence identity of a particular nucleic acid or amino acid sequence A to a particular nucleic acid or amino acid sequence B (which can also be expressed as a particular amino acid sequence A having a particular percentage of sequence identity to a particular nucleic acid or amino acid sequence B) is calculated as follows: Multiply the ratio X / Y by 100. X is the number of nucleotides or amino acid residues that a sequence alignment program (e.g., BLAST) has scored as identical in the alignment of A and B in that program, and Y is the total number of nucleic acids in B. It goes without saying that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the ratio of sequence identity of A to B is not equal to the ratio of sequence identity of B to A.

[0098] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length (e.g., naturally occurring and unnatural amino acids). The term also encompasses modified amino acid polymers; for example, those modified by disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or bonding with a labeling component.

[0099] "Pharmaceutical composition" means any composition containing a therapeutically or biologically active agent (e.g., nanoparticles containing 1 to 30 (e.g., 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, or 8 to 30 tolerogenic antigens)) suitable for administration to a target. A biologically active agent contains nanoparticles containing 1 to 30 (e.g., 8 to 30 tolerogenic antigens per nanoparticle). The 1 to 30 tolerogenic antigens associated with a particular nanoparticle may all have the same sequence identity, or the 1 to 30 tolerogenic antigens associated with a particular nanoparticle may comprise 1 to 5 different populations of tolerogenic antigens having different sequence identities. Any of these formulations can be prepared by methods well known and accepted in the art. For example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006 (each of these is incorporated herein by reference).

[0100] A "pharmaceutically acceptable diluent, excipient, carrier, or adjuvant" means a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to the target while maintaining the therapeutic properties of the administered pharmaceutical composition.

[0101] As used herein, the term “sample” is used in its broadest sense. In one sense, it means a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be taken from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Environmental samples include environmental substances such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be interpreted as limiting the types of samples to which the present invention can be applied.

[0102] As used herein, the term “subject” refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Typically, the terms “subject” and “patient” are used interchangeably herein with respect to human subjects.

[0103] As used herein, the terms “synthetic HDL,” “sHDL,” “reconstituted HDL,” or “rHDL” refer to particles structurally similar to natural HDL, consisting of lipids or lipids (preferably ApoA-I or a mimetic thereof) associated with at least one HDL protein. Typically, the components of sHDL may be derived from blood or produced by recombinant technology.

[0104] "Therapeutically effective amount" means the amount of composition administered to improve, suppress or alleviate, in a clinically relevant manner, a condition or disorder or symptom of a subject (e.g., celiac disease). Any improvement in the subject is considered sufficient to achieve treatment. Preferably, a sufficient amount to treat is an amount that reduces, inhibits or prevents the occurrence or prevention of one or more symptoms of the disease or disorder (e.g., celiac disease), or an amount that reduces the severity or duration of the subject's suffering from one or more symptoms of the disease or disorder (e.g., celiac disease) (e.g., at least about 10%, about 20%, or about 30%, more preferably at least about 50%, about 60%, or about 70%, most preferably at least about 80%, about 90%, about 95%, about 99%, or more, compared to a control subject not treated with the composition described herein). The effective amount of the pharmaceutical composition used to carry out the methods described herein (e.g., treatment of celiac disease) varies depending on the mode of administration, as well as the age, weight, and overall health condition of the person being treated. A physician or researcher may determine the appropriate dose and administration regimen.

[0105] As used herein, the term "tolerogenic antigen" refers to a molecule that can bind to an antibody or antigen receptor (in particular, one that induces an immune response) on a T cell.

[0106] As used herein, the term “solvent” refers to the medium in which a reaction takes place. The solvent may be, but is not limited to, a liquid. Types of solvents include, but are not limited to, nonpolar, polar, protic, and aprotic.

[0107] [Detailed description of the invention] The present invention relates to nanoparticles associated with multiple toxicogenic antigens (e.g., 1 to 30 toxicogenic antigens per nanoparticle (e.g., 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, or 8 to 30 toxicogenic antigens) in a manner that promotes potent immune tolerance upon administration to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder (e.g., MS or celiac disease)). The present invention further relates to a method for synthesizing such nanoparticles associated with toxicogenic antigens involved in autoimmune disorders (e.g., MS or celiac disease), and to a system and method utilizing such nanoparticles for treating a subject suffering from an autoimmune disorder (e.g., MS or celiac disease).

[0108] (Nanoparticles) The present invention is not limited to a specific type or category of nanoparticles associated with a tolerogenic antigen (e.g., complexed, bound, encapsulated, absorbed, adsorbed, mixed) for treating, preventing, or improving various types of autoimmune disorders (e.g., celiac disease).

[0109] Examples of nanoparticles include fullerene (also known as C 60 , C 70 , C 76 , C 80 , C 84Examples of nanoparticle embodiments include, but are not limited to, metal-encapsulated fullerenes containing additional atoms, ions, or clusters within a fullerene cage (buckyballs of EMI), trimetallic nitride-templated metal-encapsulated fullerenes (TNT EMEs, encapsulating highly symmetrical tetraatomic molecular clusters formed in a trimetallic nitride template within a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods (carbon nanotubes with internal metal-encapsulated fullerenes and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Embodiments of particles may include microparticles having the ability to enhance efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, and gold, silver, carbon, and iron nanoparticles.

[0110] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise a polyol polymer modified to contain a hydrophobic polymer block. As used in this disclosure, the term “hydrophobic polymer block” refers to a segment of a polymer that is hydrophobic by itself. As used herein, the term “micelle” refers to an aggregate of molecules dispersed in a liquid. A typical micelle in an aqueous solution forms an aggregate having a hydrophilic “head” region in contact with the surrounding solvent, isolating a single hydrophobic tail region at the center of the micelle. In some embodiments, the head region may be, for example, a surface region of the polyol polymer, while the tail region may be, for example, a hydrophobic polymer block region of the polyol polymer.

[0111] The present invention further encompasses the use of particles at the micrometer scale in addition to the nanometer scale. When microparticles are used, they are preferably relatively small, on the order of about 1 to 50 micrometers. For the sake of ease of consideration, the use of “nanoparticles” as herein encompasses true nanoparticles (sizes of about 1 nm to about 1000 nm), microparticles (e.g., about 1 micrometer to about 50 micrometers), or both.

[0112] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers having covalently bonded metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In these embodiments, the nanoparticles are metallic nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more of these). The nanoparticles may consist of a core, or a core and a shell as core-shell nanoparticles.

[0113] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles consist of a mixture of HDL apolipoprotein and amphiphilic lipids.

[0114] The present invention is not limited to the use of a specific type or category of HDL apolipoprotein. HDL apolipoproteins include, for example, apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apoA4), apolipoprotein Cs (apoCs), apolipoprotein M (apoM), and apolipoprotein E (apoE). In some embodiments, HDL apolipoproteins include, for example, preproapolipoproteins, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, apolipoprotein A-II xxx (apo A-II-xxx), preproApoA-1V, proApoA-1V, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-IMilano, proApoA-IMilano, ApoA-IMilano, preproApoA-IParis, proApoA-IParis, and ApoA-IParis, as well as peptide mimes of mixtures of these proteins. Preferably, the carrier particles consist of ApoA-I or ApoA-II, but the use of other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate a carrier particle mixture for therapeutic agent delivery. In some embodiments, mimics of such HDL apolipoproteins are used.

[0115] ApoA-I is synthesized in the liver and small intestine as a preproapolipoprotein, secreted as a proprotein that is rapidly broken down to produce a mature polypeptide with 243 amino acid residues. ApoA-I mainly consists of 6-8 different 22-amino acid repeats and two different 11-amino acid repeats separated by a proline linker moiety, each of which has an amphiphilic α-helix helix wheel signature separated often by a proline linker moiety, and in some cases consists of stretches composed of several residues. ApoA-I forms three types of stable complexes with lipids: small, low-lipid complexes called pre-β-1HDL; flattened, disc-shaped particles containing polar lipids (phospholipids and cholesterol) called pre-β-2HDL; and spherical particles containing both polar and nonpolar lipids, called spherical or mature HDL (HDL3 and HDL2). Most HDL in the circulating population contains both ApoA-I and ApoA-II (the second major HDL protein).

[0116] In some embodiments, ApoA-I agonists or mimics are provided. In some embodiments, such ApoA-I mimics can form amphiphilic α-helices that mimic the activity of ApoA-I and have specific activities that approach or exceed those of the native molecule. In some, the ApoA-I mimics are peptides or peptide analogs. These form amphiphilic helices (in the presence of lipids), bind to lipids, form pre-β-like or HDL-like complexes, activate lecithin-cholesterol acyltransferase (LCAT), increase serum concentrations of the HDL fraction, and promote cholesterol efflux.

[0117] The present invention is not limited to the use of a specific ApoA-I mimic. In some embodiments, any of the ApoA-I mimics described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 are used. In some embodiments, any of the ApoA-I mimics described in U.S. Patent Application Publication Nos. 20110046056 and 20130231459 are used.

[0118] In some embodiments, the "22A" ApoA-I mimic (PVLDLFRELLNELLEALKQKLK) (Sequence ID 4) (see, for example, U.S. Patent No. 7,566,695) is used. In some embodiments, one of the following ApoA-I mimics listed in Table 1, as described in U.S. Patent No. 7,566,695, is used.

[0119] [Table 1-1]

[0120] [Table 1-2]

[0121] [Table 1-3]

[0122] [Table 1-4]

[0123] [Table 1-5]

[0124] [Table 1-6]

[0125] [Table 1-7]

[0126] * indicates peptides with N-terminal acetylation and C-terminal amidation; indicates peptides with N-terminal dansylation; sp indicates peptides that exhibited solubility issues under experimental conditions. X is Aib; Z is Na; O is Orn; ~ indicates a missing amino acid.

[0127] In some embodiments, an ApoA-I mimic having the following sequence, as described in U.S. Patent No. 6,743,778, is utilized: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 255).

[0128] In some embodiments, one of the following ApoA-I imitations shown in Table 2, as described in U.S. Patent Application Publication No. 2003 / 0171277, is utilized.

[0129] [Table 2-1]

[0130] [Table 2-2]

[0131] [Table 2-3]

[0132] In some embodiments, an ApoA-I mimic having the following sequence, as described in U.S. Patent Application Publication No. 2006 / 0069030, is utilized: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333).

[0133] In some embodiments, an ApoA-I mimic having the following sequence described in U.S. Patent Application Publication No. 2009 / 0081293 is utilized: DWFKAFYDKVAEKFKEAF (Sequence ID 334); DWLKAFYDKVAEKLKEAF (Sequence ID 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (Sequence ID 336).

[0134] In some embodiments, an ApoA-I mimic having one of the following sequences is utilized: WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347), PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVL ESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354), QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (distribution) Column number 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASLL (SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0135] Amphiphilic lipids include, for example, any lipid molecule having both a hydrophobic and a hydrophilic portion. Examples include phospholipids and glycolipids. Examples of phospholipids that may be used in sHDL-tolerogenic antigen nanoparticles include 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-dimiristoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-sn-glycero-3-phosphocholine; 1,2-diarachidoyl-sn-glycero-3-phosphocholine; 1,2-dibehenoyl-sn-glycero-3-phosphocholine; and 1,2-diligceloyl -sn-glycero-3-phosphocholine;1,2-dimyristreoil-sn-glycero-3-phosphocholine;1,2-dimyristeridoyl-sn-glycero-3-phosphocholine;1,2-dipalmitreoil-sn-glycero-3-phosphocholine;1,2-dipalmiteridoyl-sn-glycero-3-phosphocholine;1,2-dipetroselenoil-sn-glycero-3-phosphocholine;1,2-dioleoil-sn-glycero-3-phosphocholine;1,2-dieridoyl-sn -Glycero-3-phosphocholine; 1,2-dieycosenoyl-sn-glycero-3-phosphocholine; 1,2-dinervonoyl-sn-glycero-3-phosphocholine; 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine; 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine; 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; 1,2-dis Thearoyl-sn-glycero-3-phosphoethanolamine; 1,2-dipalmitreoyl-sn-glycero-3-phosphoethanolamine; 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]; 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol;1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butylamide]; 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butylamide]; 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphothio Ethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide];N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidyl-ethanolamine;N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidyl-ethanolamine;N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, distearoyl;N-[(3- [maleimido-1-oxopropyl]aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, dimyristoyl; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, dioleoyl; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine Phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; phosphatidylcholine; phosphatidylinositol; phosphatidylserine; phosphatidylethanolamine; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, distearoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dioleoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl;This includes, but is not limited to, N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succinimidyloxyglutaryl)aminopropyl and polyethylene glycol-carbamyl distearoylphosphatidylethanolamine; and N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoylethanolamine.

