Peptide-associated nanoparticle composition

Abstract

The present invention relates to a composition comprising sHDL nanoparticles. More specifically, the present invention relates to sHDL nanoparticles comprising a phospholipid, an apolipoprotein mimetic, a thiol-reactive lipid, and a peptide comprising a linker portion connected to a payload portion, wherein the linker comprises cysteine ​​(C) and 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), the payload comprises a polypeptide having a length of 5 to 35 amino acids and a positive net charge at pH 7 to 12, the peptide having a negative net charge at pH 7 and an isoelectric point of 0.4 to 12, and the peptide being covalently bonded to the thiol-reactive lipid via cysteine ​​(C).

Description

Detailed description of the invention

[0001] [Technical field] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 522,816, filed on 23 June 2023, which is incorporated herein by reference in its entirety.

[0002] Statement regarding government support This invention was made with government support through grant DE030691 from the National Institutes of Health. The U.S. Government has certain rights to this invention.

[0003] Sequence List The complete text of the computer-readable sequence listing file, filed on June 18, 2024, with a file size of 671,386 bytes and filename "UM_41715_601_SequenceListing", which is submitted with this specification, is incorporated herein by reference.

[0004] The present invention relates to a composition comprising sHDL nanoparticles. More specifically, the present invention relates to sHDL nanoparticles comprising a phospholipid, an apolipoprotein mimetic, a thiol-reactive lipid, and a peptide comprising a linker portion connected to a payload portion, wherein the linker comprises cysteine ​​(C) and 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), the payload comprises a polypeptide having a length of 5 to 35 amino acids and a positive net charge at pH 7 to 12, the peptide having a negative net charge at pH 7 and an isoelectric point of 0.4 to 12, and the peptide being covalently bonded to the thiol-reactive lipid via cysteine ​​(C).

[0005] [Background technology] Peptide-based therapies have attracted considerable interest as candidates for clinical application due to their high specificity, potency, low toxicity, and good tolerability [1, 2]. Furthermore, a major advantage of peptide-based therapies is the ability to generate different specific sequences, which can provide a vast range of functional diversity. To date, more than 80 peptide-based drugs have been approved for the treatment of various diseases, including HIV infection, chronic pain, cancer, diabetes, multiple sclerosis, and osteoporosis [3]. Generally, peptides are signaling molecules that induce intracellular actions by binding to specific cell surface receptors or ion channels. However, most peptides cannot autonomously exit endolysosomes or cross cell membranes [4]. In addition, the use of peptide drugs is greatly limited by their low plasma stability, short circulation time, low oral bioavailability, and high cost of large-scale manufacturing [5].

[0006] These drawbacks of peptides are expected to be overcome by the development of novel drug delivery systems. To this end, numerous peptide / protein delivery systems and various other approaches [13-16] have been proposed, including those using lipid nanocarriers [6, 7], polymers [8, 9], mesoporous silica nanoparticles

[10] , and cell-permeable peptides [11, 12]. Surprisingly, synthetic high-density lipoprotein (sHDL) nanodiscs (NDs) have shown promise as carriers in peptide delivery studies [17, 18]. sHDL NDs possess all the advantages of lipid nanoparticles and liposomes, and furthermore, they surpass those advantages by exhibiting long-term circulation, good tolerability, and high stability

[19] . In addition, sHDL is a promising carrier for tissue penetration and high intracellular accumulation due to its ability to internalize cells via receptors and its inherently small size of approximately 10 nm, making it particularly useful for applications such as drug delivery to tumors [20-22].

[0007] Various sHDL-based ND delivery systems consisting of phospholipids and apolipoprotein A1-mimicking peptides have been developed to date [23-28]. One conventional approach involves adding a "Cys-Ser-Ser" linker to the N-terminus of a peptide and using the thiol chemical properties to bind the peptide to the lipid. The resulting peptide-lipid conjugate is then loaded onto a pre-formed ND. Although this method worked for many peptides, it has been shown that this approach is not universally applicable. For example, hydrophobic peptides and peptides with isoelectric points near the pH of the solution tend to form aggregates when incorporated into NDs [29, 30]. Furthermore, while peptides with a neutral charge tend to aggregate, peptides with isoelectric points that are strongly positively charged at pH 7 also form aggregates [31, 32]. In this regard, arginine aggregates more readily than lysine, which is attributed to the Arg side chain's higher tendency to form protein-protein interactions

[33] .

