Improved formulations of autoantigen conjugates

Abstract

The present invention relates to conjugates of a glucocorticoid receptor agonist and a self-antigen. These conjugates are useful for treating or preventing autoimmune diseases, such as rheumatoid arthritis or inflammation. These conjugates can induce tolerogenic dendritic cells and show increased efficiency in vivo compared to cell therapy.

Description

Technical Field

[0001] This invention relates to conjugates of glucocorticoid receptor agonists and autoantigens. These conjugates can be used to treat or prevent autoimmune diseases such as rheumatoid arthritis or inflammation. These conjugates can induce tolerant dendritic cells and show increased efficiency in vivo compared to cell therapy. Background Technology

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by the influx of pro-inflammatory immune cells into the synovium, leading to pain and disability. Current treatments achieve only temporary remission in a subset of RA patients and require lifelong medication, which gradually loses its effectiveness and increases in cost. Known treatments include the use of disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids, but these drugs often produce off-target side effects because they are not antigen-specific and are generally immunosuppressive.

[0003] Regulatory T cells (Tregs), mainly CD4 + T cells can restore immune tolerance by suppressing effector cells in an antigen-specific manner. In patients with autoimmune diseases, subtle changes in the function or presence of Tregs are thought to be related to disease pathogenesis. However, several studies have shown reduced Treg suppression in the synovial fluid of RA patients, while these abilities are maintained in peripheral blood. Other studies suggest a decrease in Treg numbers, which may contribute to excessive inflammation in RA. Antigen-specific Tregs can suppress this excessive inflammation by inhibiting immune cells that trigger pathological autoimmune responses while maintaining protective immune integrity.

[0004] Tolerogenic dendritic cells (tolDCs) can be used as a tool to induce antigen-specific Tregs. TolDCs are dendritic cells (DCs) that are modulated to become immune-tolerant. Whether a DC is immunostimulatory or immune-tolerant depends primarily on the environmental cues it receives, which determine whether it becomes immunogenic or tolerant.

[0005] Jansen et al. (doi: 10.3389 / fimmu.2019.02068) described using TolDCs to suppress arthritis using dexamethasone (Dex) pulses loaded with RA-specific autoantigen human proteoglycan (hPG). WO 2021038283 describes a method for treating rheumatoid arthritis that involves isolating mononuclear cells from a subject and then sequentially adding dexamethasone and the autoantigen to the cells. The cells are then transferred back to the subject. Like other cell-based therapies, such cell-based therapies are limited to specialized medical facilities, increasing treatment costs and reducing accessibility. The need for patient-specific cells also increases the burden on patients. Improvements in RA treatment are needed. Drugs with longer shelf lives are needed. Off-the-shelf drugs that do not require cell transfer are needed. Summary of the Invention

[0006] The inventors surprisingly discovered that when self-antigen peptides are conjugated with glucocorticoid receptor agonists, improved induction of immune tolerance can be achieved. For example, they found that antigen-corticosteroid complexes can induce antigen-specific immune tolerance and prevent the development and progression of conditions such as arthritis in mice in an antigen-specific manner. They also found that the representative antigen-dexamethasone complex hPG-Dex induces a tolerance phenotype in immature dendritic cells of humans and mice.

[0007] This invention provides a conjugate comprising: (i) Glucocorticoid receptor agonists, and (ii) Peptides with self-antigenicity.

[0008] Preferably, the glucocorticoid receptor agonist is (i) Corticosteroids, preferably dexamethasone, triamcinolone, methylprednisolone, prednisolone, or prednisolone, more preferably dexamethasone or prednisolone, most preferably dexamethasone, or (ii) Synthesize a nonsteroidal glucocorticoid receptor agonist, preferably dagrocorat, AZD-5423, fosdagrocorat or mapracorat.

[0009] The peptide is preferably an autoantigen associated with autoimmune diseases such as rheumatoid arthritis, spondyloarthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis, Sjögren's disease, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, idiopathic thrombocytopenic purpura, alopecia, vitiligo, type 1 diabetes, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis, more preferably associated with rheumatoid arthritis or multiple sclerosis, and most preferably associated with rheumatoid arthritis. The peptide is preferably derived from a protein selected from: human proteoglycans; insulin; proinsulin; preinsulin; proinsulin; melanin; topoisomerase 1; topoisomerase 2; glutamate decarboxylases, such as glutamate decarboxylase 2 or glutamate decarboxylase 65; collagen, such as type II collagen; citrullinated human proteoglycans; α-enolase; citrullinated α-enolase; cartilage mesothelial protein; citrullinated cartilage mesothelial protein; fibrinogen; citrullinated fibrinogen; vimentin; citrullinated vimentin; acetylcholine receptors; myelin proteins, such as myelin oligodendrocyte glycoproteins, myelin lipoproteins, or myelin basic proteins; thyroid-stimulating hormone receptors; and smooth muscle, preferably derived from human proteoglycans. In a preferred embodiment, the peptide comprises a sequence of 6 to 60, preferably 10 to 40, more preferably 12 to 20 consecutive amino acids having a sequence from the proteins identified above, wherein the consecutive sequence may have zero, one, two, or three amino acid substitutions. Preferably, the peptide has a length of 6 to 70 amino acids, more preferably 10 to 40 amino acids, and even more preferably 12 to 20 amino acids. Preferably, the peptide comprises or consists of the sequence of SEQ ID NO: 3-43 and optionally 66-68, preferably SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, and even more preferably the sequence of SEQ ID NO: 3.

[0010] In some embodiments, the glucocorticoid receptor agonist and the peptide are linked by a linker, wherein the linker is preferably a peptide linker comprising 1 to about 12 amino acids, more preferably 2 to about 8 amino acids, more preferably about 3 to about 6 amino acids, and most preferably 4 or 5 amino acids, wherein preferably, these amino acids are hydrophilic. In a preferred embodiment, the glucocorticoid receptor agonist is dexamethasone, and the peptide is an autoantigen associated with rheumatoid arthritis or multiple sclerosis, preferably associated with rheumatoid arthritis.

[0011] A composition comprising the conjugate as described above is also provided, wherein the conjugate is contained within nanoparticles. The nanoparticles are preferably micelles, polymer nanoparticles, polysaccharide nanoparticles, liposomes, lipid complexes, monolayer vesicles, multilayer vesicles, or cross-linked or hybrid variants thereof, more preferably liposomes. Preferably, the nanoparticles comprise one or more phospholipids, preferably two phospholipids, wherein these phospholipids are preferably selected from 1,2-dilauroyl-sn-glycerol-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), and 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPC). 1,2-Dimyristoyl-sn-glycerol-3-phosphate (DMPG), 1,2-dimyristoyl-sn-glycerol-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPG) 1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine (DPPS), 1,2-distearyl-sn-glycerol-3-phosphate (DSPA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphoglycerol (DSPG), 1,2-distearyl-sn-glycerol-3-phosphoserine (DSPS), and hydrogenated... Soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate serine (DOPS), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), or polymer-conjugated phospholipids, such as polyethylene glycol-modified phospholipids or polyglycerol phospholipids.

[0012] Also provided are the conjugates or compositions described above, used as pharmaceuticals. The pharmaceuticals are preferably used to treat immune or autoimmune diseases and / or inflammation, preferably for treating rheumatoid arthritis, systemic lupus erythematosus, thrombocytopenic purpura (ITP), systemic sclerosis, Graves' disease, Hashimoto's thyroiditis, vasculitis, inflammatory bowel disease, multiple sclerosis, hemolytic anemia, thyroiditis, stiff-person syndrome, pemphigus vulgaris, myasthenia gravis, lupus nephritis, Crohn's disease, or ulcerative colitis; more preferably for treating rheumatoid arthritis, type 1 diabetes mellitus, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis; and even more preferably for treating rheumatoid arthritis or multiple sclerosis. In some highly preferred embodiments, it is used to treat rheumatoid arthritis. In some highly preferred embodiments, it is used to treat multiple sclerosis. A method for treating autoimmune diseases and / or inflammation is also provided, comprising the step of administering the conjugate or composition as described above to a subject in need. Detailed Implementation

[0013] The inventors have surprisingly discovered that improved induction of immune tolerance can be achieved when self-antigen peptides are conjugated to glucocorticoid receptor agonists. For example, it has been found that antigen-corticosteroid complexes can induce antigen-specific immune tolerance and prevent the development and progression of, for example, arthritis in mice in an antigen-specific manner. The representative antigen-dexamethasone complex hPG-Dex has been found to induce a tolerance phenotype in immature dendritic cells of humans and mice. Therefore, the present invention provides a conjugate comprising: (i) Glucocorticoid receptor agonists, and (ii) Peptides with self-antigenicity.

[0014] Such conjugates are referred to herein as conjugates according to the invention. The term conjugate has its common meaning in the art and refers to any entity in which two components are bound together. Preferably, the glucocorticoid receptor agonist and the peptide are covalently bound together. In some embodiments, the conjugate comprises two or more peptides. In some embodiments, the conjugate comprises two or more glucocorticoid receptor agonists. Preferably, the conjugate comprises a single peptide and a single glucocorticoid receptor agonist. In a preferred embodiment, the conjugate consists of a single peptide and a single glucocorticoid receptor agonist. In other preferred embodiments, the conjugate consists of a single peptide, a linker, and a single glucocorticoid receptor agonist. The peptide is preferably linear.

[0015] In some embodiments, the glucocorticoid receptor agonist and the peptide are linked by a linker. Linkers are commonly used and may be present to: facilitate chemical coupling of the glucocorticoid receptor agonist and the peptide (e.g., a triazole linker); or increase the spatial distance between the glucocorticoid receptor agonist and the peptide (e.g., an alkane linker); or impart additional properties to the conjugate, such as increased water solubility (e.g., using an oligomeric glycol or polyethylene glycol linker), increased lipophilicity (e.g., using an aliphatic linker), increased positive charge (e.g., using an oligomeric lysine or polyethyleneimine linker), increased negative charge (e.g., using an oligomeric glutamic acid or oligomeric acrylic acid linker), or any other properties known to those skilled in the art.

[0016] In a preferred embodiment, the linker is a peptide linker. This is advantageous because peptide linkers can be considered biodegradable linkers. Preferably, the peptide linker comprises 1 to about 12 amino acids, more preferably 2 to about 8 amino acids, even more preferably about 3 to about 6 amino acids, and most preferably 4 or 5 amino acids. In the peptide linker, preferably, the amino acids are hydrophilic. Those skilled in the art are very familiar with the properties of amino acids. Examples of hydrophilic amino acids are lysine, serine, threonine, histidine, ornithine, and tyrosine. Other examples of hydrophilic amino acids are aspartic acid and glutamic acid. The hydrophilic peptide linker may comprise glycine or alanine. Examples of suitable linkers are peptides represented by one of SEQ ID NO: 54-65, preferably 54-62. In some embodiments, the linker is represented by one of SEQ ID NO: 60-62. In some embodiments, the linker is represented by one of SEQ ID NO: 57-59. In some embodiments, the linker is represented by one of SEQ ID NO: 63-65. Preferably, the connector is represented by one of SEQ ID NO: 54-56, more preferably SEQ ID NO: 54. Many connectors are suitable – it has been found that multiple connectors do not show different effects on the encapsulation of the conjugate in nanoparticles (such as liposomes) or on the physicochemical properties of the nanoparticles.

[0017] When a linker is present, it preferably links the N-terminus or C-terminus of the peptide to a glucocorticoid receptor agonist. In a preferred embodiment, when a linker is present, it links the N-terminus of the peptide to a glucocorticoid receptor agonist. Preferably, the linker links only the peptide to a glucocorticoid receptor agonist. Preferably, the glucocorticoid receptor agonist is linked to the linker or peptide via a reactive group. Esters of the agonist are convenient because esters can introduce a carboxylic acid into the agonist. Esters can be, for example, succinates to introduce a carboxylic acid ester, which also introduces a free carboxylic acid. Succinates and similar dicarboxylic acids are attractive esters for introducing a free carboxylic acid into the agonist. These can be conveniently used for N-terminal conjugation. The ester is biodegradable and does not remove the activity of the agonist. A suitable example of a dicarboxylic acid is HOOC-(CH2). 2-6 -COOH, one or both of the -CH2- moieties may be further substituted, for example, by being substituted with methyl or methoxy groups. Preferably, one such further substitution is present, more preferably, no such further substitution is present.

[0018] Particularly preferred conjugates are those in which the glucocorticoid receptor agonist is dexamethasone and the peptide is an autoantigen associated with rheumatoid arthritis. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate and the peptide is an autoantigen associated with rheumatoid arthritis. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone and the peptide is an autoantigen associated with rheumatoid arthritis, selected from peptides consisting of sequences represented by SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, and even more preferably SEQ ID NO: 3. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate and the peptide is an autoantigen associated with rheumatoid arthritis, selected from peptides consisting of sequences represented by SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, and even more preferably SEQ ID NO: 3.

[0019] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is an autoantigen associated with rheumatoid arthritis, selected from peptides consisting of the sequence represented by SEQ ID NO: 3-18, more preferably any of SEQ ID NO: 3-11, or even more preferably SEQ ID NO: 3, wherein the dexamethasone succinate is conjugated to the N-terminus of the peptide.

