Compositions and methods for treating autoimmune disorders

Abstract

The present invention relates to compositions comprising nanoparticles, either associated or not associated with one or more tolerogenic antigens, and compositions comprising an immunomodulatory agent (e.g., interleukin-2 (IL-2) or IL-2 variants or IL-2 / IC), as well as related methods comprising co-administration of the above compositions for the purpose of inducing expansion of regulatory T cells (Tregs) (e.g., antigen-specific regulatory Tregs). The present invention further provides a method of treating an autoimmune disorder by administering to a subject a composition comprising nanoparticles, either associated or not associated with one or more tolerogenic antigens associated with the autoimmune disorder, prior to administering to the subject a composition comprising an immunomodulatory agent.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 312,452, filed February 22, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] [Statement regarding Federally Sponsored Research and Development] This invention was made with Government support under R01 NS122536 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] [Sequence Listing] The text of the computer readable sequence listing submitted herewith, entitled "40287_601_SequenceListing", created on February 22, 2023, with a file size of 722,461 bytes, is hereby incorporated by reference in its entirety.

[0004] [Technical Field of the Invention] The invention provides compositions comprising nanoparticles, with or without association with one or more tolerogenic antigens, and compositions comprising an immunomodulatory agent (e.g., human interleukin-2 (IL-2), an IL-2 mutein, an IL-2 variant, or an IL-2:anti-IL-2 antibody immune complex (IL-2 / IC)), as well as related methods comprising co-administration of the above compositions to induce expansion of regulatory T cells (Tregs) (e.g., antigen-specific regulatory Tregs). The invention further provides methods of treating an autoimmune disorder by administering to a subject a composition comprising nanoparticles, with or without association with one or more tolerogenic antigens associated with the autoimmune disorder, prior to administering to the subject a composition comprising an immunomodulatory agent.

[0005] 2. Background of the Invention Autoimmune disorders are diseases that occur when the body's immune system attacks its own healthy tissues, organs, or other components of the body due to an immune system abnormality with no known cause. These autoimmune disorders are systemic diseases that can affect almost any part of the body, including the nervous system, digestive system, endocrine system, skin, skeletal system, and vascular tissue. Autoimmune disorders are known to affect approximately 5-8% of the world's population, but due to limitations in our understanding of autoimmune disorders and the methods for diagnosing the diseases, the reported prevalence of autoimmune disorders is lower than the actual level.

[0006] There is a need for improved compositions and methods for treating autoimmune conditions.

[0007] The present invention addresses these needs.

[0008] Summary of the Invention Experiments conducted during the course of developing embodiments of the present invention have led to the development of a new strategy for inducing high frequencies of antigen-specific Tregs in vivo without ex vivo cell manipulation. As a result of the above experiments, a new strategy was developed and optimized to combine nanodiscs with modified IL-2 to induce unprecedented amounts of antigen-specific Tregs in vivo. In particular, IL-2:anti-IL-2 antibody (clone: ​​JES6-1) immune complexes (IL-2 / IC) have been shown to selectively induce polyclonal Tregs

[14] . Administration of IL-2 / IC with free peptide or peptide-tetramers has been reported to induce antigen-specific Tregs [15,16]. However, these aforementioned attempts have resulted in rather poor antigen-specific Treg responses, with the antigen-specific Treg frequency in the CD4+ T cell compartment being less than 0.25% [15,16].

[0009] It was hypothesized that it would be desirable to deliver peptide antigens to lymphoid tissues to maximize antigen-specific Treg induction in conjunction with IL-2 / IC. In the experiments described herein, we report for the first time that lymphatic-targeted Nanodisc-mediated delivery of peptides in combination with IL-2 / IC therapy results in a significant expansion of antigen-specific Tregs compared to IL-2 / IC alone.

[0010] It was also assumed that the actual treatment method was important. The above experiments showed that it was desirable to administer Nanodiscs first (e.g., subcutaneously), followed by systemic administration of IL-2 and / or mutein / engineered IL-2. This ensured that antigen-specific Tregs were primed and generated first, and subsequent administration of mutein / engineered IL-2 caused robust proliferation of antigen-specific Tregs. For long-term maintenance of antigen-specific Tregs, administration of a maintenance dose of Nanodiscs and / or mutein / engineered IL-2 was further assumed.

[0011] Thus, the invention provides compositions comprising nanoparticles associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) or not associated with one or more tolerogenic antigens, and compositions comprising an immunomodulatory agent (e.g., human interleukin-2 (IL-2), IL-2 muteins, IL-2 variants, or IL-2:anti-IL-2 antibody immune complexes (IL-2 / IC)), as well as related methods comprising co-administration of the above compositions for the purpose of inducing expansion of regulatory T cells (Tregs) (e.g., antigen-specific regulatory Tregs). The invention further provides methods of treating an autoimmune disorder by administering to a subject a composition comprising nanoparticles associated or not associated with one or more tolerogenic antigens associated with the autoimmune disorder prior to administering to the subject a composition comprising an immunomodulatory agent.

[0012] In certain embodiments, the present invention provides a method for the proliferation and proliferation of regulatory Tregs (e.g., CD4+ Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.), administering to the subject a composition comprising one or more immunomodulatory agents (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC) capable of expanding Tregs in the subject. + CD25 high Foxp3 + In some embodiments, a composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, an antigen-specific regulatory Treg (e.g., CD4 + CD25 high Foxp3 + ) in vivo amplification is specific for a particular tissue region (eg, a particular tissue region associated with one or more autoimmune disorders).

[0013] In certain embodiments, the present invention provides a method for the proliferation and proliferation of antigen-specific regulatory Tregs (e.g., CD4+ Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.) administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, an IL-2 mutein, an IL-2 variant, or IL-2 / IC)) capable of expanding antigen-specific Tregs. + CD25 high Foxp3 +In some embodiments, the present invention provides a method for in vivo expansion of antigen-specific regulatory Tregs (e.g., CD4 Tregs) in a subject, where "antigen-specific" is specific for one or more tolerogenic antigens with which it is associated, including nanoparticles associated with one or more tolerogenic antigens. + CD25 high Foxp3 + ) in vivo amplification is specific for a particular tissue region (eg, a particular tissue region associated with one or more autoimmune disorders).

[0014] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 + The present invention provides a method for promoting potent immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of augmenting the immune system of an autoimmune disorder. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the promotion of potent immune tolerance to an antigen associated with an autoimmune disorder in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0015] In certain embodiments, the present invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + The present invention provides a method for promoting potent immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of augmenting immune responses to an autoimmune disorder, where "antigen-specific" is specific to the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the promotion of potent immune tolerance to an antigen associated with an autoimmune disorder in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0016] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 +The present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the increase in the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in the subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0017] In certain embodiments, the present invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + The present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the immune response of a subject to a particular tissue, where "antigen-specific" is specific to the one or more tolerogenic antigens associated therewith, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the increase in the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject is specific to a particular tissue.

[0018] In certain embodiments, the present invention provides a method for administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc. later) producing Tregs (e.g., CD4 + CD25 high Foxp3 + In some embodiments, the present invention provides a method of treating, preventing and / or attenuating one or more autoimmune disorders in a subject, the method comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating mTOR. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0019] In certain embodiments, the invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens (e.g., one or more tolerogenic antigens associated with an autoimmune disorder), and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 +In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders), comprising administering to a subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating the immune system of the subject's immune system, where "antigen-specific" is specific for the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0020] The methods are not limited to the treatment of any particular disorder.

[0021] In some embodiments, the disorder is an autoimmune disorder. The above method is not limited to treating a particular autoimmune disorder. Examples of autoimmune disorders include multiple sclerosis (MS), celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid gland (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye and 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 variants, bullous pemphigoid. , Dermatitis Herpetiformis, Epidermolysis Bullosa Acquisita, Systemic Sclerosis, Mixed Connective Tissue Disease, Sjogren's Syndrome, Systemic Lupus Erythematosus, Good-Basture's Syndrome, Rheumatic Heart Disease, Autoimmune Polyglandular Syndrome Type 1, Aicardi-Gautieres Syndrome, Acute Pancreatitis, Age-Related Macular Degeneration, Alcoholic Liver Disease, Liver Fibrosis, Metastases, Myocardial Infarction, Nonalcoholic Steatohepatitis (NASH), Parkinson's Disease, Polyarthritis / Fetal and Neonatal Anemia, Sepsis, and Inflammatory Bowel Disease.

[0022] In some embodiments, the disorder is a transplant-related disorder. In some embodiments, the disorder is one or more allergies. In some embodiments, the disorder is a respiratory disease (e.g., asthma). In some embodiments, the disorder is graft-versus-host disease (GvHD).

[0023] In some embodiments, the above method (e.g., administration of a composition comprising nanoparticles associated with one or more tolerogenic antigens followed by administration of a composition comprising an immunomodulatory agent capable of expanding Tregs) is further followed by administration of one or more tolerogenic antigens to a specific tissue region (e.g., a specific tissue region associated with one or more autoimmune disorders). In some embodiments, administration of one or more tolerogenic antigens to a specific tissue region is by injection and / or topical and / or subcutaneous administration. In some embodiments, administration of one or more tolerogenic antigens to a specific tissue region prevents immune tolerance in the specific tissue region.

[0024] In some embodiments, the nanoparticles are associated with an immunomodulatory agent and are not associated with a tolerogenic antigen, hi some embodiments, the nanoparticles are associated with a tolerogenic antigen and are further associated with an immunomodulatory agent.

[0025] The methods described herein are not limited to a particular type or variety of compositions comprising one or more immunomodulatory agents capable of expanding Tregs and / or antigen-specific Tregs.

[0026] In some embodiments, a composition comprising an immunomodulatory agent capable of expanding Tregs is comprised within a nanoparticle, such that the immunomodulatory agent capable of expanding Tregs is associated with the nanoparticle (e.g., thereby providing a composition comprising a nanoparticle associated with an immunomodulatory agent capable of expanding Tregs).

[0027] The above embodiments are not limited to a particular immunomodulatory agent.

[0028] In some embodiments, the one or more immunomodulatory agents are fingolimod; rapamycin; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or related ligand; trichostatin A; suberoylanilide hydroxamic acid (SAHA); statins; mTOR inhibitors; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors; corticosteroids; inhibitors of mitochondrial function; 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 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-Thioguanine (6-TG); FK506; Sanglifehrin A; Salmeterol; Mycophenolate Mofetil (MMF); Aspirin and other COX Inhibitors; Niflumic Acid; Estriol; Triptolide; OPN-305; OPN- 401;erythritol (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;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;amlexanox;Bay11-7082;BX795;CH-223191;chloroquine;CLI-095;CU-CPT9a;cyclosporine A;CTY387;gefitinib;glibenclamide;H-89;H-131;isoliquiritigenin;MCC950;MRT67307;OxPAPC;parthenolide;Pepinh-MYD;Pepinh-TRIF;polymyxin B;R406;RU.521;VX-765;YM201636;Z-VAD-FMK;and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD);tryptamine (TA);and 6-formyl indolo[3,2 b] selected from the group including AHR-specific ligands, including but not limited to carbazole (FICZ);

[0029] In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine is a human cytokine. In some embodiments, the cytokine is selected from TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL12B, IL-15, IL-21, and IL-18.

[0030] In some embodiments, the immunomodulatory agent is human IL-2. In some embodiments, the immunomodulatory agent is low dose IL-2. In some embodiments, the immunomodulatory agent is PT101 or a variant thereof. In some embodiments, the immunomodulatory agent is a mutein IL-2 and / or a variation thereof. In some embodiments, the IL-2 is any of the IL-2 cytokines, IL-2 muteins and / or IL-2 variants described in U.S. Patents 11,091,527, 11,091,526, 11,077,195, 11,077,172, 10,960,079, 10,946,068, 10,766,938, 10,722,460, 10,174,092, 10,174,091; EP Patent No. 3808764, and / or U.S. Patent Application Publication No. US20120315245.

[0031] In some embodiments, the immunomodulatory agent is an IL-2:anti-IL-2 antibody immune complex (IL-2 / IC).

[0032] In some embodiments, the IL-2 is extended pharmacokinetic (PK) IL-2. In some embodiments, the extended PK IL-2 comprises a fusion protein. In some embodiments, the fusion protein comprises an IL-2 portion and a portion selected from the group consisting of an immunoglobulin fragment, human serum albumin, and Fn3. In some embodiments, the fusion protein comprises an IL-2 portion operably linked to an immunoglobulin Fc domain. In some embodiments, the fusion protein comprises an IL-2 portion operably linked to human serum albumin. In some embodiments, the extended-PK IL-2 comprises an IL-2 portion conjugated to a non-proteinaceous polymer. In some embodiments, the non-proteinaceous polymer is polyethylene glycol.

[0033] In some embodiments, the extended-PK IL-2 is mutated to have altered affinity (e.g., higher affinity) for the IL-2Rα receptor compared to unmodified IL-2. Site-directed mutagenesis can be used to isolate IL-2 mutants that exhibit higher affinity binding to CD25, i.e., IL-2Rα, compared to wild-type IL-2. Increasing the affinity of IL-2 for IL-2Rα at the cell surface will increase receptor occupancy within a limited range of IL-2 concentrations, further increasing the local concentration of IL-2 at the cell surface.

[0034] In some embodiments, IL-2 variants are provided, which may be, but are not necessarily, substantially purified and can function as high affinity CD25 binders. IL-2 is a T cell growth factor that induces proliferation of antigen-activated T cells and stimulation of NK cells. Exemplary IL-2 variants that are high affinity binders include those described in WO2013 / 177187A2. Further exemplary IL-2 variants with increased affinity for CD25 are disclosed in U.S. Patent No. 7,569,215, the contents of which are incorporated herein by reference.

[0035] In certain embodiments, a composition comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) can expand Treg cells in a subject or sample. Indeed, the composition comprising an immunomodulatory agent can increase the ratio of Treg to non-regulatory T cells. This ratio may be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells. Tregs are typically present in human blood at 5-10% of total CD4+CD3+ T cells, but in autoimmune diseases, the percentage may be lower or higher. In a preferred embodiment, the percentage of Tregs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. The maximum fold increase in Tregs may vary depending on the specific disease, but the maximum abundance of Tregs that may be obtained by IL-2 mutein treatment is 50% or 60% of total CD4+CD3+ T cells. In a specific embodiment, a composition comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) is administered to a subject to increase the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the peripheral blood of the subject.

[0036] The methods described herein are not limited to a particular composition comprising one or more nanoparticles, either associated or unassociated with one or more tolerogenic antigens.

[0037] The composition is not limited to a specific nanoparticle. In some embodiments, the nanoparticle is an sHDL nanoparticle. In some embodiments, the average diameter of the nanoparticle is 6 to 500 nm (e.g., 7 to 20 nm, 21 to 50 nm, 51 to 100 nm, 101 to 200 nm, 201 to 300 nm, 301 to 400 nm, and 401 to 500 nm). In some embodiments, the average particle diameter of the sHDL nanoparticle is 6 to 70 nm (e.g., 7 to 10 nm, 11 to 20 nm, 21 to 30 nm, 31 to 40 nm, 41 to 50 nm, 51 to 60 nm, and 61 to 70 nm).

[0038] In some embodiments, the phospholipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-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-diglyceroyl-sn-glycero-3-phosphocholine; 1,2-dimyristoleyl-sn-glycero-3-phosphocholine; 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine; 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine; 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine; 1,2-dioleoyl-sn-glycero-3-phosphocholine; 1,2-dielaidoyl-sn-glycero-3-phosphocholine 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-dimyristoyl-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-Dipalmitoleoyl-sn-glycero-3-phosphoethanolamine;1,2-Dieleidoyl-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)butyramide];1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butyramide];1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butyramide] 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide];N-[(3-maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine;N-[(3-maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine; N-(3-Maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, distearoyl;N-[(3-Maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine;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-(Succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, distearoyl;N-(Succinimidyloxy-glutaryl)-L -α-phosphatidylethanolamine, dioleoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succinimidyloxyglutaryl)aminopropyl, and polyethylene glycol-carbamyl distearoylphosphatidyl-ethanolamine; N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoyl-ethanolamine;

[0039] In some embodiments, the HDL apolipoprotein components 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 mimetics, preproapoliprotein, preproApoA-I, proApoA I, preproApoA-II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, preproApoA I Milano, proApoA-I Milano , preproApoA-I Paris , proApoA-I Paris and mixtures thereof.

[0040] In some embodiments, the apolipoprotein mimetics include those of 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: 347), and / or . 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 any of the following: 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).

[0041] In some embodiments, the one or more tolerogenic antigens is a plurality of tolerogenic antigens.

[0042] In some embodiments, the plurality of tolerogenic antigens are tolerogenic antigens that comprise between 3 amino acids and 50 amino acids in length (e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 amino acids in length).

[0043] In some embodiments, the multiple tolerogenic antigens are tolerogenic antigens comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 375-796.

[0044] In some embodiments, the multiple tolerogenic antigens are human allograft transplantation antigens, hi some embodiments, the human allograft transplantation antigens are selected from subunits of various MHC class I and MHC class II haplotype proteins, and single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Duffy, and Ss.

[0045] In some embodiments, the plurality of tolerogenic antigens are specific for type 1 diabetes. In some embodiments, the type 1 diabetes tolerogenic antigens are selected from insulin, proinsulin, preproinsulin, glutamic acid 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, hepatoma-intestine-pancreas / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5.

[0046] In some embodiments, the tolerogenic antigen is an antigen that is associated with rheumatoid arthritis, multiple sclerosis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid gland (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye and skin disorders, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune-mediated gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, juvenile rheumatoid arthritis, ... The antibody is specific for one or more of the following autoimmune disorders: dermatitis herpetiformis, epidermolysis bullosa acquisita, systemic sclerosis, mixed connective tissue disease, Sjogren's syndrome, systemic lupus erythematosus, Good-Basture's syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Aicardi-Gautieres syndrome, acute pancreatitis age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, nonalcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

[0047] In some embodiments, the multiple tolerogenic antigens include thyroglobulin (TG), thyroid peroxidase (TPO), thyroid stimulating hormone receptor (TSHR), sodium iodide symporter (NIS), megalin, thyroid autoantigens including TSHR, insulin-like growth factor 1 receptor, calcium sensitive receptor, 21-hydroxylase, 17α-hydroxylase, and P450 side chain cleavage enzyme (P450scc), ACTH receptor, P450c21, P450c17, FSH receptor, α-enolase, pituitary specific protein factor (PGSF) 1a and 2, and iodothyronine deiodinase type 2, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, collagen II, H + ,K +-ATPase, tissue transglutaminase and gliadin, tyrosinase, tyrosinase-related protein 1 and 2, acetylcholine receptor, desmocollin 3, 1 and 4, pemfaxin, desmocollin, plakoglobin, perplakin, desmoplakin, acetylcholine receptor, BP180, BP230, plectin, laminin 5, endomysium, tissue transglutaminase, collagen VII, matrix metalloproteinase 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 proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II, and III, PM-Slc, fibrillarin, B23, U1snRNP, nuclear antigens SS-A and SS-B, fodrin, poly(ADP-ribose) polymerase, topoisomerase, nuclear proteins 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 ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic cytochrome P450 P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and type 1 interferon It comprises one or more tolerogenic antigens selected from interferon α, β and ω.

[0048] The composition is not limited to a particular tolerogenic antigen. In some embodiments, the tolerogenic antigen is a foreign antigen to which the patient develops an undesired immune response. In some embodiments, the multiple tolerogenic antigens are specific for celiac disease. In some embodiments, the tolerogenic antigen is selected from gliadin, glutenin, and fragments thereof capable of inducing 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 tolerogenic 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 the polypeptide sequence of any one of SEQ ID NOs: 375-580. In some embodiments, the tolerogenic antigen comprises a polypeptide having the polypeptide sequence of any one of SEQ ID NOs: 375-580. In some embodiments, the tolerogenic antigen comprises two or more (e.g., two, three, four, five, and six) polypeptide sequences having the sequence of any one of SEQ ID NOs: 375-580.

[0049] In some embodiments, the tolerogenic antigen is an autoantigen to which a subject (e.g., a human patient) has developed or may develop an autoimmune response. 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 refers to a variety of autoimmune diseases, where the protein or proteins causing the disease are known or can be established by routine testing. Embodiments include testing patients to identify the autoimmune protein, generating antigens for use in molecular fusion, and generating immune tolerance to the protein. Embodiments include the antigen or selecting the antigen from one or more of the following proteins: In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid 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, hepatocellular-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regenerative gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptor 1–5. In autoimmune diseases of the thyroid gland, including Hashimoto's thyroiditis and Graves' disease, the primary antigens include thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin receptor (TSHR); other antigens include sodium-iodide symporter (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 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 factors (PGSF) 1a and 2; other antigens are iodothyronine deiodinase type 2. 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-mediated gastritis, the main antigen is H. + ,K +-ATPase. In pernicious anemia, the primary antigen is intrinsic factor. In celiac disease, the primary antigens are tissue transglutaminase and gliadin. In vitiligo, the primary antigens are tyrosinase and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the primary antigen is the acetylcholine receptor. In pemphigus vulgaris and variants, the primary antigens are desmogleins 3, 1, and 4; other antigens include pemfaxin, desmocollin, plakoglobin, pelplakin, desmoplakin, and the acetylcholine receptor. In bullous pemphigoid, the primary antigens include BP180 and BP230; other antigens include plectin and laminin 5. In Dühring's dermatitis herpetiformis, the primary antigens include endomysial and tissue transglutaminase. In epidermolysis bullosa acquisita, the primary 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 PDGF receptor; other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase 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's syndrome, the main antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the main antigen is cardiac myosin.Other autoantigens defined in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and the type 1 interferons interferon α, β, and ω.

[0050] In some cases, the tolerogenic antigen is a foreign antigen to which the patient develops an unwanted autoimmune response. An example is a food antigen. An embodiment includes testing the patient to identify the foreign 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 are from peanut: conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apple: 31 kda major allergen / disease resistance protein homolog (Mal d 2), lipid transfer 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, thaumatin-like protein (Act d 2), kiwellin (Act d 5); from mustard: 2S globulin (Sin a 1), 11S globulin (Sin a 2), lipid transfer 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-gliadins, hordein, secalin, avenin; from strawberry: major strawberry allergen Fra a 1-E (Fra a 1), from banana: profilin (Mus xp 1).