[0008] Improved compositions and methods for delivering peptides are needed.

[0009] This invention addresses these requirements.

[0010] [Overview of the prefecture] Experiments conducted during the development of embodiments of the present invention have led to the development of a novel method for improving the water solubility of peptides and the productivity of peptide-loaded sHDL ND by attaching negatively or positively charged amino acids to the C-terminus or N-terminus. These experiments demonstrated that for peptides with high isoelectric points (pI), adding negatively charged amino acids (such as aspartic acid or glutamic acid) to the peptide sequence converts the net charge at pH 7 from positive to negative. This charge conversion enables efficient and stable loading of the peptide-lipid conjugate into ND, resulting in homogeneous and uniform peptide-loaded ND. Therefore, the addition of negatively or positively charged amino acids to the C-terminus or N-terminus of a peptide provides a universal method for improving peptide incorporation into ND and for improving the productivity of peptide-ND products. Furthermore, our approach demonstrates the versatility of using peptides with cysteine ​​residues not only at the N-terminus but also at the C-terminus or non-terminal positions in obtaining peptide-loaded sHDL ND. Overall, the embodiments described herein relate to novel compositions that improve the incorporation of various peptides into NDs and the manufacturability of peptide-NDS products. Furthermore, this approach is considered to be broadly applicable to the incorporation of various peptides into other nanoparticles and polymers.

[0011] Accordingly, in certain embodiments, the present invention provides compositions comprising sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a phospholipid, an apolipoprotein mimetic, a thiol-reactive lipid, and a peptide comprising a linker moiety connected to a payload moiety, wherein the s-linker comprises either [cysteine ​​(C) and optionally 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S)] or [DD or DDD if the payload contains a C amino acid], the payload comprises a polypeptide having a net positive charge at pH 7 to 12 and a net negative charge at pH 7 and an isoelectric point of 0.4 to 12, and the peptide is covalently bonded to the thiol-reactive lipid via the linker.

[0012] In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the carboxyl terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the amino terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the non-terminal position of Cys.

[0013] In some embodiments, when the payload contains a C amino acid, the peptide is covalently bonded to a thiol-reactive lipid via a linker, the linker comprising DD or DDD.

[0014] In some embodiments, the peptide includes the following formula: [Linker]-[Payload] or [Payload]-[Linker].

[0015] In some embodiments, the payload has a positive net charge at pH 7–12. In some embodiments, the payload has a net charge of 0 at pH 7–12. In some embodiments, the payload has a negative net charge at pH 7–12.

[0016] In some embodiments, the peptide has a charge less than -0.1. In some embodiments, the peptide has a charge in the range of -0.1 to -5.0.

[0017] In some embodiments, the peptide has an isoelectric point of 3.7 to 12. In some embodiments, the peptide has an isoelectric point of 0.62 to 9.78.

[0018] In some embodiments, the linker sequence is C. In some embodiments, the linker sequence is DDCDD. In some embodiments, if the payload contains the C amino acid, the linker sequence is DD or DDD.

[0019] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide dimer. In some embodiments, the peptide dimer is selected from DD, SE, SD, and EE.

[0020] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide trimer. In some embodiments, the peptide trimer is selected from DDD, EEE, and KEE.

[0021] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide tetramer. In some embodiments, the peptide tetramer is selected from DDDD (SEQ ID NO: 762) and EEEE (SEQ ID NO: 763).

[0022] In some embodiments, the payload has a charge greater than 0.1 at pH 7. In some embodiments, the payload has a charge in the range of 0.1 to 5.0. In some embodiments, the payload is a polypeptide with a length of 12 to 35 amino acids.

[0023] In some embodiments, the payload is selected from GWYRSPFSRVVHL (SEQ ID NO: 764), NTWTTSQSIAFPSK (SEQ ID NO: 765), KVAPVWVRMME (SEQ ID NO: 766), KYNKANAFL (SEQ ID NO: 767), and ASFEAQGALANIAVDKA (SEQ ID NO: 768).