[0020] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is an autoantigen associated with rheumatoid arthritis, selected from peptides consisting of sequences represented by SEQ ID NO: 3-18, more preferably any of SEQ ID NO: 3-11, or even more preferably SEQ ID NO: 3, and the dexamethasone succinate is conjugated to the N-terminus of the peptide via a peptide linker.

[0021] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is an autoantigen associated with rheumatoid arthritis, selected from peptides consisting of sequences represented by SEQ ID NO: 3-18, more preferably any of SEQ ID NO: 3-11, and even more preferably SEQ ID NO: 3, wherein the dexamethasone succinate is conjugated to the N-terminus of the peptide via a peptide linker of 3, 4, or 5 amino acids, preferably 4 amino acids, wherein the linker preferably contains 1, 2, 3, 4, or 5 lysine residues, preferably 4 lysine residues.

[0022] Particularly preferred conjugates are those in which the glucocorticoid receptor agonist is dexamethasone and the peptide is a multiple sclerosis-associated autoantigen. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate and the peptide is a multiple sclerosis-associated autoantigen. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone and the peptide is a multiple sclerosis-associated autoantigen selected from peptides consisting of sequences represented by SEQ ID NO: 35-41, more preferably any one of SEQ ID NO: 35, 38, or 40, and even more preferably SEQ ID NO: 35. In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate and the peptide is a multiple sclerosis-associated autoantigen selected from peptides consisting of sequences represented by SEQ ID NO: 35-41, more preferably any one of SEQ ID NO: 35, 38, or 40.

[0023] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is an autoantigen associated with multiple sclerosis, selected from peptides consisting of the sequence represented by SEQ ID NO: 35-41, more preferably any one of SEQ ID NO: 35, 38 or 40, even more preferably SEQ ID NO: 35, and dexamethasone succinate is conjugated to the N-terminus of the peptide.

[0024] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is an autoantigen associated with multiple sclerosis, selected from peptides consisting of the sequence represented by SEQ ID NO: 35-41, more preferably any one of SEQ ID NO: 35, 38 or 40, even more preferably SEQ ID NO: 35, and the dexamethasone succinate is conjugated to the N-terminus of the peptide via a peptide linker.

[0025] In other preferred embodiments, the glucocorticoid receptor agonist is dexamethasone succinate, and the peptide is a multiple sclerosis-associated autoantigen selected from peptides consisting of sequences represented by SEQ ID NO: 35-41, more preferably any one of SEQ ID NO: 35, 38, or 40, and even more preferably SEQ ID NO: 35, and the dexamethasone succinate is conjugated to the N-terminus of the peptide via a peptide linker of 3, 4, or 5 amino acids, preferably 4 amino acids, wherein the linker preferably contains 1, 2, 3, 4, or 5 lysine residues, preferably 4 lysine residues.

[0026] Glucocorticoid receptor agonists Glucocorticoid receptors are the receptors to which cortisol and other glucocorticoids bind. When agonists bind to glucocorticoid receptors, they can regulate gene transcription. Activated receptors can upregulate the expression of anti-inflammatory proteins in the cell nucleus or inhibit the expression of pro-inflammatory proteins in the cytosol.

[0027] Glucocorticoid receptor agonists are known in the art. Glucocorticoid receptor agonists can be corticosteroids or synthetic nonsteroidal glucocorticoid receptor agonists, preferably corticosteroids. Derivatives of the agonists, such as esters, can also be used. Suitable esters are acetates, propionates, furoates, succinates, or neopentanoates, with succinates being preferred. Succinates are attractive because they introduce a carboxylic acid moiety that can be used for conjugation with self-antigenic peptides or linkers.

[0028] Examples of suitable corticosteroids include cortisone, cortisone acetate, tododozane, deoxycorticosterone, deoxycorticosterone ester, hydrocortisone, hydrocortisone ester, prednisolone acetate, pregnenolone, pregnenolone acetate, pregnenolone succinate, chlorprednisolone, chlorprednisolone, difluoroprednisolone ester, fludrocortisone, fluoroprogesterone acetate, fluocinolone, fluorometholone, fluorometholone acetate, flusperidone, flusperidone acetate, fluoroprednisolone, fluoroprednisolone ester, chlorprednisolone, methylprednisolone, methylprednisolone ester, prednicarbamate, prednisolone, prednisolone, tecortisone, tecortisone pentanoate, aclomethasone, beclomethasone, beclomethasone ester, betamethasone, betamethasone ester, clobetasol, clobetasol Solomonone propionate, clobetasol, clocotropin, clocotropin ester, cortivazole, deshydroxymethasone, dexamethasone, dexamethasone ester, difluralasone, diflucolone, diflucolone valerate, fluclosonide, flumethasone, flucodone, flucodone ester, fluprednisolone acetate, fluticasone, fluticasone furoate, fluticasone propionate, halometasone, methylprednisolone, mometasone, mometasone furoate, peramisone, prednisolone, limexolone, triamcinolone, halometasol, ansine, budesonide, cyclosine, dexamethasone, desinesol, fluclosonide, flunisolone, fluocinolone acetonide, fluocinolone acetonide, fluocinolone acetonide, formococcal, halcinonide, triamcinolone acetonide, and triamcinolone acetonide ester.

[0029] Preferred corticosteroids include aclomethasone, beclomethasone, beclomethasone ester, betamethasone, betamethasone ester, clobetasol, clobetasol propionate, clobetasol, clocotropin, clocotropin ester, cortivazole, dexamethasone, dexamethasone ester, difluralasone, diflucolone, diflucolone valerate, fluclosonide, flumethasone, flucodone, flucotropin ester, fluprednisolone acetate, fluticasone, fluticasone furoate, fluticasone propionate, halometasone, methylprednisolone, and moxifloxacin. Mometasone, mometasone furoate, peramisone, prednisolone, limexolo, triamcinolone, and halometasone, more preferably dexamethasone, triamcinolone, methylprednisolone, prednisolone, or prednisolone, even more preferably dexamethasone or prednisolone, most preferably dexamethasone, such as dexamethasone succinate, preferably 4-{[(11β,16α)-9-fluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregnat-1,4-dien-21-yl]oxy}-4-oxobutyric acid. In some embodiments, the corticosteroid is prednisolone. In some embodiments, the corticosteroid is dexamethasone.

[0030] Examples of suitable synthetic nonsteroidal glucocorticoid receptor agonists are dagcourt (also known as PF-251802), AZD-5423 (CAS: 1034148-04-3), GSK-9027 (CAS: 1229096-88-1), fudacourt (also known as PF-4171327), or mapcort (also known as ZK-245186). Preferred nonsteroidal agonists are dagcourt, AZD-5423, fudacourt, and mapcort.

[0031] Self-antigen peptides The conjugates according to the invention comprise peptides having autoantigenic properties. Autoimmunity is an immune response system of an organism against its own healthy cells, tissues, or other normal body components. Conditions caused by this type of immune response are called autoimmune diseases, which can lead to, for example, tissue damage, inflammation, and pain. Autoimmunity refers to the presence of antibodies or T cells that react with self-proteins present in all individuals (even in normal health). Many autoimmune diseases are well known, and their mechanisms are understood. Therefore, many self-antigens are known, including self-antigen peptides. Those skilled in the art are familiar with autoimmune diseases and the associated self-antigen peptides. Self-antigen peptides may be referred to as self-antigens.

[0032] In preferred embodiments, the peptide is an autoantigen associated with, preferably associated with, rheumatoid arthritis or multiple sclerosis, systemic lupus erythematosus, spondyloarthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis, Sjögren's disease, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, idiopathic thrombocytopenic purpura, alopecia, vitiligo, type 1 diabetes mellitus, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis. It should be understood that the peptide is not necessarily autologous; it can also be a peptide that induces an autoantigen response. This is the case, for example, in celiac disease, where the intake of wheat gluten and homologous proteins from barley and rye induces a significant T-cell-mediated inflammatory response. Therefore, in some embodiments, the autoantigen peptide is autologous, or the peptide is heterologous and induces an autoantigen response. In other embodiments, the autoantigen peptide is autologous. In other embodiments, the peptide is heterologous and induces an autoantigen response.

[0033] The peptide is preferably composed of naturally occurring proteomic amino acids. It can be modified while present in the organism, for example, it can be citrullinated. Preferably, it is not modified in any other way. Preferably, it is unmodified. The peptide terminus is preferably a free terminus, or an amidated, acetylated, or methylated terminus. Notably, it is preferred that one of the termini serves as a reactive stalk for conjugating glucocorticoid receptor agonists.

[0034] Peptides can be readily derived from proteins known to be associated with autoimmune diseases. For example, peptides can be derived from proteins selected from: human proteoglycans; insulin; proinsulin; preinsulin; proinsulin; melanin; topoisomerase 1; topoisomerase 2; glutamate decarboxylases, such as glutamate decarboxylase 2 or glutamate decarboxylase 65; collagen, such as type II collagen; citrullinated human proteoglycans; α-enolase; citrullinated α-enolase; cartilage mesothelial protein; citrullinated cartilage mesothelial protein; fibrinogen; citrullinated fibrinogen; vimentin; citrullinated vimentin; acetylcholine receptors; myelin proteins, such as myelin oligodendrocyte glycoproteins, myelin lipoproteins, or myelin basic proteins; thyroid-stimulating hormone receptors; and smooth muscle. Preferably, the peptide is derived from a protein selected from: human proteoglycans, insulin, proinsulin, pre-insulin, proinsulin, acetylcholine receptors, and myelin proteins (such as myelin oligodendrocyte glycoproteins, myelin lipoproteins, or myelin basic proteins). More preferably, the peptide is derived from a protein selected from: human proteoglycans, insulin, acetylcholine receptors, or myelin oligodendrocyte glycoproteins. Most preferably, the peptide is derived from human proteoglycans. In other highly preferred embodiments, the peptide is derived from myelin oligodendrocyte glycoproteins.

[0035] The peptide derived from the protein is preferably a peptide comprising or composed of a continuous amino acid sequence from the source protein. Preferably, the peptide comprises or is composed of a sequence having 6 to 70, preferably 7 to 60, more preferably 10 to 40, and even more preferably 12 to 20 continuous amino acids from a protein known to be associated with autoimmune diseases, wherein the continuous sequence may have zero, one, two, or three amino acid substitutions. In some embodiments, the peptide comprises or is composed of a sequence having 8 to 50, preferably 9 to 35, more preferably 11 to 30 continuous amino acids, having optional substitutions as described herein.

[0036] In some embodiments, the continuous sequence may have one, two, or three amino acid substitutions. In some embodiments, the continuous sequence may have zero, one, or two amino acid substitutions. In some embodiments, the continuous sequence may have zero or one amino acid substitution. In some embodiments, the continuous sequence may have one amino acid substitution. In some embodiments, the continuous sequence has no amino acid substitutions. In a preferred embodiment, the peptide consists of a continuous sequence.

[0037] In a preferred embodiment, the peptide comprises or consists of the sequence of any one of SEQ ID NO: 3-43. In other preferred embodiments, the peptide comprises or consists of the following: SEQ ID NO: 3-43 or 66-68, preferably SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, and even more preferably the sequence of SEQ ID NO: 3. In other preferred embodiments, the peptide comprises or consists of the sequence of any one of SEQ ID NO: 3-11, 19, 20, 22, 35, 38, 40, 33, or 34.

[0038] In some embodiments, the peptide is derived from human proteoglycan, type II collagen, citrullinated human proteoglycan, citrullinated α-enolase, citrullinated cartilage interlaminar protein, citrullinated fibrinogen, or citrullinated vimentin. Such peptides are associated with rheumatoid arthritis. Such peptides preferably comprise or consist of the sequence represented by SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, and even more preferably SEQ ID NO: 3.

[0039] In some embodiments, the peptide is derived from insulin, proinsulin, pre-insulin, pre-proinsulin, glutamate decarboxylase 2, or glutamate decarboxylase 65. Such peptides are associated with type 1 diabetes. Such peptides preferably comprise or consist of the sequence represented by any one of SEQ ID NO: 19-28, more preferably SEQ ID NO: 19.

[0040] In some embodiments, the peptide is derived from an acetylcholine receptor. Such peptides are associated with myasthenia gravis. Such peptides preferably comprise or consist of any one of SEQ ID NO: 33-34, more preferably the sequence represented by SEQ ID NO: 33.

[0041] In some embodiments, the peptide is derived from myelin oligodendrocyte glycoprotein, myelin lipoprotein, or myelin basic protein. Such peptides are associated with multiple sclerosis. Such peptides preferably comprise or consist of a sequence represented by any of SEQ ID NO: 35-41. More preferably, they are associated with myelin oligodendrocyte glycoproteins, such as SEQ ID NO: 35, 38, or 40, and even more preferably SEQ ID NO: 35.

[0042] In some embodiments, the peptide is derived from the thyroid-stimulating hormone receptor. Such peptides are associated with Graves' disease. Such peptides preferably comprise or consist of the sequence represented by SEQ ID NO: 42.

[0043] In some embodiments, the peptide is derived from smooth muscle. Such peptides are associated with autoimmune hepatitis. Such peptides preferably comprise or consist of the sequence represented by SEQ ID NO: 43.