[0051] In some embodiments, the tolerogenic antigen is a multimeric tolerogenic antigen comprising the following N-terminal-C-terminal structure: (P4-L4) n4 -(P3-L3) n3 -P2-(L1-P1) n1 wherein P1, P2, P3, and P4 are each independently a tolerogenic antigen; L1, L3, and L4 are each independently a linker; and n1, n3, and n4 are each independently 0 or 1, and at least one of n1, n3, and n4 is 1.

[0052] In some embodiments, n1 is 1, n3 is 0, and n4 is 0; The tolerogenic antigen comprises the following N-terminal-C-terminal structure: P2-L1-P1

[0053] 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 a peptide linker comprising glycine (G) and serine (S) residues (GS). x , (GGS) x , or (GGGGS) x 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 comprise a different tolerogenic antigen. In some embodiments, P1 and P2 each comprise the same tolerogenic antigen.

[0054] In some embodiments, n1 is 1, n3 is 1, and n4 is 0, and the tolerogenic antigen comprises the following N-terminal to C-terminal structure: P3-L3-P2-L1-P1

[0055] In some embodiments, L1 and L3 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), 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 each independently selected from peptide linkers comprising glycine (G) and serine (S) residues. ... x , (GGS) x , or (GGGGS) x and x is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, P1, P2 and / or P3 each comprise a different tolerogenic antigen. In some embodiments, P1, P2 and P3 each comprise the same tolerogenic antigen.

[0056] In some embodiments, n1 is 1, n3 is 1, and n4 is 1; The tolerogenic antigen comprises the following N-terminal-C-terminal structure: P4-L4-P3-L3-P2-L1-P1

[0057] 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), or 1 In some embodiments, L1, L2, and L3 are each independently selected from peptide linkers comprising glycine (G) and serine (S) residues. In some embodiments, L1, L2, and L3 are each independently selected from peptide linkers comprising glycine (G) and serine (S) residues. In some embodiments, L1, L2, and L3 are each independently selected from peptide linkers comprising glycine (G) and serine (S) residues. x , (GGS) x , or (GGGGS (SEQ ID NO: 219)) x and x is an integer from 1 to 10. In some embodiments, P1, P2, P3 and / or P4 each comprise a different tolerogenic antigen. In some embodiments, P1, P2, P3 and P4 each comprise the same tolerogenic antigen.

[0058] In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle comprises a population of 1-30 (e.g., 1-10, 9-15, 12-18, 15-22, 18-25, 20-27, 22-28, or 25-30) tolerogenic antigens per nanoparticle. In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle comprises a population of 6 tolerogenic antigens per particle. In other embodiments, the number of tolerogenic antigens associated with a particular nanoparticle comprises 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 comprises 1-5 (e.g., 2, 3, 4, and 5) different tolerogenic antigens. In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle comprises 3-4 different tolerogenic antigens. In some embodiments, the population of tolerogenic antigens is specific for one to three different diseases. In certain embodiments, the population of tolerogenic antigens is specific for the same disease.

[0059] In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle comprises (i) a first population of polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 406-588 or a biologically active fragment or variant thereof, (ii) a second population of polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 406-588 or a biologically active fragment or variant thereof, and (iii) a third population of polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 406-588 or a biologically active fragment or variant thereof.

[0060] In some embodiments, (ii) a first population of polypeptides comprises the amino acid sequence of SEQ ID NO: 474, or a biologically active fragment or variant thereof, (iii) a second population of polypeptides comprises the amino acid sequence of any one of SEQ ID NOs: 406-588, or a biologically active fragment or variant thereof, and (iii) the third population of polypeptides comprises the amino acid sequence of any one of SEQ ID NOs: 406-588, or a biologically active fragment or variant thereof.

[0061] In some embodiments, the population of tolerogenic antigens associated with a particular nanoparticle comprises (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 the amino acid sequence of any one of SEQ ID NOs: 406-588, 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.

[0062] In some embodiments, the tolerogenic 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 the polypeptide sequence of SEQ ID NO: 374. In some embodiments, the tolerogenic antigen comprises a polypeptide sequence of SEQ ID NO: 374. In some embodiments, the tolerogenic antigen comprises a fragment of SEQ ID NO: 373 comprising between 6 and 12 amino acid residues in length.

[0063] In some embodiments, the tolerogenic antigen comprises an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises a pyroglutamic acid residue at the N-terminus. In some embodiments, the tolerogenic antigen comprises an acetyl group at the N-terminus. In some embodiments, the tolerogenic antigen comprises a pyroglutamic acid residue at the N-terminus and an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises an acetyl group at the N-terminus and an amide group at the C-terminus. In certain embodiments, the tolerogenic antigen comprises an N-terminus or a C-terminus modified with a cysteine ​​residue attached to a linker. In some embodiments, the tolerogenic antigen comprises an N-terminus and a C-terminus modified with a cysteine ​​residue attached to a linker.

[0064] In some embodiments of any one of the compositions described herein, the population of tolerogenic antigens is associated with the nanoparticle phospholipid 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, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated ophthalmopathy and skin disorders, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune-mediated gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, and the like. Herpetitis, Dermatitis Herpetiformis, Epidermolysis bullosa acquisita, Systemic sclerosis, Mixed connective tissue disease, Sjögren's syndrome, Systemic lupus erythematosus, Good-Basture's syndrome, Rheumatic heart disease, Autoimmune polyglandular syndrome type 1, Aicardi-Gautieres syndrome, Acute pancreatitis Age-related macular degeneration, Alcoholic liver disease, Liver fibrosis, Metastases, Myocardial infarction, Nonalcoholic steatohepatitis (NASH), Parkinson's disease, Polyarthritis / Fetal and Neonatal Anemia, Sepsis, or Inflammatory Bowel Disease.

[0065] In some embodiments, the multiple tolerogenic antigens are bound to the nanoparticle phospholipid via thiol-reactive and reduction-insensitive bonds between each tolerogenic antigen and the nanoparticle phospholipid. In fact, the thiol-reactive and reduction-insensitive bonds between the tolerogenic antigen and the nanoparticle phospholipid promote strong immune tolerance. In some embodiments, the phospholipid is N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine.

[0066] In some embodiments, the tolerogenic antigen is conjugated to the nanoparticle phospholipid via an amine-mediated interaction (e.g., N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl (DOPE-NHS)). In some embodiments, the amine-mediated interaction is 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-immolative linkage. Self-immolative linkages include linkers comprising o-dithiobenzyl, p-dithiobenzyl, β-dithiobenzyl carbamate moieties, 2,2-dimethyl-4-mercaptobutyric acid, or disulfide-carbonate-based traceless linkers.

[0067] In some embodiments, the composition does not include an adjuvant.

[0068] In certain embodiments, compositions comprising one or more immunomodulatory agents are provided.

[0069] Additional embodiments will be apparent to those skilled in the relevant art(s) based on the teachings contained herein.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS (Figure 1) HDL nanodiscs were analyzed by dynamic light scattering. Hydrodynamic size, polydispersity index (PDI), intensity and volume profiles of nanodiscs before and after loading with OVA-II peptide are shown.

[0071] (Figure 2) Nanodiscs-OVA-II were analyzed by LC-MS. Chromatograms of DOPE-MAL before and after conjugation to OVA-II peptide, conjugation efficiency of DOPE-MAL to OVA-II peptide, and loading efficiency of DOPE-OVA-II onto nanodiscs are shown.

[0072] (Figure 3) Blank nanodiscs and nanodisc-OVA-II were analyzed by GPC. The chromatograms are shown.

[0073] (Figure 4) Schematic diagram of the treatment regimen of ND-OVA-II in combination with IL-2 / IC therapy.

[0074] (Figure 5) PBMCs were analyzed by flow cytometry on day 7. Representative scatter plots are shown. Numbers in the plots represent the frequency of the parent gate.

[0075] (Figure 6A-F) PBMCs were analyzed by flow cytometry on day 7. a) % CD4 in PBMCs + T cells, b) CD4 in PBMCs + % OT-II tetramers in T cells + T cells, c) CD4 + % Foxp3 in T cells + CD25 + T regs d) CD4 + % OT-II tetramers in T cells + Foxp3 + CD25 + T regs , e) Foxp3 + CD25 + T regs and f) mean fluorescence intensity (MFI) of CD25 in OT-II tetramer. + Foxp3 + CD25+ T regs The MFI of CD25 in the cells is shown.

[0076] (Figure 7) PBMCs were analyzed by flow cytometry on day 14. Representative scatter plots are shown. Numbers in the plots represent the frequency of the parent gate.

[0077] (FIG. 8A-F) PBMCs were analyzed by flow cytometry on day 14. a) CD4 + % and number of T cells, b) CD4 in PBMCs + OT-II tetramers in T cells + % and number of T cells, c) CD4 + Foxp3 in T cells + CD25 + T regs % and number of CD4 + OT-II tetramers in T cells + Foxp3 + CD25 + T regs e) MFI of CD25, and f) Foxp3 + CD25 + T regs and OT-II tetramer + Foxp3 + CD25 + T regs Shows GITR inside.

[0078] (Figure 9A-C) Peripheral CD4 + a) Foxp3 in T cells + CD25 + T regs b) total, b) OT-II tetramer + Foxp3 + CD25 + T regs and c) OT-II tetramer. + Foxp3 - CD25 + T conv Shows the change in.

[0079] (Figure 10) Schematic diagram of the treatment regimen of ACT+ND-OVA-II combined with IL-2 / IC therapy followed by antigen challenge in the ear on day 25.

[0080] (FIG. 11A-C) 24 hours after intradermal challenge with MOG or OVA-II peptide in the left or right ear, respectively, ear tissue was analyzed by flow cytometry. a) CD4+ in live cells + b) number of CD4 cells; + CD25 + Foxp3 + number of cells, c) CD25 + Foxp3 + OT-II tetramer + The number of cells is indicated.

[0081] (FIG. 12) Schematic diagram of the therapeutic dosing regimen of ND-OVA-II in combination with wild-type IL-2 (wtIL-2) or IL-2 / IC. One dose of ND-OVA-II was initially administered on days 4, 3 or 1, and 3 doses of IL-2 / IC or 5 doses of wtIL-2 were administered daily from day 0. The same dosing regimen was repeated 10 days after the initial day in each group, respectively.

[0082] (Figure 13A-D) Peripheral blood-derived CD4 + T cells were analyzed by flow cytometry on days 5, 10, and 15 after IL-2 or IL-2 / IC treatment. + CD4 in T cells + b) CD4 + CD25 in T cells + Foxp3 + Treg cell abundance ratio, c) CD4 + OT-II tetramers in T cells + CD44 hi and d) CD25 + Foxp3 + OT-II tetramers in Treg cells + CD44 hi The ratio of cells present is shown.

[0083] (FIG. 14A-F) Peripheral blood-derived CD8 + T cells and NK cells were analyzed by flow cytometry on days 5, 10, and 15 after IL-2 or IL-2 / IC treatment. + CD8 in T cells + c) CD8 cell abundance + CD44 in T cells hi e) CD3 cell abundance - SSC in cells low CD49b + b) NK cell abundance ratio, and c) CD8 + T cell count, d) CD8 + number of CD44hi T cells, and f) SSC low CD49b + The number of NK cells is shown.

[0084] (FIG. 15A-C) A) Schematic of the treatment regimen of ACT+ND combined with IL-2 / IC therapy. Six days after the last IL-2 treatment, mice were transplanted with 3 million pre-activated BDC splenocytes and 3 million pre-activated NY8.3 splenocytes by retro-orbital injection. Diabetes incidence was monitored with OneTouch Ultra 2. B) Mice were treated with p31-ND or p31-ND+IL-2 / IC combination. C) Mice were treated with InsB-ND and InsC-ND or InsB-ND+InsC-ND+IL-2 / IC combination.

[0085] [Definition] As used herein, the term "about" means a value that is ±10% of the recited value.

[0086] As used herein, "administering" refers to a method of giving a dose of a composition (e.g., a nanoparticle or a nanoparticle associated with an antigen) (e.g., an immunomodulator) described herein to a subject. The compositions utilized in the methods described herein may be administered by any suitable route, including, for example, inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, or buccal. The compositions utilized in the methods described herein may also be administered locally 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).

[0087] As used herein, the term "associated with" refers to the state of two or more moieties (e.g., nanoparticles and one or more immunomodulatory agents) being linked by direct or indirect, covalent or non-covalent interactions. In some embodiments, the association is covalent. In some embodiments, the covalent association is mediated by a linker moiety. In some embodiments, the association is non-covalent (e.g., charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.). For example, in some embodiments, the immunomodulatory agent is intermingled with the nanoparticles. In some embodiments, the immunomodulatory agent is associated with the nanoparticles. In some embodiments, the immunomodulatory agent is encapsulated within the nanoparticles. In some embodiments, the immunomodulatory agent is absorbed to the nanoparticles. In some embodiments, the immunomodulatory agent is adsorbed to the nanoparticles. In some embodiments, the immunomodulatory agent is intermingled with the nanoparticles.

[0088] As used herein, the term "absorbed" refers to a biopolymeric agent (e.g., an antigen) that is incorporated into and stably retained within the nanoparticles and / or microparticles, i.e., within the outer surface.

[0089] As used herein, the term "admixed" refers to a biopolymeric agent (e.g., an antigen) that is dissolved, dispersed, or suspended in the nanoparticles and / or microparticles. In some cases, the biopolymeric agent may be homogenously mixed in the nanoparticles and / or microparticles.

[0090] As used herein, the term "adsorbed" refers to the attachment of a biopolymer agent (e.g., an antigen) to the outer surface of a nanoparticle and / or microparticle. The adsorption preferably occurs by electrostatic attraction. Electrostatic attraction is an attractive force or bond generated between two or more oppositely charged or ionic chemical groups. In general, adsorption is typically reversible.

[0091] As used herein, the term "mutein" is intended to include proteins and polypeptides with altered amino acid sequences that result from mutations or recombinant DNA procedures. As used herein, the term "IL-2 mutein molecule" or "IL-2 mutein" refers to an IL-2 variant that preferentially activates Treg cells.

[0092] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a conformational arrangement composed of a contiguous stretch of amino acids or distinct regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding moiety binds and forms an antigen-binding moiety-antigen complex. Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Proteins referred to herein as antigens include the full-length 33-mer polypeptide from α-gliadin (SEQ ID NO: 374) or any fragment thereof, or the CD4 + It may be any of the polypeptides disclosed in Table 3 (SEQ ID NOs: 375-405) as epitopes recognized by T cells. When referring to a particular protein herein, the term encompasses the "full-length" unprocessed protein and any form of the protein that results from processing in the cell. The term also encompasses variants of naturally occurring proteins (e.g., splice variants or allelic variants).

[0093] As used herein, the terms "autoimmune disorder" and "autoimmune disease," used interchangeably herein, refer to a medical condition in which a subject's immune system mistakenly attacks the subject's own body.

[0094] As used herein, "combination therapy" or "administered in combination" means administering two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different agents or treatments to a subject as part of a defined treatment regimen for a particular disease or condition (e.g., an autoimmune disorder (e.g., MS or celiac disease)). The treatment regimen defines the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, the two or more agents are not combined, but are administered in a sequential manner as part of a prescribed regimen. In some embodiments, the administration of two or more agents or combination treatments results in a greater reduction in symptoms or other parameters associated with the disorder than would be observed with one agent or treatment delivered alone or in the absence of the other agent. The effect of the two treatments may be partially additive, completely additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent may be effected by any suitable route, including, but not limited to, inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, buccal, or direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection, while a second therapeutic agent of the combination may be administered orally.

[0095] As used herein, the term "complexed" refers to a non-covalent interaction between a biopolymeric agent (e.g., an antigen) and a nanoparticle and / or microparticle.

[0096] As used herein, the term "conjugated" refers to the covalent association of a biopolymeric agent (e.g., an antigen) with a nanoparticle and / or microparticle.

[0097] As used herein, the term "drug" or "therapeutic agent" is meant to include any molecule, molecular complex, or substance that is administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical, and prophylactic uses. The term "drug" is further meant to include any such molecule, molecular complex, or substance that is chemically modified and / or operably linked to a biological or biocompatible structure.

[0098] The term "regulatory T cells" (also referred to as "Treg" or "Treg cells") has its general meaning in the art and is intended to refer to a subpopulation of T cells that have been characterized to "suppress" the activity of effector T cells in vitro and / or in vivo. Treg cells are therefore an important component of a healthy immune system. Regulatory T cells are involved in the management of effector T cells, regulating the immune system, maintaining tolerance to self-antigens, and eliminating autoimmune and / or inflammatory diseases. Tregs have numerous biomarkers known in the art. Regulatory T cells include two subsets that are distinguished from each other by their expression of CD45RA: "naive Tregs" that express FOXP3 and CD45RA, and "effector Tregs" that express FOXP3 and do not express CD45RA. Naive Tregs are the preferred cells for proliferation, as they are highly proliferative under stimulatory conditions and in the presence of IL-2, whereas effector Tregs are less proliferative under such conditions. Thus, FoxP3+CD4+ T cells can be divided into (1) naive / resting Treg cells with CD127lowCD25++CD45RA+FoxP3low phenotype (naive Tregs), (2) effector Tregs with CD127lowCD25+++CD45RA-FoxP3high phenotype ("effector Treg cells"), both of which are highly suppressive in vitro, and (3) non-suppressive CD4+ T cells with CD127lowCD25+++CD45RA-FoxP3low phenotype. Thus, the term "effector Treg cells" (also referred to as "eTreg cells") refers to activated Treg cells that exhibit regulatory or suppressive functions on effector T cells (which literally act as "effectors" of Treg cells).Demonstration of regulatory / suppressive function of eTreg cells can be determined by any suitable method known in the art (see Miyara, M. et al. Functional Delineation and Differentiation Dynamics of Human CD4(+) T Cells Expressing the FoxP3 Transcription Factor. Immunity 30, 899-911 (2009)). In particular, examples of the above tests are described in the Examples section. In particular, the test embodied in the Examples and FIG. 2 is considered as the in vitro standard test for evaluating regulatory T cell function.

[0099] 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. of the full-length sequence), but including, 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 of sufficient length such that a desired function of the full-length protein is maintained. For example, regulation of the alternative complement pathway in liquid phase by a fragment such as factor H is maintained. Such a fragment is a "biologically active fragment."

[0100] As used herein, the term "expanding" refers to increasing the number of cells (eg, Tregs) in a cell population or sample by cell replication.

[0101] As used herein, the term "HDL" or "high density lipoprotein" refers to high density lipoprotein. HDL contains a complex of lipids and proteins in approximately equal amounts, which functions as a transporter of cholesterol in the blood. HDL is mainly synthesized in and secreted from epithelial cells of the liver and small intestine. Immediately after secretion, HDL is in the form of a discoid particle containing apolipoprotein AI (also called apoA-I) and phospholipids as its main components, which is also called nascent HDL. In the blood, this nascent HDL receives free cholesterol from the cell membrane of peripheral cells or is produced in the hydrolysis process of other lipoproteins, and forms mature globular HDL while retaining the cholesterol ester converted from the above cholesterol in its hydrophobic center by the action of LCAT (lecithin cholesterol acyltransferase). HDL plays a vital role in the lipid metabolism process called "reverse cholesterol transport", taking up cholesterol from peripheral tissues in the blood and transporting it to the liver. Reverse cholesterol transport is thought to be one of the main mechanisms of the protective effect of HDL against atherosclerosis, and therefore high levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0102] As used herein, the term "immunomodulatory agent" refers to a compound that stimulates or suppresses the immune system. Immunomodulatory agents 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). In some embodiments, the immunomodulatory agent is an immunosuppressant.Examples of immunosuppressants include IDO, vitamin D3, cyclosporines (e.g., cyclosporine A), aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin 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;Ortho-vanillin;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;Amlexanox;Bay11-7082;BX795;CH-223191;Chloroquine;CLI-095;CU-CPT9a;Chlorosporin A;CTY387;Gefitinib;Glybenclamide;H-89;H-131;Isoliquiritige These include, but are not limited to, AHR-specific ligands including, but not limited to, nin; MCC950; MRT67307; OxPAPC; parthenolide; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU.521; VX-765; YM201636; Z-VAD-FMK; and AHR-specific ligands including, but not limited to, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD); tryptamine (TA); and 6 formyl indolo[3,2 b]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-carboxylic acid 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), trichostatin (TSA) (Reilly CM et al. J. Autoimmun 31: 123-130. 2008). Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (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 agent provided herein.

[0103] As used herein, the term "nucleic acid" refers to a nucleic acid that may be DNA or RNA (e.g., mRNA). In an embodiment, the composition includes a complement, such as a full-length complement, or a degenerate (due to the degeneracy of the genetic code) of any of the nucleic acids provided herein. In an embodiment, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In an embodiment, the expression vector may include a plasmid, a retrovirus, or an adenovirus, among others. The nucleic acid may be isolated or synthesized using standard molecular biology approaches. For example, the nucleic acid may be isolated or synthesized by using the polymerase chain reaction to produce a nucleic acid fragment that is then purified and cloned into an expression vector. Further techniques useful in the practice of the invention may be 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 Texas Southwestern Medical Center, Dallas, Cold Spring Harbor.

[0104] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures.

[0105] The term "in vivo" refers to the natural environment (eg, an animal or a cell) and to processes or reactions that occur within a natural environment.

[0106] As used herein, the term "lipid" or "lipid molecule" refers to fatty substances that are insoluble in water and include fats, oils, waxes, and related compounds. They can either be made in the blood (endogenous) or ingested in the diet (exogenous). Lipids are essential for normal body function and, whether produced from exogenous or endogenous sources, must be transported to 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 from the diet or manufactured by cells in most organs and tissues in the body, primarily the liver. Cholesterol may be found in a free form or, more frequently, bound to a fatty acid, called cholesterol esters. As used herein, "lipid" or "lipid molecule" refers to any fat-soluble compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogs, which are generally grouped into at least three classes: (1) "simple lipids," which include fats and oils, as well as waxes, (2) "lipid compounds," which include 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 and non-membrane-forming lipids.