[0024] In some embodiments, the apolipoprotein mimetic is SEQ ID NOs: 1-336, and WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347), PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354) )QTVTDYGKDLME(SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE(SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV(SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL(SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS(SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS(SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS(SEQ ID NO: 361), VVALLALLASARASEAEDASLL( SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),It is an ApoA-I mimetic having any one of the sequences PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0025] In some embodiments, the apolipoprotein mimetic has the sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4).

[0026] In some embodiments, the thiol-reactive lipid is selected from dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-di-(9Z-octadecenoyl))-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-maleimide (DOPE-Mal), and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide].

[0027] In some embodiments, the thiol-reactive lipid is DOPE-PDP.

[0028] Further embodiments will be apparent to those skilled in the art based on the teachings contained herein.

[0029] [Brief Description of the Drawings] [Figure 1] This figure shows the particle size distribution of ND-SIINFEKL (with CSS, CSE, or CEE linker) and ND-Ea (with CSS or CSE linker) measured by DLS in 10 mM phosphate buffer (pH=7.4).

[0030] [Figure 2] This shows the particle size distribution of ND-MOG38-50 (with CSS, CDD, or CDDD linker) and ND-PLP178-191 (with CSS or CDD linker) measured by DLS in 10 mM phosphate buffer (pH=7.4).

[0031] [Figure 3] This shows the particle size distribution of ND-KV11 (with CSS, CSE, or CEE linker) and ND-NRPA7 (with CSS or CEE linker) measured by DLS in 10 mM phosphate buffer (pH=7.4).

[0032] [Figure 4] This figure shows the particle size distribution of ND-mIns2 B:9-23 (without linker) and ND-HMOG186-200 (without linker or with DDD linker) measured by DLS in 10 mM phosphate buffer (pH=7.4).

[0033] [Figure 5] This shows the particle size distribution of ND-gliadin-C1 (without linker) measured by DLS in 10 mM phosphate buffer (pH=7.4).

[0034] [Modes for carrying out the invention] definition In this specification, the term "approximately" is used to mean a value within ±10% of the stated value.

[0035] As used herein, “administer” means a method of giving a subject a predetermined dose of the composition described herein. The compositions used in the methods described herein may be administered by any suitable route, including, for example, inhalation, spray, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, or buccal administration. The compositions used in the methods described herein may also be administered topically or systemically. The preferred method of administration may vary depending on various factors, such as the components of the composition to be administered and the severity of the condition being treated.

[0036] As used herein, the term “bound” refers to a state in which two or more entities (e.g., nanoparticles and one or more peptides) are linked by direct or indirect covalent or non-covalent interactions. In some embodiments, the binding is covalent. In some embodiments, the covalent binding involves a linker moiety. In some embodiments, the binding is non-covalent (e.g., charge interaction, affinity interaction, metal coordination, physical adsorption, host-guest interaction, hydrophobic interaction, π-π stacking interaction, hydrogen bonding interaction, van der Waals interaction, magnetic interaction, electrostatic interaction, dipole interaction, etc.). For example, in some embodiments, peptides are mixed with nanoparticles. In some embodiments, peptides are bound to nanoparticles. In some embodiments, peptides are encapsulated within nanoparticles. In some embodiments, peptides are absorbed within nanoparticles. In some embodiments, peptides are adsorbed onto nanoparticles. In some embodiments, peptides are mixed with nanoparticles.

[0037] As used herein, the term “absorbed” means that the peptide is taken up inside the nanoparticles and / or microparticles, i.e., inside their outer surface, and is stably retained there.

[0038] As used herein, the term “mixed” means that the peptide is dissolved, dispersed, or suspended in nanoparticles and / or microparticles. In some cases, biopolymers may be uniformly mixed in nanoparticles and / or microparticles.

[0039] As used herein, the term “adsorbed” refers to the attachment of peptides to the outer surface of nanoparticles and / or fine particles. Such adsorption is preferably caused by electrostatic attraction, which is the attractive force or bond between two or more oppositely charged chemical groups or ionic chemical groups. Generally, adsorption is usually reversible.