[0044] In some embodiments, the peptide is derived from gliadin, such as α-gliadin, γ-gliadin, or ω-gliadin. Such peptides are associated with celiac disease. Such peptides preferably comprise or consist of the sequence represented by SEQ ID NO: 66-68. In these SEQ ID NOs, preferably, 1, 2, or 3 Q residues are deamidated by tissue transglutaminase (TG2). Other peptides associated with celiac disease are known in the art; see, for example... J Immunol [Journal of Immunology] (2009) 182 (7): 4158-4166.

[0045] Examples of suitable proteins and peptides known to be associated with autoimmune diseases are shown in the table below.

[0046] The peptide preferably has a length of about 6 to about 70 amino acids, more preferably about 10 to about 40 amino acids, and even more preferably about 12 to about 20 amino acids. The peptide may have a length of about 7 to about 35, about 8 to about 30, about 9 to about 25, about 10 to about 23, about 11 to about 22, about 13 to about 21, about 14 to about 19, about 15 to about 18, or about 16 to about 17 amino acids.

[0047] Composition The present invention also provides a composition comprising the conjugate according to the invention and a pharmaceutically acceptable excipient. Such a composition is referred to herein as a composition according to the invention. Preferably, such a composition is formulated as a pharmaceutical composition. The preferred excipient is water, preferably purified water, more preferably ultrapure water. In other embodiments, the water is part of a pharmacologically acceptable buffer, such as saline, buffered saline, or more preferably phosphate-buffered saline. A preferred buffer is 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), pH 7-8, such as pH 7.2.

[0048] The composition preferably has a physiologically acceptable pH, more preferably in the range of 6 to 8 or 7 to 7.8. Further preferred excipients are adjuvants, binders, desiccants, or diluents. Further preferred compositions additionally contain other drugs for treating conditions as described elsewhere herein or for treating pain or inflammation. In this regard, preferred other drugs are immunotherapeutic agents or steroids. The composition according to the invention preferably contains a therapeutically effective amount of the compound according to the invention.

[0049] It has been found that compositions containing conjugates are advantageous. Suitable compositions are those in which the nanoparticles are micelles, polymer nanoparticles, polysaccharide nanoparticles, liposomes, lipid complexes, monolayer vesicles, multilayer vesicles, or their cross-linked or hybrid variants, preferably liposomes.

[0050] Micelles are preferably lipid micelles or polymeric micelles, with lipid micelles being the most preferred. Liposomes can be monolayered or multilayered, preferably monolayered. Examples of suitable polymeric nanoparticles are beads or polymeric vesicles, such as polymeric nanoparticles based on PLGA, PLGA-N-trimethylchitosan, PLA, PLA-PEMA, PLGA-PEG, PLGA-PEMA, or PLA-PEG, or optionally mixtures thereof. Examples of suitable polysaccharide nanoparticles are dextran nanoparticles, maltodextrin nanoparticles, and hyaluronic acid nanoparticles. Exemplary hybrid particles are lipid-polymer hybrid nanoparticles.

[0051] The nanoparticles preferably contain lipids, more preferably phospholipids. In a preferred embodiment, the nanoparticles contain one or more phospholipids, more preferably two or more. Good results are achieved when the liposomes contain conjugates.

[0052] Phospholipids preferably contain diglycerides, phosphate groups, and simple organic molecules such as choline. In particular, phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin, phosphatalol, and phosphatidylcholine lipid derivatives, wherein the length of the two hydrocarbon chains is typically between about 12-26, preferably 14-22 carbon atoms, and may have different degrees of unsaturation.

[0053] Phospholipids may contain a net negative charge or a net positive charge, or they may be neutral. In a preferred embodiment, one or more phospholipids are neutral phospholipids. Neutral phospholipids are understood herein to be phospholipids without a net charge. In a more preferred embodiment of the invention, the nanoparticles are liposomes having a net negative charge. Most preferably, the nanoparticles are liposomes and contain neutral phospholipids and negatively charged phospholipids. Preferably, the molar ratio of neutral phospholipids to negatively charged phospholipids is in the range of 10:1 to 1:2, more preferably in the range of 8:1 to 1:1, more preferably in the range of 6:1 to 2:1, and even more preferably in the range of 5:1 to 3:1, such as 4:1.

[0054] Preferably, the nanoparticles comprise one or more phospholipids, more preferably two phospholipids, wherein these phospholipids are preferably selected from 1,2-dilauroyl-sn-glycerol-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), and 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPC). 1,2-Dimyristoyl-sn-glycerol-3-phosphate (DMPG), 1,2-dimyristoyl-sn-glycerol-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPG) 1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine (DPPS), 1,2-distearyl-sn-glycerol-3-phosphate (DSPA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphoglycerol (DSPG), 1,2-distearyl-sn-glycerol-3-phosphoserine (DSPS), and hydrogenated... Phospholipids such as soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate serine (DOPS), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), or polymer-conjugated phospholipids, such as polyethylene glycol-modified phospholipids or polyglycerol phospholipids. Particularly preferred phospholipids are DSPC and DSPG.

[0055] Preferred nanoparticles further comprise sterols, such as cholesterol. In some embodiments, the nanoparticles, preferably liposomes, comprise phospholipids and sterols, preferably cholesterol. Preferably, the molar ratio of phospholipids to sterols is in the range of 1:2 to 5:1, more preferably in the range of 1:1 to 4:1, more preferably in the range of 3:2 to 3:1, and even more preferably in the range of 2:1 to 3:1, such as 5:2.

[0056] When the nanoparticles contain neutral phospholipids, negatively charged phospholipids, and sterols, their respective molar ratios are preferably in the range of 2:1:1 to 8:1:4, more preferably in the range of 6:2:3 to 6:1:3, and most preferably in the range of 6:2:3 to 5:1:3, such as 4:1:2. Highly preferred nanoparticles are liposomes, which contain or consist of 1,2-distearyl-sn-glycerol-3-phosphate choline and 1,2-distearyl-sn-glycerol-3-phosphate glycerol and cholesterol in a molar ratio of 4:1:2.

[0057] Nanoparticles, preferably liposomes, preferably have a size of 10-1000 nm, more preferably 50-500 nm, more preferably 70-400 nm, more preferably 90-350 nm, even more preferably 100-300 nm, more preferably 110-290 nm, more preferably 125-280 nm, more preferably 150-270 nm, more preferably 170-260 nm, and most preferably 180-220 nm. The size of the nanoparticles is preferably determined using a microscope or light scattering (more preferably light scattering), such as as described in the examples.

[0058] Nanoparticles, preferably liposomes, preferably have a polydispersity of about 0 to 0.15, more preferably about 0.02 to about 0.14, more preferably about 0.03 to about 0.13, and particularly preferably about 0.04 to about 0.12. In some embodiments, the polydispersity is about 0.04 to about 0.11. In some embodiments, the polydispersity is about 0.05 to about 0.11. In some embodiments, the polydispersity is about 0.03 to about 0.07.

[0059] Nanoparticles, preferably liposomes, preferably have a zeta potential of -70 to -30 mV, more preferably -60 to -40 mV, more preferably -58 to -45 mV, more preferably -56 to -48 mV, even more preferably -55 to -50 mV, and most preferably -52 to -55 mV. In some embodiments, nanoparticles, preferably liposomes, have a zeta potential of -55 to -60 mV. In some embodiments, nanoparticles, preferably liposomes, have a zeta potential of -50 to -60 mV.

[0060] In embodiments where the conjugate is encapsulated within nanoparticles, the water-soluble polymer may be conjugated with the nanoparticles. Preferably, the water-soluble polymer is conjugated with lipids such as phospholipids or cholesterol.

[0061] In embodiments of the invention, the water-soluble polymer is at least one of the following: i) a polyalkyl ether, preferably a linear polyethylene glycol (PEG), a star-shaped PEG, or a multi-arm branched PEG; ii) a homopolymer as a PEG substitute or alternative, preferably selected from the group consisting of polymethyl ethylene glycol (PMEG), polyhydroxypropylene glycol (PHPG), polypropylene glycol (PPG), polymethyl propylene glycol (PMPG), polyhydroxy propylene oxide (PHPO), polyoxazoline (POZ), and hydroxyethyl starch (HES); iii) a heteropolymer of a small alkoxy monomer, preferably comprising polyethylene glycol / polypropylene glycol (PEG / PPG). Preferably, the water-soluble polymer has a molecular weight of at least about 120 Daltons and a polymerization number of at least 6 or about 6-210. Several lipid-PEG conjugates are commercially available.

[0062] In specific embodiments of the invention, the biocompatible lipid and water-soluble polymer conjugate is a conjugate of a polymer as defined above with a phospholipid as defined above or with vitamin E or a vitamin E derivative. Preferably, the conjugate is a phospholipid-PEG conjugate, such as 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG). More preferably, the conjugate is 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-mPEG2000) or d-α-tocopherol-N-[methoxy(polyethylene glycol)-1000 (TPEG1000).

[0063] Preferably, the surface of the nanoparticles is at least partially covered by a water-soluble polymer. More preferably, the water-soluble polymer covers at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of the surface of the nanoparticles. In another embodiment, the nanoparticles have a surface composed of a water-soluble polymer. The coating on the nanoparticles preferably covers the solvent-accessible surface of the nanoparticles.

[0064] It has been found that conjugates can be encapsulated in nanoparticles, particularly liposomes, with good encapsulation efficiency. Notably, the encapsulation efficiency of the conjugates is within a narrower bandwidth compared to that of unconjugated peptides (see Figure 13). Therefore, a method for encapsulating peptides in nanoparticles is provided, the method comprising the step of forming a peptide conjugate, wherein the conjugate is as described herein, followed by the step of forming nanoparticles. Preferably, in this method, the nanoparticles are liposomes, more preferably liposomes comprising neutral phospholipids and negatively charged phospholipids, and even more preferably liposomes as described above. This method is preferably used to encapsulate at least 30%, more preferably at least 35%, and even more preferably at least 40% of the peptide in nanoparticles. The peptide is preferably as described elsewhere herein, more preferably having a net charge of at most 8, preferably at most 7, more preferably at most 4, and still more preferably at most 3 at pH 7.

[0065] Uses and methods The conjugates or compositions according to the invention are suitable for use as pharmaceuticals. The pharmaceuticals can be used to treat immune or autoimmune diseases and / or inflammation, preferably for treating autoimmune diseases and / or inflammation, more preferably for autoimmune diseases. Suitable examples of immune or autoimmune diseases and / or inflammation are rheumatoid arthritis, neuropathy, rhinitis, systemic lupus erythematosus, thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), allergic reactions, transplant rejection, graft-versus-host disease, systemic sclerosis, atopic dermatitis, Graves' disease, Hashimoto's thyroiditis, vasculitis, Aumann's syndrome, chronic renal failure, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, diabetes, acute infectious mononucleosis, HIV, herpes virus-related diseases, multiple sclerosis, hemolytic anemia, thyroiditis, stiff-person syndrome, pemphigus vulgaris, myasthenia gravis, or lupus nephritis, more preferably for the treatment of rheumatoid arthritis, type 1 diabetes, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis, and even more preferably for the treatment of rheumatoid arthritis, type 1 diabetes, myasthenia gravis, or multiple sclerosis. Good results have been achieved in the treatment of rheumatoid arthritis. Good results have also been achieved in the treatment of multiple sclerosis.

[0066] Suitable examples of autoimmune diseases and / or inflammation include rheumatoid arthritis, systemic lupus erythematosus, thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), systemic sclerosis, Graves' disease, Hashimoto's thyroiditis, vasculitis, inflammatory bowel disease, multiple sclerosis, hemolytic anemia, thyroiditis, stiff-person syndrome, pemphigus vulgaris, myasthenia gravis, lupus nephritis, Crohn's disease, or ulcerative colitis, preferably rheumatoid arthritis, systemic lupus erythematosus, thrombocytopenic purpura, and idiopathic thrombocytopenic purpura (ITP). It is indicated for the treatment of intermittent thrombocytopenic purpura (ITP), systemic sclerosis, Graves' disease, Hashimoto's thyroiditis, vasculitis, inflammatory bowel disease, multiple sclerosis, hemolytic anemia, thyroiditis, stiff-person syndrome, pemphigus vulgaris, myasthenia gravis, lupus nephritis, and more preferably rheumatoid arthritis, type 1 diabetes mellitus, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis, and even more preferably for the treatment of rheumatoid arthritis, type 1 diabetes mellitus, myasthenia gravis, or multiple sclerosis. Good results have been achieved in the treatment of rheumatoid arthritis. Good results have been achieved in the treatment of multiple sclerosis.