[0107] As used herein, the term "lipoprotein" refers to a compound structured such that water-insoluble lipids are contained within a partially water-soluble outer shell. Depending on the type of lipoprotein, the contents contain various amounts of free and esterified cholesterol, triglycerides, and apoproteins, or apoproteins. There are five main types of lipoproteins that differ in function and lipid and apoprotein content and are classified according to 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 associated with lipoproteins.

[0108] As used herein, the term "non-naturally occurring amino acid" refers to an alpha amino acid that is not naturally produced or found in mammals. Examples of non-natural amino acids include D-amino acids; amino acids with an acetylaminomethyl group attached to the sulfur atom of cysteine; pegylated amino acids; omega amino acids of the formula NH2(CH2)nCOOH (n is 2-6), neutral non-polar amino acids (e.g., sarcosine), t-butylalanine, t-butylglycine, n-methylisoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic 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, aminonorbornyl-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-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, D-α-methylalanine, D-α-methylaminoisobutyrate ... -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-thiazolidine-4-carboxylic acid, L-homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-cetyl 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, b-(benzothiazolyl-2-yl)-alanine, b-cyclopropylalanine, 5-amino-cyclohexane-1-carboxylic acid, 1-amino-cyclopentane-1- ...-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino-cyclopentane-1-carboxylic acid, 1-amino- ,5-Dimethyl-1,3-thiazolidine-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-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenyl-pyrrolidine N-methyl-2-carboxylic acid, 1-aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxy-benzoic acid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid, N-methylanthranilic 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-methylamino-benzoic acid ... Tyrthreonine, 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-(hydrogen 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 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-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl)carbamylmethylglycine, L-pyroglutamic acid, D-pyroglutamine acid, 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-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, 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, Symmetrical dimethylarginine, 3-Carboxythiomorpholine, D-1,2,3,4-Tetrahydronorharman-3-carboxylic acid, 3-Aminobenzoic acid, 3-Amino-1-carboxymethyl-pyridin-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-(31unction 3131ol(31unction 3131ole)-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, b-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, nicopetinic acid, (2S,4R ) and (2S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercapto-pyrrolidine-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 non-naturally occurring analogs thereof. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or non-naturally occurring analogs thereof. In some embodiments,It is specifically contemplated that the terminal amino group in the amino acid may be an amide group or a carbamate group.

[0109] "Percent sequence identity (%)" to a reference polynucleotide or polypeptide 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 aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for purposes of determining percentage sequence identity of nucleic acids or amino acids may be accomplished in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art may determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, percentage sequence identity values ​​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 may 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) scores as identical in its alignment of A and B, and Y is the total number of nucleic acids in B. It will be appreciated 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, then the percentage of sequence identity of A to B will not be equal to the percentage of sequence identity of B to A.

[0110] The term "protein" refers to a polymer of amino acids of any length, including naturally occurring and non-naturally occurring amino acids. The term also includes amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.

[0111] As used herein, the term "peptide" refers to a polymer in which the monomers are amino acids covalently linked through amide bonds. A peptide is two or more amino acid monomers long.

[0112] "Pharmaceutical composition" means any composition comprising a therapeutically or biologically active agent (e.g., nanoparticles comprising 1-30 (e.g., 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, or 8-30 tolerogenic antigens) suitable for administration to a subject. The biologically active agent comprises nanoparticles comprising 1-30 (e.g., 8-30 tolerogenic antigens per nanoparticle). The 1-30 tolerogenic antigens associated with a particular nanoparticle may all have the same sequence identity, or the 1-30 tolerogenic antigens associated with a particular nanoparticle may comprise 1-5 different populations of tolerogenic antigens having different sequence identities. Any of these formulations may be prepared by methods well known and accepted in the art. See, e.g., Remington: The Science and Practice of Pharmacy (21st ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is incorporated by reference herein.

[0113] By "pharmaceutical acceptable diluent, excipient, carrier, or adjuvant" is meant a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to a subject while retaining the therapeutic properties of the pharmaceutical composition being administered.

[0114] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be taken from animals (including humans) and encompass liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Environmental samples include environmental materials such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0115] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0116] As used herein, the terms "synthetic HDL," "sHDL," "reconstituted HDL," and "rHDL" refer to particles structurally similar to native HDL, composed of lipids or lipids (preferably ApoA-I or a mimetic thereof) associated with at least one protein of HDL. Typically, the components of sHDL may be derived from blood or produced by recombinant technology.

[0117] "Therapeutically effective amount" refers to the amount of a composition administered to improve, inhibit, or alleviate a condition, or a symptom of a disorder or disease (e.g., celiac disease) in a clinically relevant manner in a subject. Any improvement in a subject is considered sufficient to achieve treatment. Preferably, an amount sufficient to treat is an amount that reduces, inhibits, or prevents the occurrence or prevention of one or more symptoms of a disease or disorder (e.g., celiac disease) or reduces the severity or length of time that a subject suffers from one or more symptoms of a disease or disorder (e.g., celiac disease) (e.g., by at least about 10%, about 20%, or about 30%, more preferably at least about 50%, about 60%, or about 70%, and most preferably at least about 80%, about 90%, about 95%, about 99%, or more, compared to a control subject not treated with a composition described herein). The effective amount of the pharmaceutical composition used to practice the methods described herein (e.g., treating celiac disease) will vary depending on the mode of administration, as well as the age, weight, and general health of the subject being treated. A physician or researcher can determine the appropriate amount and administration regimen.

[0118] As used herein, the term "tolerogenic antigen" refers to a molecule that can bind to an antibody or antigen receptor on a T cell, particularly one that induces an immune response.

[0119] As used herein, the term "solvent" refers to the medium in which a reaction takes place. Solvents may be liquids, but are not limited to liquid forms. Types of solvents include, but are not limited to, non-polar, polar, protic, and aprotic.

[0120] Detailed Description of the Invention Regulatory T cells (Treg, CD4 + CD25 high Foxp3 +) play an important role in immune tolerance to autoimmune diseases [1]. In preclinical models, many studies have shown that adoptive transfer of Tregs can attenuate inflammation and restore immune tolerance. However, it remains unclear how to effectively induce and maintain high abundance of Tregs in vivo without ex vivo manipulation of Tregs, such as CAR-Tregs. Exogenous IL-2 via the high-affinity IL2R complex is required for Treg survival and proliferation [2]. Low doses of IL-2 have been shown to induce Tregs in vivo, but IL-2 may also promote effector T cells and NK cells that exacerbate inflammation [3, 4].

[0121] Recently, engineered IL-2 and mutein IL-2, such as PT101, have been developed to selectively induce Tregs without inducing effector T cells and NK cells [5-8]. In clinical studies, PT101 has been reported to induce polyclonal Tregs in humans [9]. However, previous studies have shown that antigen-specific Tregs have significantly superior therapeutic potential in targeting immune tolerance compared with polyclonal Tregs [10-13]. Therefore, new approaches are needed to induce antigen-specific Tregs in high abundance in vivo.

[0122] Experiments conducted during the course of developing embodiments of the present invention have led to the development of a new strategy for inducing high frequencies of antigen-specific Tregs in vivo without ex vivo cell manipulation. As a result of the above experiments, a new strategy was developed and optimized to combine nanodiscs with modified IL-2 to induce unprecedented amounts of antigen-specific Tregs in vivo. In particular, IL-2:anti-IL-2 antibody (clone: ​​JES6-1) immune complexes (IL-2 / IC) have been shown to selectively induce polyclonal Tregs

[14] . Administration of IL-2 / IC with free peptide or peptide-tetramers has been reported to induce antigen-specific Tregs [15,16]. However, these aforementioned attempts have resulted in rather poor antigen-specific Treg responses, with the antigen-specific Treg frequency in the CD4+ T cell compartment being less than 0.25% [15,16].

[0123] It was hypothesized that appropriate delivery of peptide antigens to lymphoid tissues is important to maximize antigen-specific Treg induction with IL-2 / IC. In the experiments described herein, we report for the first time a significant expansion of antigen-specific Tregs by combining lymphatic-targeted Nanodisc-mediated delivery of peptides with IL-2 / IC therapy compared to IL-2 / IC alone.

[0124] It was also assumed that the actual treatment method was important. The above experiments showed that it was desirable to administer Nanodiscs first (e.g., subcutaneously), followed by systemic administration of IL-2 and / or mutein / engineered IL-2. This ensured that antigen-specific Tregs were primed and generated first, and subsequent administration of mutein / engineered IL-2 caused robust proliferation of antigen-specific Tregs. For long-term maintenance of antigen-specific Tregs, administration of a maintenance dose of Nanodiscs and / or mutein / engineered IL-2 was further assumed.

[0125] Compared to CAR-Treg or other cell therapies, the Nanodiscs described herein are synthetic, well-characterized, and easy to manufacture [17, 18]. Thus, this combinatorial approach opens new doors for targeted immunotherapy for various autoimmune diseases. More broadly, it is envisioned that the Nanodiscs described herein can be combined with mutein IL-2, other endogenous or engineered cytokines, growth factors, or antibodies to achieve synergistic effects and induce Tregs.

[0126] Thus, the invention provides compositions comprising nanoparticles associated (or not) with one or more tolerogenic antigens, and compositions comprising an immunomodulatory agent (e.g., interleukin-2 (IL-2) or an IL-2 variant, or IL-2 / IC), and related methods comprising co-administration of the above compositions for the purpose of inducing expansion of regulatory T cells (Tregs) (e.g., antigen-specific regulatory Tregs). The invention further provides methods of treating an autoimmune disorder by administering to a subject a composition comprising nanoparticles associated (or not) with one or more tolerogenic antigens associated with the autoimmune disorder prior to administering to the subject a composition comprising an immunomodulatory agent.

[0127] In certain embodiments, the present invention provides a method for the proliferation and proliferation of regulatory Tregs (e.g., CD4+ Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.), administering to the subject a composition comprising one or more immunomodulatory agents (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC) capable of expanding Tregs in the subject. + CD25 high Foxp3 +In some embodiments, a composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, an antigen-specific regulatory Treg (e.g., CD4 + CD25 high Foxp3 + ) in vivo amplification is specific for a particular tissue region (eg, a particular tissue region associated with one or more autoimmune disorders).

[0128] In certain embodiments, the present invention provides a method for the proliferation and proliferation of antigen-specific regulatory Tregs (e.g., CD4+ Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.) administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, an IL-2 mutein, an IL-2 variant, or IL-2 / IC)) capable of expanding antigen-specific Tregs. + CD25 high Foxp3 + In some embodiments, the present invention provides a method for in vivo expansion of antigen-specific regulatory Tregs (e.g., CD4 Tregs) in a subject, where "antigen-specific" is specific for one or more tolerogenic antigens with which it is associated, including nanoparticles associated with one or more tolerogenic antigens. + CD25 high Foxp3 + ) in vivo amplification is specific for a particular tissue region (eg, a particular tissue region associated with one or more autoimmune disorders).

[0129] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 + The present invention provides a method for promoting potent immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of augmenting the immune system of an autoimmune disorder. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the promotion of potent immune tolerance to an antigen associated with an autoimmune disorder in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0130] In certain embodiments, the present invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 +The present invention provides a method for promoting potent immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of augmenting immune responses to an autoimmune disorder, where "antigen-specific" is specific to the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the promotion of potent immune tolerance to an antigen associated with an autoimmune disorder in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0131] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 + The present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the increase in the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in the subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0132] In certain embodiments, the present invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + The present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the immune response of a subject to one or more tolerogenic antigens, where "antigen-specific" is specific to the one or more tolerogenic antigens associated therewith, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the increase in the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0133] In certain embodiments, the present invention provides a method for administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc. later) producing Tregs (e.g., CD4 + CD25 high Foxp3 +In some embodiments, the present invention provides a method of treating, preventing and / or attenuating one or more autoimmune disorders in a subject, the method comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating mTOR. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0134] In certain embodiments, the invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens (e.g., one or more tolerogenic antigens associated with an autoimmune disorder), and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders), comprising administering to a subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating the immune system of the subject's immune system, where "antigen-specific" is specific for the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0135] The methods are not limited to the treatment of any particular disorder.

[0136] In some embodiments, the disorder is an autoimmune disorder. Examples of autoimmune disorders include multiple sclerosis (MS), celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid gland (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated eye and 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 variants, and bullous pemphigoid. , Dermatitis Herpetiformis, Epidermolysis Bullosa Acquisita, Systemic Sclerosis, Mixed Connective Tissue Disease, Sjogren's Syndrome, Systemic Lupus Erythematosus, Good-Basture's Syndrome, Rheumatic Heart Disease, Autoimmune Polyglandular Syndrome Type 1, Aicardi-Gautieres Syndrome, Acute Pancreatitis, Age-Related Macular Degeneration, Alcoholic Liver Disease, Liver Fibrosis, Metastases, Myocardial Infarction, Nonalcoholic Steatohepatitis (NASH), Parkinson's Disease, Polyarthritis / Fetal and Neonatal Anemia, Sepsis, and Inflammatory Bowel Disease.

[0137] In some embodiments, the disorder is a transplant-related disorder. In some embodiments, the disorder is one or more allergies. In some embodiments, the disorder is a respiratory disease (e.g., asthma). In some embodiments, the disorder is graft-versus-host disease (GvHD).

[0138] In some embodiments, the above method (e.g., administration of a composition comprising nanoparticles associated with one or more tolerogenic antigens followed by administration of a composition comprising an immunomodulatory agent capable of expanding Tregs) is further followed by administration of one or more tolerogenic antigens to a specific tissue region (e.g., a specific tissue region associated with one or more autoimmune disorders). In some embodiments, administration of one or more tolerogenic antigens to a specific tissue region is by injection and / or topical and / or subcutaneous administration. In some embodiments, administration of one or more tolerogenic antigens to a specific tissue region prevents immune tolerance in the specific tissue region.

[0139] In some embodiments, the nanoparticles are associated with an immunomodulatory agent and are not associated with a tolerogenic antigen, hi some embodiments, the nanoparticles are associated with a tolerogenic antigen and are further associated with an immunomodulatory agent.

[0140] In some embodiments, a composition comprising an immunomodulatory agent capable of expanding Tregs is formulated within a nanoparticle, such that the immunomodulatory agent capable of expanding Tregs is associated with the nanoparticle (e.g., thereby providing a composition comprising a nanoparticle associated with an immunomodulatory agent capable of expanding Tregs).

[0141] [Immunomodulators] The present invention provides compositions comprising one or more immunomodulatory agents. The compositions are not limited to a particular immunomodulatory agent.

[0142] In some embodiments, the one or more immunomodulatory agents are fingolimod; rapamycin; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or related ligand; trichostatin A; suberoylanilide hydroxamic acid (SAHA); statins; mTOR inhibitors; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors; corticosteroids; inhibitors of mitochondrial function; 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 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-Thioguanine (6-TG); FK506; Sanglifehrin A; Salmeterol; Mycophenolate Mofetil (MMF); Aspirin and other COX Inhibitors; Niflumic Acid; Estriol; Triptolide; OPN-305; OPN- 401;erythritol (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;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;amlexanox;Bay11-7082;BX795;CH-223191;chloroquine;CLI-095;CU-CPT9a;cyclosporine A;CTY387;gefitinib;glibenclamide;H-89;H-131;isoliquiritigenin;MCC950;MRT67307;OxPAPC;parthenolide;Pepinh-MYD;Pepinh-TRIF;polymyxin B;R406;RU.521;VX-765;YM201636;Z-VAD-FMK;and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD);tryptamine (TA);and 6-formyl indolo[3,2 b] selected from AHR-specific ligands, including but not limited to carbazole (FICZ);

[0143] In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine is a human cytokine. In some embodiments, the cytokine is selected from TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL12B, IL-15, IL-21, and IL-18.

[0144] In some embodiments, the immunomodulatory agent is human IL-2. In some embodiments, the immunomodulatory agent is low dose IL-2. In some embodiments, the immunomodulatory agent is PT101 or a variant thereof. In some embodiments, the immunomodulatory agent is a mutein IL-2 and / or a variation thereof. In some embodiments, the IL-2 is any of the IL-2 cytokines, IL-2 muteins and / or IL-2 variants described in U.S. Patents 11,091,527, 11,091,526, 11,077,195, 11,077,172, 10,960,079, 10,946,068, 10,766,938, 10,722,460, 10,174,092, 10,174,091; EP Patent No. 3808764, and / or U.S. Patent Application Publication No. US20120315245.

[0145] In some embodiments, the immunomodulatory agent is an IL-2:anti-IL-2 antibody (clone: ​​JES6-1) immune complex (IL-2 / IC).

[0146] In some embodiments, the IL-2 is Proleukin. In some embodiments, the IL-2 is 14 MIU / ml 2 Less than 12MIU / m 2 Less than 10MIU / m 2 Less than 8MIU / m 2 Less than 6MIU / m 2 Less than 4MIU / m 2 Less than or equal to 2MIU / m 2 It is administered in doses of less than 100 mg / kg.

[0147] In some embodiments, the IL-2 is extended pharmacokinetic (PK) IL-2. In some embodiments, the extended PK IL-2 comprises a fusion protein. In some embodiments, the fusion protein comprises an IL-2 portion and a portion selected from the group consisting of an immunoglobulin fragment, human serum albumin, and Fn3. In some embodiments, the fusion protein comprises an IL-2 portion operably linked to an immunoglobulin Fc domain. In some embodiments, the fusion protein comprises an IL-2 portion operably linked to human serum albumin. In some embodiments, the extended-PK IL-2 comprises an IL-2 portion conjugated to a non-proteinaceous polymer. In some embodiments, the non-proteinaceous polymer is polyethylene glycol.

[0148] In certain embodiments, the extended-PK IL-2 is mutated to have altered affinity (e.g., higher affinity) for the IL-2Rα receptor compared to unmodified IL-2. Site-directed mutagenesis can be used to isolate IL-2 mutants that exhibit higher affinity binding to CD25, i.e., IL-2Rα, compared to wild-type IL-2. Increasing the affinity of IL-2 for IL-2Rα at the cell surface will increase receptor occupancy within a limited range of concentrations of IL-2, further increasing the local concentration of IL-2 at the cell surface.

[0149] In certain embodiments, IL-2 variants are provided, which may be, but are not necessarily, substantially purified and can function as high affinity CD25 binders. IL-2 is a T cell growth factor that induces proliferation of antigen-activated T cells and stimulation of NK cells. Exemplary IL-2 variants that are high affinity binders include those described in WO2013 / 177187A2. Further exemplary IL-2 variants with increased affinity for CD25 are disclosed in U.S. Patent No. 7,569,215, the contents of which are incorporated herein by reference.

[0150] An IL-2 variant may be at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical in amino acid sequence to wild-type IL-2 (in its precursor or preferably mature form). Mutations may consist of changes in the number or content of amino acid residues. For example, an IL-2 variant may have more or fewer amino acid residues than wild-type IL-2. Alternatively, or in addition, an IL-2 variant may contain a substitution of one or more amino acid residues present in wild-type IL-2.

[0151] The operations required to generate IL-2 mutants are routine in the art and can be performed by those of skill in the art without resorting to undue experimentation. For example, mutations consisting of the substitution of one or more amino acid residues in IL-2 can be generated using PCR-assisted mutagenesis techniques (e.g., known in the art and / or described herein for generating IL-2 mutants). Mutations consisting of deletions or additions of amino acid residues to the IL-2 polypeptide can also be generated using standard recombinant techniques. In the case of deletions or additions, the nucleic acid molecule encoding IL-2 is simply digested with the appropriate restriction endonuclease. The resulting fragments can be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation is facilitated if both ends of the nucleic acid molecule contain complementary nucleotides that overlap each other, although blunt-ended fragments can also be ligated. The nucleic acids generated by PCR can also be used to generate a variety of mutant sequences.

[0152] In addition to producing IL-2 variants through expression of nucleic acid molecules modified by recombinant molecular biology techniques, IL-2 variants can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by one of skill in the art.

[0153] As noted above, IL-2 can also be prepared as a fusion or chimeric polypeptide comprising IL-2 and a heterologous polypeptide (i.e., a polypeptide that is not IL-2). The heterologous polypeptide can increase the circulating half-life of the chimeric polypeptide in vivo, thus further enhancing the properties of IL-2.

[0154] In certain embodiments, the chimeric polypeptide may comprise a polypeptide that functions as an antigenic tag, such as IL-2 and a FLAG sequence. The FLAG sequence is recognized by a highly specific anti-FLAG antibody that is biotinylated as described herein (see also Blanar et al., Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In certain embodiments, the chimeric polypeptide further comprises a C-terminal c-myc epitope tag.

[0155] The chimeric polypeptides can be constructed using only conventional molecular biology techniques, which are well within the capabilities of a person skilled in the art.

[0156] In certain embodiments, a composition comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) can expand Treg cells in a subject or sample. Indeed, the composition comprising an immunomodulatory agent can increase the ratio of Treg to non-regulatory T cells. This ratio may be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells. Tregs are typically present in human blood at 5-10% of total CD4+CD3+ T cells, but in autoimmune diseases, the percentage may be lower or higher. In a preferred embodiment, the percentage of Tregs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. The maximum fold increase in Tregs may vary depending on the specific disease, but the maximum abundance of Tregs that may be obtained by IL-2 mutein treatment is 50% or 60% of total CD4+CD3+ T cells. In a specific embodiment, a composition comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) is administered to a subject to increase the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the peripheral blood of the subject.

[0157] The compositions comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) are also useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject, as they preferentially expand Tregs over other cell types. This ratio may be measured by determining the ratio of CD3+FOXP3+ cells to CD16+ and / or CD56+ lymphocytes that are CD19- and CD3-.

[0158] [Nanoparticles] The present invention is not limited to a particular type or variety of nanoparticles that are associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, mixed) or not associated with a tolerogenic antigen for treating, preventing or ameliorating various types of autoimmune disorders (e.g., celiac disease).

[0159] Examples of nanoparticles include fullerenes (also known as C 60 , C 70 , C 76 , C 80 , C 84 ), endohedral metallofullerenes containing additional atoms, ions, or clusters within the fullerene cage, buckyballs (EMI), trimetallic nitride templated endohedral metallofullerenes (TNT EMEs, encapsulating highly symmetrical four-atom 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 metallofullerenes and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Particle embodiments may include microparticles capable of enhancing efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymeric microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold, silver, carbon, and iron nanoparticles.