[0136] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0137] Generally, the sHDL nanoparticles formed in this way are spherical or disc-shaped and have a diameter of approximately 5 nm to 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more homogeneous preparation.

[0138] The above compositions are not limited to specific tolerogenic antigens involved in autoimmune diseases (e.g., MS or celiac disease).

[0139] (Tolerogenic antigen) The present invention includes a composition comprising nanoparticles associated with multiple tolerogenic antigens (e.g., 1 to 30 tolerogenic antigens (e.g., 8 to 30 tolerogenic antigens per nanoparticle)) involved in autoimmune diseases (e.g., MS or celiac disease), a method for treating autoimmune diseases (e.g., MS or celiac disease), and a method for utilizing the above nanoparticles. In the present invention, tolerogenic antigens are antigens that have been identified as being involved in autoimmune diseases (e.g., MS or celiac disease). In one embodiment, the tolerogenic antigen is approximately 3 to 50 amino acids long (for example, approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long). In some embodiments, the tolerogenic antigen is a single tolerogenic antigen with a length of approximately 3 to 50 amino acids.

[0140] In celiac disease, the primary antigens are tissue transglutaminases and gliadins (e.g., α-, γ-, ω-gliadins). Any antigen identified as a tissue transglutaminase or gliadin antigen may be used.

[0141] In some embodiments, the antigen associated with the nanoparticles comprises a gliadin polypeptide (e.g., a full-length gliadin polypeptide or any epitope of a gliadin polypeptide). In some embodiments, the antigen associated with the nanoparticles comprises a 33-mer polypeptide derived from α2-gliadin. In some embodiments, the 33-mer gliadin polypeptide has at least 90% (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the polypeptide sequence of LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 374). In some embodiments, the antigen associated with the nanoparticles comprises an epitope of the 33-mer gliadin polypeptide. The epitope of the 33-mer gliadin polypeptide may be a polypeptide of any length shorter than the 33-mer polypeptide. For example, an epitope may have a length of 25-3 (e.g., 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3) amino acid residues, 20-5 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5) amino acid residues, 12-6 (e.g., 12, 11, 10, 9, 8, 7, or 6) amino acid residues, or 9 amino acid residues. Further examples of 33-gliadin epitopes that may be associated with nanoparticles include any one of the epitopes listed in Table 3, including SEQ ID NOs. 375-405. In some embodiments, the tolerogenic antigen associated with nanoparticles may include any one of the antigens listed in Table 4, including SEQ ID NOs. 406-580. In some embodiments, the polypeptide sequence includes a polypeptide sequence having at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, or 100%) sequence identity with any one of sequence numbers 375 to 580.In some embodiments, the tolerogenic antigen associated with the nanoparticles may include an antigen comprising two or more polypeptides (e.g., 2, 3, 4, 5, or 6) having any two polypeptide sequences of SEQ ID NOs.375–580. In some embodiments, the multiple tolerogenic antigens associated with the nanoparticles (e.g., 1–30 (e.g., 6–30 or 8–30 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)) per nanoparticle have the same identity as all other tolerogenic antigens associated with the nanoparticles. In some embodiments, the multiple tolerogenic antigens associated with the nanoparticles comprise a population of 2–10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different antigen sequences involved in the same disease. For example, nanoparticles may associate with 3-8 (e.g., 3, 4, 5, 6, 7, or 8), 4-6 (e.g., 4, 5, or 6), or 3-4 different polypeptide antigen sequences. In some embodiments, nanoparticles may associate with (i) a first polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-580 or a biologically active fragment or variant thereof, (ii) a second polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-580 or a biologically active fragment or variant thereof, and (iii) a third polypeptide population comprising any one amino acid sequence of SEQ ID NOs. 406-580 or a biologically active fragment or variant thereof. In some examples, the first, second, and third polypeptide populations have different amino acid sequences.In some embodiments, nanoparticles may be associated with (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476) or a biologically active fragment or variant thereof. In some embodiments, nanoparticles may be associated with (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478) or a biologically active fragment or variant thereof. In some embodiments, nanoparticles may be associated with (i) a first polypeptide comprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 506) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 507) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508) or a biologically active fragment or variant thereof. In some embodiments, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include a C-terminal amide group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include an N-terminal pyro-E residue and a C-terminal amide group.In some embodiments, nanoparticles may be associated with (i) a first polypeptide comprising the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511) or a biologically active fragment or variant thereof. In some embodiments, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include an N-terminal acetyl group. In some embodiments described herein, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include a C-terminal amide group. In some embodiments described herein, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include an N-terminal acetyl group and a C-terminal amide group. In any embodiment described herein, the group of antigens associated with the nanoparticles may be completely or partially deamidated. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may contain an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may contain an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may contain an N-terminal amide group. In some embodiments described herein, the immunotolerogenic antigen associated with the nanoparticles may contain a C-terminal amide group.

[0142] [Table 3]

[0143] [Table 4-1]

[0144] [Table 4-2]

[0145] [Table 4-3]

[0146] In some embodiments, the tolerogenic antigen is a bioactive fragment of SEQ ID NO: 474. In some examples, the bioactive fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 512. In some examples, the bioactive fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 580.

[0147] In some cases, the tolerogenic antigen is the bioactive fragment of SEQ ID NO: 475. In some cases, the bioactive fragment of SEQ ID NO: 475 contains a polypeptide comprising the sequence of SEQ ID NO: 542.

[0148] In some embodiments, the tolerogenic antigen is a bioactive fragment of SEQ ID NO: 476. In some examples, the bioactive fragment of SEQ ID NO: 476 comprises a polypeptide containing the sequence of SEQ ID NO: 563.

[0149] In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 375). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPQPELPY (SEQ ID NO: 376). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQ (SEQ ID NO: 377). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FRPEQPYPQ (SEQ ID NO: 378). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQQ (SEQ ID NO: 379). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IQPEQPAQL (SEQ ID NO: 380). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPYPQ (SEQ ID NO: 381). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SQPEQEFPQ (SEQ ID NO: 382). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQEFPQ (SEQ ID NO: 383). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPFPQ (SEQ ID NO: 384). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFCQ (SEQ ID NO: 385). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPEQPQ (SEQ ID NO: 386). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPF (SEQ ID NO: 387). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPW (SEQ ID NO: 388). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 389). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FSQQQESPF (SEQ ID NO: 390).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQ (SEQ ID NO: 391). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 392). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPY (SEQ ID NO: 393). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQ (SEQ ID NO: 394). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 395). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEEPF (SEQ ID NO: 396). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEQPF (SEQ ID NO: 397). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 398). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 399). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPFPQ (SEQ ID NO: 400). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPYPE (SEQ ID NO: 401). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 402). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 403). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQE (SEQ ID NO: 404). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 405). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPW (SEQ ID NO: 406). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPV (SEQ ID NO: 407).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPW (SEQ ID NO: 408). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPIPV (SEQ ID NO: 409). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPFPQ (SEQ ID NO: 410). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPQLPYPQ (SEQ ID NO: 411). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPELPYPQ (SEQ ID NO: 412). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYPQ (SEQ ID NO: 413). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQ (SEQ ID NO: 414). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSQ (SEQ ID NO: 415). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSQ (SEQ ID NO: 416). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFCQ (SEQ ID NO: 417). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFCQ (SEQ ID NO: 418). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYSQ (SEQ ID NO: 419). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYSQ (SEQ ID NO: 420). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQQQCSPVAMPQRLAR (SEQ ID NO: 421). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYLQ (SEQ ID NO: 422). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYLQ (SEQ ID NO: 423).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPQQPFPQ (SEQ ID NO: 424). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPEQPFPQ (SEQ ID NO: 425). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQQQPLPP (SEQ ID NO: 426). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQEQPLPP (SEQ ID NO: 427). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPQQPEQPFPL (SEQ ID NO: 428). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQQGYYPISPQQSGQ (SEQ ID NO: 429). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQPGYYPTSPQQIGQ (SEQ ID NO: 430). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PGQGQSGYYPTSPQQS (SEQ ID NO: 431). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPQQPQLP (SEQ ID NO: 432). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPEQPQLP (SEQ ID NO: 433). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence GQGQSGYYPTSPQQSG (SEQ ID NO: 434). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QYEVIRSLVLRTLPNM (SEQ ID NO: 435). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QVDPSGQVQWPQ (SEQ ID NO: 436). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QVDPSGEVQWPQ (SEQ ID NO: 437). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPL (SEQ ID NO: 438). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPL (SEQ ID NO: 439).The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPY (SEQ ID NO: 440). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPY (SEQ ID NO: 441). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPQQPQPY (SEQ ID NO: 442). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPEQPQPY (SEQ ID NO: 443). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFPQQPIPQQPQPY (SEQ ID NO: 444). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPQQPQPY (SEQ ID NO: 445). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQPY (SEQ ID NO: 446). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPQQPFPQ (SEQ ID NO: 447). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPEQPFPQ (SEQ ID NO: 448). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPIPQQPQPYPQQP (SEQ ID NO: 449). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQQPFPQQPQPY (SEQ ID NO: 450). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSW (SEQ ID NO: 451). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSW (SEQ ID NO: 452). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQPYPQQP (SEQ ID NO: 453). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPQ (SEQ ID NO: 454). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPQ (SEQ ID NO: 455).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPQ (SEQ ID NO: 456). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPQ (SEQ ID NO: 457). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPTPI (SEQ ID NO: 458). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPTPI (SEQ ID NO: 459). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPQQPFPQ (SEQ ID NO: 460). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPEQPFPQ (SEQ ID NO: 461). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPEQI (SEQ ID NO: 462). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPEQI (SEQ ID NO: 463). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQQI (Sequence ID 464). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPQQI (Sequence ID 465). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (Sequence ID 466). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (Sequence ID 467). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (Sequence ID 464). The antigen includes a polypeptide containing (SEQ ID NO. 468). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO. 469). The tolerogenic antigen includes a polypeptide containing the amino acid sequence QPFPQPQQQLPL (SEQ ID NO. 470). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence QPFPQPEQQLPL (SEQ ID NO. 471). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence LFPLPQQPFPQ (SEQ ID NO. 472). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence LFPLPEQPFPQ (SEQ ID NO. 473). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO. 474). In some embodiments, the tolerogenic antigen includes a polypeptide containing the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO. 475). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 479). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFLPQLPYPQ (SEQ ID NO: 480). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QAFPQPQQTFPH (SEQ ID NO: 481). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence TPIQPQQPFPQ (SEQ ID NO: 482). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 483). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFTQPQQPTPI (SEQ ID NO: 484).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQLQQPQQP (SEQ ID NO: 485). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence VAHAIIMHQQQQQQQE (SEQ ID NO: 486). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SYPVQPQQPFPQ (SEQ ID NO: 487). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPQPFPQQPVPQQP (SEQ ID NO: 488). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPWQPQQPFPQ (SEQ ID NO: 489). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 490). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFQPQQPFPQ (SEQ ID NO: 491). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence NPLQPQQPFPLQPQPP (SEQ ID NO: 492). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PLQPQQPFPLQPQPPQ (SEQ ID NO: 493). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PNPLQPQQPFPLQ (SEQ ID NO: 494). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence TIPQQPQQPFPL (SEQ ID NO: 495). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSQQPQQPFPL (SEQ ID NO: 496). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSEQPQQPFPL (SEQ ID NO: 497). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence YSPYQPQQPFPQ (SEQ ID NO: 498). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QLPLQPQQPFPQ (SEQ ID NO: 499). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPQQPFPLQPQQPVP (SEQ ID NO: 500).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IIPQQPQQPFPL (SEQ ID NO: 501). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQIIPQQPQQP (SEQ ID NO: 502). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FLLQPQQPFSQ (SEQ ID NO: 503). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IISQQPQQPFPL (SEQ ID NO: 504). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQRPQQPFPQ (SEQ ID NO: 505). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 506). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 507). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELP (SEQ ID NO: 512). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYP (SEQ ID NO: 513). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPY (SEQ ID NO: 514). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELP (SEQ ID NO: 515). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQP (SEQ ID NO: 516).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQ (SEQ ID NO: 517). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYP (SEQ ID NO: 518). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPY (SEQ ID NO: 519). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELP (SEQ ID NO: 520). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQPQ (SEQ ID NO: 521). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQP (SEQ ID NO: 522). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYP (SEQ ID NO: 523). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 524). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELP (SEQ ID NO: 525). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQPQ (SEQ ID NO: 526). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQP (SEQ ID NO: 527). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQ (SEQ ID NO: 528). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYP (SEQ ID NO: 529). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPY (SEQ ID NO: 530). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELP (SEQ ID NO: 531). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQPQ (SEQ ID NO: 532). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQP (SEQ ID NO: 533).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQ (SEQ ID NO: 534). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYP (SEQ ID NO: 535). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPY (SEQ ID NO: 536). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQPQ (SEQ ID NO: 537). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQP (SEQ ID NO: 538). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYP (SEQ ID NO: 539). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQPQ (SEQ ID NO: 540). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQ (SEQ ID NO: 541). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPF (SEQ ID NO: 542). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFP (SEQ ID NO: 543). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPF (SEQ ID NO: 544). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPW (SEQ ID NO: 545). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFP (SEQ ID NO: 546). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPF (SEQ ID NO: 547). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPWQ (SEQ ID NO: 548). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFP (SEQ ID NO: 549). The tolerogenic antigen contains a polypeptide comprising the amino acid sequence QPEQPFPWQP (SEQ ID NO: 550).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQ (SEQ ID NO: 551). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPW (SEQ ID NO: 552). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFP (SEQ ID NO: 553). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPF (SEQ ID NO: 554). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQP (SEQ ID NO: 555). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQ (SEQ ID NO: 556). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPW (SEQ ID NO: 557). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFP (SEQ ID NO: 558). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQP (SEQ ID NO: 559). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQ (SEQ ID NO: 560). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQP (SEQ ID NO: 561). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPWQ (SEQ ID NO: 562). In some embodiments... In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQ (SEQ ID NO: 563). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQP (SEQ ID NO: 564). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQ (SEQ ID NO: 565). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQP (SEQ ID NO: 566). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYP (SEQ ID NO: 567). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPY (SEQ ID NO: 568). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQP (SEQ ID NO: 569). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQ (SEQ ID NO: 570). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQQ (SEQ ID NO: 571). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQ (SEQ ID NO: 572). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYP (SEQ ID NO: 573). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPY (SEQ ID NO: 574). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQQ (SEQ ID NO: 575). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYPQ (SEQ ID NO: 576). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYP (SEQ ID NO: 577). In some embodiments, the tolerogenic antigen comprises a polypeptide having the amino acid sequence EQPIPEQPQPYPQQ (SEQ ID NO: 578).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQ (SEQ ID NO: 579). The tolerogenic antigen comprises a polypeptide containing the amino acid sequence PDLP (SEQ ID NO: 580). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQ (SEQ ID NO: 581). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQP (SEQ ID NO: 582). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQPQ (SEQ ID NO: 583). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQP (SEQ ID NO: 584). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQ (SEQ ID NO: 585). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYP (SEQ ID NO: 586). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPY (SEQ ID NO: 587). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQP (SEQ ID NO: 588).