[0067] This invention provides a sustained systemic effect. A preferred use is for reducing CD11c levels in subjects. + CD86 + The proportion of dendritic cells is preferably compared to PBS treatment. A preferred use is for increasing CD4 count. + CD25 + FoxP3 + The number of cells, or used to increase CD4 + PD-1 + The amount of T cells, or used to simultaneously increase CD4 + CD25 + FoxP3 + Cell count and CD4 + PD-1 + The number of T cells. This increase is preferably in the spleen. A preferred use is for reducing the amount of IgG1 or IgG2, or both IgG1 and IgG2, that are specific to the self-antigen peptide contained in the conjugate. In particular, compositions wherein the conjugate is contained in liposomes are suitable for use as a medicament for reducing the amount of IgG1 that is specific to the self-antigen peptide contained in the conjugate. A preferred use is to reduce IgG1 without reducing IgG2. A further preferred use is for increasing IL10 expression. A further preferred use is for increasing... IDO Gene expression. A further preferred use is for increasing LAP protein expression. Particularly preferred is for increasing IL10 expression, increasing... IDO The use of [this technology] to increase gene expression and protein expression of LAP. A further preferred use is for inducing antigen-specific CD49b [expression].+ LAG-3 + Tr1 cells. A further preferred use is for inducing tolDCs with a phenotype that promotes tolerance in T cells, characterized by cytokine secretion and gene expression profiles. A preferred use is for inducing tolerant DCs. A further preferred use is for inducing antigen-specific Tregs. The above uses provide additional therapeutic strategies for treating, preventing, or improving autoimmune diseases, possibly when combined with the following... The present invention also provides a method for treating autoimmune diseases and / or inflammation, the method comprising the step of administering a conjugate or composition according to the invention to a subject in need. Preferably, an effective dose is administered. This use can be for treating diseases and / or inflammation, alleviating symptoms of diseases and / or inflammation, preventing diseases and / or inflammation, or improving diseases and / or inflammation. Preferably, it treats, alleviates, prevents, or improves at least one symptom. Symptoms may be pain or swelling.

[0068] Generally, the conjugates and compositions according to the invention can be administered orally or via a parenteral route, typically by injection or infusion. "Parenteral route" means administration by injection other than enteral and local administration, and includes intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0069] The subject is preferably a mammal, such as a primate, cow, horse, camel, dog, cat, or mouse. Primates are particularly preferred subjects, such as humans or non-human primates. Most preferably, the subject is a human. The subject is preferably a subject in need of treatment, such as a subject suffering from an autoimmune disease or inflammation, or a subject susceptible to an autoimmune disease or inflammation. The need for treatment may be for the cure or relief of symptoms of an autoimmune disease or inflammation, but it can also be preventative treatment. In a preferred embodiment, the treatment is primary preventative treatment for the prevention of the onset of symptoms of an autoimmune disease. The inventors have found that the invention is also particularly suitable for secondary prevention; therefore, in other preferred embodiments, the treatment is secondary preventative treatment for the prevention of symptom recurrence after early treatment. In some embodiments, the subject is a young subject, preferably an adolescent subject, more preferably a newborn subject. In other preferred embodiments, the subject is an elderly person. The elderly subject is preferably over 50 years of age, more preferably over 60, even more preferably over 65, still more preferably over 70, and most preferably over 75. Alternatively, the subject may be over 30 years old, preferably over 35 years old, more preferably over 40 years old, and most preferably over 45 years old.

[0070] General definition Unless otherwise stated, each embodiment described herein may be combined together. All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0071] In the context of this invention, a decrease or increase in the parameter to be evaluated means a change corresponding to at least 5% in the value of the parameter. More preferably, the decrease or increase in the value means a change of at least 10%, or even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In the latter case, it may be that there is no longer a detectable value associated with this parameter.

[0072] As described in this document, the use of a compound or composition as a medicine can also be interpreted as its use in the manufacture of a medicine. Similarly, whenever a compound or composition is used as a medicine, it can also be used in the manufacture of a medicine or in a method thereof.

[0073] In this document and its claims, the verb “comprising” and its inflections are used in their non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. Furthermore, the reference to an element by the indefinite article “a” or “an” does not preclude the possibility of more than one element unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article “a” or “an” generally means “at least one”. The word “about” or “approximately” when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be 1% of a given value (10) plus or minus that value.

[0074] In the context of this invention, cells or samples can be cells or samples derived from samples obtained from a subject. Such obtained samples can be samples previously obtained from a subject. Such samples can be obtained from human subjects. Such samples can be obtained from non-human subjects. Attached Figure Description

[0075] Figure 1 - Free or encapsulated hPG-Dex in liposomes induced a tolerance phenotype in Balb / c BMDCs and human moDCs. Immature BMDCs or moDCs were stimulated overnight with LPS and hPG, hPG-Dex, hPG liposomes (Lip), or hPG-Dex Lip. (A) IL1B and (B) IDO The relative expression, based on HPRTExpression and normalization relative to the hPG group were measured by qPCR. The concentrations (pg / mL) of (C) IL-10 and (D) IL-12p70 in the supernatant of BMDC were measured using ELISA. Live CD11c + I TLR2 in BMDCs as measured by flow cytometry in the population + %, (F) LAP + %, (G) CD86 + % and (H) CD40 + %, and (I) CD86 in moDC + % and (J) CD40 + %. Average (+SD) p<0.01, p<0.001, p < 0.0001, determined by two-way ANOVA and Tukey multiple comparison test.

[0076] Figure 2 - Dexamethasone increases BMDC uptake by liposomes. (A) Liposomes were prepared as described above, with the addition of 0.02 mol% 1,1-bis(octadecyl-3,3,3,3-tetramethylindole-dicarbocyanine). Immature BMDCs were stimulated overnight with LPS and fluorescently labeled hPG liposomes (Lip) or hPG-Dex Lip. Fluorescently labeled CD11c levels in the liposomes were measured by flow cytometry. + (B) Immature BMDCs were stimulated overnight with LPS and hPG, hPG-Dex, hPG Lip, or hPG-Dex Lip. Active CD11c was measured using flow cytometry. + Cellular MerTK% % Mean + SD p < 0.0001, determined by t-test or one-way ANOVA and Tukey multiple comparison test.

[0077] Figure 3 - Antigen-specific T cell migration via hPG-Dex liposomes in vitro and in vivo. Immature BMDCs were stimulated overnight with hPG + LPS, hPG-Dex + LPS, hPG liposomes (Lip) + LPS, or hPG-Dex Lip + LPS. Cells were washed and CFSE-labeled CD4+ from hPG-TCR mice were added.+ T cells were co-incubated with BMDCs for 3 days. CFSE - CD4 + (A) CD25 in T cells + FoxP3 + Cellular percentage, (B) CD49b + LAG-3 + Cell % and (C) Tbet + Cell percentage, measured by flow cytometry. To Thy1.1 - Balb / c mice were intramuscularly injected with hPG protein, followed by intravenous injection of 500,000 Thy1.1 molecules. + hPG-TCR CD4 + T cells. One day later, mice were intravenously injected with 1 nmol hPG or 1 nmol hPG-Dex encapsulated in liposomes. Three days later, the mice were sacrificed, and the spleens were isolated for flow cytometry. (Thy1.1) + CD4 + (D)CD25 in T cells + FoxP3 + Cell percentage and I CD49b + LAG-3 + Cell percentage. Mean (+SD) p<0.05, p<0.01, p<0.001, p < 0.0001, determined by one-way ANOVA and Tukey multiple comparison test (in vitro) or Bonferroni multiple comparison test (in vivo).

[0078] Figure 4 -Free and encapsulated OVA 323K4 -Dex induces antigen-specific CD25 in vivo. + Foxp3+ Treg. To CD45.1 + Bl6 mice were intravenously injected with 500,000 OT-II CD45.2. + CD4 + T cells. One day later, mice were subcutaneously injected with 85 nmol OVA encapsulated in liposomes. 323 85 nmol OVA 323 -Dex or 1 nmol OVA 323-Dex. Seven days later, mice were sacrificed, and spleens were isolated for flow cytometry. Cells were stained with Viakrome, CD45.2, CD4, CD25, and Foxp3, and measured by flow cytometry. Mean +SD, p<0.05, p<0.01, p < 0.001, determined by one-way ANOVA and Bonferroni multiple comparison test.

[0079] Figure 5 - hPG-Dex liposomes inhibited the development of arthritis in mice. Arthritis was induced in female Balb / c mice by intraperitoneal injection of a mixture of 2 mg DDA and 250 μg human proteoglycan on days 0 and 21. (A) In the prevention model, mice were treated on day 17 by intravenous injection of PBS, hPG-Dex tolDC, or hPG-Dex liposomes. (B) In the cure model, mice were recruited after arthritis was established and administered intravenous injections of PBS, hPG-Dex liposomes, or OVA on days 0 and 7. 323 -Dex liposomes were used for treatment. Mean ± SEM, compared with the PBS group p<0.05, p<0.01, p<0.001, p < 0.0001, and compared with hPG-Dex tolDC, † p < 0.05, †† p < 0.01, and compared with OVA 323 Compared to -Dex liposomes, ## p<0.01, confirmed by two-way ANOVA and Bonferroni multiple comparison test. (C) anti-hPG IgG1 and (D) IgG2a antibodies in mouse serum were measured 25 days after the first injection. OD values ​​for each plate were normalized based on calibration curves. Mean +SD, p<0.05, p < 0.0001, determined by one-way ANOVA and Bonferroni multiple comparison test.

[0080] Figure 6- hPG-Dex liposomes enhance tolerance-inducing responses in arthritic mice. Arthritis was induced in female Balb / c mice by intraperitoneal injection of a mixture of 2 mg DDA and 250 μg human proteoglycan on days 0 and 21. Mice were recruited after arthritis was established and administered PBS, hPG-Dex liposomes, or OVA intravenously on days 0 and 7. 323 Mice were treated with Dex liposomes. Mice were sacrificed on day 25 and their organs were separated for analysis. (A) CD11c + CD86 + DC、(B)CD11c + PD-L1 + DC、(C)CD4 + PD-1 + T cells, (D)CD4 + CD25 + FoxP3 + Treg, I CD4 + RORγT + Th17 and (F) CD4 + Tbet + Th1 cells, % of all viable cells in the spleen, measured by flow cytometry. (G) in mouse paws. MPO, (H) IL1B and (I) IL10 The expression, using the Pfaffl method based on HPRT Expression was normalized and measured by qPCR. Mean + SD. p<0.05, p<0.01, p < 0.0001, determined by one-way ANOVA and Bonferroni multiple comparison test.

[0081] Figure 7 - Encapsulation efficiency of hPG (left), dexamethasone (middle), or hPG-Dex conjugate (right) in liposomes. The conjugation of Dex to hPG was found to increase the encapsulation of both components.

[0082] Figure 8 - Variations in conjugate design do not affect encapsulation efficiency (DSPC:DSPG:CHOL (4:1:2 molar ratio) liposomes). (A) Encapsulation of three different OVAs 323-Dex conjugates (using connectors KKKK (SEQ ID NO: 54) or EEEE (SEQ ID NO: 57) or SSSS (SEQ ID NO: 63)). Encapsulation efficiency remained high for all options. (B) Using the same three conjugates, the liposome size remained around 200 nm. (C) Using the same three conjugates, the polydispersity of the liposomes remained around 0.04–0.07. (D) Using the same three conjugates, the zeta potential of the liposomes remained around -55 to -60 mV.

[0083] Figure 9 -With OVA 323 Conjugated dexamethasone and prednisolone increase antigen-specific Tregs in vitro. Immature BMDCs were treated with LPS and free OVA. 323 +Dexamethasone, free OVA 323 Stimulation overnight with prednisolone, or a conjugated dexamethasone or prednisolone. After incubation, cells were washed and CD4 from OT-II mice were added. + T cells were co-incubated with BMDCs for 3 days. CD25 levels were measured by flow cytometry. + FoxP3 + Cell percentage. Mean (+SD) p<0.01, p<0.001, p < 0.0001, determined by one-way ANOVA and Tukey multiple comparison test.

[0084] Figure 10 -with free OVA 323 In comparison, antigen (here, OVA) 323 Conjugation with a glucocorticoid receptor agonist (Dex, using an E4 or K4 linker) increases antigen-specific Tregs in vivo. Encapsulation of the conjugate allows for a 200-fold dose reduction. Conjugation results in increased efficacy of either linker. Mean +SD, p<0.05, p<0.001, p < 0.0001, determined by one-way ANOVA and Bonferroni multiple comparison test.

[0085] Figure 11 - Variations in the selection of autoantigen peptides do not affect the encapsulation efficiency in DSPC:DSPG:CHOL (4:1:2 molar ratio) liposomes. (A) Encapsulation of four different Dex conjugates (using N-terminal linkers SEQ ID NO: 54), hPG = human proteoglycan (SEQ ID NO: 3), MOG = myelin oligodendrocyte glycoprotein (SEQ ID NO: 35), Ins = insulin (SEQ ID NO: 19), and AChR = acetylcholine receptor (SEQ ID NO: 33). Encapsulation efficiency remained high for all options. (B) Using the same four conjugates, the liposome size remained around 200 nm. (C) Using the same four conjugates, the polydispersity of the liposomes remained around 0.05–0.11. (D) Using the same four conjugates, the zeta potential of the liposomes remained around -50 to -60 mV.

[0086] Figure 12 -Stability of liposomes over time. Liposomes were stored at 4°C in 10 mM phosphate buffer (pH 7.4) for up to 40 months. Periodic DLS measurements revealed no change in liposome size. The conjugates tested were OVA-Dex and hPG-Dex, both of which have a linker (SEQ ID NO: 54). Peptides with linkers were also tested without Dex.