[0040] As used herein, the term "mutein" is intended to include proteins and polypeptides resulting from mutation or recombinant DNA methods, in which the amino acid sequence has been altered.

[0041] As used herein, “combination therapy” or “combined administration” means that two or more different drugs or treatments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) are administered to a subject as part of a prescribed treatment regimen for a particular disease or condition. The treatment regimen specifies the dose and duration of administration of each drug such that the effects of the separate drugs on the subject overlap. In some embodiments, the administration of two or more drugs is simultaneous or parallel, and the drugs may be co-formulated. In some embodiments, the two or more drugs are not co-formulated and are administered sequentially as part of a given regimen. In some embodiments, the combined administration of two or more drugs or treatments results in a greater reduction of symptoms or other disease-related parameters than would be observed if one drug or treatment were administered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be carried out by any suitable route, including, but is not limited to, inhalation, spray, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, buccal, or direct absorption via mucosal tissue. The therapeutic agents may be administered by the same or different routes. For example, the first therapeutic agent used in combination may be administered by intravenous injection, and the second therapeutic agent used in combination may be administered orally.

[0042] As used herein, the terms “drug” or “therapeutic agent” include any molecule, molecular complex, or substance administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraception, cosmetic, nutritional supplement, pharmaceutical, and preventive uses. The term “drug” further includes any such molecule, molecular complex, or substance that is chemically modified and / or functionally bound to a biological or biocompatible structure.

[0043] As used herein, the term “fragment” refers to a fragment containing less than 100% of the amino acid sequence of the full-length reference protein (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but containing, for example, 5, 10, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, or more amino acids. The fragment may be of sufficient length to maintain the desired function of the full-length protein.

[0044] As used herein, the term “to increase” refers to increasing the number of cells in a cell population or sample by cell replication.

[0045] As used herein, the terms “HDL” or “high-density lipoprotein” refer to high-density lipoprotein. HDL contains a complex of nearly equal amounts of lipids and proteins that functions as a transporter of cholesterol in the blood. HDL is primarily synthesized and secreted by epithelial cells of the liver and small intestine. Immediately after secretion, HDL is in the form of disc-shaped particles containing apolipoprotein AI (also called apoA-I) and phospholipids as its main components, and is also called nascent HDL. In the blood, this nascent HDL accepts free cholesterol from the cell membranes of peripheral cells or free cholesterol produced during the hydrolysis of other lipoproteins to form mature spherical HDL, which holds cholesterol esters converted from cholesterol by the action of LCAT (lecithin cholesterol acyltransferase) in its hydrophobic center. HDL plays a crucial role in a lipid metabolic process called “reverse cholesterol transport,” which collects cholesterol from peripheral tissues into the blood and transports it to the liver. Since reverse cholesterol transport is considered one of the main mechanisms of HDL's protective effect against atherosclerosis, high levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0046] As used herein, the term “nucleic acid” may be DNA or RNA such as mRNA. In embodiments, the composition comprises a complement, such as a full-length complement, or a degenerate (due to degeneracy of the genetic code) of any of the nucleic acids provided herein. In embodiments, the nucleic acid is an expression vector that can be transcribed upon transfection into a cell line. In embodiments, the expression vector may include, among other things, a plasmid, a retrovirus, or an adenovirus. Nucleic acids can be isolated or synthesized using standard molecular biology approaches, for example, by generating nucleic acid fragments using polymerase chain reaction, purifying them, and cloning them into an expression vector. Further techniques useful in carrying out the present invention can be found in Current Protocols in Molecular Biology 2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas, Cold Spring Harbor.

[0047] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within it. An in vitro environment may consist of, but is not limited to, a test tube and a cell culture.

[0048] The term "in vivo" refers to natural environments (e.g., animals or cells) and processes or reactions that occur within those natural environments.