[0160] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles include a polyol polymer that has been modified to contain a hydrophobic polymer block. The term "hydrophobic polymer block" as used in this disclosure refers to a segment of a polymer that is hydrophobic by itself. The term "micelle" as used herein refers to an assembly of molecules dispersed in a liquid. A typical micelle in an aqueous solution forms an assembly with a hydrophilic "head" region in contact with the surrounding solvent, isolating a single hydrophobic tail region in 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.

[0161] The present invention further encompasses the use of particles on 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 ease of discussion, the use of "nanoparticles" 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.

[0162] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers with covalently bound metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In these embodiments, the nanoparticles are metal nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). The nanoparticles may include a core, or a core and a shell as core-shell nanoparticles.

[0163] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles are composed of a mixture of HDL apolipoproteins and amphipathic lipids.

[0164] The present invention is not limited to the use of a particular type or variety of HDL apolipoproteins. 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, the HDL apolipoprotein is selected from, e.g., preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, apolipoprotein A-II xxx (apo A-II-xxx), preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-I Milano, proApoA-I Milano, ApoA-I Milano, preproApoA-I paris, proApoA-I paris, and ApoA-I paris, as well as peptidomimetics of mixtures of these proteins. Preferably, the carrier particles are composed of ApoA-I or ApoA-II, although the use of other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate carrier particle mixtures for delivery of therapeutic agents. In some embodiments, mimetics of such HDL apolipoproteins are used.

[0165] ApoA-I is synthesized by the liver and small intestine as a preproapolipoprotein that is secreted as a proprotein that is rapidly degraded to generate a mature polypeptide with 243 amino acid residues. ApoA-I consists primarily of 6-8 different 22 amino acid repeats separated by linker moieties that are proline, and two different 11 amino acid repeats, each of which has a helical wheel signature of an amphipathic α-helix separated by linker moieties that are often proline, and in some cases, consists of a stretch composed of several residues. ApoA-I forms three types of stable complexes with lipids: small lipid-poor complexes called pre-β-1 HDL; flattened discoidal particles containing polar lipids (phospholipids and cholesterol) called pre-β-2 HDL; 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).

[0166] In some embodiments, ApoA-I agonists or mimetics are provided. In some embodiments, such ApoA-I mimetics can form amphipathic α-helices that mimic the activity of ApoA-I and have specific activity that approaches or exceeds that of the natural molecule. In some, ApoA-I mimetics are peptides or peptide analogs. They form amphipathic helices (in the presence of lipids), bind lipids, form pre-β-like or HDL-like complexes, activate lecithin-cholesterol acyltransferase (LCAT), increase serum concentration of HDL fraction, and promote cholesterol efflux.

[0167] The present invention is not limited to the use of a specific ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetics described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 are utilized. In some embodiments, any of the ApoA-I mimetics described in U.S. Patent Application Publication Nos. 20110046056 and 20130231459 are utilized.

[0168] In some embodiments, the "22A" ApoA-I mimetic is used (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (see, e.g., U.S. Pat. No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1, described in U.S. Pat. No. 7,566,695, are utilized.

[0169] [Table 1-1]

[0170] [Table 1-2]

[0171] [Table 1-3]

[0172] [Table 1-4]

[0173] [Table 1-5]

[0174] [Table 1-6]

[0175] [Table 1-7]

[0176] * indicates a peptide that is N-terminally acetylated and C-terminally amidated; indicates a peptide that is N-terminally dansylated; sp indicates a peptide that showed solubility problems under the experimental conditions; X is Aib; Z is Nal; O is Orn; ∼ indicates a deleted amino acid.

[0177] In some embodiments, an ApoA-I mimetic is utilized as described in U.S. Pat. No. 6,743,778, having the following sequence: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 255).

[0178] In some embodiments, any of the following ApoA-I mimetics shown in Table 2, described in US Patent Application Publication No. 2003 / 0171277, are utilized.

[0179] [Table 2-1]

[0180] [Table 2-2]

[0181] [Table 2-3]

[0182] In some embodiments, an ApoA-I mimetic is utilized, as described in US Patent Application Publication No. 2006 / 0069030, having the following sequence: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333).

[0183] In some embodiments, ApoA-I mimetics are utilized having the following sequences as described in U.S. Patent Application Publication No. 2009 / 0081293: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0184] In some embodiments, an ApoA-I mimetic is utilized having one of the following sequences: 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), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 35 4), 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), 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).

[0185] Amphipathic lipids include, for example, any lipid molecule that has both hydrophobic and hydrophilic portions. Examples include phospholipids or glycolipids. Examples of phospholipids that may be used in the sHDL-tolerogenic antigen nanoparticles include 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-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-diglyceroyl -sn-glycero-3-phosphocholine;1,2-dimyristoleoyl-sn-glycero-3-phosphocholine;1,2-dimyristeridoyl-sn-glycero-3-phosphocholine;1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine;1,2-dipalmiteroyl-sn-glycero-3-phosphocholine;1,2-dipetroselenoyl-sn-glycero-3-phosphocholine;1,2-dioleoyl-sn-glycero-3-phosphocholine;1,2-dielideoyl-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-dimyristoyl-sn-glycero-3-phosphoethanolamine;1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine;1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine;1,2-dis 1,2-Dipalmitoleoyl-sn-glycero-3-phosphoethanolamine;1,2-Dieleidoyl-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)butyramide];1,2-Dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidophenyl)butyramide];1,2-Dihexadecanoyl-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide];1,2-Di-(9Z-octadecenoyl)-sn-glycero-3-phosphothioethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] Ethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide];N-[(3-maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine;N-[(3-maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine;N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine, distearoyl;N-[(3- 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, dipalmitoyl Photidylethanolamine, 1-palmitoyl-2-oleoyl;Phosphatidylcholine;Phosphatidylinositol;Phosphatidylserine;Phosphatidylethanolamine;N-(Succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, distearoyl;N-(Succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl;N-(Succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl;Examples of phosphatidylethanolamine include, but are not limited to, N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succinimidyloxyglutaryl)aminopropyl, and polyethylene glycol-carbamyl distearoylphosphatidyl-ethanolamine; N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoyl-ethanolamine;

[0186] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2-250 (eg, 10-200, 20-100, 20-50, 30-40).

[0187] Typically, the sHDL nanoparticles so formed are spherical or discoidal and have a diameter of about 5 nm to about 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 homogenous preparation.

[0188] The compositions are not limited to particular tolerogenic antigens involved in autoimmune diseases (eg, MS or celiac disease).

[0189] [Tolerogenic antigens] The present invention includes compositions comprising nanoparticles associated with multiple tolerogenic antigens (e.g., 1-30 tolerogenic antigens (e.g., 8-30 tolerogenic antigens per nanoparticle)) involved in an autoimmune disease (e.g., MS or celiac disease) and methods of treating an autoimmune disease (e.g., MS or celiac disease), as well as methods utilizing the nanoparticles. In the present invention, a tolerogenic antigen is an antigen identified as being involved in an autoimmune disease (e.g., MS or celiac disease). In some embodiments, the tolerogenic antigen is about 3 amino acids to about 50 amino acids in length (e.g., 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 in length). In some embodiments, the tolerogenic antigen is a single tolerogenic antigen that is about 3 amino acids to about 50 amino acids in length.

[0190] In celiac disease, the primary antigens are tissue transglutaminase and gliadin (e.g., α-, γ-, ω-gliadins). Any antigen identified as a tissue transglutaminase or gliadin antigen may be used.

[0191] In some embodiments, the antigen associated with the nanoparticle 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 nanoparticle comprises a 33mer polypeptide from α2-gliadin. In some embodiments, the 33mer 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 nanoparticle comprises an epitope of a 33mer gliadin polypeptide. The epitope of a 33mer gliadin polypeptide may be any length polypeptide shorter than the 33mer polypeptide. For example, the epitope may comprise 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 acids in length. Further examples of epitopes of 33-gliadin that may be associated with the nanoparticles include any one of the epitopes listed in Table 3, including SEQ ID NOs: 375-405. In some embodiments, the tolerogenic antigens associated with the nanoparticles may include any one of the antigens listed in Table 4, including SEQ ID NOs: 406-580. In some embodiments, the polypeptide sequence has 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 to any one of SEQ ID NOs: 375-580.In some embodiments, the tolerogenic antigens associated with the nanoparticles may include antigens comprising two or more (e.g., 2, 3, 4, 5, or 6) polypeptides having any two of the 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)) tolerogenic antigens per nanoparticle) have the same identity as all other tolerogenic antigens associated with the nanoparticle. In some embodiments, the multiple tolerogenic antigens associated with the nanoparticles include 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, the nanoparticles may be associated with 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8), 4 to 6 (e.g., 4, 5, or 6), or 3 to 4 different polypeptide antigen sequences. In some embodiments, the nanoparticles may be associated with (i) a first polypeptide population comprising the amino acid sequence of any one of SEQ ID NOs: 406-580 or a biologically active fragment or variant thereof, (ii) a second polypeptide population comprising the amino acid sequence of any one of SEQ ID NOs: 406-580 or a biologically active fragment or variant thereof, and (iii) a third polypeptide population comprising the amino acid sequence of any one 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, the 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, the 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, the 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, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 comprise an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 comprise 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 comprise an N-terminal pyroE residue and a C-terminal amide group.In some embodiments, the 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, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 comprise an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 comprise a C-terminal amide group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 comprise an N-terminal acetyl group and a C-terminal amide group. In any of the embodiments described herein, the population of antigens associated with the nanoparticles may be fully or partially deamidated. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal amide group. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise a C-terminal amide group.

[0192] [Table 3]

[0193] [Table 4-1]

[0194] [Table 4-2]

[0195] [Table 4-3]

[0196] In some embodiments, the tolerogenic antigen is a biologically active fragment of SEQ ID NO: 474. In some examples, the biologically active fragment of SEQ ID NO: 474 comprises a polypeptide comprising the sequence of SEQ ID NO: 512. In some examples, the biologically active fragment of SEQ ID NO: 474 comprises a polypeptide comprising the sequence of SEQ ID NO: 580.

[0197] In some examples, the tolerogenic antigen is a biologically active fragment of SEQ ID NO: 475. In some examples, the biologically active fragment of SEQ ID NO: 475 comprises a polypeptide comprising the sequence of SEQ ID NO: 542.

[0198] In some embodiments, the tolerogenic antigen is a biologically active fragment of SEQ ID NO: 476. In some examples, the biologically active fragment of SEQ ID NO: 476 comprises a polypeptide comprising the sequence of SEQ ID NO:563.

[0199] In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 375). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PYPQPELPY (SEQ ID NO: 376). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPELPYPQ (SEQ ID NO: 377). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FRPEQPYPQ (SEQ ID NO: 378). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQSFPEQQ (SEQ ID NO: 379). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence IQPEQPAQL (SEQ ID NO: 380). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPEQPYPQ (SEQ ID NO: 381). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence SQPEQEFPQ (SEQ ID NO: 382). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQEFPQ (SEQ ID NO: 383). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPEQPFPQ (SEQ ID NO: 384). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFCQ (SEQ ID NO: 385). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPFPEQPQ (SEQ ID NO: 386). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 387). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFPW (SEQ ID NO: 388). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFSEQEQPV (SEQ ID NO: 389). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FSQQQESPF (SEQ ID NO: 390).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPIPEQPQ (SEQ ID NO: 391). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFPQ (SEQ ID NO: 392). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 393). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQ (SEQ ID NO: 394). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFPQ (SEQ ID NO: 395). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PYPEQEEPF (SEQ ID NO: 396). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PYPEQEQPF (SEQ ID NO: 397). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFSEQEQPV (SEQ ID NO: 398). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EGSFQPSQE (SEQ ID NO: 399). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPQQPFPQ (SEQ ID NO: 400). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPQQPYPE (SEQ ID NO: 401). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QGYYPTSPQ (SEQ ID NO: 402). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EGSFQPSQE (SEQ ID NO: 403). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQSFPEQE (SEQ ID NO: 404). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QGYYPTSPQ (SEQ ID NO: 405). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFPW (SEQ ID NO: 406). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPIPV (SEQ ID NO: 407).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFPW (SEQ ID NO: 408). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPIPV (SEQ ID NO: 409). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPFPQ (SEQ ID NO: 410). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LPYPQPQLPYPQ (SEQ ID NO: 411). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LPYPQPELPYPQ (SEQ ID NO: 412). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQLPYPQ (SEQ ID NO: 413). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYPQ (SEQ ID NO: 414). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFSQ (SEQ ID NO: 415). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFSQ (SEQ ID NO: 416). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFCQ (SEQ ID NO: 417). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFCQ (SEQ ID NO: 418). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQLPYSQ (SEQ ID NO: 419). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYSQ (SEQ ID NO: 420). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQQQCSPVAMPQRLAR (SEQ ID NO: 421). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQLPYLQ (SEQ ID NO: 422). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYLQ (SEQ ID NO: 423).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQFIQPQQPFPQ (SEQ ID NO: 424). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQFIQPEQPFPQ (SEQ ID NO: 425). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LERPWQQQPLPP (SEQ ID NO: 426). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LERPWQEQPLPP (SEQ ID NO: 427). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPQQPEQPFPL (SEQ ID NO: 428). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QGQQGYYPISPQQSGQ (SEQ ID NO: 429). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QGQPGYYPTSPQQIGQ (SEQ ID NO: 430). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PGQGQSGYYPTSPQQS (SEQ ID NO: 431). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQTFPQQPQLP (SEQ ID NO: 432). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQTFPEQPQLP (SEQ ID NO: 433). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence GQGQSGYYPTSPQQSG (SEQ ID NO: 434). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QYEVIRSLVLRTLPNM (SEQ ID NO: 435). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QVDPSGQVQWPQ (SEQ ID NO: 436). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QVDPSGEVQWPQ (SEQ ID NO: 437). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFPL (SEQ ID NO: 438). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFPL (SEQ ID NO: 439).The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPIPY (SEQ ID NO: 440). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPIPY (SEQ ID NO: 441). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPVPQQPQPY (SEQ ID NO: 442). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPVPEQPQPY (SEQ ID NO: 443). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPFPQQPIPQQPQPY (SEQ ID NO: 444). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPIPQQPQPY (SEQ ID NO: 445). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPIPEQPQPY (SEQ ID NO: 446). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQFPQPQQPFPQ (SEQ ID NO: 447). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQFPQPEQPFPQ (SEQ ID NO: 448). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPIPQQPQPYPQQP (SEQ ID NO: 449). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPFPQQPFPQQPQPY (SEQ ID NO: 450). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFSW (SEQ ID NO: 451). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFSW (SEQ ID NO: 452). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPQQPQPYPQQP (SEQ ID NO: 453). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPIPQ (SEQ ID NO: 454). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPIPQ (SEQ ID NO: 455).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPFPQ (SEQ ID NO: 456). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFPQ (SEQ ID NO: 457). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQPTPI (SEQ ID NO: 458). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPTPI (SEQ ID NO: 459). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PAPIQPQQPFPQ (SEQ ID NO: 460). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PAPIQPEQPFPQ (SEQ ID NO: 461). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPQQPEQI (SEQ ID NO: 462). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPEQPEQI (SEQ ID NO: 463). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPQQPQQI (SEQ ID NO: 464). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPEQPQQI (SEQ ID NO: 465). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 466). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 467). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: No. 468). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: 469). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPQQQLPL (SEQ ID NO: 470). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQQLPL (SEQ ID NO: 471). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LFPLPQQPFPQ (SEQ ID NO: 472). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LFPLPEQPFPQ (SEQ ID NO: 473). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 479). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPFLPQLPYPQ (SEQ ID NO: 480). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QAFPQPQQTFPH (SEQ ID NO: 481). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence TPIQPQQPFPQ (SEQ ID NO: 482). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 483). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFTQPQQPTPI (SEQ ID NO: 484).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQLQQPQQP (SEQ ID NO: 485). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence VAHAIIMHQQQQQQQE (SEQ ID NO: 486). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence SYPVQPQQPFPQ (SEQ ID NO: 487). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQQPQPFPQQPVPQQP (SEQ ID NO: 488). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPWQPQQPFPQ (SEQ ID NO: 489). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 490). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPFQPQQPFPQ (SEQ ID NO: 491). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence NPLQPQQPFPLQPQPP (SEQ ID NO: 492). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PLQPQQPFPLQPQPPQ (SEQ ID NO: 493). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PNPLQPQQPFPLQ (SEQ ID NO: 494). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence TIPQQPQQPFPL (SEQ ID NO: 495). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence SFSQQPQQPFPL (SEQ ID NO: 496). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence SFSEQPQQPFPL (SEQ ID NO: 497). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence YSPYQPQQPFPQ (SEQ ID NO: 498). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QLPLQPQQPFPQ (SEQ ID NO: 499). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPQQPFPLQPQQPVP (SEQ ID NO: 500).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence IIPQQPQQPFPL (SEQ ID NO:501). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQIIPQQPQQP (SEQ ID NO:502). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FLLQPQQPFSQ (SEQ ID NO:503). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence IISQQPQQPFPL (SEQ ID NO:504). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQRPQQPFPQ (SEQ ID NO:505). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO:506). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO:507). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PELP (SEQ ID NO: 512). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPELPYP (SEQ ID NO: 513). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPELPY (SEQ ID NO: 514). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELP (SEQ ID NO: 515). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PELPYPQP (SEQ ID NO:516).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPELPYPQ (SEQ ID NO:517). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPELPYP (SEQ ID NO:518). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELPY (SEQ ID NO:519). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELP (SEQ ID NO:520). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PELPYPQPQ (SEQ ID NO:521). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPELPYPQP (SEQ ID NO:522). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELPYP (SEQ ID NO:523). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO:524). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELP (SEQ ID NO: 525). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPELPYPQPQ (SEQ ID NO: 526). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPELPYPQP (SEQ ID NO: 527). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELPYPQ (SEQ ID NO: 528). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPYP (SEQ ID NO: 529). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPY (SEQ ID NO: 530). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELP (SEQ ID NO: 531). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPELPYPQPQ (SEQ ID NO: 532). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELPYPQP (SEQ ID NO:533).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPYPQ (SEQ ID NO:534). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYP (SEQ ID NO:535). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELPY (SEQ ID NO:536). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPELPYPQPQ (SEQ ID NO:537). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPYPQP (SEQ ID NO:538). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELPYP (SEQ ID NO:539). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPELPYPQPQ (SEQ ID NO:540). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELPYPQ (SEQ ID NO:541). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPEQPF (SEQ ID NO: 542). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPEQPFP (SEQ ID NO: 543). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPF (SEQ ID NO: 544). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPEQPFPW (SEQ ID NO: 545). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFP (SEQ ID NO: 546). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPEQPF (SEQ ID NO: 547). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPEQPFPWQ (SEQ ID NO: 548). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPEQPFP (SEQ ID NO: 549). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPEQPFPWQP (SEQ ID NO:550).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFPWQ (SEQ ID NO:551). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPEQPFPW (SEQ ID NO:552). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPFP (SEQ ID NO:553). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPF (SEQ ID NO:554). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PQPEQPFPWQP (SEQ ID NO:555). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPEQPFPWQ (SEQ ID NO:556). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPFPW (SEQ ID NO:557). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFP (SEQ ID NO:558). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence FPQPEQPFPWQP (SEQ ID NO: 559). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPFPWQ (SEQ ID NO: 560). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PFPQPEQPFPWQP (SEQ ID NO: 561). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPEQPFPWQ (SEQ ID NO: 562). In some embodiments. In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQ (SEQ ID NO: 563). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQP (SEQ ID NO: 564). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQP (SEQ ID NO: 565). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQP (SEQ ID NO: 566). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQPYP (SEQ ID NO: 567). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQPY (SEQ ID NO: 568). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQP (SEQ ID NO: 569). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQ (SEQ ID NO: 570). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQPYPQQ (SEQ ID NO: 571). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQPYPQ (SEQ ID NO: 572). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQPYP (SEQ ID NO: 573). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQPY (SEQ ID NO: 574). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQPYPQQ (SEQ ID NO: 575). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQPYPQ (SEQ ID NO: 576). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQPYP (SEQ ID NO: 577). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQPYPQQ (SEQ ID NO:578).In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQ (SEQ ID NO: 579). The tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PDLP (SEQ ID NO: 580). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PELPYPQ (SEQ ID NO: 581). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYPQP (SEQ ID NO: 582). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPFPQPELPYPQPQ (SEQ ID NO: 583). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence LQPFPQPELPYPQP (SEQ ID NO: 584). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PIPEQPQPYPQ (SEQ ID NO: 585). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence QPIPEQPQPYP (SEQ ID NO: 586). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence EQPIPEQPQPY (SEQ ID NO: 587). In some embodiments, the tolerogenic antigen comprises a polypeptide comprising the amino acid sequence PEQPIPEQPQP (SEQ ID NO: 588).

[0200] In some embodiments, the tolerogenic antigen comprises a human allograft transplantation antigen, examples of which include, but are not limited to, subunits of various MHC class I and MHC class II haplotype proteins, and single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Duffy, and Ss.

[0201] In some embodiments, the tolerogenic antigen is an autoantigen to which a subject (e.g., a human patient) has developed or may develop an autoimmune response. 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 refers to a variety of autoimmune diseases, where the protein or proteins causing the disease are known or can be established by routine testing. Embodiments include testing patients to identify the autoimmune protein, generating antigens for use in molecular fusion, and generating immune tolerance to the protein. Embodiments include the antigen or selecting the antigen from one or more of the following proteins: In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid 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, hepatocellular-entero-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regenerative gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptor 1–5. In autoimmune diseases of the thyroid gland, including Hashimoto's thyroiditis and Graves' disease, the primary antigens include thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin receptor (TSHR); other antigens include sodium-iodide symporter (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 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 factors (PGSF) 1a and 2; other antigens are iodothyronine deiodinase type 2. 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-mediated gastritis, the main antigen is H. + ,K +-ATPase. In pernicious anemia, the primary antigen is intrinsic factor. In celiac disease, the primary antigens are tissue transglutaminase and gliadin. In vitiligo, the primary antigens are tyrosinase and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the primary antigen is the acetylcholine receptor. In pemphigus vulgaris and variants, the primary antigens are desmogleins 3, 1, and 4; other antigens include pemfaxin, desmocollin, plakoglobin, pelplakin, desmoplakin, and the acetylcholine receptor. In bullous pemphigoid, the primary antigens include BP180 and BP230; other antigens include plectin and laminin 5. In Dühring's dermatitis herpetiformis, the primary antigens include endomysial and tissue transglutaminase. In epidermolysis bullosa acquisita, the primary 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 PDGF receptor; other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase 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's syndrome, the main antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the main antigen is cardiac myosin.Other autoantigens defined in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and the type 1 interferons interferon α, β, and ω.