[0150] In some embodiments, the tolerogenic antigens include human allograft antigens. Examples of human allograft antigens include, but are not limited to, subunits of various MHC class I and MHC class II haplotype proteins, as well as single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Duffy, and Ss.

[0151] In some embodiments, a tolerogenic antigen is an autoantigen that a subject (e.g., a human patient) has developed or may develop an autoimmune response to. Examples include proinsulin (e.g., for subjects with or at risk of developing diabetes), collagen (e.g., for subjects with or at risk of developing rheumatoid arthritis), and myelin basic protein (e.g., for subjects with or at risk of developing multiple sclerosis). There are many proteins that are human autoimmune proteins, and the term is used in relation to various autoimmune diseases, and such proteins or the proteins that cause such diseases may be known or established by routine testing. Embodiments include testing a patient to identify an autoimmune protein, creating an antigen for use in molecular fusion, and creating immune tolerance to a protein. Embodiments include including an antigen or selecting an antigen from one or more of the following proteins: In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamate decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2β (IA-2β); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-enteropancreatic / pancreatic-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreaticoduodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5. In autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves' disease, the main antigens include thyroglobulin (TG), thyroid peroxidase (TPO), and thyrotropin receptor (TSHR); other antigens include sodium-iodine cotransporter (NIS) and megalin.In thyroid-associated eye and skin disorders, the antigen is insulin-like growth factor 1 receptor, in addition to thyroid autoantigens including TSHR. In hypoparathyroidism, the main antigen is calcium-sensitive receptor. In Addison's disease, the main antigens include 21-hydroxylase, 17α-hydroxylase, and P450 side-chain cleavage enzyme (P450scc); other antigens include ACTH receptor, P450c21, and P450c17. In premature ovarian failure, the main antigens include FSH receptor and α-enolase. In autoimmune hypothyroidism or autoimmune pituitary disease, the main antigens include pituitary-specific protein factor (PGSF) 1a and 2; other antigens include type 2 iodothyronine deiodinase. In multiple sclerosis, the main antigens include myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein. In rheumatoid arthritis, the main antigen is collagen II. In immune gastritis, the main antigen is H. + ,K +-ATPase. In pernicious anemia, the main antigen is intrinsic factor. In celiac disease, the main antigens are tissue transglutaminase and gliadin. In vitiligo, the main antigens are tyrosinase and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the main antigen is acetylcholine receptor. In pemphigus vulgaris and its variants, the main antigens are desmoglein 3, 1 and 4; other antigens include pemfaxine, desmocolin, placoglobin, perplakin, desmoplakin, and acetylcholine receptor. In bullous pemphigoid, the main antigens include BP180 and BP230; other antigens include plectin and laminin 5. In herpetiform dermatitis of Duhring, the main antigens include endomysium and tissue transglutaminase. In acquired epidermolysis bullosa, the main antigen is collagen VII. In systemic sclerosis, the main antigens include matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, and the PDGF receptor; other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere protein, topoisomerase I, nucleolar protein, RNA polymerases I, II, and III, PM-Slc, fibrillarin, and B23. In mixed connective tissue disease, the main antigen is U1snRNP. In Sjögren's syndrome, the main antigens are nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase, and topoisomerase. In systemic lupus erythematosus, the main antigens include nuclear proteins including SS-A, high-mobility group box 1 (HMGB1), nucleosomes, histone proteins, and double-stranded DNA. In Goodpasture syndrome, the main antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the main antigen is cardiac myosin.Other autoantigens identified in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, liver P450 cytochromes P450 1A2 and 2A6, SOX-9, SOX-10, calcium-sensitive receptor proteins, and type 1 interferons interferon α, β, and ω.

[0152] In some cases, a tolerogenic antigen is an exogenous antigen that causes a patient to develop an undesirable autoimmune response. An example is a food antigen. Embodiments include testing a patient to identify an exogenous antigen, creating a molecular fusion containing the antigen, and treating the patient to develop immune tolerance to the antigen or food. Examples of such foods and / or antigens are provided below. Examples include: from peanuts: conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apples: 31kDa major allergen / disease resistance protein homolog (Mal d 2), lipid transport protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1); from milk: α-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c 1, Act d 1), phytocystatin, tomatin-like protein (Act d 2), kiuelin (Act d 5); from mustard: 2S globulin (Sin a 1), 11S globulin (Sin a 2), lipid transport protein (Sin a 3), profilin (Sin a 4); from celery: profilin (Api g 4) High molecular weight glycoprotein (Api g 5); from shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform; from wheat and / or other cereals: high molecular weight glutenin, low molecular weight glutenin, α- and γ-gliadin, hordein, secarin, avenin; from strawberries: major strawberry allergen Fra a 1-E (Fra a 1); from bananas: profilin (Mus xp 1). In some embodiments, the tolerogenic antigen is one antigenic peptide from SEQ ID NOs. 589-742 (Table 5).

[0153] [Table 5-1]

[0154] [Table 5-2]

[0155] [Table 5-3]

[0156] [Table 5-4]

[0157] [Table 5-5]

[0158] [Table 5-6]

[0159] [Table 5-7]

[0160] [Table 5-8]

[0161] [Table 5-9]

[0162] In some cases, the autoimmune disease is type 1 diabetes, and the tolerogenic antigens are derived from carboxypeptidase H, chromagranin A, glutamate decarboxylase, Imogen-38, insulin, insulinoma antigens-2 and 2β, islet-associated glucose-6-phosphatase catalytic subunit-associated protein (IGRP), islet β-cell antigen, or proinsulin.

[0163] In some cases, the autoimmune disease is MS, and the tolerogenic antigens are derived from α-enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, or S100-β.

[0164] In some cases, the autoimmune disease is rheumatoid arthritis, and the tolerogenic antigens are derived from citrullinated proteins, collagen II, heat shock proteins, gpl30-RAPS, or human cartilage glycoprotein 39.

[0165] In some cases, the autoimmune disease is systemic lupus erythematosus, and the tolerogenic antigens are derived from the Sm antigen of the La antigen, nucleosome histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, glycoprotein gp70, or the U-1 small ribonucleoprotein complex.

[0166] In some cases, the autoimmune disease is scleroderma, and the tolerogenic antigen is derived from fibrillarin or small nucleolar protein (snoRNP).

[0167] In some embodiments, the autoimmune disease is Graves' disease, and the tolerogenic antigen is derived from the thyroid-stimulating factor receptor (TSH-R).

[0168] In some cases, the autoimmune disease is biliary cirrhosis, and the tolerogenic antigen is derived from pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2).

[0169] In some embodiments, the autoimmune disease is alopecia areata, and the tolerogenic antigen is derived from a hair follicle antigen.

[0170] In some cases, the autoimmune disease is ulcerative colitis, and the tolerogenic antigen is derived from human tropomyosin isoform 5 (hTM5).

[0171] In some cases, tolerogenic antigens include 17-hydroxylase, 21-hydroxylase, ADAMTS13, annexin A5, apoH, AQP4, aromatic carboxylase, basement membrane type IV collagen, BP-1, BP-2, carbonic anhydrase, carboxypeptidase H, cardiolipin, cardiolipin, chromogranin A, complement component 3, desmoglein 3, enolase, epithelial transglutaminase, GD1a, gliadin, glutamate receptor, glutamate decarboxylase, glycoprotein. Quality IIb-IIIa or Ib-IX, GMCSF, gpIIb-IIIa or 1b-IX, GQ1b, GQ1b, histidine-tRNA, histone, HPA-1a, HPA-5b, HSP60, HSP70, HSP90, Hu, IA-2β, IAPP, ICA69, IFN-γ, IGRP, IL-1, insulin, insulinoma antigen-2, interferon ω, Jo1, keratin, Kir4.1, LA, LKM-1, LKM-1, LKM-2, LKM-3, LP, major peripheral myelin protein P0, Mi-2, muscarinic acetylcholine receptor M1, MuSK protein, hypocretin, myelin-related protein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin-related oligodendrocyte basic protein, cardiac myosin, myeloperoxidase, neurofilament, nicotinic acetylcholine receptor, orexin, outer surface protein (OSP), p62, phosphatidylserine, proteolipid The antigen is derived from an antigen selected from the group consisting of protein (PLP), pyruvate dehydrogenase, Q-type calcium channel, Ro, sc170, signal recognition peptide, SMA, soluble liver antigen, sp100, synaptogagmine, thyroglobulin, thyroid peroxidase, tissue transglutaminase, TNF-α, topoisomerase, transglutaminase, type XVII collagen, U1-RNP, voltage-gated calcium channel, Yo, ZnT8, or β2-glycoprotein I.

[0172] The tolerogenic antigen is hInsB10-18 (HLVEALYLV (SEQ ID NO: 743)), hIGRP 228-236 (LNIDLLWSV(SEQ ID NO: 744)), hIGRP 265-273 (VLFGLGFAI (SEQ ID NO: 745)), IGRP 206-214 (VYLKTNVFL (SEQ ID NO: 746)), NRP-A7 (KYNKANAFL (SEQ ID NO: 747)), NRP-I4 (KYNIANVFL (SEQ ID NO: 748)), NRP-V7 (KYNKANVFL (SEQ ID NO: 749)), YAI / Db (FQDENYLYL (SEQ ID NO: 750)) and / or INS B 15-23 The present invention may further include, but is not limited to, (LYLVCGERG (SEQ ID NO: 751)) and the peptides and proteins disclosed in U.S. Patent Application Publication No. 20050202032.