[0087] Figure 13 - Conjugates exhibit predictable encapsulation efficiency (EE). (A) EE of a series of peptides in DSPC:DSPG:CHOL liposomes reveals a significant positive correlation between peptide charge and EE. (B) EE of a series of peptide-Dex conjugates in the same liposome is independent of the net charge of the peptide.

[0088] Figure 14 - MOG-Dex conjugate prevents the development of EAE in mice (as a model of multiple sclerosis). Mice were injected with dexamethasone-conjugated MOG in liposomes, or with a control (empty liposomes, saline, or OVA). 323(Conjugates were used instead). Four days later, EAE was induced. Two days after EAE induction, mice were injected again with MOG-Dex or controls. (A) Mice were scored daily according to a five-point standardized rating scale for clinical symptoms: 0, no signs; 1, loss of tail tension; 2, tail relaxation; 3, mild hind limb paralysis; 4, hind limb paralysis; 5, death. Notably, only MOG-Dex showed almost complete prevention of EAE, while the three controls showed similarly worse results. (B) Mice were weighed daily. Notably, only MOG-Dex showed healthy weight characteristics, while the three controls showed similarly worse results. The legend for (B) is the same as that shown in (A).

[0089] Example Example 1. Materials and Methods Synthesis and characterization of Dex-peptide conjugates Preloaded Fmoc-Lys(Boc)-Wang resin, Fmoc-Arg(Pbf)-Wang resin, 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids, and trifluoroacetic acid (TFA) were purchased from Novabiochem GmbH (Hornbrunn, Germany). Peptide-grade dimethylformamide (DMF), dichloromethane (DCM), piperidine, N,N'-diisopropylcarbodiimide (DIC), and HPLC-grade acetonitrile were purchased from Biosolve BV (The Hague, Netherlands). Ethyl cyanohydroxyiminoacetate (Oxyma pure) was purchased from Manchester Organics Ltd (Cheshire, UK). Triisopropylsilane (TIPS), BioUltra grade ammonium bicarbonate, succinic anhydride, 4-dimethylaminopyridine (DMAP), and pyridine were purchased from Sigma-Aldrich Chemie BV (Zwindrecht, Netherlands). Dex was purchased from Acros Organics BV (The Hague, Netherlands).

[0090] Dex-peptide conjugates were synthesized using the previously described synthetic method. Briefly, the peptide epitope sequence was synthesized via microwave-assisted Fmoc-based chemical synthesis using an H12 liberty blue peptide synthesizer (CEM, USA). Dex succinate was coupled to the N-terminus of the peptide, as with other Fmoc-protected amino acids. The peptide was cleaved from the resin using TFA / water / TIPS (95 / 2.5 / 2.5) and the side-chain protecting groups were removed. The peptide was purified by preparative HPLC using a Reprosil-Pur C18 column (10 μm, 250 × 22 mm). Mass spectrometry (MS) analysis in positive mode using a Bruker microTOF-Q instrument confirmed the identity of the synthesized product. The epitopes were derived from hPG and ovalbumin (OVA) antigens having the sequences ATEGRVRVNSAYQDK (SEQ ID NO: 3) and ISQAVHAAHAEINEAGR (SEQ ID NO: 2), respectively. A lysine tetramer linker (SEQ ID NO: 54) was added to the N-terminus of the sequence to link the peptide to dexamethasone. The Dex-peptide conjugate was cleaved and purified as described above for the peptide. Unless otherwise stated, the linker (where present) is located at the N-terminus.

[0091] Liposome preparation and characterization Liposomes were prepared using an established membrane dehydration-rehydration method. Phospholipids 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC) and 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPG) were purchased from Avanti Polar Lipids, Birmingham, AL, USA. Cholesterol (CHOL) was purchased from Sigma-Aldrich. Briefly, a total of 180 mg of DSPC:DSPG:CHOL dry powder in a 4:1:2 molar ratio was weighed and transferred to a dry 100 mL round-bottom flask. The lipids were dissolved in 8 mL of chloroform and 8 mL of methanol. The solvent was evaporated under vacuum in a rotary evaporator at 40 °C for 1 h, followed by evaporation under a nitrogen stream at RT for 30 min. The resulting lipid membrane was treated with 2000 μg of hPG, hPG-Dex, or OVA dissolved in 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, pH 7.2) buffer. 323-Dex was rehydrated to a total volume of 4 mL and homogenized by rotation in a 40°C water bath for 1 h. For empty liposomes, the liposomes were rehydrated with 4 mL of 10 mM HEPES buffer. The resulting suspension was fractionated by high-pressure extrusion on a heated plate set to 60°C (LIPEX extruder, Northern Lipids Inc., Burnaby, British Columbia, Canada) by passing the dispersion four times through stacked 400 nm and 200 nm pore size membranes (Whatman® Nucleopore™, GE Healthcare, Amersham, UK). To separate unencapsulated cargo from the liposomes, the liposomes were ultracentrifuged at 55,000 rpm (70.1 Ti rotor) for 35 min at 4°C. This was repeated three times. The liposomes were stored at 4°C, and their stability was measured periodically. The liposomes were used for in vitro experiments within 2 months and for in vivo experiments within 2 weeks.

[0092] The Z-mean diameter and polydispersity index (PDI) of liposomes were measured by dynamic light scattering (DLS) using a NanoZS Zetasizer (Malvern Ltd., Malvern, UK). For this purpose, 10 μL of liposomes were diluted in 990 μL of HEPES buffer at pH 7.2. The zeta potential was measured by laser Doppler electrophoresis (Malvern Ltd.) using a universal dip cell. To determine the loaded hPG, hPG-Dex, and OVA... 323 or OVA 323 -Dex concentration was determined using RP-UPLC. For this purpose, 20 μL of liposome suspension was dissolved in 180 μL of methanol, and the sample was vortexed. The injection volume was 7.5 μL, and a 1.7 μm BEH C18 column (2.1 × 50 mm, Waters ACQUITY UPLC, Waters, Massachusetts, USA) was used. Column and sample temperatures were 40 °C and 20 °C, respectively. The mobile phase consisted of Milli-Q water containing 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B). For separation, the mobile phase was applied at a flow rate of 0.25 mL / min with a linear gradient of 5% to 95% solvent B for 10 min. Peptide content was detected by absorbance at 280 nm and by TUV detector at 240 nm using an ACQUITY UPLC (Waters ACQUITY UPLC, Waters, Massachusetts, USA). 39 Dex was detected at the site. The peptide concentration was calculated based on the corresponding calibration curve of the antigen-Dex complex dissolved in Milli-Q water.

[0093] mice For bone marrow isolation, 8-week-old male and female Balb / cAnNCrl background WT mice were purchased from Charles River Laboratories. Tyh1.1 + hPG-TCR transgenic mice were bred in-house at the central animal laboratory of Utrecht University, the Netherlands. For the study of proteoglycan-induced arthritis (PGIA), 16-week-old female Balb / cAnNCrl mice were purchased from Charles River Laboratories. Mice were randomly assigned to experimental groups based on weight or arthritis score using RandoMice. Humane endpoints were adhered to, and the animals with arthritis were provided with easily accessible water and food, as well as additional soft bedding to alleviate their discomfort. Animals were kept under standard conditions in the animal facility, and all experiments were approved by the relevant animal testing committee.

[0094] Isolation, culture, and stimulation of mouse bone marrow-derived dendritic cells (BMDCs) Bone marrow homogenates isolated from the femur and tibia of Balb / cAnNCrl WT mice were seeded at a cell density of 450,000 cells / mL in 2 mL of IMDM (Gibco, Thermo Fisher Scientific) in 6-well plates supplemented with 10% FCS (fetal bovine serum; Bodinco, Alkmaar, Netherlands), 100 units / mL penicillin (Gibco, Thermo Fisher Scientific, Lansmael, Netherlands), 100 μg / mL streptomycin (Gibco, Thermo Fisher Scientific, Lansmael, Netherlands), and 0.5 µM β-mercaptoethanol (Gibco, Thermo Fisher Scientific, Lansmael, Netherlands). Cells were cultured for 6 days at 37°C and 5% CO2 in the presence of 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF, internally produced). On day 2, IMDM and 20 ng / mL GM-CSF were added to the wells. Additional GM-CSF (20 ng / mL) was added on day 5. On day 6, cells were harvested by scraping and counted. For flow cytometry of DCs and further co-culture with T cells, cells were transferred to 96-well F-plates at 50,000 cells / well. Cell adhesion was allowed for 2 hours. Cells were matured in the presence of 10 ng / mL lipopolysaccharide (LPS, O111:B4; Sigma-Aldrich) and treated with free or encapsulated hPG, or free or encapsulated hPG-Dex (200 μL / well). In all cases, the peptide concentration was 1 μg / mL. For the Dex-containing group, the concentration was 0.18 μg / mL Dex. After 16 h, DCs were harvested for phenotypic characterization by flow cytometry. For qPCR and ELISA, cells were seeded at 600,000 cells / well in 48-well F-bottom plates. Cell adhesion was allowed for 2 hours. Cells were stimulated with the same conditions and concentrations as for flow cytometry, with a total volume of 600 μL / well.

[0095] Human monocyte isolation, monocyte-derived dendritic cell culture and stimulation Peripheral blood mononuclear cells (PBMCs) were obtained from healthy human donors at the Sanquin Blood Center (Amsterdam, Netherlands). PBMCs were isolated using a Ficoll gradient, followed by mononuclear cell isolation using anti-CD14 microbeads (Miltenyi Biotech) according to the manufacturer's instructions. Mononuclear cells were seeded at 2,000,000 cells / mL in 2 mL of RPMI (Gibberco) in 6-well plates supplemented with 5% FCS (Bodinco, Alkmaar, Netherlands), 100 units / mL penicillin (Gibberco, Thermo Fisher Scientific, Lansmer, Netherlands), and 100 μg / mL streptomycin (Gibberco, Thermo Fisher Scientific, Lansmer, Netherlands). To induce monocyte differentiation into dendritic cells (DCs), 50 ng / mL hGM-CSF (Miltenyi Biotech) and 50 ng / mL hIL-4 (Miltenyi Biotech) were added. On day 3 of culture, fresh medium and cytokines were added. On day 6, cells were harvested by scraping, counted, and transferred at 50,000 cells / well to 96-well F plates. Cell adhesion was allowed for 2 hours. Cells were matured in the presence of 100 ng / mL lipopolysaccharide (LPS, O111:B4; Sigma-Aldrich) and treated with free or encapsulated hPG, or free or encapsulated hPG-Dex (200 μL / well). In all cases, the peptide concentration was 1 μg / mL. For the Dex-containing group, the concentration was 0.18 μg / mL Dex. After 16 h, DCs were harvested for phenotypic characterization by flow cytometry.

[0096] Enriching CD4 from mouse spleen + T cells co-cultured with BMDC From Thy1.1 + Spleens were isolated from hPG-TCR mice. Single-cell suspensions of spleen cells were obtained by homogenizing the spleens through a 70 μM filter (Falcon, Corning, NY, USA). Red blood cells were lysed using ammonium chloride-potassium (ACK) lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA; pH 7.3). Anti-CD4+ was separated magnetically using Dynabeads™ (sheep anti-rat IgG, Thermo Fisher Scientific) and anti-CD8 (YTS169), anti-CD11b (M1 / 70), anti-MHCII (M5 / 114), and anti-B220 (RA3-6B2, all in-house manufactured). + T cells underwent negative selection. Following the manufacturer's protocol (Thermo Fisher Scientific), enriched CD4 cells were labeled with carboxyfluorescein succinimide ester (CFSE, 0.5 nM). +T cells. BMDCs were plated into 96-well F substrate plates (50,000 cells / well) and stimulated as described above. After 16 h of stimulation, cells were washed four times with 200 μL PBS / well to remove any free stimulants. CFSE-labeled CD4+ cells at 100,000 cells / well suspended in 200 μL RPMI (Gibberellic Acid) were then added. + T cells were incubated with RPMI supplemented with 5% FCS (Bodinco, Alkmaar, Netherlands), 100 units / mL penicillin (Gibco, Thermo Fisher Scientific, Lansmael, Netherlands) and 100 μg / mL streptomycin (Gibco, Thermo Fisher Scientific, Lansmael, Netherlands) for 3 days. CD4 cells were then harvested. + T cells are used for phenotypic characterization via flow cytometry.

[0097] Inflammatory adoptive metastasis in the body As mentioned above, from Thy1.1 + CD4 purified from the spleen and lymph nodes of hPG-TCR transgenic mice + T cells. At t0, WT Balb / cAnNCrl mice received an intramuscular injection of 50 μL PBS containing 100 μg hPG protein to induce a strong inflammatory response against hPG. Two hours later, the mice received 500,000 CD4+ cells via the tail vein. + T cells. Sixteen hours later, mice were intravenously immunized with 200 μL PBS, 1 nmol of free hPG, 1 nmol of hPG liposomes, or 1 nmol of hPG-Dex liposomes. Three days post-immunization, mice were sacrificed, and spleens were removed and processed as described above.