[0202] In some cases, the tolerogenic antigen is a foreign antigen against which the patient has developed an unwanted autoimmune response. An example is a food antigen. An embodiment includes testing the patient to identify the foreign 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 are from peanut: conaratin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apple: 31 kDa major allergen / disease resistance protein homologue (Mal d 2), lipid transfer 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, thaumatin-like protein (Act d 2), kiwellin (Act d 5); from mustard: 2S globulin (Sin a 1), 11S globulin (Sin a 2), lipid transfer 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-gliadins, hordein, secalin, avenin; from strawberry: major strawberry allergen Fra a 1-E (Fra a 1), from banana: profilin (Mus xp 1). In some embodiments, the tolerogenic antigen is an antigenic peptide of any one of SEQ ID NOs: 589-742 (Table 5).

[0203] [Table 5-1]

[0204] [Table 5-2]

[0205] [Table 5-3]

[0206] [Table 5-4]

[0207] [Table 5-5]

[0208] [Table 5-6]

[0209] [Table 5-7]

[0210] [Table 5-8]

[0211] [Table 5-9]

[0212] In some instances, the autoimmune disease is type 1 diabetes and the tolerogenic antigen is derived from carboxypeptidase H, chromagranin A, glutamic acid decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-associated glucose-6-phosphatase catalytic subunit-related protein (IGRP), islet beta cell antigen, or proinsulin.

[0213] In some instances, the autoimmune disease is MS and the tolerogenic antigen is derived from alpha-enolase, aquaporin-4, beta-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, or S100-beta.

[0214] In some instances, the autoimmune disease is rheumatoid arthritis and the tolerogenic antigen is derived from citrullinated proteins, collagen II, heat shock proteins, gpl30-RAPS, or human cartilage glycoprotein 39.

[0215] In some instances, the autoimmune disease is systemic lupus erythematosus and the tolerogenic antigen is derived from La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, glycoprotein gp70, or Sm antigen of the U-1 small ribonucleoprotein complex.

[0216] In some instances, the autoimmune disease is scleroderma and the tolerogenic antigen is derived from fibrillarin or a small nucleolar protein (snoRNP).

[0217] In some embodiments, the autoimmune disease is Graves' disease and the tolerogenic antigen is derived from the thyroid stimulating factor receptor (TSH-R).

[0218] In some instances, the autoimmune disease is biliary cirrhosis and the tolerogenic antigen is derived from pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2).

[0219] In some embodiments, the autoimmune disease is alopecia areata and the tolerogenic antigen is derived from a hair follicle antigen.

[0220] In some instances, the autoimmune disease is ulcerative colitis and the tolerogenic antigen is derived from human tropomyosin isoform 5 (hTM5).

[0221] In some instances, the tolerogenic antigen is 17-hydroxylase, 21-hydroxylase, ADAMTS13, annexin A5, apoH, AQP4, aromatic acid 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, glutamic acid decarboxylase, glycoprotein gpIIb-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-associated protein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated 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, synaptogagumin, thyroglobulin, thyroid peroxidase, tissue transglutaminase, TNF-α, topoisomerase, transglutaminase, collagen XVII, U1-RNP, voltage-dependent calcium channel, Yo, ZnT8, β2 glycoprotein I, or β2 glycoprotein I.

[0222] 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 (LYLVCGERG (SEQ ID NO: 751)), as well as the peptides and proteins disclosed in U.S. Patent Application Publication No. 20050202032.

[0223] In certain embodiments, the peptide antigen used to treat type I 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 (SEQ ID NO: 755); IA-2 172-180 , SLSPLQAEL (SEQ ID NO: 756); IA-2 482-490 , SLAAGVKLL (SEQ ID NO:757); IA-2 805-813 , VIVMLTPLV (SEQ ID NO:758); ppIAPP 5-13 , KLQVFLIVL (SEQ ID NO: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 (SEQ ID NO:763); IGRP 222-230 , YLLLRVLNI (sequence number 764); IGRP 228-236 , LNIDLLWSV (sequence number 744); IGRP 265-273, VLFGLGFAI (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 (SEQ ID NO: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 , SLYQLENYC (SEQ ID NO: 783), or a combination thereof.

[0224] In a further embodiment, tolerogenic antigens associated with multiple sclerosis (MS) are used and may include: MAG 287-295 , SLLLELEEV (SEQ ID NO: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 (sequence number 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.

[0225] In some examples, tolerogenic antigens associated with systemic lupus erythematosus may be used, including, but not limited to, FIEWNKLRFRQGLEW (SEQ ID NO: 796). In some examples, tolerogenic antigens comprising a polypeptide having the sequence of SEQ ID NO: 796 include at least one amino acid moiety that is a D amino acid.

[0226] (Multimeric tolerogenic antigens) In certain embodiments, the tolerogenic antigen provided herein is a multimeric tolerogenic antigen. 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 wherein P1, P2, P3, and P4 are each independently selected from any tolerogenic antigen described herein (e.g., any of the tolerogenic antigens in Tables 3-5); L1, L3, and L4 are each independently a linker; and n1, n3, and n4 are each independently 0 or 1, and at least one of n1, n3, and n4 is 1.

[0227] In some examples, n1 is 1, n3 is 0, and n4 is 0, and the tolerogenic antigen comprises the following N-terminal-C-terminal structure: P2-L1-P1

[0228] In some examples, the peptide linker comprises 2-200 amino acids (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 135-150), or 145-200 amino acids (e.g., 145-160, 155-170, 165-180, 175-190, or 185-200). In some embodiments, the peptide linker comprises glycine (Gly) and serine (Ser) amino acids. In some examples, the peptide linker comprises GS (Gly) and GS (Ser) amino acids. x , (GGS) x , (GGGGS (sequence number 797)) x , (GGSG) x , (SGGG) x wherein x is an integer from 1 to 10. In certain embodiments, the linker comprises the amino acid sequence of (GGGGS (SEQ ID NO: 797)) x wherein x is an integer from 2 to 5. In some examples, P2 and P1 are different tolerogenic antigens. In some examples, P2 and P1 are the same tolerogenic antigen.

[0229] In some examples, n1 is 1, n3 is 1, and n4 is 0, and the tolerogenic antigen comprises the following N-terminal-C-terminal structure: P3-L3-P2-L1-P1

[0230] In some examples, the peptide linkers each independently comprise 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 linkers comprise glycine (Gly) and serine (Ser) amino acids. In some examples, the peptide linkers comprise GS (Ser) amino acids. x , (GGS) x , (GGGGS (sequence number 797)) x , (GGSG) x , (SGGG) x wherein x is an integer from 1 to 10. In certain embodiments, the linker comprises the amino acid sequence of (GGGGS (SEQ ID NO: 797)) x wherein 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.

[0231] In some examples, n1 is 1, n3 is 1, and n4 is 1, and the tolerogenic antigen comprises the following N-terminal to C-terminal structure: P4-L4-P3-L3-P2-L1-P1

[0232] In some examples, the peptide linkers each independently comprise 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 linkers comprise glycine (Gly) and serine (Ser) amino acids. In some examples, the peptide linkers comprise GS (Ser) amino acids. x , (GGS) x , (GGGGS (sequence number 797)) x , (GGSG) x , (SGGG) x wherein x is an integer from 1 to 10. In certain embodiments, the linker comprises the amino acid sequence of (GGGGS (SEQ ID NO: 797)) x wherein x is an integer from 2 to 5. In some examples, P4, P3, P2 and / or P1 are different tolerogenic antigens. In some examples, P4, P3, P2 and / or P1 are the same tolerogenic antigen.

[0233] In some embodiments, the tolerogenic antigen is associated with the nanoparticle phospholipid in a manner that promotes strong 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)).

[0234] In some embodiments, the tolerogenic antigen is bound to the nanoparticle phospholipid via a thiol-reactive and reduction-insensitive bond between the tolerogenic antigen and the nanoparticle phospholipid. In fact, the thiol-reactive and reduction-insensitive bond between the tolerogenic antigen and the nanoparticle phospholipid promotes strong immune tolerance. In some embodiments, for example, the phospholipid is N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine.

[0235] In some embodiments, the tolerogenic antigen is attached to the nanoparticle phospholipid via an amine-mediated interaction. 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-collapsing linkage. Self-collapsing linkages include linkers comprising o-dithiobenzyl, p-dithiobenzyl, β-dithiobenzyl carbamate moieties, 2,2-dimethyl-4-mercaptobutyric acid, or disulfide-carbonate-based traceless linkers.

[0236] In some embodiments, the number of tolerogenic antigens associated with a particular nanoparticle is any amount that promotes strong 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).

[0237] Tolerogenic antigens can be prepared by many techniques known in the art, depending on the nature of the molecule.Polynucleotide, polypeptide, and carbohydrate antigens can be isolated from the cells of the type of subject to be treated in which they are enriched.Short peptides are conveniently prepared by amino acid synthesis.Proteins longer than known sequences can be prepared by synthesizing the coding sequence, or by PCR amplifying the coding sequence from natural sources or vectors, and then expressing the coding sequence in suitable bacterial or eukaryotic host cells.

[0238] In some embodiments of the compositions described herein, the nanoparticles having multiple tolerogenic antigens comprise a linker between the tolerogenic antigen and the nanoparticle. 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 are modified with a terminal cysteine ​​residue attached to a linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen are modified with a terminal C(S) npolypeptide, n is 1-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) serine residues, and the terminal serine residue is attached to a linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen are modified by a terminal CSS polypeptide attached 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 tolerogenic antigen are modified by a terminal cysteine ​​residue attached to a maleimide linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen are modified by a terminal cysteine ​​residue attached to a pyridyl linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen are modified by a terminal CSS polypeptide attached to a maleimide linker. In some embodiments, the N-terminus and / or C-terminus of the tolerogenic antigen are modified by a terminal CSS polypeptide attached to a pyridyl linker. The linker may be attached at a first end to a modified nucleoside or nucleotide (eg, Cys and Ser) on a nucleobase or sugar moiety and at a second end to a payload (eg, a lipid (eg, a phospholipid)).

[0239] 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 of sufficient length so as not to interfere with the polypeptide sequence or the lipid moiety. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl groups, each of which may be optionally substituted. The linker may include, for example, a synthetic group derived from a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker may include one or more amino acid residues (e.g., D- or L-amino acid residues). Further examples of useful linkers include linkers that include electrophiles. Examples of electrophiles include Michael addition agents (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, and are suitable for reacting with nucleophilic substituents present in antibodies, antigen-binding fragments, proteins, peptides, and small molecules (e.g., amine and thiol moieties).

[0240] In the present invention, the linker between the multimeric tolerogenic antigens (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), or a polypeptide comprising 145-200 amino acids (e.g., 145-160, 155-170, 165-180, 175-190, or 185-200). In some embodiments, the linker between the tolerogenic antigens (e.g., L1, L3, and / or L4) of the multimer is a polypeptide comprising 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- In some embodiments, the linker between the tolerogenic antigens (e.g., L1, L3, and / or L4) of the multimer 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).

[0241] Suitable peptide linkers are known in the art and include, for example, peptide linkers that include flexible amino acid residues such as glycine and serine. In certain embodiments, the linker may include a GS, GGS, GGGGS (SEQ ID NO: 797), GGSG (SEQ ID NO: 798), or SGGG (SEQ ID NO: 799) motif (e.g., multiple or repeated motifs). In certain embodiments, the linker may include 2-12 amino acids that include a 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 include 3-12 amino acids that include a GGS motif, for example, GGS, GGSGGS (SEQ ID NO: 805), GGSGGSGGS (SEQ ID NO: 806), and GGSGGSGGSGGS (SEQ ID NO: 807). In yet other embodiments, the linker may comprise 4 to 12 amino acids including a GGSG (SEQ ID NO: 808) motif, e.g., GGSGGGSG (SEQ ID NO: 809), or GGSGGGSGGGSG (SEQ ID NO: 810). In other embodiments, the linker may comprise a GGGGS (SEQ ID NO: 797) motif, e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 811). In certain embodiments, the linker is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 812).

[0242] In a preferred embodiment, the peptide linker (e.g., L1, L3, and / or L4) is a peptide linker comprising any one of the amino acid sequences (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, and (SGGG)x, where x is an integer of 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 comprises the amino acid sequence (GGGGS) x wherein x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0243] In some embodiments, the peptide linker comprises at least four glycine residues (e.g., 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 In some embodiments, the linker has 4-30 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 that includes only glycine residues can, for example, be unglycosylated (e.g., O-linked glycosylation, also referred to as O-glycosylation) or can be less glycosylated (e.g., less O-glycosylated) than, for example, a linker that includes one or more serine residues (e.g., less O-glycosylated with glycans such as xylose, mannose, sialic acid, fucose (Fuc), and / or galactose (Gal) (e.g., xylose).

[0244] In some embodiments, a linker that includes only glycine residues may, for example, be not O-glycosylated (e.g., O-xylosylated) or may be less O-glycosylated (e.g., less O-xylosylated) than, for example, a linker that includes one or more serine residues.

[0245] In some embodiments, a linker that includes only glycine residues may not undergo proteolysis or may have a reduced rate of proteolysis compared to, for example, a linker that includes one or more serine residues.

[0246] In certain embodiments, the linker can include a GGGG (SEQ ID NO: 813) motif, e.g., GGGGGGGG (SEQ ID NO: 814), GGGGGGGGGGGG (SEQ ID NO: 815), GGGGGGGGGGGGGGGG (SEQ ID NO: 816), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 817). In certain embodiments, the linker can include a GGGGG (SEQ ID NO: 818) motif, e.g., GGGGGGGGGG (SEQ ID NO: 819), GGGGGGGGGGGGGGGG (SEQ ID NO: 820), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 821). In certain embodiments, the linker is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 822).

[0247] In other embodiments, the linker can also contain amino acids other than glycine and serine, such as GENLYFQSGG (SEQ ID NO: 823), SACYCELS (SEQ ID NO: 824), RSIAT (SEQ ID NO: 825), RPACKIPNDLKQKVMNH (SEQ ID NO: 826), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 827), AAANSSIDLISVPVDSR (SEQ ID NO: 828), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 829).

[0248] [Tolerogenic antigen variants] In certain embodiments, amino acid sequence variants of the tolerogenic antigens of the present invention are contemplated. For example, it may be desired to improve the tolerogenicity and / or other biological properties of the tolerogenic antigen. The amino acid sequence variants of the tolerogenic antigen may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the tolerogenic antigen or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion and / or substitution of, residues in the amino acid sequence of the tolerogenic antigen. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics, for example, inducing antigen tolerance.

[0249] In certain embodiments, tolerogenic antigen variants are provided that have one or more amino acid substitutions. Conservative substitutions are provided in Table 6 under the heading of "preferred substitutions". More substantial changes are provided in Table 6 under the heading of "exemplary substitutions" and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the tolerogenic antigen of interest and screened for a desired activity, e.g., to retain / improve tolerogenic antigenicity.

[0250] [Table 6]

[0251] 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) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0252] Non-conservative substitutions involve exchanging a member of one of these classes for a different class.

[0253] A useful method for identifying residues or regions of a tolerogenic antigen that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Tolerogenic antigen variants may be screened to determine whether they contain the desired properties.

[0254] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0255] In some embodiments, the tolerogenic antigen comprises an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises a pyroglutamic acid residue at the N-terminus. In some embodiments, the tolerogenic antigen comprises an acetyl group at the N-terminus. In some embodiments, the tolerogenic antigen comprises a pyroglutamic acid residue at the N-terminus and an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises an acetyl group at the N-terminus and an amide group at the C-terminus. In certain embodiments, the tolerogenic antigen comprises an N-terminus or a C-terminus modified with a cysteine ​​residue attached to a linker. In some embodiments, the tolerogenic antigen comprises an N-terminus and a C-terminus modified with a cysteine ​​residue attached to a linker.

[0256] In some embodiments of any one of the compositions described herein, the population of tolerogenic antigens is associated with the nanoparticle phospholipid 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, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated ophthalmopathy and skin disorders, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary disease, immune-mediated gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, and the like. Herpetitis, Dermatitis Herpetiformis, Epidermolysis bullosa acquisita, Systemic sclerosis, Mixed connective tissue disease, Sjögren's syndrome, Systemic lupus erythematosus, Good-Basture's syndrome, Rheumatic heart disease, Autoimmune polyglandular syndrome type 1, Aicardi-Gautieres syndrome, Acute pancreatitis Age-related macular degeneration, Alcoholic liver disease, Liver fibrosis, Metastases, Myocardial infarction, Nonalcoholic steatohepatitis (NASH), Parkinson's disease, Polyarthritis / Fetal and Neonatal Anemia, Sepsis, or Inflammatory Bowel Disease.

[0257] In some embodiments, the multiple tolerogenic antigens are bound to the nanoparticle phospholipid via thiol-reactive and reduction-insensitive bonds between each tolerogenic antigen and the nanoparticle phospholipid. In fact, the thiol-reactive and reduction-insensitive bonds between the tolerogenic antigen and the nanoparticle phospholipid promote strong immune tolerance. In some embodiments, the phospholipid is N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine.

[0258] In some embodiments, the tolerogenic antigen is conjugated to the nanoparticle phospholipid via an amine-mediated interaction (e.g., N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl (DOPE-NHS)). In some embodiments, the amine-mediated interaction is 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-immolative linkage. Self-immolative linkages include linkers comprising o-dithiobenzyl, p-dithiobenzyl, β-dithiobenzyl carbamate moieties, 2,2-dimethyl-4-mercaptobutyric acid, or disulfide-carbonate-based traceless linkers.

[0259] [Characteristics of nanoparticles] The nanoparticles of the invention may be characterized for size and uniformity by any suitable analytical technique, including atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, LC-MS / MS, 13These analytical methods include, but are not limited to, C 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 gating electrophoresis. These analytical methods ensure the homogeneity of the sHDL nanoparticle population and are important in production quality control for eventual use in in vivo applications.

[0260] 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, particle size distribution and zeta potential are determined by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0261] [How to use] In certain embodiments, the present invention provides a method for expanding regulatory Tregs (e.g., CD4 Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.), administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, an IL-2 mutein, an IL-2 variant, or IL-2 / IC)) capable of expanding Tregs in the subject. + CD25 high Foxp3 + In some embodiments, a composition comprising one or more nanoparticles is associated with (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled with) an mTOR inhibitor (e.g., rapamycin or a variant thereof).

[0262] In certain embodiments, the present invention provides a method for the proliferation and proliferation of antigen-specific regulatory Tregs (e.g., CD4+ Tregs) in a subject, comprising administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year later, etc.) administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, an IL-2 mutein, an IL-2 variant, or IL-2 / IC)) capable of expanding antigen-specific Tregs. + CD25 high Foxp3 + The present invention provides a method for in vivo amplification of one or more tolerogenic antigens, wherein "antigen-specific" is specific for one or more tolerogenic antigens with which it is associated, including nanoparticles that are associated with one or more tolerogenic antigens.

[0263] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 + The present invention provides a method for promoting strong immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of augmenting an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof).

[0264] In certain embodiments, the present invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + The present invention provides a method for promoting potent immune tolerance to an antigen associated with an autoimmune disorder in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, an IL-2 mutein, an IL-2 variant, or IL-2 / IC)) capable of proliferating an immune response to an autoimmune disorder, wherein "antigen-specific" is specific for the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens.

[0265] In certain embodiments, the present invention provides a method for detecting Tregs (e.g., CD4 T cells) by administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) detecting Tregs (e.g., CD4 T cells) and / or Tregs (e.g., CD4 T cells) in a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder ... + CD25 high Foxp3 + The present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the T cell proliferation and proliferation of the subject. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof).

[0266] In certain embodiments, the present invention provides a method for administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens, and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 + CD25 high Foxp3 + In one embodiment, the present invention provides a method for increasing the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells in a subject, comprising administering to the subject a composition comprising one or more immunomodulatory agents (human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)) capable of expanding the expression of CD3+FOXP3+ cells relative to CD3+FOXP3- cells in a population of T cells in a subject, where "antigen-specific" is specific for the one or more tolerogenic antigens associated therewith, including nanoparticles associated with one or more tolerogenic antigens.

[0267] In certain embodiments, the present invention provides a method for administering a composition comprising one or more nanoparticles to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc. later) producing Tregs (e.g., CD4 + CD25 high Foxp3 +In some embodiments, the present invention provides a method of treating, preventing and / or attenuating one or more autoimmune disorders in a subject, the method comprising administering to the subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating mTOR. In some embodiments, the composition comprising one or more nanoparticles is associated (e.g., complexed, bound, encapsulated, absorbed, adsorbed, intermingled) with an mTOR inhibitor (e.g., rapamycin or a variant thereof). In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific to a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0268] In certain embodiments, the invention provides a method for the production of antigen-specific Tregs (e.g., CD4 T cells) by administering to a subject (e.g., a human subject suffering from or at risk of suffering from an autoimmune disorder, e.g., celiac disease) a composition comprising one or more nanoparticles associated with one or more tolerogenic antigens (e.g., one or more tolerogenic antigens associated with an autoimmune disorder), and then (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) generating antigen-specific Tregs (e.g., CD4 T cells ... + CD25 high Foxp3 + In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders), comprising administering to a subject a composition comprising one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) capable of proliferating the immune system of the subject's immune system, where "antigen-specific" is specific for the one or more tolerogenic antigens with which it is associated, including nanoparticles associated with the one or more tolerogenic antigens. In some embodiments, the treatment, prevention and / or attenuation of one or more autoimmune disorders in a subject is specific for a particular tissue region (e.g., a particular tissue region associated with one or more autoimmune disorders).