[0049] As used herein, the terms “lipid” or “lipid molecule” refer to water-insoluble fatty substances, including fats, oils, waxes, and related compounds. Lipids may be produced in the blood (endogenous) or ingested through diet (exogenous). Lipids are essential for normal bodily functions and, whether produced from exogenous or endogenous sources, need to be transported for use by cells and subsequently released. The production, transport, and release of lipids for use by cells is called lipid metabolism. There are several classes of lipids, but two major classes are cholesterol and triglycerides. Cholesterol is ingested through diet and can be produced by cells in most organs and tissues of the body, primarily in the liver. Cholesterol is found in its free form or, more commonly, in forms known as cholesterol esters, combined with fatty acids. As used herein, “lipid” or “lipid molecule” refers to any lipophilic compound. Non-exclusive examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogues. Lipid compounds are generally classified into at least three classes: (1) "simple lipids" including oils and waxes, (2) "complex lipids" including phospholipids and glycolipids, and (3) "derived lipids" such as steroids. Lipids or lipid molecules suitable for use in the present invention include both membrane-forming lipids and non-membrane-forming lipids.

[0050] As used herein, the term “lipoprotein” refers to a compound having a structure in which an insoluble lipid is contained within a partially soluble shell. Depending on the type of lipoprotein, the contents include varying amounts of free and esterified cholesterol, triglycerides, and apoproteins or apolipoproteins. There are five main types of lipoproteins that differ in function and the lipid and apoprotein content within them, and they are classified in order of increasing density as follows: (i) chylomicrons and chylomicron remnants, (ii) very low-density lipoproteins (VLDL), (iii) medium-density lipoproteins (IDL), (iv) low-density lipoproteins (LDL), and (v) high-density lipoproteins (HDL). Cholesterol circulates in the bloodstream as particles bound to lipoproteins.

[0051] As used herein, the term “unnatural amino acid” means an α-amino acid that is not produced or found naturally in mammals. Examples of unnatural amino acids include D-amino acids; amino acids having an acetylaminomethyl group bonded to the sulfur atom of cysteine; pegylated amino acids; and amino acids with the formula NH2(CH2). nExamples include omega amino acids of COOH (wherein n is 2 to 6); neutral nonpolar amino acids, such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid; 2-carboxypiperazine; piperazine-2-carboxylic acid; 2-amino-4-phenylbutanoic acid; 3-(2-naphthyl)alanine, and hydroxyproline. Other amino acids include α-aminobutyric acid, α-amino-α-methylbutyric acid, aminocyclopropanecarboxylate, aminoisobutyric acid, aminonorbornylcarboxylate, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylnorvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-norleucine, α-methylaminoisobutyric acid, α-methylcyclohexylalanine, D-α-methylalanine, D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartic acid, D-α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine, D- α-methylisoleucine, D-α-methylleucine, D-α-methyllysine, D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-methylserine, DN-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, DN-methylalanine, DN-methylarginine, DN-methylasparagine, DN-methylaspartic acid, DN-methylcysteine, DN-methylglutamine, DN-methylglutamic acid, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, N-methylcyclohexylalanine, DN-methylornithine, N-methylglycine, N-methylaminoisobutyric acid,N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyltyrosine, DN-methylvaline, γ-aminobutyric acid, Lt-butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α-methylaspartic acid, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-methylleucine, L-α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine Nin, L-α-methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-diphenylethyl)carbamylmethylglycine, 1-carboxy-1-(2,2-diphenylethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine, 4-phenylphenylalanine, L-citrulline, α-cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidinedione-4-carboxylic acid, L - Homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-serine, meta-tyrosine, no-tyrosine, LN,N',N''-trimethyllysine, homolysine, norlysine, N-glycanasparagine, 7-hydroxy-1,2,3,4-tetrahydro-4-fluorophenylalanine, 4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3 -Amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-cyclohexaneacetic acid, D / L-allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutanecarboxylic acid, 2 or 3 or 4-aminocyclohexanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetraline-2-carboxylic acid, azetidine-3-carboxylic acid,4-benzyl-pyrrolidine-2-carboxylic acid, tert-butylglycine, β-(benzothiazolyl-2-yl)-alanine, β-cyclopropylalanine, 5,5-dimethyl-1,3-thiazolidined-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S) and (2S,4R)-4-(2-naphthylmethoxy)-pyrrolidine-2-carboxylic acid, (2S,4S) and (2S,4R)4-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenyl-pyrrolidine-2- Carboxylic acid, (2S,4S)-4-amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1-aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxybenzoic acid, 3,5-diaminobenzoic acid, 2-methylaminobenzoic acid, N-methylantranilic acid, LN-methylalanine, LN-methylarginine, LN-methylasparagine, LN-methylaspartic acid, LN-methylcysteine, LN-methylglutamine, LN- Methylglutamic acid, LN-methylhistidine, LN-methylisoleucine, LN-methyllysine, LN-methylnorleucine, LN-methylornithine, LN-methylthreonine, LN-methyltyrosine, LN-methylvaline, LN-methyl-t-butylglycine, L-norvaline, α-methyl-γ-aminobutyric acid, 4,4'-biphenylalanine, α-methylcyclopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminoethyl) Minopropyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine, N-(2-carbamylethyl)glycine, N-(carbamylmethyl)glycine, N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cyclododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine,N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N-(hydroxyethyl)glycine, N-(imidazolylethyl)glycine, N-(3-indolylethyl)glycine, N-methyl-γ-aminobutyric acid, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN-methylthreonine, N-(1-methylethyl)glycine, N-methyl-naphthylalanine Lanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine, L-α-methyl-t-butylglycine, L-methylethylglycine, L-α-methylglutamic acid, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, LN-methyl- Mophenylalanine, N-(N-(3,3-diphenylpropyl)carbamylmethylglycine, L-pyroglutamic acid, D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamic acid, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine, L-dimethyldopa or L-dimethoxyphenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptidine Glycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, O-glycan-threonine, ortho-tyrosine, LN,N'-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantylalanine, symmetric dimethylarginine, 3-carboxythiomorpholine,D-1,2,3,4-tetrahydronorharmann-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethylpyridine-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-aminotetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylic acid, β-(indole-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, β-cyclobutylalanine, allylglycine, diaminopropionic acid, homo-cyclohexylalanine, (2S,4R)-4- Examples include hydroxypiperidine-2-carboxylic acid, octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, nipecotinic acid, (2S,4R) and (2S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercaptopyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and selenocysteine. In some embodiments, amino acid residues may be charged or polar. Examples of charged amino acids include alanine, lysine, aspartic acid, or glutamic acid, or their unnatural analogs. Examples of polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or their unnatural analogues. In some embodiments, it is specifically conceivable that the terminal amino group of the amino acid may be an amide group or a carbamate group.