[0098] In vivo preventive arthritis study To induce arthritis in mice, female Balb / c mice were intraperitoneally injected with a mixture of 2 mg dimethyl dioctadecyl ammonium bromide (DDA) and 250 μg human proteoglycan on days 0 and 21. For mice treated with hPG-Dex tolDCs, BMDCs were cultured in 6-well plates as described above. On day 6, 40 μg / mL hPG-Dex and 10 ng / mL LPS were added to the cells. DCs were harvested 16 hours later. DC viability, purity, and phenotype were confirmed by flow cytometry before injection into mice. On day 17, the cells were intravenously injected with 200 μL PBS, 200 μL of 1 × 10⁻⁶ PBS, and 100 μL of 1 × 10⁻⁶ PBS. 6Mice were treated with hPG-DextolDC (equivalent to 20 nmol hPG-Dex) or 200 μL of hPG-Dex liposomes in PBS (2 nmol hPG-Dex). From day 21 to day 55, two researchers independently determined arthritis scores three times a week in a blinded manner using a visual scoring system based on paw swelling and redness. At the end of the experiment, mice were euthanized by cervical dislocation.

[0099] In vivo curative arthritis research To induce arthritis in mice, female Balb / c mice were intraperitoneally injected twice with a mixture of 2 mg DDA and 250 μg human proteoglycan, as described above. Arthritis scores were determined three times weekly, as described above. Mice were recruited for the experiment (day -1) when they had a score >2 for two consecutive scoring moments. On days 0 and 7, mice were intravenously injected via the tail vein with 200 μL PBS, 200 μL hPG-Dex liposomes in PBS (2 nmol hPG-Dex), or 200 μL OVA-Dex liposomes in PBS (2 nmol OVA). 323 Mice were treated with β-Dex. Mice were scored within 25 days after recruitment. At the end of the experiment, mice were euthanized by cervical dislocation. Spleens were collected for flow cytometry, paws were collected for qPCR, and blood was collected in 0.8 mL z-serum separation tubes (Greiner Bio-One, Kremsmünster, Austria). Serum was separated from cells by centrifuging the blood samples at 10,000 × g for 5 min at 4 °C, collected in separate tubes, and stored at -20 °C.

[0100] Stimulated BMDC ELISA Stimulate BMDCs as described above and harvest the supernatant for direct use in ELISA or store at -80°C for future analysis. IL-10 (U-CyTech, Utrecht, Netherlands) and IL-12p70 (9A5 and C17.8, BD Biosciences) in the supernatant were measured by ELISA according to the manufacturer's instructions. In short, F-bottom Coster assay 96-well plates (Corning, Kennebunk, Maine, USA) were coated overnight at 4°C with capture antibodies. The plates were thoroughly washed with 0.01% Tween-20 in PBS and blocked with 1% BSA in PBS at RT for 30 min. Subsequently, the plates were washed, and the (diluted) samples and standard curves were incubated at RT for 2 h. Then, the plates were washed, and the biotinylated detection antibody and streptavidin-HRP (BD Biosciences) were incubated at RT for 1 h. Finally, the plate was washed and the sample was reacted with the TMB substrate solution (BioLegend). The reaction was terminated with 2N H2SO4 solution, and the plate was measured using an iMark™ microplate absorbance reader (Bio-Rad). Cytokine concentrations were calculated based on the corresponding calibration curves prepared with purified cytokines.

[0101] qPCR of stimulated BMDC BMDCs were stimulated as described above, and 350 μL of RLT buffer (Qiagen Benelux BV, Venlo, Netherlands) was added to the cells. Lysates were used directly for mRNA extraction or stored at -80°C for future analysis. Total mRNA was extracted from stimulated BMDCs using the RNeasy kit (Qiagen) according to the manufacturer's instructions. DNase treatment was performed on a column (Qiagen). The mRNA extraction yield was measured using Nanodrop (Thermo Fisher Scientific). Transcription to cDNA was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories B.V., Vinedal, Netherlands). PCR and real-time detection were performed using Bio-Rad MyiQ iCycler (Bio-Rad). Amplification was performed using IQ™ SYBR Green® Supermix (Bio-Rad) with primers specific to the following at a final concentration of 0.25 µM: IL1B (5'-TCC ATC TTC TTC TTT GGG TAT TG-3' (SEQ ID NO: 44) and 5'-TTC CCG TGG ACC TTC CAG-3' SEQ ID NO: 45) and indoleamine 2,3-dioxygenase 1 (IDO)(5'-GCA GAC TGT GTC CTG GCA AAC T-3' (SEQ ID NO: 46) and 5'-AGA GAC GAG GAA GAAGCC CTT G-3' (SEQ ID NO: 47)) and hypoxanthine-guanine phosphoribosyltransferase ( HPRT (5'-CTGGTG AAA AGG ACC TCT CG-3' (SEQ ID NO: 48) and 5'-TGA AGT ACT CAT TAT AGT CAA GGGCA-3' (SEQ ID NO: 49)). The following PCR procedure was used: pre-soaking at 95°C for 3 min, [denaturation at 95°C for 20 sec, annealing at 59°C for 30 sec] repeated 40 times. Melting curves and primer efficiencies were measured for each sample. For each sample, mRNA expression was compared with detected levels. HPRT C t The values ​​are normalized and expressed relative to the average value of DC incubated with hPG + LPS.

[0102] qPCR of claw Harvest the paws of each mouse and combine them in a 6-well plate containing ice-cold sterile PBS. Remove the skin using scissors and forceps, and shake the paws to release synovial fluid. Pass the resulting suspension through a 70 μM filter (Falcon, Corning Incorporated, NY, USA) and centrifuge to pellet the cells. After removing the supernatant, lyse the cells using 350 μL RLT buffer (Qiagen Benallux GmbH, Venlo, Netherlands). Extract total mRNA immediately using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Transcription to cDNA was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories B.V., Vinedal, Netherlands). PCR and real-time detection were performed using Bio-Rad MyiQ iCycler (Bio-Rad). Amplification was performed using IQ™ SYBR Green® Supermix (Bio-Rad) with primers specific to the following at a final concentration of 0.25 µM: MPO (5'-GCT ACC CGC TTC TCC TTC TT-3' (SEQ ID NO: 50) and 5'-GGT TCT TGA TTC GAG GGT CA-3' (SEQ ID NO: 51)), IL1B (SEQ ID NO 44 and 45) IL10(5'-GGT TGC CAA GCC TTA TCG GA-3' (SEQ ID NO: 52) and 5'-ACC TGC TCC ACT GCCTTG CT-3' (SEQ ID NO: 53)) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) (SEQ ID NO 48 and 49'). The following PCR procedure was used: pre-soaking at 95°C for 3 min, [denaturation at 95°C for 20 sec, annealing at 59°C for 30 sec] repeated 40 times. Melting curves and primer efficiencies were measured for each sample. Using the PBS group as a control, the gene expression ratio of each gene of interest to HPRT was calculated using the Pfaffl method.

[0103] ELISA of serum anti-hPG IgG1 and IgG2a ELISA 96-well plates (Corning) were coated overnight with hPG (5 µg / mL per well) in 0.1 M carbonate buffer (pH = 9.5). The wells were then blocked at RT for 2 hours with blocking buffer consisting of 1.5% milk powder (Campina, Zalterbomer, Netherlands) dissolved in 1X PBS. Mouse serum was added to the wells at different dilutions (IgG1: 1:12500, 1:25000, 1:50000; IgG2a: 1:500, 1:2500, 1:12500). Each plate contained a standard curve consisting of serum from mice that had reached the humane endpoint of arthritis development (PGIA-induced, treatment-free), with dilutions of 0, 1:6250, 1:12500, 1:25000, 1:50000, and 1:100000 for IgG1, and dilutions of 0, 1:250, 1:500, 1:1000, 1:2000, and 1:4000 for IgG2a. Two hours later, IgG1-HRP (X56; BD Biosciences) and IgG2a-HRP (19-15; BD Biosciences) antibodies were added to wells in blocking buffer at a dilution of 1:1000. After incubation at RT for 1 hour, the wells were washed and TMB (Thermo Fisher Scientific) was added. The reaction was terminated using 2M H2SO4. ELISA data were read at 450 nm using an iMark™ microplate absorbance reader (Bio-Rad Laboratories). Background signal was subtracted (550 nm), and serum levels of anti-hPG IgG1 and IgG2a were calculated using a standard curve.

[0104] Flow cytometry Stimulate BMDCs or moDCs as described above and harvest them. Wash three times with 200 μL 4 mM EDTA and once with 200 μL PBS to remove any free antigens or liposomes, and transfer them to V-bottom 96-well plates. Harvest co-cultured CFSE-labeled CD4+. + T cells were extracted and transferred to V-bottom 96-well plates. For spleen cells from in vivo experiments, 2 × 10⁶ cells were transferred to each plate. 6 One spleen cell was plated in a 96-well U-shaped plate.

[0105] Cell suspensions were blocked with 10 μg / mL Fc Block (2.4G2, in-house manufactured) for 15 min. BMDCs were stained with a mixture of monoclonal antibodies against CD11c-APC (N418, eBioscience, Thermo Fisher Scientific), TLR2-FITC (6C2, eBioscience, Thermo Fisher Scientific), CD86-FITC (GL1, BD Bioscience), CD40-PE (3 / 23, BD Bioscience), LAP-PE (TW7-16B4, eBioscience, Thermo Fisher Scientific), MerTK-APC (2B10C42, Baijin Biotech), and ViaKrome808 (Beckman Coulter, Indianapolis, Indiana, USA) in FACS buffer (1X PBS supplemented with 2% FCS, 0.01% sodium azide and 2 mM EDTA). moDC was stained with a mixture of monoclonal antibodies in FACS buffer containing CD11c-PE (MJ4-27G12, Miltenyi Biotechnology), CD40 PE-Cy7 (5C3, ElectroBio), CD86-BB515 (FUN-1, BD Biosciences), and ViaKrome808 (Beckman Coulter, Indianapolis, WA, USA). The CD4 antibody was tested using a mixture of monoclonal antibodies in FACS buffer containing CD4-BV785 (RM4-5, Baijin Biotech, USA), LAG-3-PE (eBioC9B7W, ElectroBio Sciences, Thermo Fisher Scientific, USA), CD49b-APC-Cy7 (DX5, Baijin Biotech, USA), CD25-PerCP-Cy5.5 (PC61.5, ElectroBio Sciences, Thermo Fisher Scientific, USA), and ViaKrome808 (Beckman Coulter, Indianapolis, Indiana, USA). +T cell staining. After incubation at 4°C in the dark for 30 min, cells were washed with PBS and fixed and permeabilized using a FoxP3 transcription factor staining device (Electronic Biosciences, San Diego, California, USA). Subsequently, intracellular staining was performed on cells using FoxP3-eFluor450 (FJK-16s, Electron Biosciences, Thermo Fisher Scientific) and T-Bet-APC (4B10, Electron Biosciences, Thermo Fisher Scientific) according to the manufacturer's instructions. Spleen cells from adoptive transfer experiments were stained with CD4-BV510 (RM4-5, Bio-Tech, USA), Thy1.1-PerCP-Cy5.5 (HIS51, ElectroBioscience), Thy1.1-FITC (HIS51, ElectroBioscience), LAG-3-APC (C9B7W, ElectroBioscience), CD49b-APC-Cy7 (DX5, Bio-Tech), PD-L1-BV650 (10F.9G2, Bio-Tech), CD11c-FITC (N418, ElectroBioscience), CD86-PE-Cy5 (GL1, ElectroBioscience), and ViaKrome808 (Beckman Coulter, Indianapolis, Indiana, USA) in FACS buffer. After incubation at 4°C in the dark for 30 min, cells were washed with PBS and fixed and permeabilized using a FoxP3 transcription factor staining apparatus (Electronic Biosciences, San Diego, CA, USA). Subsequently, intracellular staining was performed using FoxP3-eFluor450 (FJK-16s, Electron Biosciences, Thermo Fisher Scientific) and T-bet-APC (4B10, Electron Biosciences) according to the manufacturer's instructions. Spleen cells from the curative arthritis research experiment were stained with a mixture of monoclonal antibodies in FACS buffer containing CD4-BV785 (RM4-5, Baijin Biotech, USA), CD25-PerCPCy5.5 (PC61.5, Electron Biosciences, Thermo Fisher Scientific), and ViaKrome808 (Beckman Coulter, Indianapolis, Indiana, USA). After incubating in the dark at 4°C for 30 min, the cells were washed with PBS and fixed and permeabilized using a FoxP3 transcription factor staining apparatus (Electronic Biosciences, San Diego, California, USA).Subsequently, intracellular staining was performed on cells using FoxP-eFluor450 (FJK-16s, ElectroBioSciences, Thermo Fisher Scientific), RORγT-PE (AFKJS-9, ElectroBioSciences, Thermo Fisher Scientific), GATA-3-PE-Cy7 (TWAJ, ElectroBioSciences, Thermo Fisher Scientific), and T-Bet-APC (4B10, ElectroBioSciences, Thermo Fisher Scientific) according to the manufacturer's instructions. After incubation at 4°C in the dark for 30 min, cells were washed and resuspended in 100 μL PBS for measurement. To ensure accurate analysis, relevant single staining and fluorescence minus one (FMO) controls were used. Samples were measured on a Beckman Coulter Cytoflex LX at the Faculty of Veterinary Medicine at Utrecht University's Flow Cytometry and Cell Sorting Facility. The total measurement volume was 85 μL per sample, and the measurement rate was 60 μL / min. Data were analyzed using FlowJo software v.10.7 (FlowJo LLC, Ashland, Oregon).