[0269] The immune system can be categorized into 101unctionnal 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 significant infection. The adaptive immune system responds to molecular structures, called antigens, of invading organisms. There are two types of adaptive immune responses: humoral and cellular. In humoral immune responses, antibodies secreted into bodily fluids by B cells bind to antigens from pathogens, leading to their elimination through various mechanisms, for example, bacteriolysis via complement. In cellular immune responses, T cells are activated that can destroy other cells. For example, if proteins associated with a disease (e.g., MS or celiac disease) are present in a cell, 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 surface of the cell, where they are presented to the body's molecular defense mechanisms, in particular, T cells. Cytotoxic T cells recognize these antigens and kill the antigen-bearing cells.

[0270] The molecules that transport and present peptides on the cell surface are called proteins of the major histocompatibility complex (MHC), known in humans as the human leukocyte antigen (HLA) complex. MHC proteins are divided into two types, called MHC class I and MHC class II. The structures of the proteins of the two MHC classes are very similar, but they have very different functions. Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells. MHC class I proteins are loaded with antigens, usually derived from endogenous proteins or pathogens present inside the cell, and then presented to I or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. They mainly present peptides that are processed from external antigen sources, i.e., from outside the cell, to T helper (Th) cells. Most of the peptides bound by MHC class I proteins are derived from cytoplasmic proteins produced in the organism's own healthy host cells and do not usually stimulate an immune response. Thus, cytotoxic T lymphocytes that recognize such class I self-peptide-presenting MHC molecules are either deleted in the thymus (central tolerance) or deleted or inactivated, i.e., tolerized, after release from the thymus (peripheral tolerance). MHC molecules can stimulate an immune response when they present peptides to non-tolerized T lymphocytes. Cytotoxic T lymphocytes have both T cell receptors (TCRs) and CD8 molecules on their surface. The T cell receptors can recognize and bind peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind a specific MHC / peptide complex.

[0271] Experiments conducted during the course of developing embodiments of the present invention have led to the development of a new strategy for inducing high frequencies of antigen-specific Tregs in vivo without ex vivo cell manipulation. As a result of the above experiments, a new strategy was developed and optimized to combine nanodiscs with modified IL-2 to induce unprecedented amounts of antigen-specific Tregs in vivo. In particular, IL-2:anti-IL-2 antibody (clone: ​​JES6-1) immune complexes (IL-2 / IC) have been shown to selectively induce polyclonal Tregs

[14] . Administration of IL-2 / IC with free peptide or peptide-tetramers has been reported to induce antigen-specific Tregs [15,16]. However, these aforementioned attempts have resulted in rather poor antigen-specific Treg responses, with the antigen-specific Treg frequency in the CD4+ T cell compartment being less than 0.25% [15,16].

[0272] It was hypothesized that appropriate delivery of peptide antigens to lymphoid tissues is important to maximize antigen-specific Treg induction with IL-2 / IC. In the experiments described herein, we report for the first time a significant expansion of antigen-specific Tregs by combining lymphatic-targeted Nanodisc-mediated delivery of peptides with IL-2 / IC therapy compared to IL-2 / IC alone.

[0273] It was also assumed that the actual treatment method was important. The above experiments showed that Nanodiscs should be administered first (e.g. subcutaneously), followed by systemic administration of IL-2 and / or mutein / engineered IL-2. This ensured that antigen-specific Tregs were primed and generated first, and the subsequent administration of mutein / engineered IL-2 caused robust proliferation of antigen-specific Tregs. For long-term maintenance of antigen-specific Tregs, administration of a maintenance dose of Nanodiscs and / or mutein / engineered IL-2 was further assumed.

[0274] The methods are not limited to the treatment of any particular disorder.

[0275] In some embodiments, the disorder is an autoimmune disorder. The method is not limited to treating a particular autoimmune disorder. Examples of autoimmune diseases include multiple sclerosis (MS). Celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), autoimmune diseases of the thyroid gland (Hashimoto's disease, Graves' disease), thyroid-associated eye and 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 variants, bullous pemphigoid, dermatitis herpetiformis of Duhring, acquired These conditions include, but are not limited to, epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjogren's syndrome, systemic lupus erythematosus, Goodpasture's syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Aicardi-Gautieres syndrome, acute pancreatitis age-related macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, nonalcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

[0276] In some embodiments, the disorder is a transplant-related disorder. In some embodiments, the disorder is one or more allergies. In some embodiments, the disorder is a respiratory disease (e.g., asthma). In some embodiments, the disorder is graft-versus-host disease (GvHD).

[0277] In some embodiments, the above methods for treating or preventing an autoimmune disorder further comprise co-administering (eg, simultaneously or at different times) an additional therapeutic agent. Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologics (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 metalloprotease inhibitors. In some embodiments, therapeutic agents include but are not limited to infliximab, adalimumab, etanercept, or parenteral or oral gold.In some instances, the therapeutic agent is an immunomodulatory or immunosuppressive agent (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; inhibitors of mitochondrial function such as rotenone; P38 inhibitors; 6Bio, dexamethasone, TCPA-1, IKK NF-κβ inhibitors such as NF-κB 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 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, cyclosporines such as cycloporine A, aromatic hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic 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;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;amlexanox;Bay11-7082;BX795;CH-223191;chloroquine;CLI-095;CU-CPT9a;cyclosporine A;CTY387;gefitinib;glibenclamide;H-89;H-131;isoliquiritigenin;MCC950;MRT67307;OxPAPC;parthenolide;Pepinh-MYD;Pepinh-TRIF;polymyxin B;R406;RU.521;VX-765;YM201636;Z-VAD-FMK;and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD);tryptamine (TA);and 6-formyl indolo[3,2 b] AHR-specific ligands, including but not limited to carbazole (FICZ).In certain embodiments, the immunosuppressant is fingolimod; rapamycin; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or related ligand; trichostatin A; and / or suberoylanilide hydroxamic acid (SAHA).

[0278] The methods are not limited to the particular mode of administering a composition comprising nanoparticles, with or without association with a tolerogenic antigen, and / or a composition comprising an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)). Indeed, any acceptable method known to one of skill in the art may be used to administer any composition to a subject. Administration may be local (i.e., to a particular area, physiological system, tissue, organ, or cell type) or systemic. The compositions may be administered by a number of routes, including, but not limited to, oral, inhalation (nose or lung), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, subcutaneous, sublingual, or rectal techniques. Injection may be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, injection may be at multiple locations.

[0279] Administration of the formulations can be accomplished by any acceptable method that allows an effective amount of any composition to achieve its desired effect. The particular mode selected will depend on factors such as the particular formulation, the severity of the condition of the subject being treated, and the dosage required to induce an effective immune response. As generally used herein, an "effective amount" refers to an amount that is sufficient to induce an effective immune response in a subject to an antigen-specific regulatory Treg (e.g., CD4 + CD25 high Foxp3 +) in vivo and / or promote strong immune tolerance to antigens associated with autoimmune disorders and / or induce an immune response in the subject being treated. The actual effective amount of any composition may vary according to the particular antigen or combinations utilized, the particular composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the individual being vaccinated.

[0280] Pharmaceutical Compositions Where clinical applications are contemplated, in some embodiments of the invention, compositions comprising nanoparticles, with or without association with a tolerogenic antigen, and / or one or more immunomodulatory agents (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) are prepared as part of a pharmaceutical composition in a form appropriate for the intended application. Generally, this entails preparing 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 invention, straight compositions comprising nanoparticles, with or without association with a tolerogenic antigen, and / or an immunomodulatory agent (e.g., a human cytokine (e.g., IL-2, IL-2 mutein, IL-2 variant, or IL-2 / IC)) may be administered using one or more of the routes described herein.

[0281] In a preferred embodiment, the compositions are used in conjunction with appropriate salts and buffers to effect delivery of the compositions in a stable manner that allows for uptake by target cells. Buffers are also used when any composition is introduced into a patient.

[0282] The aqueous composition comprises an effective amount of sHDL nanoparticles for cells dispersed in a pharma- ceutically acceptable carrier or aqueous medium. Such a composition is also referred to as an inocula.

[0283] The term "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like. Except insofar as any conventional media or agent is incompatible with the vector or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0284] In some embodiments of the invention, active compositions include classical pharmaceutical preparations. Administration of these compositions according to the invention is via any common route, so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal, subcutaneous, or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0285] The active composition may also be administered parenterally or intraperitoneally or intratumorally.The solution of the active compound as a free base or a pharma-ceutically acceptable salt is prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils.Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0286] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of 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 (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it may be preferable to include isotonic agents (for example, sugars or sodium chloride). Prolonged absorption of the injectable composition can be achieved by the use of agents that delay absorption (for example, aluminum monostearate and gelatin) in the composition.

[0287] Any composition is mixed in the required amount in a suitable solvent with various other components as listed above, if necessary, and then sterilized by filtration to prepare sterile injection solution.Generally, dispersion is prepared by mixing various sterilized active ingredients with a sterile medium (basic dispersion medium and other components as listed above).In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active ingredient and any additional desired ingredient from the solution previously sterilized and filtered.

[0288] Upon formulation, any composition is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution is suitably buffered, if necessary, and the diluent is first rendered isotonic with sufficient saline or glucose. These particular 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 at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the invention, the active particles or agents are formulated in the 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, etc. per dose. Multiple doses may be administered.

[0289] Further formulations suitable for other modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may be used. Suppositories are solid dosage forms of various weights and shapes that are usually medicated for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the fluid of the cavity. Generally, suppositories may contain traditional binders and carriers, such as polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Vaginal suppositories or pessaries are usually spherical or ovoid, each weighing about 5 g. Vaginal medications are available in various physical forms (e.g., creams, gels, or liquids), which deviates from the classical concept of a suppository. The composition may also be formulated as an inhalant.

[0290] [kit] In some embodiments, the invention also provides kits that include compositions comprising one or more nanoparticles with or without associated with one or more tolerogenic antigens, and / or compositions comprising one or more immunomodulatory agents described herein (e.g., human cytokines (e.g., IL-2, IL-2 muteins, IL-2 variants, or IL-2 / IC)). In some embodiments, the kits include one or more reagents and tools necessary to produce any of the compositions, as well as methods of using any of the compositions.

[0291] [Example] 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. Use of pronouns such as "we," "our," and "I" refers to the subject of the present invention.

[0292] [Example 1] [Preparation of antigen-loaded nanodiscs] DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. DMPC and ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) powder were mixed and hydrated with 10 mM sodium phosphate buffer (DMPC:22A=2:1, mass ratio), then subjected to cycles of heating and cooling to obtain blank synthetic HDL nanodiscs, which were then sonicated for 5 min at room temperature. To load antigen peptides into blank nanodiscs, the cysteine-terminated antigen peptides were first conjugated with DOPE-MAL (antigen peptide:DOPE-MAL=2:1, molar ratio). DOPE-peptide was then added to the blank nanodiscs (22A:antigen peptide = 5:1, mass ratio) and incubated for 1 h at room temperature with gentle agitation on an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce). The antigen peptide used in this study was OVA-II (OVA323-339) peptide, CSS-ISQAVHAAHAEINEAGR. The peptide loading efficiency was measured by LC-MS. Nanodiscs-OVA-II were analyzed by gel permeation chromatography (GPC) with a TSKgel G3000SWxl column (7.8 mm i.d. × 30 cm, Tosoh Bioscience LLC). The hydrodynamic size and zeta potential of the nanodisc samples were measured by dynamic light scattering (DLS, Zetasizer Nano ZSP).

[0293] [Preparation of mouse IL-2:anti-IL-2 immune complex (IL-2 / IC)] IL-2 / anti-IL-2 mAb immune complexes (IL-2 / IC) were prepared as previously reported

[15] . 0.1 mg of anti-IL-2 mAb (JES6-1A12, InVivoMAb, BioX-Cell, Lebanon, NH) was mixed in vitro with recombinant mouse IL-2 (20 μg, PeproTech, Rocky Hill, NJ) in 2 mL of HBSS (Mediatech, Herndon, VA) and incubated for 10 min at room temperature. The resulting IL-2 / IC was then administered in a single injection dose of 0.1 mL containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb.

[0294] [Animal experiments] Animals were cared for in accordance with federal, state, and local guidelines. All work with animals was in accordance with and approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Michigan, Ann Arbor. Seven-week-old C57BL / 6 female mice were randomly divided into five groups, with five mice in each group. Mice in groups 1, 2, and 3 were injected subcutaneously at the base of the tail with 0.1 mL of nanodisc-OVA-II (containing 0.1 mg of OVA-II peptide) on days 0, 7, and 14. Additionally, group 1 received 0.1 mL of freshly prepared IL-2 / IC solution (above) intraperitoneally (i.p.) on days 1, 2, and 3 after each nanodisc-OVA-II injection. Group 2 received 0.1 mL of freshly prepared IL-2 / IC solution (above) intraperitoneally (i.p.) on days 3, 4, and 5 after each nanodisc-OVA-II injection. Mice in group 3 did not receive IL-2 / IC. Mice in group 4 received IL-2 / IC alone on days 1, 2, and 3 of weeks 0, 1, and 2 (similar to group 1 without nanodisc-OVA-II injection). Group 5 received PBS. Peripheral blood was collected for flow cytometry analysis on days 7, 14, 21, and 28.

[0295] [Flow cytometry analysis] At the indicated time points, 150–200 uL of blood was collected from individual mice into EDTA-coated tubes. For red blood cell lysis, blood volumes were transferred to 1.5 mL Eppendorf tubes. 1 mL of ACK lysis buffer was added to each blood volume to lyse red blood cells, and the tubes were shaken at room temperature for 5 min. After 5 min, the tubes were spun at 600 g for 5 min, and the supernatant was discarded. The pellet was resuspended in 1 mL of ACK lysis buffer and immediately spun at 600 g for 5 min. The supernatant was discarded, and the PBMCs were resuspended in PBS, transferred to a 96-well plate, and washed once with PBS before proceeding with FACS staining. For surface staining, cells were first incubated with eBioscience Fixable Viability Dye eFluor450 in PBS for 10 min at room temperature in the dark. PBMCs were washed with 200 uL of PBS and spun at 600 g for 5 min. The supernatant was discarded and samples were suspended in Fc block (anti-CD16 / 32) in FACS buffer (PBS + 1% BSA). Blocking was allowed to proceed for 10 minutes at room temperature in the dark before OT-II tetramers were added to the wells at a final dilution of 1:40 in FACS buffer. OT-II tetramers (NIH Tetramer Core Facility, Atlanta, GA) were incubated with PBMCs for 1 hour at room temperature in the dark. Twenty minutes before the end of the OT-II tetramer incubation, surface antibodies were added to the samples at a final dilution of 1:100. After tetramer and surface incubation, PBMCs were washed with 200 uL of FACS buffer and spun at 600 g for 5 minutes. For intracellular staining of Foxp3, eBioscienc™ Foxp3 / Transcription Factor Staining Buffer Set was used according to the kit instructions. To measure total cell counts, 50uL of Life Technologies Absolute Counting Beads were added to fully stained PBMC samples before harvesting on a BioRad ZE5 analyzer. FSC files were analyzed using FlowJo.

[0296] [Characterization of antigen-loaded nanodiscs] [Synthesis and characterization of HDL nanodiscs-OVA-II] Blank HDL nanodiscs showed an average diameter of 8.9 ± 2.1 nm (PDI = 0.137), whereas OVA-II-loaded HDL nanodiscs showed an average diameter of 10.4 ± 2.6 nm (PDI = 0.095, Figure 1). The loading efficiency of OVA-II in sHDL nanodiscs was approximately 99% as quantified by LC-MS (Figure 2). In addition, the as-synthesized blank nanodiscs and nanodisc-OVA-II were further characterized by gel permeation chromatography (GPC) analysis, and nanodisc-OVA-II showed a single homogenous peak with a short retention time in GPC analysis (0.8 mL / min, PBS as mobile phase, column: TSKgel G3000SWxl, Tosoh Bioscience).

[0297] [Analysis of antigen-specific T cells in mice] C57BL / 6 mice were administered nanodisc-OVA-II (ND-OVA-II) sc and treated with IL-2 / IC via the ip route using the dosing regimen shown in Figure 4. Control groups included either ND-OVA-II, IL-2 / IC, or PBS treatment only. Systemic immune responses were measured by flow cytometry on days 7 and 14 after the first ND-OVA-II administration.

[0298] By day 7, animals in groups 2 and 4 that received ND-OVA-II in combination with IL-2 / IC or IL-2 / IC on days 3, 4, and 5 (D3, 4, and 5) had significantly increased CD4+ expression in PBMCs. + The animals in group 1, which received ND-OVA-II in combination with IL-2 / IC on days 1, 2, and 3 (D1, 2, and 3), showed an increase in the proportion of T cells (Figure 5 and Figure 6a). + antigen-specific CD4 + The abundance of CD4 T cells was significantly higher in mice in groups 1, 2, and 4 that received ND-OVA-II in combination with IL-2 / IC or IL-2 / IC alone, compared with mice in groups 3 and 5 that were treated with ND-OVA-II or PBS (Fig. 5, Fig. 6b). + CD25 +Foxp3 + Regulatory T cells (T regs ) showed a 5-fold increase in overall proliferation (Figure 6c). Importantly, ND-OVA-II+IL-2 / IC (D1, 2, 3) showed no significant increase in proliferation compared with OT-II tetramers. + CD25 + Foxp3 + T regs The most striking feature was that these OVA-specific T regs is T regs In mice in group 2 treated with ND-OVA-II+IL-2 / IC (D3, 4, 5), the proliferation of antigen-specific Tregs was low (Fig. 6d), but these cells expressed significantly more CD25 Tregs than Tregs induced with ND-OVA-II+IL-2 / IC (D1, 2, 3) or IL-2 / IC alone. + Foxp3 + T regs and OT-II tetramer + CD25 + Foxp3 + T regs (Fig. 6e, f) showed significantly higher CD25 expression overall.

[0299] By day 14, mice administered IL-2 / IC (D1, 2, 3) had increased CD4+ T cells in PBMC compared to other groups (Figure 7, Figure 8a). ND-OVA-II administered in combination with IL-2 / IC on days 3, 4, and 5 (D3, 4, 5), or IL-2 / IC, increased CD4+ T cells in PBMC. + As on day 7, animals treated with ND-OVA-II+IL-2 / IC (D1, 2, 3) showed a further expansion of antigen-specific OT-II tetramer+CD4+T cells, reaching approximately 20% of CD4+T cells (Fig. 7, Fig. 8b). regsOverall, there was a 5-fold and 10-fold increase in ND OVA-II+IL-2 / IC (D1, 2, 3) and ND-OVA-II+IL-2 / IC (D3, 4, 5), respectively, compared with ND-OVA-II and PBS controls (Figure 7, Figure 8c). As on day 7, animals treated with ND-OVA-II+IL-2 / IC (D1, 2, 3) showed increased antigen-specific T cells. regs has shown further proliferation and is now known as T regs Approximately 22% of the total showed positive staining for OT-II tetramer (FIG. 8d).

[0300] In addition to assessing the abundance of cell populations, the day 14 samples were counted by adding Life Technologies Absolute Counting Beads to the flow samples. ND-OVA-II+IL-2 / IC (D3, 4, 5) animals averaged approximately 99,000 CD4 + CD25 + Foxp3 + Cells are counted and regs The total number of antigen-specific T cells was the highest in ND-OVA-II+IL-2 / IC (D1, 2, 3) mice (Fig. 8c). regs The highest number of CD4 + CD25 + Foxp3 + OT-II tetramer + Cells were counted (Fig. 8d). Furthermore, CD25 and GITR expression were analyzed by Treg and OT-II tetramer analysis. + T regs Similar to the data on day 7, ND-OVA-II+IL-2 / IC (D3, 4, 5) treatment significantly reduced the overall and OT-II tetramer levels compared to all other groups. + T regs Among them, the expression of both CD25 and GITR was highest (Fig. 8e, f).

[0301] A summary of these findings is shown in Figure 9. ND-OVA-II+IL-2 / IC (D1, 2, 3) treatment increased the T cell population by approximately 30% of CD4+ T cells in PBMCs on both days 7 and 14. regs ND-OVA-II+IL-2 / IC (D1, 2, 3) treatment increased antigen-specific OT-II tetramer counts in CD4+ T cells in PBMCs on days 7 and 14, respectively. + Foxp3 + CD25 + T regs ND-OVA-II+IL-2 / IC (D1, 2, 3) treatment also significantly increased antigen-specific OT-II tetramer counts in total CD4+ T cells on days 7 and 14, respectively. + Foxp3 - CD25 + T conv increased from about 1% to about 3.3% (Figure 9c).

[0302] On the other hand, ND-OVA-II+IL-2 / IC (D3, 4, 5) treatment increased the T regs The total number of CD4+ T cells in PBMCs increased from approximately 35% on day 7 to approximately 50% on day 14 (FIG. 9a). ND-OVA-II+IL-2 / IC (D3, 4, 5) treatment increased antigen-specific OT-II tetramer counts in CD4+ T cells in PBMCs on days 7 and 14, respectively. + Foxp3 + CD25 + T regs ND-OVA-II+IL-2 / IC (D3, 4, 5) treatment also significantly increased antigen-specific OT-II tetramer counts in total CD4+ T cells on days 7 and 14, respectively. + Foxp3 - CD25 + T conv increased from about 0.1% to about 0.6% (Figure 9c).

[0303] ND-OVA-II+IL-2 / IC (D3, 4, 5) animals showed significantly higher antigen-specific T cell proliferation compared to ND-OVA-II+IL-2 / IC (D1, 2, 3) and IL-2 / IC (D3, 4, 5) only groups. regs The number and percentage of CD4 + T in cells regs Significant overall proliferation was observed (Fig. 9a), as well as high expression of CD25 and GITR (Fig. 6e, f; Fig. 8e, f). Increased expression of CD25 and GITR was consistent with T cells stimulated by ND-OVA-II + IL-2 / IC (D3, 4, 5) treatment. regs This suggests that it may be more functional.

[0304] [Example 2] Tregs are potent mediators of immune tolerance in inflamed tissues. However, how to promote and control Treg migration to specific tissues remains unclear. It was thought that applying Treg alloantigens to local tissues could recruit circulating Tregs to specific tissues. We expect that peptide-MHC-II complexes displayed on antigen-presenting cells in local tissues would trigger T cell responses, which would lead to the secretion of chemokines and recruit Tregs to the tissues. Therefore, this approach could be beneficial to recruit Tregs to local tissues and mediate local immune tolerance.