[0173] In a particular embodiment, the peptide antigen used for the treatment of type 1 diabetes is GAD65 114123 VMNILLQYVV(SEQ ID NO: 752);GAD65 536-545 ,RMMEYGTTMV(SEQ ID NO: 753);GFAP 143-151 ,NLAQTDLATV(SEQ ID NO: 754);GFAP 214-222 , QLARQQVHV (Sequence ID 755); IA-2 172-180 ,SLSPLQAEL(Sequence ID 756);IA-2 482-490 , SLAAGVKLL(SEQ ID NO: 757);IA-2 805-813 ,VIVMLTPLV(sequence code 758);ppIAPP 5-13 ,KLQVFLIVL(sequence code 759);ppIAPP 9-17 , FLIVLSVAL(SEQ ID NO: 760); IGRP 152-160 , FLWSVFMLI (SEQ ID NO: 761); IGRP 211-219 , NLFLFLFAV(SEQ ID NO: 762); IGRP 215-223 FLFAVGFYL (Sequence ID 763); IGRP 222-230 ,YLLLRVLNI(Sequence ID 764);IGRP 228-236 , LNIDLLWSV(SEQ ID NO: 744); IGRP 265-273VLFGLGFAI (SEQ ID NO: 745); IGRP 293-301 RLLCALTSL (SEQ ID NO: 765); proinsulin L2-10 ALWMRLLPL (SEQ ID NO: 766); proinsulin L3-11 LWMRLLPLL (SEQ ID NO: 767); Proinsulin L6-14 , RLLPLLALL (SEQ ID NO: 768); proinsulin B5-14 HLCGSHLVEA (SEQ ID NO: 769); proinsulin B10-18 HLVEALYLV (Sequence ID 743); Proinsulin B14-22 ALYLVCGER (SEQ ID NO: 770); proinsulin B15-24 LYLVCGERGF (SEQ ID NO: 771); proinsulin B17-25 , LVCGERGFF (SEQ ID NO: 772); proinsulin B18-27 VCGERGFFYT (SEQ ID NO: 773); proinsulin B20-27 GERGFFYT (SEQ ID NO: 774); proinsulin B21-29 ERGFFYTPK (SEQ ID NO: 775); proinsulin B25-C1 FYTPKTRRE (SEQ ID NO: 776); Proinsulin B27-C5 TPKTRREAEDL (SEQ ID NO: 777); proinsulin C20-28 SLQPLALEG (SEQ ID NO: 778); proinsulin C25-33 ALEGSLQKR (SEQ ID NO: 779); proinsulin C29-A5 SLQKRGIVEQ (SEQ ID NO: 780); proinsulin A1-10 GIVEQCCTSI (SEQ ID NO: 781); proinsulin A2-10 , IVEQCCTSI (SEQ ID NO: 782); proinsulin A12-20 This includes SLYQLENYC (sequence number 783), or a combination of these.

[0174] In a further embodiment, a tolerogenic antigen associated with multiple sclerosis (MS) may be used, and may include: MAG 287-295 ,SLLLELEEV(Sequence ID 784);MAG 509-517, LMWAKIGPV (SEQ ID NO: 785); MAG 556-564 , VLFSSDFRI (SEQ ID NO: 786); MBP 110-118 , SLSRFSWGA (SEQ ID NO: 787); MOG 114-122 , KVEDPFYWV (SEQ ID NO: 788); MOG 166-175 , RTFDPHFLRV (SEQ ID NO: 789); MOG 172-180 , FLRVPCWKI (SEQ ID NO: 790); MOG 179-188 , KITLFVIVPV (SEQ ID NO: 791); MOG 188-196 , VLGPLVALI (SEQ ID NO: 792); MOG 181-189 , TLFVIVPVL (SEQ ID NO: 793); MOG 205-214 , RLAGQFLEEL (SEQ ID NO: 794); PLP 80-88 , FLYGALLLA (SEQ ID NO: 795), or a combination thereof.

[0175] In some examples, tolerogenic antigens associated with systemic lupus erythematosus can be used, including but not limited to FIEWNKLRFRQGLEW (SEQ ID NO: 796). In some examples, the tolerogenic antigen comprising a polypeptide having the sequence of SEQ ID NO: 796 comprises at least one amino acid moiety that is a D-amino acid.

[0176] (Multimeric tolerogenic antigen) In certain embodiments, the tolerogenic antigens provided herein are multimeric tolerogenic antigens. In one example, the multimeric tolerogenic antigen comprises two or more tolerogenic antigens (e.g., tolerogenic antigens described herein) linked by a linker (e.g., a peptide linker). In some examples, the tolerogenic antigen comprises the following N-terminal - C-terminal structure: (P4 - L4) n4 -(P3 - L3) n3 -P2-(L1 - P1) n1 In the formula, P1, P2, P3, and P4 are each independently selected from any tolerogenic antigen described herein (for example, any tolerogenic antigen in Tables 3-5); L1, L3, and L4 are each independently linkers; and n1, n3, and n4 are each independently 0 or 1, with at least one of n1, n3, and n4 being 1.

[0177] In some cases, n1 is 1, n3 is 0, n4 is 0, and the tolerogenic antigen contains the following N-terminal-C-terminal structure: P2-L1-P1

[0178] In some examples, the peptide linker comprises 2 to 200 amino acids (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150), or 145 to 200 amino acids (e.g., 145 to 160, 155 to 170, 165 to 180, 175 to 190, or 185 to 200). In some embodiments, the peptide linker comprises glycine (Gly) and serine (Ser) amino acids. In some examples, the peptide linker comprises (GS) x (GGS) x , (GGGGS(Sequence ID 797)) x (GGSG) x (SGGG) x It contains one of the amino acid sequences, where x is an integer from 1 to 10. In a particular embodiment, the linker is (GGGGS(SEQ ID NO: 797)) x The amino acid sequence is such that x is an integer between 2 and 5. In some cases, P2 and P1 are different tolerogenic antigens. In some cases, P2 and P1 are the same tolerogenic antigen.

[0179] In some examples, n1 is 1, n3 is 1, n4 is 0, and the tolerogenic antigen contains the following N-terminal-C-terminal structure: P3-L3-P2-L1-P1

[0180] In some examples, each peptide linker independently contains 2 to 200 amino acids (e.g., 5 to 50 (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150), or 145 to 200 amino acids (e.g., 145 to 160, 155 to 170, 165 to 180, 175 to 190, or 185 to 200)). In some examples, the peptide linker contains glycine (Gly) and serine (Ser) amino acids. In some examples, the peptide linker contains (GS) x , (GGS) x , (GGGGS (SEQ ID NO: 797)) x , (GGSG) x , (SGGG) x and includes any one of the amino acid sequences, where x is an integer from 1 to 10. In certain embodiments, the linker contains the amino acid sequence of (GGGGS (SEQ ID NO: 797)) x and x is an integer from 2 to 5. In some examples, P3, P2, and / or P1 are different tolerogenic antigens. In some examples, P3, P2, and / or P1 are the same tolerogenic antigen.

[0181] In some examples, n1 is 1, n3 is 1, n4 is 1, and the tolerogenic antigen contains the following N-terminal - C-terminal structure: P4-L4-P3-L3-P2-L1-P1

[0182] In some examples, peptide linkers are peptide linkers containing 2 to 200 amino acids (e.g., 5 to 50 (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50)), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100)), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150)), or 145 to 200 amino acids (e.g., 145 to 160, 155 to 170, 165 to 180, 175 to 190, or 185 to 200). In some examples, peptide linkers contain glycine (Gly) and serine (Ser) amino acids. In some examples, peptide linkers contain (GS) x (GGS) x , (GGGGS(Sequence ID 797)) x (GGSG) x (SGGG) x It contains one of the amino acid sequences, where x is an integer from 1 to 10. In a particular embodiment, the linker is (GGGGS(SEQ ID NO: 797)) x The amino acid sequence is such that x is an integer between 2 and 5. In some cases, P4, P3, P2, and / or P1 are different tolerogenic antigens. In some cases, P4, P3, P2, and / or P1 are the same tolerogenic antigen.

[0183] In some embodiments, the tolerogenic antigen is bound to nanoparticle phospholipids in a manner that promotes potent immune tolerance upon administration to subjects (e.g., human subjects with or at risk of developing autoimmune disorders (e.g., MS or celiac disease)).

[0184] In some embodiments, the tolerogenic antigen is bound to the nanoparticle phospholipid via thiol-reactive and reduction-insensitive binding between the tolerogenic antigen and the nanoparticle phospholipid. Indeed, the thiol-reactive and reduction-insensitive binding between the tolerogenic antigen and the nanoparticle phospholipid promotes potent immunotolerance. In some embodiments, for example, the phospholipid is N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine.

[0185] In some embodiments, tolerogenic antigens are bound to nanoparticle phospholipids via amine-mediated interactions. In some embodiments, for example, the amine-mediated interaction is via an amine-reactive phospholipid (e.g., N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl (DOPE-NHS)). In some embodiments, the amine-mediated interaction is via an amine-reactive phospholipid having a self-destructive linkage. The self-destructive linkage includes a linker containing o-dithiobenzyl, p-dithiobenzyl, a β-dithiobenzylcarbamate moiety, 2,2-dimethyl-4-mercaptobutyric acid, or a disulfide-carbonate-based traceless linker.

[0186] In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle is any amount that promotes potent immune tolerance upon administration to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder (e.g., MS or celiac disease)). In some embodiments, the amount of tolerogenic antigens associated with a particular nanoparticle is 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

[0187] Tolerogenic antigens can be prepared by many techniques known in the art, depending on the molecular properties. Polynucleotides, polypeptides, and carbohydrate antigens can be isolated from the type of cells being treated in which they are concentrated. Short peptides can be conveniently prepared by amino acid synthesis. Proteins longer than known sequences can be prepared by synthesizing the coding sequence or by PCR amplification of the coding sequence from a natural source or vector, and then expressing the coding sequence in a suitable bacterial or eukaryotic host cell.

[0188] [Linker] In some embodiments of the compositions described herein, nanoparticles having multiple tolerogenic antigens include a linker between the tolerogenic antigens and the nanoparticles. In some embodiments, the linker refers to a covalent bond or bond between the tolerogenic antigen and the phospholipid group of the nanoparticle. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen is modified by a terminal cysteine ​​residue bound to the linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen is terminal C(S) nModified by a polypeptide, where n is a serine residue of 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), and the terminal serine residue is bound to a linker. In some embodiments, the N-terminus and / or C-terminus of the toxicogenic antigen is modified by a terminal CSS polypeptide bound to a linker. In some embodiments, the linker is a thiol-reactive crosslinker. In some embodiments, the linker is a maleimide linker. In some embodiments, the linker is a pyridyl linker. In some embodiments, the N-terminus and / or C-terminus of the toxicogenic antigen is modified by a terminal cysteine ​​residue bound to a maleimide linker. In some embodiments, the N-terminus and / or C-terminus of the toxicogenic antigen is modified by a terminal cysteine ​​residue bound to a pyridyl linker. In some embodiments, the N-terminus and / or C-terminus of the toxicogenic antigen is modified by a terminal CSS polypeptide bound to a maleimide linker. In some embodiments, the N-terminus and / or C-terminus of the toxicogenic antigen is modified by a terminal CSS polypeptide bound to a maleimide linker. The linker may be bound at its first end to a modified nucleoside or nucleotide (e.g., Cys and Ser) on a nucleic acid base or sugar moiety, and at its second end to a payload (e.g., a lipid (e.g., a phospholipid)).

[0189] The linker may be a chemical linker known to those skilled in the art. Alternatively, the linker may be a peptide linker. The linker may be long enough so as not to interfere with the polypeptide sequence or lipid moiety. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, amide groups, amino groups, ether groups, thioether groups, ester groups, alkylene groups, heteroalkylene groups, aryl groups, or heterocyclyl groups (each of which may be optionally substituted). The linker may, for example, contain synthetic groups derived from synthetic polymers (e.g., polyethylene glycol (PEG) polymers). In some embodiments, the linker may contain one or more amino acid residues (e.g., D- or L-amino acid residues). Further examples of useful linkers include those containing electrophiles. Examples of electrophiles include Michael adducts (e.g., maleimide), activated esters, electron-deficient carbonyl compounds, and aldehydes, which are suitable for reactions with nucleophilic substituents present in antibodies, antigen-binding fragments, proteins, peptides, and small molecules (e.g., amine and thiol moieties).