[0106] OVA for use with liposome encapsulation 323 Mice used for adoptive transfer assays with -Dex Eight-week-old female C57BL / 6-Ly5.1 and C57BL / 6-Tg(TcraTcrb)425Cbn / Crl(OTII) mice used for the adoption transfer experiment were purchased from Charles River Laboratories.

[0107] OVA encapsulated with liposomes 323 -Dex In vivo adoptive transfer According to the manufacturer's instructions (Mitianyi, Netherlands), use CD4. + T-cell enrichment kit for purifying CD4 from OT-II transgenic mice + T cells. On day -1, all CD45.1+ Ly5.1 mice received 500,000 CD4 cells intravenously via the tail vein. + T cells. On day 0, 85 nmol OVA encapsulated in liposomes was administered. 323 85 nmol OVA 323 -Dex or 1 nmol OVA 323 Mice were subcutaneously immunized with dex via injection into the left and right ventral sides (50 μL each). Seven days post-immunization, mice were sacrificed, and the spleens were removed and processed as described above. For FACS analysis, 2 × 10⁻⁶ cells were used.6 Spleen cells were plated in 96-well U-shaped plates. Cells were blocked with Fc Block (2.4G2, in-house produced) for 15 min. The cells were plated with a mixture of monoclonal antibodies against CD4-BV785 (RM4-5, Baijin Biotech, USA), CD45.2-PerCP-Cy5.5 (104, Electronic Biosciences), and CD25-BV650 (PC61, Baijin Biotech), and ViaKrome808 (Beckman Coulter, Indianapolis, . INSTORE, USA) in FACS buffer (1X PBS supplemented with 2% FCS and 2 mM EDTA). + T cell staining. After incubation in the dark at 4°C for 30 min, cells were washed with PBS and fixed and permeabilized using a FoxP3 transcription factor staining device (Electronic Biosciences, San Diego, CA). Subsequently, intracellular staining was performed using a FoxP-eFluor450 (FJK-16s, Electron Biosciences, Thermo Fisher Scientific) according to the manufacturer's instructions. Finally, cells were washed and resuspended in 100 μL PBS for measurement. To ensure accurate analysis, relevant single staining and fluorescence minus one (FMO) controls were used. Samples were measured on a Beckman Coulter Cytoflex LX at the Flow Cytometry and Cell Sorting Facility of the Faculty of Veterinary Medicine, Utrecht University. Data were analyzed using FlowJo software v.10.7 (FlowJo LLC, Ashland, Oregon).

[0108] Dosage determination of encapsulated or unencapsulated conjugates To CD45.1 + Bl6 mice were intravenously injected with 500,000 OT-II CD45.2. + CD4 + T cells. One day later, mice were subcutaneously injected with OVA encapsulated in liposomes. 323 OVA 323E4 -Dex, OVA 323K4 -Dex or OVA 323K4 -Dex. Seven days later, the mice were sacrificed and the spleens were isolated for flow cytometry. Cells were stained with Viakrome, CD45.2, CD4, CD25, and Foxp3, and measured by flow cytometry.

[0109] Statistical analysis Statistical analysis was performed using GraphPad Prism v.9.3.1. Details of the analysis are shown in the legend.

[0110] Example 2. Conjugates induce tolerant DCs, which in turn induce antigen-specific Tregs. Dexamethasone, whether free or encapsulated in liposomes, induces a tolerogenic phenotype in dendritic cells in vitro.

[0111] Arthritis-associated MHC-II autoantigen hPG and ovalbumin-derived MHC-II-restricted OVA 323-339 The antigen is extended with a linker to conjugate with Dex (thus forming hPG-Dex and OVA, respectively). 323 -Dex). The antigen was encapsulated in anionic DSPG liposomes. The liposomes were less than 200 nm in size and negatively charged. The LE of the antigen-Dex complex was between 46.5% and 49.6% (see Table 1). The conjugate without linkers had a loading efficiency of approximately 10%.

[0112] Table 1: Physicochemical characterization of Dex-loaded liposomes a Z-average diameter (Z ave ), mean ± SD, n = 3.

[0113] b LE% (loading efficiency) is calculated as the total amount of peptides before extrusion divided by the total amount of peptides after purification. 100%.

[0114] Liposomes were found to be particularly stable (see Figure 12 And it does not leak. Figure 12 Ultracentrifugation of the liposomes tested and measurement of the supernatant by UPLC showed no cargo leakage.

[0115] To evaluate tolerance induction to free or encapsulated Dex, immature BMDCs were stimulated overnight with LPS and free or encapsulated hPG and hPG-Dex. A free hPG control was coupled with the same linker used to conjugate hPG to Dex. Compared to the hPG control, when BMDCs were incubated with hPG-Dex or hPG-Dex encapsulated in liposomes, IL1B Gene expression and IL-12p70 secretion were significantly reduced. Encapsulating the antigen in Dex-free liposomes had the same effect. Figure 1 A and D). Free and encapsulated hPG-Dex increases Toll-like receptor 2 (TLR2) ( Figure 1 E) and latency-related proteins (LAP, the membrane-bound form of TGF-β) Figure 1 The expression of F). This is consistent with the reduced expression of the co-stimulatory molecules CD86 and CD40 in BMDC and moDC. Figure 1G, H, I, J). Surprisingly, encapsulation in liposomes enhanced the tolerogenic capacity of hPG-Dex, such as increased IDO gene expression (G, H, I, J). Figure 1 B) Release of IL-10 ( Figure 1 As demonstrated in C). We also observed that hPG-Dex liposomes were taken up more efficiently by BMDC compared to hPG liposomes without Dex. Figure 2 A), this may be due to an increase in the phagocytic receptor MerTK ( Figure 2 B).

[0116] Dex-linked hPGs induce antigen-specific Tregs in vitro and in vivo.

[0117] As shown above, the conjugate (here: hPG-Dex liposomes) can induce tolDCs with a phenotype that promotes cytokine secretion and gene expression profiles that enhance tolerance in T cells. Next, the effects of these tolDCs on antigen-specific T cells were assessed in vitro and in vivo. Both free and encapsulated hPG-Dex pulsed DCs increased antigen-specific CD25 levels. + FoxP3 + Treg ( Figure 3 A). Only the encapsulated hPG-Dex adds CD49b. + LAG-3 + Tr1 cells ( Figure 3 B). Both free and encapsulated hPG-Dex reduce antigen-specific T-bet. + Th1 cells, although free hPG-Dex is more effective than encapsulated hPG-Dex. In the primary adoptive transfer model, OVA 323 -Dex liposomes can amplify antigen-specific CD25 + FoxP3 + Treg ( Figure 4 In an inflammatory adoptive transfer model, intravenous injection of hPG liposomes induces antigen-specific CD25. + FoxP3 + The same applies to Treg and hPG-Dex liposomes, although to a lesser extent. Figure 3 D). Compared to free hPG antigen and Dex-free hPG liposomes, hPG-Dex liposomes do indeed significantly enhance antigen specificity to CD49b. + LAG-3 + Tr1 cells ( Figure 3 E). This indicates that hPG-Dex liposomes can induce a strong Tr1 response even in an inflammatory environment, which is hypothesized to be necessary for suppressing the response in arthritis models.

[0118] hPG-Dex liposomes inhibit the development of arthritis in mice. Based on the results, we hypothesize that hPG-Dex liposomes will provide optimal protection in an arthritis model. To evaluate the preclinical efficacy of hPG-Dex liposomes, we used a PGIA mouse model. First, we conducted a prophylactic study in which mice were intravenously injected with PBS (1 × 10⁻⁶ ppm) before developing arthritis. 6 One hPG-Dex tolDC (equivalent to 20 nmol of hPG-Dex), or hPG-Dex liposomes (2 nmol) Figure 5 A). Mice treated with hPG-Dex liposomes developed significantly less arthritis compared to mice treated with PBS and even hPG-Dex pulsed tolDC. Figure 5 A). In addition, 100% of mice in the PBS group developed arthritis (score of 2 or higher), while 75% and 27% of mice in the hPG-Dex pulsed tolDC and hPG-Dex liposome groups, respectively, developed arthritis (Table E1).

[0119] Table E1: Additional data from the PGIA mouse study All data are displayed as average or average ± SD.

[0120] a At the end of the study.

[0121] Next, we evaluated whether hPG-Dex liposomes could prevent the progression of arthritis in mice with persistent inflammation. Figure 5 B). In this model, we observed differences compared to PBS and OVA. 323 Compared to -Dex liposomes, they did indeed stabilize arthritis in mice. Figure 5 B). Compared to the day of the first injection, PBS and OVA... 323 In the hPG-Dex liposome group, 100% of mice had increased arthritis scores, while 33% of mice treated with hPG-Dex liposomes had lower scores at the end of the experiment compared to at the start (Table E1). After sacrifice, anti-hPG IgG1 and IgG2a levels were measured in mouse serum. Compared to other groups, mice receiving hPG-Dex liposomes generally had lower levels of anti-hPG IgG1 but not lower levels of IgG2a. Figure 5 (C and D).

[0122] Further analysis of the spleens of mice sacrificed on day 25 after the first injection of liposomes or in the control group showed that, in mice receiving hPG-Dex liposomes, CD11c levels were significantly lower compared to other groups. + CD86 + DC% decrease ( Figure 6 A). This is related to CD25. + FoxP3 + and PD-1 + The increase in regulatory T cells was consistent ( Figure 6 C and D). PD-L1, RORγT + and Tbet + The group was slightly affected. Figure 6 (B, E, and F). Interestingly, in the mouse's paw, IL10 The expression of [a substance] was enhanced in mice receiving hPG-Dex liposomes. Figure 6 I), and MPO and IL1B non-significant reduction ( Figure 6 G and H).

[0123] MOG-Dex liposomes inhibit the development of multiple sclerosis in mice. Encapsulating the MOG-Dex conjugate in liposomes prevented the development of EAE (autoimmune encephalomyelitis) in mice. Eleven-week-old mice were intravenously injected with MOG (SEQ ID NO: 35) conjugated with dexamethasone in DSPC:DSPG:CHOL liposomes. Parallel, other mice were injected with OVA conjugated with dexamethasone in DSPC:DSPG:CHOL liposomes. 323 Alternatively, an equal volume of DSPC:DSPG:CHOL liposomes or saline solution can be injected. Four days later, following the manufacturer's guidelines (Hooke Laboratories, Lawrence, USA), 200 µg of recombinant human myelin oligodendrocyte glycoprotein MOG, emulsified in 100 µl of complete Freund's adjuvant supplemented with 4 mg / ml Mycobacterium tuberculosis (H37RA strain), is injected subcutaneously at the tail root. 35-55 EAE was induced in mice. 0.1 ml of pertussis toxin was injected intraperitoneally into mice within 2 h and 24 h afterward. Two days after EAE induction, mice were again injected with liposomes or as a control. Mice were weighed and scored daily according to a five-point standardized clinical symptom rating: 0, no signs; 1, loss of tail tone; 2, relaxed tail; 3, mild hind limb paralysis; 4, hind limb paralysis; 5, death.

[0124] discuss Restoring immune tolerance is associated with the treatment of autoimmune and chronic inflammatory diseases. Current clinical trials are using dendritic cells (DCs) pulsed with immunomodulators and disease-associated antigens (DAAs) to achieve this (Bell et al. 2015). Despite encouraging results, the production of these tolDCs requires specialized research centers and is costly, making them inaccessible to the large number of patients in need of treatment. Therefore, a strategy to overcome these limitations is needed. Nanoparticles, including liposomes, are promising drug delivery mediators that can exceed the need for in vitro cultured tolDCs (Benne et al. 2022). Here, we prepare liposomes for delivering conjugates to induce antigen-specific immune tolerance in vitro, in vivo, and in preclinical models of RA. We conjugate Dex with our antigen of interest to prevent APCs from taking up Dex in a non-antigen-specific environment, thereby preventing non-antigen-specific effects. Furthermore, we hypothesize that liposomes promote more efficient uptake of antigen-Dex conjugates by APCs than free antigen-Dex conjugates, which allows us to significantly reduce the required dose, thereby further minimizing side effects.