[0305] Moreover, this approach may be broadly applicable to autoimmune diseases where the autoantigens are yet to be identified. Although the autoantigens in some autoimmune diseases are clearly defined, the autoantigens in many autoimmune diseases are not. Therefore, it would be beneficial to develop an antigen-nonspecific approach to induce Tregs in the general circulation and promote their infiltration into specific tissues or organs.

[0306] In Example 1, we reported that the combination of antigen-loaded nanodiscs and IL-2 / IC treatment led to robust induction of antigen-specific Tregs in the blood circulation. In this Example (Example 2), we show that antigen-specific Tregs in the blood circulation are recruited to the ear dermis tissue to which the antigen is applied. Therefore, it is believed that the combination of antigen-nanodiscs and IL-2 / IC or mutein IL-2 treatment, followed by application of autoantigen (either by injection or topical application), can promote Treg infiltration into specific tissues and organs.

[0307] [Preparation of antigen-loaded nanodiscs] DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. DMPC and ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) powder were mixed and hydrated with 10 mM sodium phosphate buffer (DMPC:22A=2:1, mass ratio), then subjected to cycles of heating and cooling to obtain blank synthetic HDL nanodiscs, which were then sonicated for 5 min at room temperature. To load antigen peptides into blank nanodiscs, the cysteine-terminated antigen peptides were first conjugated with DOPE-MAL (antigen peptide:DOPE-MAL=2:1, molar ratio). DOPE-peptide was then added to the blank nanodiscs (22A:antigen peptide = 5:1, mass ratio) and incubated for 1 h at room temperature with gentle agitation on an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce). The antigen peptide used in this study was OVA-II (OVA323-339) peptide, CSS-ISQAVHAAHAEINEAGR. The peptide loading efficiency was measured by LC-MS. Nanodiscs-OVA-II were analyzed by gel permeation chromatography (GPC) with a TSKgel G3000SWxl column (7.8 mm i.d. × 30 cm, Tosoh Bioscience LLC). The hydrodynamic size and zeta potential of the nanodisc samples were measured by dynamic light scattering (DLS, Zetasizer Nano ZSP).

[0308] [Preparation of mouse IL-2:anti-IL-2 immune complex (IL-2 / IC)] IL-2 / anti-IL-2 mAb immune complexes (IL-2 / IC) were prepared as previously reported [1]. 0.1 mg of anti-IL-2 mAb (JES6-1A12, InVivoMAb, BioX-Cell, Lebanon, NH) was mixed in vitro with recombinant mouse IL-2 (20 μg, PeproTech, Rocky Hill, NJ) in 2 mL of HBSS (Mediatech, Herndon, VA) and incubated for 10 min at room temperature. The resulting IL-2 / IC was then administered in a single injection dose of 0.1 mL containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb.

[0309] [Animal experiments] Animals were cared for in accordance with federal, state, and local guidelines. All work with animals was in accordance with and approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Michigan, Ann Arbor. Seven-week-old C57BL / 6 female mice were randomly divided into three groups, five mice per group. On day 0, mice were injected by retroorbital injection with 2.8 × 10 6 CD4 +They received adoptive cell transfer (ACT) of T cells. Mice in groups 1 and 2 were then injected subcutaneously at the base of the tail with 0.1 mL of nanodisc-OVA-II (containing 0.1 mg of OVA-II peptide) on days 0, 7, and 14. Additionally, group 1 received 0.1 mL of freshly prepared IL-2 / IC solution (above) intraperitoneally (ip) on days 3, 4, and 5 after each nanodisc-OVA-II injection. Group 2 received 0.1 mL of freshly prepared IL-2 / IC solution (above) intraperitoneally (ip) on days 3, 4, and 5 after each nanodisc-OVA-II injection. Mice in group 2 received only ACT+nanodisc-OVA-II treatment, and mice in group 3 received ACT+PBS as a control group. On day 25, mice were anesthetized by isoflurane inhalation and 10 mg of OVA-II peptide or irrelevant MOG35-55 peptide in PBS was administered intradermally to the left and right ears using a 29G needle syringe (BD SAFETYGLIDE 0.5ML insulin syringe 29G x 0.5"). 24 hours later, mice were euthanized and cell suspensions were prepared from the ears. Briefly, ears were removed, divided into dorsal and ventral halves, and cartilage was removed. Skins were cut into small pieces and digested for 90 min at 37°C in a solution of RPMI1640 containing 2% FBS, 5 mg / ml DNaseI (Sigma-Aldrich), and 3 mg / ml collagenase type III (Sigma-Aldrich). The cell suspension was passed through a 70um strainer and washed before flow cytometry analysis.

[0310] [Flow cytometry analysis] For surface staining, whole cells from each ear digest were incubated with eBioscience Fixable Viability Dye eFluor450 in PBS for 10 min at room temperature in the dark. Samples were washed with 200uL of PBS and spun at 600g for 5 min. Supernatant was discarded and cells were suspended in Fc block (anti-CD16 / 32) in FACS buffer (PBS+1% BSA). Blocking was allowed to proceed for 10 min at room temperature in the dark before OT-II tetramers were added to the wells at a final dilution of 1:40 in FACS buffer. OT-II tetramers (NIH Tetramer Core Facility, Atlanta, GA) were incubated with PBMCs for 1 h at room temperature in the dark. Twenty min before the end of the OT-II tetramer incubation, surface antibodies were added to samples at a final dilution of 1:100. After tetramer and surface incubation, cells were washed with 200uL of FACS buffer and spun at 600g for 5 min. For intracellular staining of Foxp3, eBioscienc™ Foxp3 / Transcription Factor Staining Buffer Set was used according to the kit instructions. To measure total cell number, 25uL of Life Technologies Absolute Counting Beads were added to fully stained samples before harvesting on a BioRad ZE5 analyzer. FSC files were analyzed using FlowJo.

[0311] [Analysis of antigen-specific T cells after antigen challenge in the ear] C57BL / 6 mice were injected with 2.8 × 10 6 CD4 +Mice were administered OVA-II T cells (ACT). One day later, nanodisc-OVA-II (ND-OVA-II) was administered sc and treated with IL-2 / IC via the ip route using the dosing regimen shown in Figure 10. Control groups included either ACT+ND-OVA-II or ACT+PBS treatment. On day 25 after the first ND vaccination, mice were challenged with 10ug of OVA-II peptide intradermally in the right ear or with an irrelevant MOG peptide in the left ear. After 24 hours, both ears were harvested, processed into single cell suspensions, and stained for flow cytometry analysis. In mice treated with ACT+ND-OVA-II+IL-2 / IC (D3, 4, 5), the ear challenged with OVA-II specific peptide had an average of 13,000 total CD4 + In addition, the OVA-II peptide-challenged ears contained 10,000 CD4 T cells in mice treated with either ACT+ND-OVA-II+IL-2 / IC (D3, 4, 5) or ACT+ND-OVA-II, whereas only 4,300 were found in the MOG-challenged ears (Figure ​(Figure11A). 11A). Furthermore, the OVA-II peptide-challenged ears contained 10,000 CD4 T cells in mice treated with either ACT+ND-OVA-II+IL-2 / IC (D3, 4, 5) or ACT+ND-OVA-II, whereas only 4,300 were found in the MOG-challenged ears (Figure ​(Figure11A). 11A). + CD25 + Foxp3 + T regs and antigen-specific OT-II-tetramer + CD4 + CD25 + Foxp3 + T regs In mice treated with ACT+ND-OVA-II+IL-2 / IC (Days 3, 4, and 5), the total number of CD4 + CD25 + Foxp3 + T regs and OT-II tetramer + T regs The number of antigen-specific T cells migrating to ear tissue was increased 3.2- and 3.8-fold, respectively, in OVA-II-treated ears compared to ears treated with the irrelevant MOG peptide (Fig. 11B, C). Compared with ACT+ND-OVA-II or ACT+PBS controls challenged with OVA-II peptide, ACT+ND-OVA-II+IL-2 / IC (D3, 4, 5) mice had significantly fewer antigen-specific T cells migrating to ear tissue.regs The total number of β-terminal ...

[0312] [Example 3] [material and method] [Preparation of antigen-loaded nanodiscs] DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION and Avanti Polar Lipids, respectively. DMPC and ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) powder were mixed and hydrated with 10 mM sodium phosphate buffer (DMPC:22A=2:1, mass ratio), then subjected to cycles of heating and cooling and sonicated for 2 min at room temperature to obtain blank synthetic HDL nanodiscs (NDs). To load antigen peptides into blank nanodiscs, cysteine-terminated antigen peptides were first conjugated with DOPE-MAL (antigen peptide:DOPE-MAL=1.5:1, molar ratio). DOPE-peptide was then added to the blank nanodiscs (22A:antigen peptide = 5:1, mass ratio) and incubated for 1 h at room temperature on an orbital shaker with gentle agitation. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce). The antigen peptides used in this study were OVA-II (OVA323-339) peptide, CSS-ISQAVHAAHAEINEAGR. Peptide loading efficiency was measured by LC-MS.

[0313] [Preparation of mouse IL-2:anti-IL-2 immune complex (IL-2 / IC)] IL-2 / anti-IL-2 mAb immune complexes (IL-2 / IC) were prepared as previously reported

[15] . 1 mg of anti-IL-2 mAb (JES6-1A12, InVivoMAb, BioX-Cell, Lebanon, NH) was mixed in vitro with 0.2 mg of recombinant mouse IL-2 (PeproTech, Rocky Hill, NJ) in 20 mL of HBSS (Mediatech, Herndon, VA) and incubated for 10 min at room temperature. The resulting IL-2 / IC was then administered to experimental animals in a single injection volume of 0.1 mL containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb.

[0314] [Animal experiments] Animals were cared for in accordance with federal, state, and local guidelines. All work with animals was in accordance with and approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Michigan, Ann Arbor. Seven-week-old C57BL / 6 female mice were randomly divided into six groups, with five mice in each group. For group 1 (ND-OVA_D-3,+7+IL2 / IC_D3,4,5), 0.1 mL of ND-OVA (containing 0.1 mg of OVA-II peptide) was injected subcutaneously at the base of the tail of each mouse on days 3 and 7; 0.1 mL of IL2 / IC (containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb) was injected intraperitoneally (ip) into each mouse on days 0, 1, 2, and 10, 11, 12. For group 2 (ND-OVA_D-4,+6+IL2 / IC_D4,5,6), 0.1 mL of ND-OVA (containing 0.1 mg of OVA-II peptide) was injected subcutaneously at the base of the tail of each mouse on days 4 and 6; 0.1 mL of IL2 / IC (containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb) was injected intraperitoneally (ip) of each mouse on days 0, 1, 2 and D10, 11, 12. For group 3 (ND-OVA_D-3,+7+IL2 / IC_D3,4,5,6,7), 0.1 mL of ND-OVA (containing 0.1 mg of OVA-II peptide) was injected subcutaneously at the base of the tail of each mouse on days 3 and 7; 0.1 mL of IL2 (containing 1 μg of mouse IL-2) was injected intraperitoneally (ip) of each mouse on days 0, 1, 2, 3, 4 and 10, 11, 12, 13, 14. For group 4 (ND-OVA_D-1,+9+IL2 / IC_D1,2,3,4,5), 0.1 mL of ND-OVA (containing 0.1 mg of OVA-II peptide) was injected subcutaneously at the base of the tail of each mouse on days 1 and 9; 0.1 mL of IL2 (containing 1 μg of mouse IL-2) was injected intraperitoneally (ip) of each mouse on days 0, 1, 2, 3, 4 and 10, 11, 12, 13, 14.For group 5 (IL2 / IC_D3,4,5), 0.1 mL of IL2 / IC (containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb) was intraperitoneally (ip) injected into each mouse on days 0, 1, 2 and 10, 11, 12. For group 6 (IL2 / IC_D1,2,3,4,5), 0.1 mL of IL2 (containing 1 μg of mouse IL-2) was intraperitoneally (ip) injected into each mouse on days 0, 1, 2, 3, 4 and 10, 11, 12, 13, 14 as shown in Figure 12. A 29G needle syringe (BD SAFETYGLIDE 0.5ML insulin syringe 29G x 0.5") was used for injection. On days 5, 15, and 25, mouse peripheral blood was collected for flow cytometry analysis.

[0315] [Flow cytometry analysis] At the indicated time points, 150–200 uL of blood was collected from individual mice into EDTA-coated tubes. For red blood cell lysis, blood volumes were transferred to 1.5 mL Eppendorf tubes. 1 mL of ACK lysis buffer was added to each blood volume to lyse red blood cells, and the tubes were shaken at room temperature for 5 min. After 5 min, the tubes were spun at 600 g for 5 min, and the supernatant was discarded. The pellet was resuspended in 1 mL of ACK lysis buffer and immediately spun at 600 g for 5 min. The supernatant was discarded, and the PBMCs were resuspended in PBS, transferred to a 96-well plate, and washed once with PBS before proceeding with FACS staining. For surface staining, cells were first incubated with eBioscience Fixable Viability Dye eFluor450 in PBS for 10 min at room temperature in the dark. PBMCs were washed with 200 uL of PBS and spun at 600 g for 5 min. The supernatant was discarded and samples were suspended in Fc block (anti-CD16 / 32) in FACS buffer (PBS + 1% BSA). Blocking was allowed to proceed for 10 minutes at room temperature in the dark before OT-II tetramers were added to the wells at a final dilution of 1:40 in FACS buffer. OT-II tetramers (NIH Tetramer Core Facility, Atlanta, GA) were incubated with PBMCs for 1 hour at room temperature in the dark. Twenty minutes before the end of the OT-II tetramer incubation, surface antibodies were added to the samples at a final dilution of 1:100. After tetramer and surface incubation, PBMCs were washed with 200 uL of FACS buffer and spun at 600 g for 5 minutes. For intracellular staining of Foxp3, eBioscienc™ Foxp3 / Transcription Factor Staining Buffer Set was used according to the kit instructions. To measure total cell counts, 50uL of Life Technologies Absolute Counting Beads were added to fully stained PBMC samples before harvesting on a BioRad ZE5 analyzer. FSC files were analyzed using FlowJo.

[0316] [result] [Optimization of dosing regimen and comparison of ND+wtIL2 vs. ND+IL2 / IC] C57BL / 6 mice were administered nanodisc-OVA-II (ND-OVA) sc and treated with IL-2 immune complexes (IL-2 / IC) or wild type IL-2 protein (IL-2) by ip route using the dosing schedule shown in Figure 12. We found in Example 1 that IL-2 / IC injections close to the first ND-OVA vaccination can expand OT-II tetramer+Foxp3-CD25+Tconv cells (Example 1, Figure 9c). To optimize ND vaccination and IL-2 / IC combination therapy, two dosing schedules were tested: IL-2 / IC administered for three consecutive days starting on day 3 (group 1) or day 4 (group 2) after vaccination. These two dosing schedules were compared to ND vaccination + wtIL-2 protein therapy. The half-life of IL-2 protein in vivo is significantly shorter than IL-2 / IC. Therefore, we included five doses of IL-2 starting on day 3 (group 3) or day 1 (group 4) after ND-OVA vaccination. Control groups included IL-2 / IC (group 5) and IL-2 (group 6) treatment only (Figure 12). Systemic immune responses were measured by flow cytometry on days 5, 15, and 25 after the first injection of IL-2 / IC or IL-2.

[0317] Over the course of the vaccine regimen, CD4+ T cell abundance in lymphocytes was similar between vaccine groups, with IL-2 / IC only subjects showing a decline in CD4 T cells on day 15 (Figure 13a). Mice from groups 1 and 2 receiving ND-OVA+IL-2 / IC on D3, 4, 5 or D4, 5, 6 demonstrated nearly identical CD4+ T cell responses in PBMCs.

[0318] On days 5 and 15, animals receiving ND-OVA+IL-2 / IC on D3, 4, 5 or D4, 5, 6 had significantly higher CD4+CD25+Foxp3+regulatory T cells (T regs ) (Figure 13b). Importantly, the combination of ND-OVA+IL-2 / IC significantly increased T regsThe overall proliferation rate was approximately 1.6-fold, whereas the T regs Ten days after the last vaccination cycle, the abundance of T in animals treated with IL-2 / IC was not increased by more than 10% (Fig. 13b). regs returned to baseline (Fig. 13b).

[0319] T regs In addition to the overall response, animals in groups 1 and 2 elicited stronger antigen-specific OT-II responses compared to all other groups. After the first vaccine cycle, the overall OT-II tetramer counts of CD4+ T cells were significantly higher than those of the control group. + and OT-II tetramer + Foxp3 + CD25 + T regs The total number of CD4+ cells accounted for approximately 0.76% and 0.44%, respectively (Fig. 13c, d). The second round of vaccination further increased the total and T reg The antigen-specific cells further increased to approximately 2.5% and approximately 1.5%, respectively, a 3.3-fold increase from day 5 (Figure 13c, d). Particularly by day 15, antigen-specific OT-IIT in ND-OVA+IL-2 / IC mice regs was increased approximately 55-fold compared to mice treated with ND-OVA+IL-2 or IL-2 alone (FIG. 13d).

[0320] To examine the impact of the vaccine regimen on other immune cell subsets, we measured the abundance and absolute numbers of CD8+ T cells, activated CD8+CD44hi T cells, and NK cells in PBMCs (Figure 14). The abundance of CD8+ cells averaged 40-50% from days 5 to 25 and was similar between groups (Figure 14a). On day 15, two of four mice in the IL-2 / IC-only group had a significant increase in the abundance of CD8+ T cells above 64%, while the other two mice had abundances in the average range. The absolute numbers of CD8+ T cells ranged from 0.38 to 1.0 × 106 per 2 mL of blood from days 5 to 25 (Figure 14b). The abundance of activated CD8+CD44hi ranged from 24 to 60% of total CD8+ T cells, and the absolute numbers ranged from 0.10 to 0.33 × 106 per 2 mL of blood from days 5 to 25 (Figure 14c, d). Finally, the abundance and absolute numbers of NK cells were 16–42% and 1.6–2.8 × 106 cells per 2 mL of blood, respectively (Fig. 14e, f).

[0321] These results highlight three important findings: (1) the addition of ND to IL-2 / IC treatment significantly improved T regs (2) IL-2 protein can increase the overall abundance ratio of T regs Total and antigen-specific T regs (3) Delaying IL-2 / IC injection from D3, 4, 5 to D4, 5, 6 after ND-OVA vaccination did not inhibit T regs Total or antigen-specific T regs The results show no significant effect on the proliferation of

[0322] [Example 4] [material and method] [Preparation of antigen-loaded nanodiscs] DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. DMPC and ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) powder were mixed and hydrated with 10 mM sodium phosphate buffer (DMPC:22A=2:1, mass ratio), then subjected to cycles of heating and cooling to obtain blank synthetic HDL nanodiscs, which were then sonicated for 5 min at room temperature. To load antigen peptides into blank nanodiscs, the cysteine-terminated antigen peptides were first conjugated with DOPE-MAL (antigen peptide:DOPE-MAL=2:1, molar ratio). DOPE-peptide was then added to the blank nanodiscs (22A:antigen peptide = 5:1, mass ratio) and incubated for 1 h at room temperature with gentle agitation on an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce). The antigen peptides used in this study were p31 peptide (YVRPLWVRME), InsB9-23 peptide, and full-length C peptide. The peptide loading efficiency was measured by LC-MS. Nanodiscs were analyzed by gel permeation chromatography (GPC) with a TSKgel G3000SWxl column (7.8 mm i.d. × 30 cm, Tosoh Bioscience LLC). The hydrodynamic size and zeta potential of the nanodisc samples were measured by dynamic light scattering (DLS, Zetasizer Nano ZSP).

[0323] [Preparation of mouse IL-2:anti-IL-2 immune complex (IL-2 / IC)] IL-2 / anti-IL-2 mAb immune complexes (IL-2 / IC) were prepared as previously reported

[15] . 0.1 mg of anti-IL-2 mAb (JES6-1A12, InVivoMAb, BioX-Cell, Lebanon, NH) was mixed in vitro with recombinant mouse IL-2 (20 μg, PeproTech, Rocky Hill, NJ) in 2 mL of HBSS (Mediatech, Herndon, VA) and incubated for 10 min at room temperature. The resulting IL-2 / IC was then administered in a single injection dose of 0.1 mL containing 1 μg of mouse IL-2 and 5 μg of IL-2 mAb.

[0324] [Animal experiments] Animals were cared for in accordance with federal, state, and local guidelines. All work with animals was performed in accordance with and approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Michigan, Ann Arbor. 7-9 week-old NOD male mice were randomly divided into four groups, with 5-10 mice in each group. On day 0, mice in groups 1 and 2 were subcutaneously injected with 0.1 mL of Nanodisc-p31 (containing 0.1 mg of p31 peptide) at the base of the tail. Additionally, mice in group 1 were intraperitoneally (ip) administered 0.1 mL of as-prepared IL-2 / IC solution (above) on days 3, 4, and 5 after each Nanodisc-p31 injection. Mice in group 2 received only Nanodisc-p31 treatment, while mice in groups 3 and 4 received IL-2 / IC and PBS as controls, respectively. On day 11, mice were intravenously injected with 2 million pre-activated BDC2.5 splenocytes and 2 million pre-activated NY8.3 splenocytes, respectively. Mice were then monitored for diabetes onset using a OneTouch Ultra2 glucose meter via the tail vein, and animals with blood glucose levels above 250 mg / dl for two consecutive times were considered diabetic.

[0325] [result] [Therapeutic effect of ND combined with IL-2 / IC in transplanted T1D models] Using the dosing regimen shown in Figure 15A, NOD mice were subcutaneously administered nanodisc-p31 (ND-p31) followed by IL-2 / IC via ip route. Control groups included either PBS or IL-2 / IC or ND only. Six days after the last IL-2 / IC treatment, mice were transplanted with 3 million preactivated BDC2.5 splenocytes and 3 million preactivated NY8.3 splenocytes by retro-orbital injection (Figure 15A). In mice treated with IL-2 / IC, the transfer of diabetogenic T cells accelerated the onset of diabetes similar to the PBS group (Figure 15B). Furthermore, one mouse only treated with p31-ND was protected from diabetes. In stark contrast, over 70% of mice receiving the combination of p31-ND+IL-2 / IC were protected from diabetes.