[0190] In the present invention, the linker between the tolerogenic antigens of the multimer (e.g., L1, L3, and / or L4) is 2 to 200 amino acids (e.g., 5 to 50 (e.g., 5 to 20, 15 to 30, 25 to 40, or 35 to 50), 45 to 100 (e.g., 45 to 60, 55 to 70, 65 to 80, 75 to 90, or 85 to 100), 95 to 150 (e.g., 95 to 110, 105 to 120, 115 to 130, 125 to 140, or 135 to 150), Alternatively, it may be a peptide linker containing 145-200 amino acids (e.g., 145-160, 155-170, 165-180, 175-190, or 185-200). In some embodiments, the linker between multimerized tolerogenic antigens (e.g., L1, L3, and / or L4) is a polypeptide containing at least 12 amino acids, for example, 12-200 amino acids (e.g., 12-200, 12-180, 12-160, 12 ~140, 12~120, 12~100, 12~90, 12~80, 12~70, 12~60, 12~50, 12~40, 12~30, 12~20, 12~19, 12~18, 12~17, 12~16, 12~15, 12~14, or 12~13 amino acids) (e.g., 14~200, 16~200, 18~200, 20~200, 30~200, 40~200, 50~200, 60~200, 70~200, 80~200, 90~200, 100) The amino acids are ~200, 120~200, 140~200, 160~200, 180~200, or 190~200. In some embodiments, the linker between the tolerogenic antigens of the multimer (e.g., L1, L3, and / or L4) is a polypeptide comprising 12 to 30 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids).

[0191] Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the linker may contain motifs (e.g., multiple or repeating motifs) of GS, GGS, GGGGS (SEQ ID NO: 797), GGSG (SEQ ID NO: 798), or SGGG (SEQ ID NO: 799). In certain embodiments, the linker may contain 2 to 12 amino acids containing the GS motif, for example, GS, GSGS (SEQ ID NO: 800), GSGSGS (SEQ ID NO: 801), GSGSGSGS (SEQ ID NO: 802), GSGSGSGSGS (SEQ ID NO: 803), or GSGSGSGSGSGS (SEQ ID NO: 804). In certain other embodiments, the linker may contain 3 to 12 amino acids containing the GGS motif, for example, GGS, GGSGGS (SEQ ID NO: 805), GGSGGSGGS (SEQ ID NO: 806), and GGSGGSGGSGGS (SEQ ID NO: 807). In yet another embodiment, the linker may contain 4 to 12 amino acids including the GGSG (SEQ ID NO: 808) motif, for example, GGSGGGSG (SEQ ID NO: 809) or GGSGGGSGGGSG (SEQ ID NO: 810). In yet another embodiment, the linker may contain the GGGGS (SEQ ID NO: 797) motif, for example, GGGGSGGGGSGGGGS (SEQ ID NO: 811). In a particular embodiment, the linker is SGGGSGGGSGGGSGGGGGGG (SEQ ID NO: 812).

[0192] In a preferred embodiment, the peptide linker (e.g., L1, L3, and / or L4) is a peptide linker comprising one of the amino acid sequences (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, or (SGGG)x, where x is an integer from 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5). In a preferred embodiment, the peptide linker is an amino acid sequence (GGGGS) x The expression has such that x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0193] In some embodiments, the peptide linker is a glycine residue (for example, at least four glycine residues (for example, 4-200, 4-180, 4-160, 4-140, 4-40, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 glycine residues) (e.g., 4-200, 6-200, 8-200, 10-200, 12-200, 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200) It contains only glycine residues. In some embodiments, the linker has 4 to 30 glycine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycine residues). In some embodiments, a linker containing only glycine residues may not be glycosylated (e.g., O-linked glycosylation, also called O-glycosylation), or may have a lower degree of glycosylation (e.g., a lower degree of O-glycosylation) (e.g., a lower degree of O-glycosylation by glycans such as xylose, mannose, sialic acid, fucose (Fuc), and / or galactose (Gal) (e.g., xylose)).

[0194] In some embodiments, a linker containing only glycine residues may not be O-glycosylated (e.g., O-xylosylated), or may have a lower degree of O-glycosylation (e.g., a lower degree of O-xylosylation) compared to a linker containing one or more serine residues.

[0195] In some embodiments, linkers containing only glycine residues may not undergo proteolysis, or may undergo a lower rate of proteolysis compared to, for example, linkers containing one or more serine residues.

[0196] In a particular embodiment, the linker may include the motif of GGGG (SEQ ID NO: 813), for example, GGGGGGGG (SEQ ID NO: 814), GGGGGGGGGGGG (SEQ ID NO: 815), GGGGGGGGGGGGGGGG (SEQ ID NO: 816), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 817). In a particular embodiment, the linker may include the motif of GGGGG (SEQ ID NO: 818), for example, GGGGGGGGGG (SEQ ID NO: 819), GGGGGGGGGGGGGGGG (SEQ ID NO: 820), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 821). In a particular embodiment, the linker is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 822).

[0197] In other embodiments, the linker may also include amino acids other than glycine and serine, such as GENLYFQSGG (SEQ ID NO: 823), SACYCELS (SEQ ID NO: 757), RSIAT (SEQ ID NO: 824), RPACKIPNDLKQKVMNH (SEQ ID NO: 825), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 826), AAANSSIDLISVPVDSR (SEQ ID NO: 827), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 828).

[0198] [Tolerogenic antigen variant] In certain embodiments, amino acid sequence variants of the tolerogenic antigen of the present invention are intended. For example, it may be desirable to improve the tolerogenicity and / or other biological properties of the tolerogenic antigen. Amino acid sequence variants of the tolerogenic antigen may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the tolerogenic antigen, or by peptide synthesis. Such modifications include, for example, deletions of residues in the amino acid sequence of the tolerogenic antigen, and / or insertions and / or substitutions therein. Any combination of deletions, insertions, and substitutions can be used to arrive at the final construct, provided that the final construct has the desired characteristics, such as inducing antigen resistance.

[0199] In certain embodiments, tolerogenic antigen variants having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table 6 under the heading "Preferred Substitutions." More substantial changes are provided in Table 6 under the heading "Exemplary Substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into an immunotolerogenic antigen of interest and screened for desired activity, for example, to maintain / improve immunotolerogenic antigenicity.

[0200] [Table 6]

[0201] Amino acids may be grouped according to common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Electrically neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0202] Non-conservative substitution involves replacing one member of one of these classes with one of a different class.

[0203] A useful method for identifying residues or regions of tolerogenic antigens that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substitutes them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively, or even further, the crystal structure of the antigen-antibody complex may be used to identify contact sites between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Tolerogenic antigen variants may be screened to determine whether they possess the desired properties.

[0204] Amino acid sequence insertions include amino- and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0205] [Treatment methods for autoimmune disorders] As described herein, in certain embodiments, the present invention provides a method for treating autoimmune disorders (e.g., MS, celiac disease) by administering a composition comprising nanoparticles associated with a tolerogenic antigen related to an autoimmune disease (e.g., MS or celiac disease) via binding (e.g., 1 to 30 tolerogenic antigens per nanoparticle) as described herein, to a subject (e.g., a human subject who has or is at risk of developing an autoimmune disease (e.g., MS or celiac disease)).

[0206] The immune system can be classified into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against infection, and most potential pathogens are rapidly neutralized by this system before they can cause a significant infection. The adaptive immune system reacts to molecular structures called antigens of invading organisms. There are two types of adaptive immune responses: humoral immune responses and cellular immune responses. In humoral immune responses, antibodies secreted into the body fluids from B cells bind to antigens derived from pathogens, leading to the elimination of pathogens through various mechanisms, such as complement-mediated lysis. In cellular immune responses, T cells, which can destroy other cells, are activated. For example, if proteins associated with a disease (e.g., MS or celiac disease) are present in cells, they are proteolytically fragmented into peptides within the cell. Certain cellular proteins then attach to the antigens or peptides formed in this manner and transport them to the cell surface, where they are presented to the body's molecular defense mechanisms, particularly T cells. Cytotoxic T cells recognize these antigens and kill the cells that possess them.

[0207] Molecules that transport and present peptides to the cell surface are called major histocompatibility complex (MHC) proteins, known in humans as human leukocyte antigen (HLA) complexes. MHC proteins are classified into two types: MHC class I and MHC class II. Although the structures of the two MHC class proteins are very similar, they have very different functions. MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. MHC class I proteins are loaded with antigens, usually derived from endogenous proteins or pathogens present within the cell, and are then presented to naive or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. They primarily present peptides that are processed by T helper (Th) cells from external antigen sources, i.e., from outside the cell. Most peptides bound to MHC class I proteins originate from cytoplasmic proteins produced in the organism's own healthy host cells and do not usually stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize such class I self-peptide-presenting MHC molecules are deleted in the thymus (central immune tolerance) or, after being released from the thymus, are deleted or inactivated, i.e., tolerated (peripheral immune tolerance). MHC molecules can stimulate an immune response when they present peptides to intolerant T lymphocytes. Cytotoxic T lymphocytes have both a T cell receptor (TCR) and a CD8 molecule on their surface. The T cell receptor can recognize and bind peptides that have formed complexes with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0208] In some embodiments, the compositions and methods described herein for treating celiac disease, comprising nanoparticles associated with a plurality of tolerogenic antigens involved in celiac disease (e.g., 1 to 30 tolerogenic antigens per particle). In the case of celiac disease, MHC class II protein haplotypes, HLA-DQ2 and HLA-DQ8 are involved in the onset of this disease. Having the HLA-DQ2 and HLA-DQ8 haplotypes does not require that the subject also have celiac disease, but these haplotypes are necessary for celiac disease to occur. This is because HLA-DQ2 and HLA-DQ8 have a greater affinity for deamidated polypeptides. The 33-mer gliadin polypeptide is deamidated by the TG2 enzyme, and thus the deamidated 33-mer gliadin polypeptide binds to HLA-DQ2 or HLA-DQ8 to form an HLA-DQ2-gliadin or HLA-DQ8-gliadin complex. This complex activates host-gluten specific CD4 + T cells, stimulates B cells, and then produces anti-gliadin and anti-TG2 antibodies. Activation of T cells causes cytokine production, leading to increased IFNγ production, inflammation and damage to the small intestine, and causing mucosal intestinal lesions.

[0209] To prevent an autoimmune response, the tolerogenic antigen attaches to HLA-DQ2 or HLA-DQ8 by competitive affinity binding within the endoplasmic reticulum before being presented on the cell surface. Here, the affinity of an individual peptide antigen is directly linked to its amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. If the sequence of such a peptide is known, the immune system against diseased cells can be manipulated, for example, using a peptide vaccine.

[0210] In some embodiments, a subject suffering from any of the autoimmune diseases (e.g., celiac disease) described herein follows a strict gluten-free diet. In some embodiments of any of the methods described herein, a subject suffering from celiac disease does not follow a gluten-free diet.

[0211] Furthermore, experiments conducted during the development of embodiments for the present invention determined that reverse vaccination with nanoparticles having tolerogenic antigens conjugated to a lipid moiety (e.g., by a thiol-reactive or autoimmune linker) is an effective strategy for the treatment of autoimmune conditions (e.g., MS). It was further demonstrated that such therapeutic compositions are optimized at a ratio of 1 - 30 (e.g., 6 - 30, 7 - 30, or 8 - 30) tolerogenic antigens per nanoparticle.