[0125] BMDC and moDC exhibited a tolerance-inducing phenotype after stimulation with free hPG-Dex. Figure 1 This indicates that the binding of Dex to antigens (such as hPG) surprisingly did not impair Dex function. Similarly, it was found that encapsulating hPG-Dex into liposomes surprisingly did not impair the immunomodulatory effects of Dex. Figure 1 and Figure 2 Conversely, compared to free hPG-Dex, stimulation of DCs with hPG-Dex liposomes increased IDO Gene expression ( Figure 1 B) Release of IL-10 ( Figure 1 C) and protein expression of LAP ( Figure 1 F). Interestingly, the addition of Dex enhanced some of the tolerogenic properties of DSPG-liposomes (F). Figure 1 Furthermore, Dex stimulates liposome uptake, possibly due to an increase in the phagocytic receptor MerTK ( ). Figure 2 This increased uptake of inherently tolerogenic liposomes, along with the tolerogenic properties of Dex, may explain why Dex-containing liposomes exhibit a more favorable tolDC phenotype. Furthermore, encapsulation is shown to be more effective for conjugates compared to single components. Figure 7 ), and shows that aspects of the conjugate design (such as the choice of connectors) do not affect this encapsulation ( Figure 8 ).

[0126] We used TCR-specific transgenic CD4 +T-cell studies showed that Dex conjugation to antigens via the adapter does not prevent MHC presentation of DCs and subsequent TCR recognition by antigen-specific T cells, as we observed an effective T-cell response. Most notably, BMDCs treated with hPG-Dex liposomes showed increased CD49b levels in in vitro co-culture experiments. + LAG-3 + Tr1 count ( Figure 3 (B) While free hPG-Dex and Dex-free hPG liposomes do not induce Tr1 cells. In an in vivo inflammatory adoptive transfer model, hPG-Dex liposomes were confirmed to induce antigen-specific CD49b. + LAG-3 + Tr1 cells ( Figure 3 E).

[0127] Because hPG-Dex liposomes induce effective Tr1 responses, and these responses are important for protecting against arthritis (Volz et al. 2013), we tested these liposomes in a mouse model of PGIA arthritis. Compared with mice receiving hPG-DextolDC and PBS, hPG-Dex liposomes significantly reduced arthritis development (…). Figure 5 A). It should be noted that dexamethasone-loaded antigen-induced tolDC is a known therapy for rheumatoid arthritis (Jansen et al. 2019), but the present invention produces better results ( Figure 5 A). Importantly, hPG-Dex liposomes prevented further progression of arthritis in mice with an already identified disease. Figure 5 B). Furthermore, the lack of arthritis development in mice receiving hPG-Dex liposomes was more pronounced than in previous studies of tolDC therapy using pulses of arthritis-related drugs in mouse models (Jansen et al. 2019; Hilkens et al. 2013).

[0128] Mechanistically, we showed that mice treated with hPG-Dex liposomes had a significantly lower proportion of CD11c compared to the PBS group. + CD86 + DC% ( Figure 6 A). Meanwhile, these mice had significantly higher levels of CD4 in their spleens. + CD25 + FoxP3 + and CD4 + PD-1 + T cells ( Figure 6 (C and D). This indicates that the therapy has some durable systemic effects, which appear to be antigen-specific, because during the administration of OVA... 323This effect was not observed in mice with -Dex liposomes. + CD25 + FoxP3 + and CD4 + PD-1 + T cells may help prevent arthritis, or it could be a result of reduced inflammation in mice. Nguyen et al. also observed CD86 levels in the spleen of mice treated with nanoparticles. + APC reduction and Foxp3 + CD4 + The increase in T cells is consistent with protection against experimental autoimmune encephalomyelitis (EAE, see Nguyen et al. 2022). Interestingly, we observed an increase in T cells in the paws of mice treated with hPG-Dex liposomes. IL10 The expression of was significantly increased ( Figure 6 I), which could explain why these mice had lower arthritis scores than the control group.

[0129] Liposomes have been used as delivery vehicles to target Dex to inflamed joints, thereby reducing arthritis symptoms through Dex's broad-spectrum anti-inflammatory properties. To test the antigen specificity of our treatment and the efficacy of liposomal Dex delivery in arthritic mice, we used OVA 323 -Dex liposome therapy in mice, OVA 323 It is an MHC-II antigen unrelated to the disease. We did not observe OVA in any assays. 323 -Changes between Dex liposomes and PBS ( Figure 5 and 6 This is expected, as liposome functionalization via PEGylation, peptides (Meka et al. 2019), or other small molecules on the liposome surface is necessary to achieve liposome accumulation in inflamed joints through the inflammation-associated leaky vascular system. Furthermore, studies reporting Dex delivery to inflamed sites via nanoparticles have reported doses of Dex that are 5 to 30 times higher than those used in this study (0.1–1.2 mg / kg vs. 0.02 mg / kg), and often require more than two injections. Therefore, we can conclude that the effect observed in our study is not due to the accumulation of Dex liposomes in the joint (which affects inflamed tissue), but rather to antigen-specific bias in T-cell responses to arthritis.

[0130] Mice treated with MOG-Dex liposomes maintained a healthier weight and scored particularly low on standardized rating scales for EAE-related clinical symptoms (Figure 14). Saline, empty liposomes, or conjugates containing substances unrelated to multiple sclerosis (i.e., OVA) were also used. 323Negative controls for MOG-Dex liposomes showed comparable results to each other, but differed from those treated with MOG-Dex. Negative controls all showed an overall increase in the five-point score and an overall decrease in body weight following EAE induction. This confirms that MOG-Dex liposomes have beneficial effects related to their peptides, rather than to Dex delivery.

[0131] Our conjugates offer a promising strategy for inhibiting the development of diseases such as arthritis or multiple sclerosis. The results presented in this article highlight the therapeutic potential of DSPG-containing liposomes loaded with antigen-Dex in immunotherapy for autoimmune diseases.

[0132] References Bell GM, Anderson AE, Diboll J, et al. Autologous tolerogenicdendritic cells for rheumatoid and inflammatory arthritis. Ann Rheum Dis .2017;76(1):227-234. doi:10.1136 / annrheumdis-2015-208456 Benne N, ter Braake D, Stoppelenburg AJ, Broere F. Nanoparticles for Inducing Antigen-Specific T Cell Tolerance in Autoimmune Diseases. Front Immunol . 2022;13. doi:10.3389 / fimmu.2022.864403 Volz T, Skabytska Y, Guenova E, et al. Nonpathogenic bacteriaalleviating atopic dermatitis inflammation induces IL-10-producing dendritic cells and regulatory Tr1 cells. Journal of Investigative Dermatology . 2014;134(1):96-104. doi:10.1038 / jid.2013.291 Jansen MAA, Spiering R, Ludwig IS, van Eden W, Hilkens CMU, Broere F.Matured tolerogenic dendritic cells effectively inhibit autoantigen specificCD4+ T cells in a murine arthritis model. Front Immunol . 2019;10(AUG). doi:10.3389 / fimmu.2019.02068 Hilkens CMU, Isaacs JD. Tolerogenic dendritic cell therapy forrheumatoid arthritis: Where are we now? Clin Exp Immunol . 2013;172(2):148-157.doi:10.1111 / cei.12038 Nguyen TL, Choi Y, Im J, et al. Immunosuppressive biomaterial-basedtherapeutic vaccine to treat multiple sclerosis via re-establishing immunetolerance. Nat Commun . 2022;13(1). doi:10.1038 / s41467-022-35263-9 Meka RR, Venkatesha SH, Acharya B, Moudgil KD. Peptide-targetedliposomal delivery of dexamethasone for arthritis therapy. Nanomedicine . 2019;14(11):1455-1469. doi:10.2217 / nnm-2018-0501。

Claims

1. A conjugate comprising: (i) Glucocorticoid receptor agonists, and (ii) Peptides with self-antigenicity.

2. The conjugate according to claim 1, wherein the glucocorticoid receptor agonist is (i) Corticosteroids, preferably dexamethasone, triamcinolone, methylprednisolone, prednisolone, or prednisolone, more preferably dexamethasone or prednisolone, most preferably dexamethasone, or (ii) Synthesize a nonsteroidal glucocorticoid receptor agonist, preferably dagcourt, AZD-5423, fudacourt or mapcort.

3. The conjugate according to claim 1 or 2, wherein the peptide is associated with an autoimmune disease, such as rheumatoid arthritis, spondyloarthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis, Sjögren's disease, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, idiopathic thrombocytopenic purpura, alopecia, vitiligo, type 1 diabetes mellitus, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis, preferably an autoantigen associated with rheumatoid arthritis or multiple sclerosis.

4. The conjugate according to any one of claims 1-3, wherein the peptide is derived from a protein selected from: human proteoglycan; insulin; insulin precursor; proinsulin; proinsulin; melanin; topoisomerase 1; topoisomerase 2; glutamate decarboxylase, such as glutamate decarboxylase 2 or glutamate decarboxylase 65; collagen, such as type II collagen; citrullinated human proteoglycan; α-enolase; citrullinated α-enolase; cartilage mesothelial protein; citrullinated cartilage mesothelial protein; fibrinogen; citrullinated fibrinogen; vimentin; citrullinated vimentin; acetylcholine receptor; myelin proteins, such as myelin oligodendrocyte glycoprotein, myelin lipoprotein or myelin basic protein; thyroid-stimulating hormone receptor and smooth muscle, preferably derived from human proteoglycan.

5. The conjugate according to any one of claims 1-4, wherein the peptide comprises a sequence of 6 to 60, preferably 10 to 40, more preferably 12 to 20 consecutive amino acids having a sequence from the protein of claim 4, wherein the consecutive sequence may have zero, one, two or three amino acid substitutions.

6. The conjugate according to any one of claims 1-5, wherein the peptide has a length of 6 to 70 amino acids, preferably 10 to 40 amino acids, more preferably 12 to 20 amino acids.

7. The conjugate according to any one of claims 1-6, wherein the peptide comprises or is composed of the following: The sequence is SEQ ID NO: 3-43, preferably SEQ ID NO: 3-18, more preferably any one of SEQ ID NO: 3-11, or even more preferably the sequence of SEQ ID NO:

3.

8. The conjugate according to any one of claims 1-7, wherein the glucocorticoid receptor agonist is attached to the end of the peptide.

9. The conjugate according to claim 8, wherein the glucocorticoid receptor agonist is linked to the N-terminus of the peptide.

10. The conjugate according to any one of claims 1-9, wherein the glucocorticoid receptor agonist and the peptide are linked by a linker, wherein the linker is preferably a peptide linker comprising 1 to about 12 amino acids, more preferably 2 to about 8 amino acids, more preferably about 3 to about 6 amino acids, and most preferably 4 or 5 amino acids, wherein preferably, these amino acids are hydrophilic.

11. The conjugate according to any one of claims 1-10, wherein the glucocorticoid receptor agonist is dexamethasone, and wherein the peptide is an autoantigen associated with rheumatoid arthritis or multiple sclerosis.

12. A composition comprising the conjugate according to any one of claims 1-11, wherein the conjugate is contained in nanoparticles.

13. The composition of claim 12, wherein the nanoparticles are micelles, polymer nanoparticles, polysaccharide nanoparticles, liposomes, lipid complexes, monolayer vesicles, multilayer vesicles, or cross-linked or hybrid variants thereof.

14. The composition of claim 12, wherein the nanoparticles are liposomes.

15. The composition according to any one of claims 12-14, wherein the nanoparticles comprise one or more phospholipids, preferably two phospholipids, wherein these phospholipids are preferably selected from 1,2-dilauroyl-sn-glycerol-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dimyristoyl Dimylmyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1,2-dimylmyl-sn-glycerol-3-phosphate glycerol (DMPG), 1,2-dimylmyl-sn-glycerol-3-phosphate serine (DMPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ester (DPPA), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycerol- 3-Glyceryl phosphate (DPPG), 1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine (DPPS), 1,2-distearyl-sn-glycerol-3-phosphate ester (DSPA), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate glycerol (DSPG), 1,2-distearyl-sn-glycerol-3-phosphoserine (DSPG) S), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate serine (DOPS), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), or polymer-conjugated phospholipids, such as polyethylene glycol phospholipids or polyglycerol phospholipids.

16. The conjugate according to any one of claims 1-11 or the composition according to any one of claims 12-15, for use as a pharmaceutical product.

17. The conjugate or composition for use according to claim 16, wherein the medicament is used to treat immune or autoimmune diseases and / or inflammation, preferably for treating rheumatoid arthritis, systemic lupus erythematosus, thrombocytopenic purpura (ITP), systemic sclerosis, Graves' disease, Hashimoto's thyroiditis, vasculitis, inflammatory bowel disease, multiple sclerosis, hemolytic anemia, thyroiditis, stiff-person syndrome, pemphigus vulgaris, myasthenia gravis, lupus nephritis, Crohn's disease, or ulcerative colitis, more preferably for treating rheumatoid arthritis, type 1 diabetes mellitus, myasthenia gravis, multiple sclerosis, Graves' disease, or autoimmune hepatitis.

18. The conjugate or composition for use according to claim 16, wherein the medicament is used to treat rheumatoid arthritis or multiple sclerosis.

19. The conjugate or composition for use according to claim 16, wherein the medicament is for treating rheumatoid arthritis, and wherein the peptide is an autoantigen associated with rheumatoid arthritis.

20. The conjugate or composition for use according to claim 16, wherein the medicament is for treating multiple sclerosis, and wherein the peptide is an autoantigen associated with multiple sclerosis.

21. A method for treating autoimmune diseases and / or inflammation, the method comprising administering to a subject in need the conjugate according to any one of claims 1-11 or the composition according to any one of claims 12-15.

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