[0326] The same experimental setting was also used to evaluate the therapeutic effect of ND+IL-2 / IC combination with InsB9-23 and Ins-C peptides (Figure 15A). Transfer of diabetogenic BDC2.5 and NY8.3 T cells in mice treated with IL-2 / IC or PBS resulted in hyperglycemia within one week (Figure 15C). In InsB-ND+InsC-ND treatment, one mouse was protected from diabetes. In stark contrast, more than 70% of mice receiving InsB-ND+InsC-ND+IL-2 / IC combination were not diabetic.

[0327] Taken together, these results indicate that a single round of ND therapy in combination with IL-2 / IC results in potent bystander suppression and robust efficacy against disease in an adoptive transfer model of T1D.

[0328] [Equivalents] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore considered to be illustrative in all respects, rather than limiting, of the invention described herein. The scope of the present invention is therefore indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0329] [Incorporated by reference] The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes. The following documents are incorporated by reference in their entirety: 1.Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. cell 133, 775-787, 2008. 2.Shevach, EM Application of IL-2 therapy to target T regulatory cell function. Trends in immunology 33, 626-632, 2012. 3.Hirakawa, M., Matos, T., Liu, H., Koreth, J., Kim, HT, Paul, NE, Murase, K., Whangbo, J., Alho, AC & Nikiforow, S. Low-dose IL-2 selectively activates subsets of CD4+ Tregs and NK cells. JCI insight 12016. 4.Rosenzwajg, M., Lorenzon, R., Cacoub, P., Pham, H.P., Pitoiset, F., El Soufi, K., RIbet, C., Bernard, C., Aractingi, S. & Banneville, B. Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial. Annals of the rheumatic diseases 78, 209-217, 2019. 5.Higginson-Scott, N., Otipoby, K.L. & Viney, J. PT101: A Treg selective agonist IL-2 mutein therapy for autoimmunity. (Am Assoc Immnol, 2020). 6.Ghelani, A., Bates, D., Conner, K., Wu, M.-Z., Lu, J., Hu, Y.-L., Li, C.-M., Chaudhry, A. & Sohn, S.J. Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins. Frontiers in Immunology 11, 1106, 2020. 7.Peterson, L.B., Bell, C.J., Howlett, S.K., Pekalski, M.L., Brady, K., Hinton, H., Sauter, D., Todd, J.A., Umana, P. & Ast, O. A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease. Journal of autoimmunity 95, 1-14, 2018. 8.Khoryati, L., Pham, M.N., Sherve, M., Kumari, S., Cook, K., Pearson, J., Bogdani, M., Campbell, D.J. & Gavin, M.A. An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice. Science immunology 52020. 9.globenewswire.com / en / news-release / 2021 / 01 / 04 / 2152594 / 0 / en / Pandion-Therapeutics-Announces-Positive-Top-Line-Phase-1a-Clinical-Data-Showing-PT101-was-Well-Tolerated-and-Selectively-Expanded-Regulatory-T-cells.html. 1 / 20 / 2022, 2022. 10.Mills, K.H. & McGuirk, P. Antigen-specific regulatory T cells-their induction and role in infection. Seminars in immunology. 11.Legoux, F.P., Lim, J.-B., Cauley, A.W., Dikiy, S., Ertelt, J., Mariani, T.J., Sparwasser, T., Way, S.S. & Moon, J.J. CD4+ T cell tolerance to tissue-restricted self antigens is mediated by antigen-specific regulatory T cells rather than deletion. Immunity 43, 896-908, 2015. 12.Tang, Q., Henriksen, K.J., Bi, M., Finger, E.B., Szot, G., Ye, J., Masteller, E.L., McDevitt, H., Bonyhadi, M. & Bluestone, J.A. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. The Journal of experimental medicine 199, 1455-1465, 2004. 13.Spence, A., Purtha, W., Tam, J., Dong, S., Kim, Y., Ju, C.-H., Sterling, T., Nakayama, M., Robinson, W.H. & Bluestone, J.A. Revealing the specificity of regulatory T cells in murine autoimmune diabetes. Proceedings of the National Academy of Sciences 115, 5265-5270, 2018. 14.Webster, K.E., Walters, S., Kohler, R.E., Mrkvan, T., Boyman, O., Surh, C.D., Grey, S.T. & Sprent, J. In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression. Journal of Experimental Medicine 206, 751-760, 2009. 15.Manirarora, J.N. & Wei, C.-H. Combination therapy using IL-2 / IL-2 monoclonal antibody complexes, rapamycin, and islet autoantigen peptides increases regulatory T cell frequency and protects against spontaneous and induced type 1 diabetes in nonobese diabetic mice. The Journal of Immunology 195, 5203-5214, 2015. 16.Izquierdo, C., Ortiz, A.Z., Presa, M., Malo, S., Montoya, A., Garabatos, N., Mora, C., Verdaguer, J. & Stratmann, T. Treatment of T1D via optimized expansion of antigen-specific Tregs induced by IL-2 / anti-IL-2 monoclonal antibody complexes and peptide / MHC tetramers. Scientific reports 8, 1-14, 2018. 17. Kuai, R., Li, D., Chen, YE, Moon, JJ & Schwendeman, A. High-density lipoproteins: nature's multifunctional nanoparticles. ACS Nano 10, 3015-3041, 2016. 18. Kuai, R., Ochyl, LJ, Bahjat, KS, Schwendeman, A. & Moon, JJ Designer vaccine nanodiscs for personalized cancer immunotherapy. Nat Mater 16, 489-496, 2017. [Brief description of the drawings]

[0330] [Figure 1] Hydrodynamic size, polydispersity index (PDI), intensity and volume profiles of nanodiscs before and after loading with OVA-II peptide are shown. [Diagram 2] Chromatograms of DOPE-MAL before and after conjugation to OVA-II peptide, the conjugation efficiency of DOPE-MAL to OVA-II peptide, and the loading efficiency of DOPE-OVA-II onto nanodiscs are shown. [Diagram 3] Blank Nanodisc and Nanodisc-OVA-II were analyzed by GPC and the chromatograms are shown. [Figure 4] FIG. 1 shows a schematic diagram of the treatment regimen of ND-OVA-II in combination with IL-2 / IC therapy. [Diagram 5] PBMCs were analyzed by flow cytometry on day 7 and representative scatter plots are shown. [Figure 6]A) % CD4+ T cells in PBMCs, B) % OT-II tetramer+ T cells in CD4+ T cells in PBMCs, C) % Foxp3+CD25+ Tregs in CD4+ T cells, D) % OT-II tetramer+Foxp3+CD25+ Tregs in CD4+ T cells, E) Mean fluorescence intensity (MFI) of CD25 in Foxp3+CD25+ Tregs, and F) MFI of CD25 in OT-II tetramer+Foxp3+CD25+ Tregs. [Figure 7] PBMCs were analyzed by flow cytometry on day 14 and representative scatter plots are shown. [Figure 8] A) % and number of CD4+ T cells in PBMC, B) % and number of OT-II tetramer+ T cells among CD4+ T cells in PBMC, C) % and number of Foxp3+CD25+ Tregs among CD4+ T cells, D) % and number of OT-II tetramer+Foxp3+CD25+ Tregs among CD4+ T cells, E) MFI of CD25, and F) GITR among Foxp3+CD25+ Tregs and OT-II tetramer+Foxp3+CD25+ Tregs. [Figure 9] Changes in A) total Foxp3+CD25+ Tregs, B) OT-II tetramer+ Foxp3+CD25+ Tregs, and C) OT-II tetramer+ Foxp3-CD25+ Tconv in peripheral CD4+ T cells on days 7 and 14 are shown. [Figure 10] Schematic diagram of the treatment regimen of ACT+ND-OVA-II in combination with IL-2 / IC therapy followed by antigen challenge in the ear on day 25. [Figure 11] A) Number of CD4+ cells among live cells, B) Number of CD4+CD25+Foxp3+ cells, C) Number of CD25+Foxp3+OT-II tetramer+ cells are shown. [Figure 12] FIG. 1 is a schematic diagram of the therapeutic dosing regimen of ND-OVA-II in combination with wild-type IL-2 (wtIL-2) or IL-2 / IC. [Figure 13]A) The ratio of CD4+ cells among CD3+ T cells, B) The ratio of CD25+Foxp3+ Treg cells among CD4+ T cells, C) The ratio of OT-II tetramer+CD44hi cells among CD4+ T cells, and D) The ratio of OT-II tetramer+CD44hi cells among CD25+Foxp3+ Treg cells. [Figure 14] The figures show, in 2 mL of blood, A) the ratio of CD8+ cells among CD3+ T cells, C) the ratio of CD44hi cells among CD8+ T cells, E) the ratio of SSClowCD49b+ NK cells among CD3- cells, B) the number of CD8+ T cells, D) the number of CD8+CD44hi T cells, and F) the number of SSClowCD49b+ NK cells. [Figure 15] A) Schematic diagram of the treatment regimen of ACT+ND combined with IL-2 / IC therapy. B) Results of mice treated with p31-ND or p31-ND+IL-2 / IC combination. C) Results of mice treated with InsB-ND and InsC-ND or InsB-ND+InsC-ND+IL-2 / IC combination.

Claims

1. 1. A composition comprising nanoparticles, A composition for use in combination with a composition comprising an immunomodulatory agent capable of expanding regulatory T cells (Tregs) in a subject.

2. the nanoparticles are associated with one or more tolerogenic antigens; Administration of the composition comprising nanoparticles associated with one or more tolerogenic antigens followed by administration of the composition comprising an immunomodulatory agent capable of expanding Tregs results in one or more of the following: Treatment, prevention, and / or attenuation of one or more disorders in said subject; promoting strong immune tolerance to antigens associated with autoimmune disease in said subject; In vivo expansion of Tregs (e.g., CD3+ FOXP3+ cells) in the subject; and an increase in the ratio of CD3+FOXP3+ cells to CD3+FOXP3− cells within a T cell population within the subject; 10. The composition of claim 1, wherein the antigen is specific for one or more tolerogenic antigens associated with the nanoparticles that are associated with one or more tolerogenic antigens. (a) the nanoparticles are associated with an immunomodulatory agent capable of expanding Tregs, such that the composition comprising the immunomodulatory agent capable of expanding Tregs is comprised within the nanoparticles; or (b) The composition of claim 2, wherein the composition comprising the nanoparticles is administered prior to, but not simultaneously with (e.g., 1 second, 2 seconds, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, etc.) administration of the composition comprising the immunomodulatory agent capable of expanding Tregs.

4. the nanoparticles are sHDL nanoparticles comprising a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic; (a) The phospholipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-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-dilignoceroyl-sn-glycero -3-phosphocholine; 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine; 1,2-dimyristerideoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; 1,2-dipalmiteroyl-sn-glycero-3-phosphocholine; 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine; 1,2-dioleoyl-sn-glycero-3-phosphocholine; 1,2-dielide 1,2-Dipentadecanoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-Dimethicone-3-phosphocholine; 1,2-Dimethicone-3-phosphoethanolamine ... -phosphoethanolamine; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine; 1,2-dielideyl-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-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]; 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]; 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; 1,2-Di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; N-[(3-maleimido-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidylethanolamine; N-[(3-maleimido-1-oxopropyl)aminopropyl N-(3-maleimido-1-oxopropyl)-aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine; 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 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-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, distearoyl; N-(succinimidyloxy) N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dioleoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succinimidyloxy-glutaryl)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succinimidyloxyglutaryl)aminopropyl, and polyethylene glycol-carbamyl distearoylphosphatidylethanolamine;N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoyl-ethanolamine; (b) The HDL apolipoprotein component is selected from 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 mimic, preproapoliprotein, preproApoA-I, proApoA I, preproApoA-II, and proApoA. II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, preproApoA I Milano, proApoA-I Milano, preproApoA-I Paris, proApoA-I Paris and mixtures thereof, (c) The composition of any one of claims 1 to 3, wherein the apolipoprotein mimetic is described by any of SEQ ID NOs: 1 to 336 or 341 to 373.

5. The composition of any one of claims 1 to 3, wherein the one or more tolerogenic antigens are specific for type 1 diabetes.

6. 6. The composition of claim 5, wherein the tolerogenic antigen is selected from insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular-enteric-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-associated glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5.

7. The tolerogenic antigen may be any of rheumatoid arthritis, multiple sclerosis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes, autoimmune diseases of the thyroid gland, thyroid-associated ophthalmopathy and skin disorders, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypothyroidism, autoimmune pituitary diseases, immune-mediated gastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and variants, bullous pemphigoid, Dühring's dermatitis herpetiformis, epidermolysis bullosa acquisita, systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, systemic erythema, 4. The composition of any one of claims 1 to 3, wherein the composition is specific for at least one or more of the following disorders: erythematosus, Goodbacher's syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Aicardi-Gautieres syndrome, acute pancreatitis 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, inflammatory bowel disease, transplant-related disorders, one or more allergies, respiratory diseases (e.g., asthma), and GVHD.

8. The one or more tolerogenic antigens are selected from the group consisting of thyroglobulin (TG), thyroid peroxidase (TPO), thyroid stimulating hormone receptor (TSHR), sodium iodide symporter (NIS), megalin, thyroid autoantigens including TSHR, insulin-like growth factor 1 receptor, calcium-sensing receptor, 21-hydroxylase, 17α-hydroxylase, and P450 side chain cleavage enzyme (P450scc), ACTH receptor, P450c21, P450c17, FSH receptor, α-enolase, pituitary-specific protein factor (PGSF) 1a and 2, and type 2 iodothyronine deiodinase, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, collagen II, H ＋ , K ＋ -ATPase, tissue transglutaminase and gliadin, tyrosinase, tyrosinase-related protein 1 and 2, acetylcholine receptor, desmoglein 3, 1 and 4, pemfaxin, desmocollin, plakoglobin, pelplakin, desmoplakin, acetylcholine receptor, BP180, BP230, plectin, laminin 5, endomysium, tissue transglutaminase, collagen VII, matrix metalloproteinase 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, centromeric proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II, and III, PM-Slc, fibrillarin, B23, U1snRNP, nuclear antigens SS-A and SS-B, fodrin, poly(ADP-ribose) polymerase, topoisomerase, nuclear proteins 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 ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, and hepatic cytochrome P450 4. The composition of any one of claims 1 to 3, comprising one or more tolerogenic antigens selected from P450 1A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and type 1 interferons interferon α, β, and ω.

9. The composition of any one of claims 1 to 3, wherein said one or more tolerogenic antigens are specific for celiac disease.

10. The composition according to any one of claims 1 to 3, wherein the tolerogenic antigen is selected from gliadin, glutenin, and fragments thereof capable of inducing an immune response.

11. The composition according to any one of claims 1 to 3, wherein the tolerogenic antigen is selected from gliadin or a fragment thereof.

12. The composition of any one of claims 1 to 3, wherein the tolerogenic antigen is selected from the group consisting of alpha, gamma, and omega gliadin or fragments thereof.

13. The composition of any one of claims 1 to 3, wherein the tolerogenic antigen comprises a polypeptide having at least 90% sequence identity to the polypeptide sequence of any one of SEQ ID NOs: 375-580.

14. 14. The composition of claim 13, wherein the tolerogenic antigen comprises a polypeptide having at least 90% sequence identity to any one of the polypeptide sequences of SEQ ID NOs: 375-580.

15. 15. The composition of claim 14, wherein the tolerogenic antigen comprises a polypeptide having the polypeptide sequence of any one of SEQ ID NOs: 375-580.

16. 16. The composition of claim 15, wherein the tolerogenic antigen comprises two or more polypeptide sequences having the sequence of any one of SEQ ID NOs: 375-580.

17. The tolerogenic antigen is a multimeric tolerogenic antigen comprising the following N-terminal-C-terminal structure: (P ４ -L ４ ) ｎ４ -(P ３ -L ３ ) ｎ３ -P ２ -(L １ -P １ ) ｎ１ In the formula, P １ , P ２ , P ３ , and P ４ are each independently a tolerogenic antigen; L １ , L ３ , and L ４ are each independently a linker; and n １ , n ３ , and n ４ are each independently 0 or 1, and n １ , n ３ , and n ４ at least one of is 1; (a) n 1 is 1, n 3 is 0, and n 4 is 0, and the tolerogenic antigen comprises the following N-terminal-C-terminal structure: P 2 -L 1 -P 1 (b) L 1 is a peptide linker containing 2 to 200 amino acids; (c) L 1 is a peptide linker containing 5 to 50 amino acids; (d) L 1 is a peptide linker containing glycine (G) and serine (S) residues; (e) L 1 is a peptide linker comprising the amino acid sequence (GS) x , (GGS) x , or (GGGGS) x , where x is an integer from 1 to 10; (f) P 1 and P 2 each comprise a different tolerogenic antigen; (g) P 1 and P 2 each contain the same tolerogenic antigen; or (h) The composition of any one of claims 1 to 3, wherein L 1 and L 3 are each independently selected from peptide linkers containing from 2 to 200 amino acids.

18. n １ is 1, n ３ is 1, n ４ is 0, The tolerogenic antigen comprises the following N-terminal-C-terminal structure: P ３ -L ３ -P ２ -L １ -P １ L 1 and L 3 are each independently selected from a peptide linker comprising the amino acid sequence of (GS) x , (GGS) x , or (GGGGS) x ; 18. The composition of claim 17, wherein x is an integer from 1 to 10.

19. L １ and L ３ are each independently selected from peptide linkers comprising 5 to 50 amino acids.

20. L １ and L ３ are each independently selected from peptide linkers comprising glycine (G) and serine (S) residues.

21. the nanoparticles are associated with an immunomodulatory agent and are not associated with a tolerogenic antigen; or 3. The composition of claim 1 or 2, wherein the nanoparticles are associated with a tolerogenic antigen and further associated with an immunomodulatory agent.

22. The immunomodulatory agent may be selected from the group consisting of fingolimod; rapamycin; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid 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; inhibitors of mitochondrial function; NF-κβ inhibitors; adenosine receptor agonists; prostaglandins. Protein 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 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; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); oxidized ATP IDO; Vitamin D3; Cyclosporine; Aryl hydrocarbon receptor inhibitors; Resveratrol; Azathioprine (Aza); 6-mercaptopurine (6-MP); 6-thioguanine (6-TG); FK506; Sanglifehrin A; Salmeterol; Mycophenolate mofetil (MMF); Aspirin and other COX inhibitors; Niflumic acid; Estriol; Triptolide; OPN-305; OPN- 401; eritoran (E5564); 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; 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; amlexanox; Bay11-7082; BX795; CH-223191; chloroquine; CLI-095; CU-CPT9a; cyclosporin A; CTY387; gefitinib; glibenclamide; H-89; H-131; isoliquiritigenin; MCC950; MRT67307; OxPAPC; parthenolide; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU. 521; VX-765; YM201636; Z-VAD-FMK; and one or more of the AHR-specific ligands including, but not limited to, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD); tryptamine (TA); and 6-formylindolo[3,2 b]carbazole (FICZ).

23. 22. The composition of claim 21, wherein the immunomodulatory agent is a cytokine.

24. 24. The composition of claim 23, wherein the cytokine is a human cytokine.

25. 24. The composition of claim 23, wherein the cytokine is selected from TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL-12B, IL-15, IL-21 and IL-18, and any variations / muteins thereof.

26. 22. The composition of claim 21, wherein the immunomodulatory agent is selected from human IL-2 or a variant thereof, low-dose IL-2 or a variant thereof, PT101 or a variant thereof, an IL-2 mutein, and an IL-2:anti-IL-2 antibody immune complex (IL-2 / IC).

27. 22. The composition of claim 21, wherein the immunomodulatory agent is selected from the group consisting of extended pharmacokinetic (PK) IL-2, extended PK IL-2 including fusion proteins (e.g., immunoglobulin fragments, human serum albumin, Fn3), an IL-2 moiety operably linked to an immunoglobulin Fc domain, an IL-2 moiety conjugated to a non-protein polymer (e.g., polyethylene glycol), and an IL-2 mutein that functions as a high affinity CD25 binder.

28. The one or more immunomodulatory agents may be selected from the group consisting of fingolimod; rapamycin; 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid 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; inhibitors of mitochondrial function; NF-κβ inhibitors; adenosine receptor agonists; prostaglandins; PGE2 agonists; phosphodiesterase inhibitors; 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; autophagy inhibitors; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); oxidized ATP IDO; Vitamin D3; Cyclosporine; Aryl hydrocarbon receptor inhibitors; Resveratrol; Azathioprine (Aza); 6-mercaptopurine (6-MP); 6-thioguanine (6-TG); FK506; Sanglifehrin A; Salmeterol; Mycophenolate mofetil (MMF); Aspirin and other COX inhibitors; Niflumic acid; Estriol; Triptolide; OPN-305; OPN- 401; eritoran (E5564); 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; 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; amlexanox; Bay11-7082; BX795; CH-223191; chloroquine; CLI-095; CU-CPT9a; cyclosporin A; CTY387; gefitinib; glibenclamide; H-89; H-131; isoliquiritigenin; MCC950; MRT67307; OxPAPC; parthenolide; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU. 521; VX-765; YM201636; Z-VAD-FMK; and AHR-specific ligands including, but not limited to, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD); tryptamine (TA); and 6-formylindolo[3,2 b]carbazole (FICZ).

29. the immunomodulator is a cytokine; 2. The composition of claim 1, wherein the cytokine is a human cytokine or is a cytokine selected from TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL-12B, IL-15, IL-21 and IL-18, and any variations and / or muteins thereof.

30. 2. The composition of claim 1, wherein the immunomodulatory agent is selected from human IL-2 or a variant thereof, low-dose IL-2 or a variant thereof, PT101 or a variant thereof, or a mutein IL-2 or a variant thereof.

31. 2. The composition of claim 1, wherein the immunomodulatory agent is an IL-2:anti-IL-2 antibody immune complex (IL-2 / IC).

32. The composition of claim 1 , wherein the subject is a human subject.

33. 33. The composition of claim 32, wherein the subject is a human subject suffering from or at risk of suffering from an autoimmune disorder.

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