[0052] The "amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity (%). Alignment for the purpose of determining the amino acid sequence identity (%) can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the maximum alignment over the full length of the sequences to be compared. For example, the sequence identity value can be generated using the sequence comparison computer program BLAST. As an example, the sequence identity of a particular nucleic acid or amino acid sequence A to, or with, or in comparison to, a particular nucleic acid or amino acid sequence B (which can also be expressed as a particular nucleic acid or amino acid sequence A having a particular sequence identity to, or with, or in comparison to, a particular nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) (However, X is the number of nucleotides or amino acids scored as identical matches within the alignment of A and B in a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It will be recognized that if the lengths of nucleic acid or amino acid sequence A and nucleic acid or amino acid sequence B are not equal, the sequence identity rate of A to B will not be equal to the sequence identity rate of B to A.)

[0053] The term "protein" refers to polymers of amino acids of any length (e.g., natural and unnatural amino acids). This term also encompasses polymers of modified amino acids (e.g., formation of disulfide bonds, glycosylation, acetylation, phosphorylation, lipidation, or binding with labeling components).

[0054] As used herein, the term "peptide" refers to a polymer in which monomers are amino acids covalently bonded to one another via amide bonds. A peptide has a monomer length of two or more, often more, amino acids.

[0055] "Pharmaceutical composition" means any composition containing a peptide suitable for administration to a target. Any formulation can be prepared by methods well known and accepted in the art. For example, Remington: The Science and Practice of Pharmacy (21), which is incorporated herein by reference in its entirety. st See also, ed. AR Gennaro, Lippincott Williams & Wilkins, 2005, and Encycle of Pharmaceutical Technology, ed. J. Swarbrick, Informat Healthcare, 2006.

[0056] "Pharmacologically acceptable diluent, excipient, carrier, or adjuvant" means a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to the subject while maintaining the therapeutic properties of the pharmaceutical composition when administered together.