[0212] The methods are not limited to treating specific types of autoimmune disorders. Examples of such autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, diabetes (e.g., type 1 diabetes), autoimmune thyroid diseases (Hashimoto's disease, Graves' disease), thyroid-associated ophthalmopathy, thyroid-associated skin disorders, hypoparathyroidism, Addison's disease, premature ovarian insufficiency, autoimmune hypothyroidism, autoimmune pituitary diseases, immune gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, dermatitis herpetiformis Duhring, epidermolysis bullosa acquisita, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodpasture's syndrome, rheumatic heart disease, autoimmune polyendocrine syndrome type 1, Aicardi-Goutières syndrome, age-related macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

[0213] In some embodiments, the above methods for treating or preventing autoimmune disorders further encompass the administration of additional therapeutic agents (e.g., simultaneously or at different times). Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biological agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent includes, but is not limited to, infliximab, adalimumab, etanercept, or parenteral or oral gold.In some cases, the therapeutic agent is an immunomodulator or immunosuppressant (e.g., statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors such as trichostatin A; corticosteroids; mitochondrial function inhibitors such as rotenone; P38 inhibitors; 6Bio, dexamethasone, TCPA-1, IKK) NF-κβ inhibitors such as VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2) such as misoprostol; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors (PDE4) such as rolipram; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB inhibitors such as TGX-221; autophagy inhibitors such as 3-methyladenine; aromatic hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATP such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cycloporine such as cycloporine A, aromatic hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, triptolide; OPN-305; OPN-401; Elistran (E5664); TAK-242; Cpn10; NI-0101; 1A6; AV411; IR S-954(DIV-1079);IMO-3100;CPG-52363;CPG-52364;OPN-305;ATNC05;NI-0101;IMO-8400;Hydroxychloroquine;CU-CPT22;C29;Ortho-vanillin;SSL3 protein;OPN-305;SsnB;Byzantine;(+)-N-phenethylnoroxymorphone;VB3323;Monosaccharide 3;(+)-naltrexone and (+)-naloxone;HT52;HTB2;Compound 4a;CNTO2424;TH1020;INH-ODN;E6446;AT791;CpG ODN 2088;ODN TTAGG;COV08-0064;2R9;GpG oligonucleotide;2-aminopurine;Anlexanox;Bay11-7082;BX795;CH-223191;Chloroquine;CLI-095;CU-CPT9a;Cyclosporine A;CTY387;Gefinitib;Glibenclamide;H-89;H-131;Isoliquitigenin;MCC950;MRT67307;OxPAPC;Parthenolide;Pepinh-MYD;Pepinh-TRIF;Polymyxin B;R406;RU.521;VX-765;YM201636;Z-VAD-FMK;and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD);Tryptamine (TA);and 6-formylindrol[3,2 b) AHR-specific ligands, including but not limited to carbazoles (FICZ).In certain embodiments, the immunosuppressant is fingolimod; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or related ligand; trichostatin A; and / or suberoylanilide hydroxamic acid (SAHA).

[0214] Such methods are not limited to specific embodiments of administering a composition comprising nanoparticles associated with a tolerogenic antigen. In fact, the composition may be administered to a subject using any acceptable method known to those skilled in the art. Administration may be topical (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic. The composition can be administered by many routes, including but not limited to oral, inhalation (nose or lung), intravenous, intraperitoneal, intramuscular, percutaneous, subcutaneous, topical, sublingual, or rectal methods. Injections may be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, injections may be performed at multiple locations.

[0215] The administration of a formulation can be achieved by any acceptable method that allows an effective amount of the composition to achieve its desired effect. The specific mode of selection depends on factors such as the specific formulation, the severity of the condition of the subject being treated, and the dose required to induce an effective immune response. As commonly used herein, “effective amount” is the amount that can induce an immune response in the subject being treated. The actual effective amount of such a composition may be modified according to the specific antigen or combination used, the specific composition being formulated, the mode of administration, and the individual age, weight, condition, and route of administration and disease or disorder being vaccinated.

[0216] [Characterization of nanoparticles] The nanoparticles of the present invention may be characterized for size and uniformity by any suitable analytical technique. These include atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, LC-MS / MS, 13This includes, but is not limited to, 13C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC) (equipped with multi-angle laser light scattering, dual UV and refractive index detectors), capillary electrophoresis, and GET electrophoresis. These analytical methods are crucial for ensuring the uniformity of the sHDL nanoparticle population and for production quality control for final use in in vivo applications.

[0217] In some embodiments, sHDL-tolerogenic antigen nanoparticles are analyzed using gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides. In some embodiments, the particle size distribution and zeta potential are determined by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0218] [Pharmaceutical Compositions] When clinical application is intended, in some embodiments of the present invention, sHDL nanoparticles are prepared as part of a pharmaceutical composition in a suitable form for the intended application. Generally, this requires the preparation of a composition that is essentially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the present invention, a straight sHDL nanoparticle formulation may be administered using one or more of the routes described herein.

[0219] In a preferred embodiment, sHDL nanoparticles are used in conjunction with appropriate salts and buffers to deliver the composition in a stable manner that allows for uptake by target cells. Buffers are also used when the sHDL nanoparticles are introduced into a patient. Aqueous compositions contain an effective amount of sHDL nanoparticles for cells dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inocula. The expression “pharmaceutically acceptable” means molecular entities and compositions that, when administered to animals or humans, do not produce harmful, allergic, or other undesirable reactions. As used herein, “pharmaceutically acceptable carrier” includes any and all of the following: solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders. Any conventional culture medium or drug is intended for use in the therapeutic composition unless it is incompatible with the vector or cells of the present invention. Supplementary active ingredients may also be incorporated into the composition.

[0220] In some embodiments of the present invention, the active composition comprises a classic pharmaceutical preparation. Administration of these compositions according to the present invention is via any common route, as long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal, or topical administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0221] Active sHDL nanoparticles may also be administered parenterally, intraperitoneally, or intratumorally. Solutions of compounds having activity as free bases or pharmaceutically acceptable salts are prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0222] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injection solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents (e.g., sugars or sodium chloride). Extension of absorption of the injection composition can be achieved by the use of absorption-delaying agents (e.g., aluminum monostearate and gelatin) in the composition.

[0223] A sterile injection solution is prepared by incorporating the required amount of active sHDL nanoparticles, along with various other components listed above as needed, in a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile medium (a basic dispersion medium and other required components from those listed above). In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques to produce active ingredient powders and any further desired components from a pre-sterilized filtered solution.

[0224] During prescription, sHDL nanoparticles are administered in a manner compatible with the drug formulation and in a therapeutically effective amount. The formulation is readily administered in various dosage forms, such as injection solutions and drug-release capsules. For parenteral administration in aqueous solutions, for example, the solution is buffered as appropriate if necessary, and the diluent is first isotonicized with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous solution, or injected into the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, active particles or drugs are formulated in a therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 milligrams, or even about 10 milligrams per dose. Multiple doses may be administered.

[0225] Further formulations suitable for other modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may also be used. Suppositories are solid dosage forms of various weights and shapes, typically administered for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the fluid of the lumen. Generally, suppositories may contain traditional binders and carriers, e.g., polyalkylene glycols or triglycerides; such suppositories may be formed from a mixture containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Vaginal suppositories or pessaries are usually spherical or oval in shape, each weighing about 5 g. Vaginal medications are available in various physical forms (e.g., creams, gels, or liquids), which deviates from the classic concept of suppositories. sHDL nanoparticles may also be formulated as inhalants.

[0226] 〔kit〕 In some embodiments, the present invention also provides kits comprising compositions comprising nanoparticles associated with toxicogenic antigens via thiol-reactive and reduction-insensitive binding as described herein. In some embodiments, the kits include one or more reagents and tools necessary to generate sHDL nanoparticles, as well as methods for using such sHDL nanoparticles.

[0227] [Examples] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope. <Example 1. Production of sHDL nanodiscs by myelin-based peptide for treatment of multiple sclerosis> This example demonstrates that myelin-based peptide-bonded nanodiscs promote immune tolerance to multiple sclerosis / experimental autoimmune encephalomyelitis (EAE).

[0228] Multiple sclerosis (MS) is an autoimmune disease caused by an autoimmune reaction against axons and myelin sheaths of the central nervous system (CNS), resulting in axonal loss and demyelination (Figure 1). Current treatments for MS are primarily based on immunosuppressive therapies, which have unintended side effects on the overall immune response and can cause significant toxicity. To develop a delivery platform that can efficiently deliver MS antigens in a tolerogenic manner, we conducted the following experiments. In summary, these experiments generated myelin oligodendrocyte glycoprotein (MOG) peptide antigens in synthetic high-density lipoprotein (HDL) nanodiscs, demonstrating that HDL-MOG exhibits potent efficacy against experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS.

[0229] HDL nanodiscs were prepared as described above. Briefly, DMPC was dissolved in chloroform and evaporated under a nitrogen stream and vacuum for at least 1 hour. The resulting lipid film was hydrated in 10 mM sodium phosphate buffer and sonicated in a plating bath sonicator for 10 minutes. ApoA1 mimetic peptide 22A, dissolved in endotoxin-free water, was added to the above mixture (22A:DMPC = 1:2 by weight ratio) to obtain nanodiscs. To load MOG antigen peptides onto the nanodiscs, each antigen peptide was reacted with 4-(4-maleimidophenyl)-butyrate DOPE in dimethylformamide (DMF) for 3 hours, diluted 10-fold with endotoxin-free water, and removed by lyophilization. The lipid-peptide conjugates were dissolved in DMSO and added to the pre-formed sHDL, and incubated at room temperature for 30 minutes. Unreacted MOG peptides were removed by using a Zeba Spin Desalting column (Pierce) according to the manufacturer's instructions. The binding efficiency of MOG peptides was determined by LC-MS and gel permeation chromatography (GPC). EAE was induced in female C57BL / 6 mice by inoculation with 100 μg of complete Freund's adjuvant MOG on day 0 after administration of pertussis toxin. Subsequently, for therapeutic studies, sHDL-MOG, MOG, or PBS was subcutaneously injected into the base of the mouse tail on the indicated days. The body weight of the mice was measured and scored daily according to the following guidelines: 0, no signs of disease; 1, loss of tone in the tail; 2, hindlimb paresis; 3, hindlimb paralysis; 4, quadriplegia; and 5, mortality.

[0230] MOG-modified sHDL exhibited a uniform disk-like morphology with an average diameter of 11±1 nm. The loading efficiency of MOG35-55 in HDL was approximately 90% as quantified by HPLC / MS. In the experiment, EAE was first induced on day 0 using either MOG35-55 ("mild EAE") or MOG1-125 ("severe EAE"), and animals were treated on days 2, 9, and 16 (Figures 2 and 3A). SC administration of HDL-MOG significantly inhibited the symptoms of EAE, with mean pathological scores remaining below 1 in both mild and severe EAE conditions (Figures 2, 3B, 3C). In contrast, mice administered PBS or free MOG peptides died within 20 days, and all animals progressed to a pathological score of 5 (Figures 3B and 3C). Next, experiments were conducted in which EAE was induced on day 0, and HDL-MOG treatment was delayed until day 15, when mice showed a pathological score of 4 (Figure 4A). Subcutaneous HDL-MOG administration (sc) on days 15, 22, and 29 significantly reversed the symptoms of EAE in mice, with EAE scores of 1 by day 35 for both mild and severe EAE conditions (Figures 4B and 4C). Next, the experiment directly compared the efficacy of HDL-MOG with FTY720, also known as fingolimod, an FDA-approved treatment widely used in MS patients. FTY720 was administered orally daily for 30 days, starting on either day 15 (Figure 5A) or day 30 (Figure 5B). When HDL-MOG was administered subcutaneously (sc) on days 15, 22, and 29, mice showed significantly reduced EAE symptoms, and scores remained below 1 for 85 days (Figure 5A). In contrast, mice treated with FTY720 for 30 days maintained an EAE score above 2, but when FTY720 treatment was discontinued, the mice exhibited severe EAE symptoms and had to be euthanized by day 55 (Figure 5A). Similarly, when HDL-MOG was administered subcutaneously (sc) on days 30, 37, and 44, the mice showed significantly reduced EAE symptoms, and the score remained below 1 for 85 days (Figure 5A).In contrast, mice that were treated with FTY720 for 30 days starting on day 30 had a decrease in EAE scores to 3 and 1, respectively, at doses of 0.3 mg / Kg and 1 mg / Kg of FTY720 (Figure 5B). However, when the FTY720 treatment was stopped, the mice developed severe EAE symptoms and had to be euthanized by day 90. Finally, this mechanistic study showed that HDL-MOG treatment decreased the production of IFN-γ and IL-17 among lymphocytes located in the central nervous system compared to treatment with free MOG peptide (Figure 6). This suggests that this HDL-based strategy may have inhibited the self-antigen-specific Th1 and Th17 responses that are known to be important in the development of EAE and MS.

[0231] <Example 2. Visualization of cellular uptake of HDL-MOG or free MOG peptide by bone marrow-derived dendritic cells (BMDC) and microglia.> To obtain bone marrow-derived dendritic cells (BMDC), bone marrow was flushed from the femurs and tibias of 5- to 6-week-old C57BL / 6 mice. The bone marrow cells were plated at 1 × 10 6 cells per dish in RPMI 1640 supplemented with 10% FBS, 55 μM β-mercaptoethanol, 5 ng / ml GM-CSF, and 100 U / ml penicillin (BMDC medium). On days 3 and 5, half of the culture medium was replaced with fresh medium. After 8 days, the BMDC were harvested and plated at 1 × 10 6 cells per well in a 12-well plate.