[0057] As used herein, the term “sample” is used in its broadest sense. In a sense, this term includes specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and may include liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Environmental samples include environmental substances such as surface materials, soil, water, crystals, and industrial samples. However, these examples should not be construed as limiting the types of samples to which the present invention is applicable.

[0058] As used herein, the term “subject” refers to any animal (e.g., mammal) including, but not limited to, humans, non-human primates, rodents, etc., that is the subject of a particular treatment. Generally, the terms “subject” and “patient” are used interchangeably herein in reference to human subjects.

[0059] As used herein, the terms “synthetic HDL,” “sHDL,” “reconstituted HDL,” and “rHDL” refer to particles structurally similar to natural HDL, composed of at least one HDL protein, preferably ApoA-I, or a lipid associated with its mimetic counterpart. Generally, the components of sHDL may be derived from blood or produced by recombinant technology.

[0060] "Therapeutic dose" means the amount of composition administered to clinically meaningfully improve, suppress, or alleviate the condition or symptoms of a disease or illness (e.g., celiac disease) of the subject. Any improvement in the subject is considered sufficient to achieve a therapeutic effect. Preferably, a therapeutic dose is an amount that reduces, suppresses, or prevents the onset of the disease or disorder or one or more symptoms, or that reduces the severity of one or more symptoms of the disease or disorder, or the length of time the subject has such symptoms (e.g., at least about 10%, about 20%, or about 30%, more preferably at least about 50%, about 60%, or about 70%, most preferably at least about 80%, about 90%, about 95%, about 99%, or more, compared to a control subject not treated with the composition described herein). The therapeutic dose of the pharmaceutical composition used to carry out the methods described herein will vary depending on the mode of administration and the age, weight, and overall health status of the subject being treated. The appropriate dose and dosage regimen can be determined by the physician or researcher.

[0061] As used herein, the term “solvent” refers to the medium in which the reaction takes place. The solvent may, but is not limited to, a liquid. Classifications of solvents include, but are not limited to, nonpolar, polar, protic, and aprotic solvents.

[0062] Experiments conducted during the development of embodiments of the present invention have led to the development of a novel method for improving the water solubility of peptides and the productivity of peptide-loaded sHDL ND by attaching negatively or positively charged amino acids to the C-terminus or N-terminus. These experiments demonstrated that for peptides with high isoelectric points (pI), adding negatively charged amino acids (such as aspartic acid or glutamic acid) to the peptide sequence converts the net charge at pH 7 from positive to negative. This charge conversion enables efficient and stable loading of the peptide-lipid conjugate into ND, resulting in homogeneous and uniform peptide-loaded ND. Therefore, the addition of negatively or positively charged amino acids to the C-terminus or N-terminus of a peptide provides a universal method for improving peptide incorporation into ND and for improving the productivity of peptide-ND products. Furthermore, our approach demonstrates the versatility of using peptides with cysteine ​​residues not only at the N-terminus but also at the C-terminus or non-terminal positions in obtaining peptide-loaded sHDL ND. Overall, the embodiments described herein relate to novel compositions that improve the incorporation of various peptides into NDs and the manufacturability of peptide-NDS products. Furthermore, this approach is considered to be broadly applicable to the incorporation of various peptides into other nanoparticles and polymers.

[0063] Accordingly, in certain embodiments, the present invention provides compositions comprising sHDL nanoparticles. In some embodiments, the HDL nanoparticles comprise a phospholipid, an apolipoprotein mimetic, a thiol-reactive lipid, and a peptide comprising a linker moiety connected to a payload moiety, wherein the s-linker comprises either [cysteine ​​(C) and optionally 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S)] or [DD or DDD if the payload contains a C amino acid], the payload comprises a polypeptide having a net positive charge at pH 7 to 12 and a net negative charge at pH 7 and an isoelectric point of 0.4 to 12, and the peptide is covalently bonded to the thiol-reactive lipid via the linker.

[0064] In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the carboxyl terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the amino terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the non-terminal position of Cys.

[0065] In some embodiments, when the payload contains a C amino acid, the peptide is covalently bonded to a thiol-reactive lipid via a linker, the linker comprising DD or DDD.