[0232] Mixed glial cell cultures were collected from the cerebral cortex of neonatal (P0-2) C57BL6 / J mice, as previously described. The cortex and brainstem were separated, and the blood vessels and meninges were carefully isolated. The tissue was then digested enzymatically (0.05% trypsin-EDTA and 25 mg / ml DNase I) and washed twice with 1% BSA in PBS. The cells were resuspended in DMEM (10% FBS, 1% penicillin / streptomycin, 0.5 mM 2-mercaptoethanol). Mixed glial cells were cultured in poly-D-lysine coated flasks and grown at 37°C and 5% CO2. After 10 days, microglia cells were isolated from the underlying astrocyte layer by shaking the flask. The cell cultures were analyzed by flow cytometry and contained more than 95% microglia (CD11b+, CD45+) cells.

[0233] The internalization of fluorescent HDL-MOG by BMDCs or microglia was visualized using a confocal microscope. BMDCs or microglia were visualized at 1 × 10⁶ 6 Cells were seeded in 35 mm Petri dishes (MatTek) and incubated with free CSS-MOG-K (FITC) or sHDL-CSS-MOG-K (FITC). The cells were then washed three times with PBS, fixed with 4% paraformaldehyde, washed, and permeabilized with 0.1% Triton-X solution. Actin filaments were stained with AlexaFluor 647-Phalloidin, MHC-II with MHC-II-Texas red, and nuclei with DAPI. Samples were imaged using a 63X oil immersion lens on a Nikon A-1 spectral confocal microscope.

[0234] As shown in Figure 7, BMDCs and microglia strongly internalized HDL-MOG-FITC. In contrast, we observed minimal signals of free MOG-FITC peptide in BMDCs and microglia. This suggests that APCs such as DCs and microglia efficiently phagocytose sHDL-MOG.

[0235] <Example 3. In vivo distribution of HDL-MOG and free MOG peptides after SC administration in mice.> Tetramethylrhodamine (TMR, excitation / emission ~540 / 560 nm)-modified MOG peptide (CSSGWYRSPFSRVVHL-TMR, MOG-TMR SEQ ID NO: 829) was prepared by reacting TMR-NHS and the MOG peptide according to the manufacturer's instructions. MOG-TMR was purified using HPLC and reacted with DOPE-MAL in DMF to produce DOPE-Mal-MOG-TMR (MOG-TMR). Next, the DMF solution was diluted with water, lyophilized, dissolved in DMSO, and added to pre-prepared HDL to prepare HDL-MOG-TMR. The binding of MOG-TMR to DOPE-MAL and the incorporation of MOG-TMR into HDL were measured by HPLC / MS as described above. For lymph node inflow studies, naive female C57BL / 6 mice or EAE-induced mice were subcutaneously administered (sc) via the tail base (100 μl) of HDL-MOG-TMR or free MOG-TMR-containing antigen peptide (100 μg / mouse). After 24, 96, and 196 hours, the animals were euthanized, organs were collected, and TMR signals were measured using the IVIS optical imaging system (Caliper Life Sciences). Inguinal lymph nodes and spinal cord were dissected into small pieces, passed through a 70 μm cell strainer, washed twice, stained with the indicated antibodies, and subsequently analyzed by flow cytometry.

[0236] SC administration of free MOG-TMR peptide in naive mice resulted in minimal TMR signaling in inguinal dLNs after 1 day (Figure 8A). The low signal intensity may be due to systemic dissemination of the low molecular weight peptide. In contrast, sHDL-MOG-TMR showed a significantly increased TMR signal in dLNs (Figure 8A), and CD11c + DC, B220 + B cells, and F4 / 80 +This was accompanied by significant cellular uptake of Ag by macrophages. Similarly, in EAE-induced mice, the inventors also observed a significant enhancement of Ag uptake compared to free MOG-TMR peptide after administration of HDL-MOG-TMR (Figure 8B). Interestingly, the inventors detected accumulation of HDL-MOG-TMR in the spinal cord of EAE-induced mice and CD11c + DC, B220 + B cells, and F4 / 80 + We detected their uptake among macrophages. In contrast, no accumulation of HDL-MOG-TMR was observed in the spinal cord of naive mice, indicating that EAE-mediated damage to the blood-brain barrier enabled HDL-MOG infiltration into the central nervous system (CNS).

[0237] To further quantify the in vivo distribution of HDL-MOG, the inventors used positron emission tomography (PET) images. Copper-64( 64 Cu was synthesized using the on-site cyclotron (GE PETtrace) method. 64 CuCl2 (74 MBq) was diluted with 0.3 ml of 0.1 M sodium acetate buffer (pH 5.0) and mixed with 0.5 mg of HDL-MOG. Mixing was carried out at 37°C for 30 minutes with continuous shaking. Subsequently, 5 μL of 0.1 M EDTA (ethylenediaminetetraacetic acid) was added to the solution and shaken for 5 minutes to allow nonspecific binding to occur. 64 Copper was removed. The obtained HDL-MOG-NOTA- 64 Cu was purified by centrifugal filtration (10 kDa). Then, 5-8 MBq of HDL-MOG-NOTA- was administered to C57BL / 6 mice. 64 Cu or MOG-NOTA- 64 Cu was administered subcutaneously (sc), and PET imaging was performed over time using a micro-PET / micro-CT Inveon rodent model scanner (Siemens Medical Solutions USA, Inc.) (Figure 9A). Quantitative PET data for major organs are shown as the percentage of injection volume per gram of tissue (%IDg-1). Free 64Subcutaneous (sc) administration of Cu-labeled MOG resulted in rapid systemic dissemination while minimizing antigen residue at the injection site or lymph nodes up to 24 hours later (Figure 9B). On the other hand, HDL-MOG-NOTA- 64 Subcutaneous (sc) administration of Cu showed results at 24 hours, as well as at the injection site, in multiple inflow regions of LNs. 64 This resulted in a significant accumulation of Cu-labeled MOG (Figure 9B). The mice were euthanized after 24 hours, and the radioactivity of major organs was quantified (Figure 9C). According to the PET imaging dataset, the inventors performed HDL-MOG-NOTA- 64 Regarding the Cu group, MOG-NOTA- 64 Compared to the Cu group, the levels were significantly higher in the lymph nodes in the inflow area and in other issues (including the spine). 64 Cu signaling was detected. These results suggest that HDL significantly enhances antigen delivery to inflowing lymph nodes, and that a subset of HDL forms deposits at the injection site, enabling continuous, systemic delivery to major organs.

[0238] <Example 4. Effects of HDL-MOG treatment on inflammatory cytokines and inflammatory cytokine producers in EAE mice.> In short, female C57BL / 6 mice were given MOG during full Freund's adjuvant (CFA) treatment. 35-55 EAE-inducing mice were subcutaneously injected with an emulsion of (MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO: 830) and pertussis toxin via intraperitoneal (ip) injection (120 ng / dose on days 0 and 2). EAE-inducing mice were treated with PBS, free MOG, HDL-MOG, or HDL-M30 (an unrelated CD4+ T cell epitope derived from B16F10 tumor cells), and CNS tissue was collected on day 40 (Figure 10A). CNS tissue was collected from mice after intracardiac perfusion with PBS. Spinal cord was collected, homogenized in 10 ml of PBS containing 1% BSA, and pelletized at 800 × g for 5 minutes. The cell pellet was treated with 3 ml of collagenase A (1 mg / ml) and DNase in HBSS. The cells were resuspended in 1 mg / ml and incubated in a 37°C water bath for 30 minutes. The samples were pelletized at 800 × g, resuspended in 27% Percoll, and centrifuged at 800 × g for 10 minutes. The myelin / fragment layer and Percoll were removed, the cell pellet was stained, and analyzed by flow cytometry. Spleen immune cells were isolated by homogenization using a 70 μm strainer (BD Falcon). RBCs were lysed using ACK lysis buffer. The cells were washed in 25 ml of PBS, centrifuged at 800 × g, resuspended in FACS buffer, and stained with antibody. For cell surface staining, the cells were resuspended in PBS with a fixable viability dye (BV510) for 10 minutes. The cells were then washed twice with FACs buffer and resuspended in Fc block (anti-CD16 / 32; 100 ng / ml). Before adding brefelzin A (BFA) (5 μg / ml) for 4 hours for exovivo restimulation, cells were subjected to MOG (Modified Oxygen Graft). 35-55 The cells were incubated with the appropriate solution for 96 hours. For intracellular staining, cells were stained for a surface marker, fixed / permeabilized, and stained with antibody on ice for 30 minutes. After two washes, the cells were resuspended in FACS buffer and analyzed by flow cytometry. Data were collected using a Cytek Aurora flow cytometer with FCS expression software (V7).

[0239] On day 40, CNS tissue was isolated and MOG was administered in exovivo. 35-55Cells were pulsed with peptides, followed by ELISA measurements of IL-17, IFN-γ, and GM-CSF (Figure 10B). Animals treated with PBS, free MOG peptide, or HDL-M30 had high levels of IL-17, IFN-γ, and GM-CSF in the CNS (Figure 10B) and showed strong inflammation. In complete contrast, HDL-MOG treated animals had significantly reduced levels of IL-17, IFN-γ, and GM-CSF in the CNS (Figure 10B), suggesting antigen-specific immune tolerance induced by HDL-MOG treatment. In parallel, we performed intracellular cytokine staining on CD4+ T cells derived from exovivo-stimulated CNS cells (Figure 10C) or spleen cells (Figure 10D), with or without free MOG peptide. As shown in Figure 10B, treatment with PBS, free MOG peptide, or HDL-M30 resulted in a high frequency of CD4+ T cells producing IL-17, IFN-gamma, and GM-CSF in both the CNS and spleen. In stark contrast, HDL-MOG-treated mice showed a significantly reduced frequency of CD4+ T cells producing IL-17, IFN-gamma, and GM-CSF in both the CNS and spleen (Figures 10C-10D).

[0240] To further confirm these results, the inventors treated EAE mice as shown in Figure 11, collected fresh CNS central nervous system tissue on day 40, and performed ELISA without restimulation with ex vivo. Correlating with the results shown in Figures 4A-4D, HDL-MOG treatment significantly reduced the concentrations of IL-17, IFN-gamma, and GM-CSF, while increasing IL-10 levels in the CNS (Figure 11). However, mice treated with free MOG or HDL-M30 had similar levels of IL-17, IFN-gamma, GM-CSF, and IL-10 as EAE mice treated with PBS.

[0241] In summary, these studies demonstrated that HDL-MOG treatment induces antigen-specific immune tolerance in systemic compartments as well as in the CNS, which is the peripheral site of inflammation.

[0242] <Example 5. Effects of HDL-MOG treatment on regulatory T cells (Tregs) in EAE mice.> EAE-inducing mice were treated as shown in Figure 12A, and the frequency of Tregs was quantified in the CNS. Cells derived from the CNS were stained with anti-CD25, anti-CD4, and MOG tetramers, subsequently fixed / permeabilized, and intracellularly stained with anti-Foxp3. The stained cells were then analyzed by flow cytometry. HDL-MOG treatment significantly increased the frequency of CD25+Foxp3+ Tregs in the CNS (Figure 12B), and the inventors also validated this using MOG tetramers (Figure 12C), demonstrating that HDL-MOG induces MOG-specific Tregs in the CNS. On the other hand, mice treated with free MOG peptide or HDL-M30 had basal levels of Tregs, similar to EAE mice treated with PBS (Figures 12A-12B).

[0243] <Example 6. The inventors studied the effect of HDL-MOG on the treatment outcomes of regulatory T cells (Tregs).> EAE-inducing mice were subcutaneously administered PBS or HDL-MOG on days 15, 22, and 29, and a subset of the animals were also intraperitoneally administered anti-CD25 IgG to deplete Tregs at specified time points (Figure 13). EAE mice treated with HDL-MOG showed a dramatic improvement in EAE symptoms (Figure 13), as previously shown. When anti-CD25 was administered on days 35 and 37, the mice rapidly relapsed, exhibiting an EAE >3 score by day 50 (Figure 13), indicating that anti-CD25-mediated Treg depletion induced relapse. Interestingly, administration of anti-CD25 on days 21, 23, 35, and 37 yielded similar results to mice administered anti-CD25 only on days 35 and 37. These results suggest that Tregs play a crucial role in HDL-MOG-mediated immune tolerance and are important for the long-term control of the disease.

[0244] <Example 7. sHDL nanodiscs using various immunomodulatory drugs> HDL nanodiscs loaded with various immunomodulatory drugs have been formulated. In particular, FTY720, ITE, TSA, SAHA, and rapamycin (Rapa) have been reported to exhibit potent immunomodulatory properties. FTY720 (also known as fingolimod) is an oral drug that can sequester T cells in lymphoid tissue (Chung and Harung, Clin. Neuropharmacol 33: 91-101, 2010). AhR activation by 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or related ligands has been reported to expand Tregs and promote immune tolerance (Yeste A, et al. Proc. Natl. Acad. Sci. USA 109: 11270-11275, 2012; Quintana FJ, et al Proc. Natl. Acad. Sci. USA 107: 20768-20773, 2010). Trichostatin A (TSA) can increase the frequency of Tregs and enhance the immunosuppressive function of Tregs (Reilly CM et al. J. Autoimmun 31: 123-130. 2008). The histone deacetylase inhibitor suberoilanilide hydroxamic acid (SAHA) has been shown to induce Treg cells (Lucas JL, et al. Cell Immunol 257: 97-104, 2009). Rapamycin (Rapa) has been shown to induce immunosuppression when administered concurrently with biological agents containing exogenous enzymes (Maldonado, RA, et al. Proc. Natl. Acad. Sci. USA 112:E156-165, 2015).

[0245] To fabricate HDL nanodiscs carrying immunomodulatory drugs, 22A and DMPC were dissolved in acetic acid and freeze-dried overnight as described above. The drug (FTY720, ITE, TSA, or SAHA) was dissolved in chloroform, added to the freeze-dried powder, and subsequently dried overnight in a vacuum oven. The dried samples were rehydrated with 10 mM phosphate buffer. After three heat-cooling cycles, the drug-loaded HDLs were processed. The concentrations of each drug were measured by HPLC-MS as shown in Figures 14-17 for FTY720, ITE, TSA, or SAHA. Standard curves were created using the free drugs. High-efficiency drug encapsulation efficiencies of ~90%, ~95%, ~80%, and ~100% were achieved for FTY720, ITE, TSA, and SAHA, respectively, as shown in Figure 18. Particle size was also analyzed by dynamic light scattering (DLS) of the samples. As shown in Figure 18, HDL-FTY720, HDL-ITE, and HDL-TSA exhibited hydrodynamic sizes of approximately 10 nm, 12 nm, and 9 nm, respectively. Furthermore, the HDL-FTY720 sample was analyzed using gel permeation chromatography (GPC) (Figure 19). The GPC chromatogram showed that HDL-FTY720 was relatively uniform and that the thank-blank HDL nanodiscs eluted quickly, indicating successful encapsulation of FTY720 within the HDL nanodiscs.

[0246] The inventors also encapsulated Rapa in HDL. Analysis by dynamic light scattering and gel permeation chromatography revealed that the HDL-Rapa exhibited a uniform size distribution with an average hydrodynamic size of ~10 nm (Figure 20).

[0247] <Example 8. Effectiveness of HDL-FTY720 in an EAE mouse model> The therapeutic potential of HDL-FTY720 in an EAE model was tested. EAE was initiated as described above. Briefly, EAE was induced in mice as described above. Mice were intraperitoneally administered PBS or HDL-FTY720 (1 mg / kg) on ​​days 14, 21, and 28. Mice were scored daily and assigned EAE scores as described above. EAE-induced mice treated with HDL-FTY720 showed rapid improvement in symptoms, with the peak EAE score of 4 at day 14 decreasing to EAE score <2 by day 30 (Figure 21). These results suggest that HDL-FTY720 can be used for systemic delivery of FTY720.

[0248] <Example 9: sHDL with CD4+ T cell epitope> To validate their approach, the inventors synthesized HDL nanodiscs loaded with other CD4+ T cell epitopes. They synthesized phospholipids conjugated to one of the following: Eα chain 52-68 peptide (EA, ASFEAQGALANIAVDKA (SEQ ID NO: 831)), ovalbumin-II 323-339 peptide (OVA-II, ISQAVHAAHAEINEAGR (SEQ ID NO: 832)), and type II collagen 250-270 peptide (CIA, GPKGQTGKPGIAGFKGEQGPK (SEQ ID NO: 833)), and modified each with a CSS-peptide at its N-terminus. The antigen-lipid conjugates were loaded onto HDL nanodiscs as described above and analyzed by HPLC / MS and DLS. EA, OVA-II, and CIA peptides were conjugated to MPB-phospholipids with approximately 95% conjugation efficiency (Figures 22-25). Antigen-lipid conjugates of EA, OVA-II, and CIA peptides were efficiently loaded onto HDL nanodiscs with loading efficiencies of ~90%, ~84%, and ~78%, respectively (Figures 22-25). HDL-EA, HDL-OVA-II, and HDL-CIA exhibited hydrodynamic sizes of 13 nm, 15 nm, and 10 nm, respectively (Figures 22-25).

[0249] <Example 10. Production of sHDL nanodiscs using gliadin peptides for the treatment of celiac disease> HDL nanodiscs are prepared by dissolving dipalmitoylphosphatidylcholine (DMPC) in chloroform, followed by evaporating the chloroform under vacuum using a nitrogen stream for at least 1 hour. The resulting lipid film is rehydrated in 10 mM sodium phosphate buffer and sonicated in a bath sonicator for 10 minutes. Nanodiscs are then obtained by adding ApoA1 mimetic peptide 22A, dissolved in endotoxin-free water, to the mixture in a 1:2 (w / w) ratio of ApoA1 mimetic peptide 22A to DMPC. Gliadin peptides are loaded onto the nanodiscs by reacting each antigen peptide with 4-(4-maleimidophenyl)-butyrate DOPE in dimethylformamide (DMF) for 3 hours. After diluting the reaction mixture 10-fold with endotoxin-free water, the DMF is removed by lyophilization. The lipid-peptide conjugates are dissolved in DMSO and added to the pre-formed sHDL, and incubated at room temperature for 30 minutes. Unreacted gliadin peptides are removed using a Zeba Spin Desalting column (Pierce) according to the manufacturer's instructions. The binding efficiency of the gliadin peptides is determined using LC-MS and gel permeation chromatography (GPC). Nanodiscs loaded with gliadin peptides provide an efficient means of delivering gliadin peptides to dendritic cells and other antigen-presenting cells during in vivo administration. The nanodiscs enable antigen processing and presentation on antigen-presenting cells so that immune tolerance to celiac disease is induced. Patients with celiac disease may be treated with these nanodiscs via subcutaneous administration. Patients may receive weekly doses for up to 6 weeks or longer, followed by a maintenance dose after at least 3 months.

[0250] <Other Embodiments> Various modifications and variations of the disclosed information will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. While the disclosure has been described in relation to specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. In fact, various modifications of the described aspects for carrying out the disclosure that are apparent to those skilled in the art are intended to be within the scope of the disclosure. Other embodiments are described in the claims. [Brief explanation of the drawing]

[0251] [Figure 1] This image shows T cell activation. [Figure 2] This graph shows the pathological scores for EAE induced by free MOG (100 μg / administered) and HDL-MOG nanodiscs (100 μg / administered). [Figure 3A] This is a schematic diagram showing the treatment plan. [Figure 3B] This graph shows the pathological scores for EAEs induced by MOG35-55. [Figure 3C] This graph shows the pathological scores for EAE induced by MOG1-125. [Figure 4A] This is a schematic diagram showing the treatment plan. [Figure 4B] This graph shows the pathological scores for EAEs induced by MOG35-55. [Figure 4C] This graph shows the pathological scores for EAE induced by MOG1-125. [Figure 5A] This graph shows that HDL-MOG nanodiscs exhibit stronger efficacy than FTY720. [Figure 5B] This graph shows that HDL-MOG nanodiscs exhibit stronger efficacy than FTY720. [Figure 6A] This is a schematic diagram showing the treatment plan. [Figure 6B]This graph shows the amounts of GM-CSF, IFN-γ, and IL-17 released after ex vivo treatment of CNS tissue with MOG35-55 peptide. [Figure 7] These are images of BMDCs and microglia after incubation with HDL-MOG-FITC or MOG-FITC peptide and visualization of antigen uptake. [Figure 8A] This image shows the experimental design and results for naive mice. [Figure 8B] This image shows the experimental design and results for EAE-induced mice. [Figure 9A] This is a schematic diagram showing the treatment plan for EAE-induced mice administered HDL-MOG-NOTA-64Cu or MOG-NOTA-64Cu for in vivo distribution studies. [Figure 9B] This is a photograph showing 24-hour positron emission tomography (PET) images of an EAE-induced mouse. [Figure 9C] This graph shows the quantitative analysis of 64Cu signaling in major organs 24 hours after injection. [Figure 10A] This is a schematic diagram of EAE-inducing mice treated with PBS, free MOG, HDL-M30, or HDL-MOG. [Figure 10B] This graph shows the results from ELISA from the CNS, where cells were collected on day 40, processed individually, restimulated ex vivo with MOG peptide, and quantified for IL-17, IFN-gamma, and GM-CFS levels. [Figure 10C] This graph shows the results of intracellular cytokine staining in the CNS, investigating the frequency of CD4 T cells secreting IL-17, IFN-gamma, and GM-CFS in exovivo restimulation with or without MOG peptide. [Figure 10D]This graph shows the results of intracellular cytokine staining in spleen cells, investigating the frequency of CD4 T cells secreting IL-17, IFN-gamma, and GM-CFS in exovivotive restimulation with or without MOG peptide. [Figure 11] The experimental design and results of EAE-induced mice treated with PBS, free MOG, HDL-M30, or HDL-MOG are presented. [Figure 12A] This is a schematic diagram of EAE-inducing mice treated with PBS, free MOG, HDL-M30, or HDL-MOG. [Figure 12B] This graph shows the frequency of CD25+Foxp3+Tregs (Figure 12B) and MOG-tetramer+Foxp3+Tregs in the CNS. [Figure 12C] This graph shows the frequency of CD25+Foxp3+Tregs (Figure 12B) and MOG-tetramer+Foxp3+Tregs in the CNS. [Figure 13] The experimental design for EAE-inducing mice treated with PBS or HDL-MOG is described. [Figure 14] This is an HPLC chromatogram that quantifies the amount of HDL-FTY720 loaded compared to a free drug standard. [Figure 15] This is an HPLC chromatogram that quantifies the amount of ITE loaded into HDL (HDL-ITE) by comparing it with a free drug standard. [Figure 16] This is an HPLC chromatogram that quantifies the amount of TSA loaded into HDL (HDL-TSA) by comparing it with a free drug standard. [Figure 17] This is an HPLC chromatogram that quantifies the amount of SAHA loaded into HDL (HDL-SAHA) by comparing it with a free drug standard. [Figure 18] This table shows the drug loading efficiency for FTY72-, ITE, TSA, and SAHA in HDL nanodiscs, quantified by HPLC / MS. [Figure 19]This is a gel permeation chromatography showing an HDL-FTY720 compared to an empty HDL nanodisk. [Figure 20] This graph shows HDL loaded with rapamycin (Rapa), analyzed by DLS and gel permeation chromatography. [Figure 21] This graph shows EAE-induced mice on day 0, and mice that received intraperitoneal administration of HDL-FTY720 (1 mg / kg of FTY720) on days 14, 21, and 28. [Figure 22] HPLC chromatograms of HDL nanodiscs loaded with EA peptides, empty HDL, lipid-EA conjugates, and MPB lipids. [Figure 23] HPLC chromatograms of HDL nanodiscs loaded with OVA-II peptide, empty HDL, lipid-OVA-II conjugates, and MPB lipids. [Figure 24] HPLC chromatograms of HDL nanodiscs loaded with CIA peptides, empty HDL, lipid-CIA conjugates, and MPB lipids. [Figure 25] This table shows the antigen binding efficiency for forming antigen-lipid conjugates (EA, OVA-II, and CIA), the antigen-lipid loading efficiency in HDL, and the hydrodynamic size of antigen-loaded HDL.

Claims

[Claim 1] A composition, The composition comprises sHDL nanoparticles associated with multiple tolerogenic antigens, in such a manner that the resulting composition can promote potent immune tolerance to antigens associated with autoimmune diseases when administered to a target. The sHDL nanoparticles are a composition comprising a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic.

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