[0066] In some embodiments, the peptide includes the following formula: [Linker]-[Payload] or [Payload]-[Linker].

[0067] In some embodiments, the payload has a positive net charge at pH 7–12. In some embodiments, the payload has a net charge of 0 at pH 7–12. In some embodiments, the payload has a negative net charge at pH 7–12.

[0068] In some embodiments, the peptide has a charge less than -0.1. In some embodiments, the peptide has a charge in the range of -0.1 to -5.0.

[0069] In some embodiments, the peptide has an isoelectric point of 3.7 to 12. In some embodiments, the peptide has an isoelectric point of 0.62 to 9.78.

[0070] In some embodiments, the linker sequence is C. In some embodiments, the linker sequence is DDCDD. In some embodiments, if the payload contains the C amino acid, the linker sequence is DD or DDD.

[0071] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide dimer. In some embodiments, the peptide dimer is selected from DD, SE, SD, and EE.

[0072] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide trimer. In some embodiments, the peptide trimer is selected from DDD, EEE, and KEE.

[0073] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide tetramer. In some embodiments, the peptide tetramer is selected from DDDD (SEQ ID NO: 762) and EEEE (SEQ ID NO: 763).

[0074] In some embodiments, the payload has a charge greater than 0.1 at pH 7. In some embodiments, the payload has a charge in the range of 0.1 to 5.0. In some embodiments, the payload is a polypeptide with a length of 12 to 35 amino acids.

[0075] In some embodiments, the payload is selected from GWYRSPFSRVVHL (SEQ ID NO: 764), NTWTTSQSIAFPSK (SEQ ID NO: 765), KVAPVWVRMME (SEQ ID NO: 766), KYNKANAFL (SEQ ID NO: 767), and ASFEAQGALANIAVDKA (SEQ ID NO: 768).

[0076] In some embodiments, the apolipoprotein mimetic is SEQ ID NOs: 1-336, and WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347), PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354) )QTVTDYGKDLME(SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE(SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV(SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL(SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS(SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS(SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS(SEQ ID NO: 361), VVALLALLASARASEAEDASLL( SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),It is an ApoA-I mimetic having one of the following sequences: PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0077] In some embodiments, the apolipoprotein mimetic has the sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4).

[0078] In some embodiments, the thiol-reactive lipids are dioleyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate](DOPE-PDP), 1,2-di-(9Z-octadecenoyl))-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide]. Selected from [Cylamide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-maleimide (DOPE-Mal), and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide].

[0079] In some embodiments, the thiol-reactive lipid is DOPE-PDP.

[0080] In certain embodiments, the present invention provides a method for treating, preventing, and / or alleviating a disease, comprising administering a composition described herein to a subject (e.g., a human subject suffering from or at risk of suffering from a disease or medical condition).

[0081] These methods are not limited to treating specific diseases.

[0082] In some embodiments, the disease is an autoimmune disease. These methods are not limited to the treatment of specific autoimmune diseases. Examples of autoimmune disorders, but are not limited to, multiple sclerosis (MS), celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody-associated disease, diabetes mellitus (e.g., type 1 diabetes), thyroid autoimmune diseases (e.g., Hashimoto's disease, Graves' disease), thyroid eye disease and thyroid dermatitis, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypophysitis, pituitary autoimmune disease, immunogastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and others. Related diseases include bullous pemphigoid, dermatitis herpetiformis of Dueling, acquired epidermolysis bullosa, systemic scleroderma, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodpasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Goutier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

[0083] In some embodiments, the disease is a transplant-related disease. In some embodiments, the disease is one or more allergies. In some embodiments, the disease is a respiratory disease (e.g., asthma). In some embodiments, the disease is a graft-versus-host disease (GvHD).

[0084] In some embodiments, such methods for treating or preventing autoimmune disorders further include the concomitant administration of additional therapeutic agents (e.g., simultaneously or at different times). Examples of such therapeutic agents, but not limited to these, include disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biological agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent may include, but is not limited to, infliximab, adalimumab, etanercept, or parenteral or oral gold preparations.In some cases, the therapeutic agent is an immunomodulator or immunosuppressant (e.g., statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors such as trichostatin A; corticosteroids; mitochondrial function inhibitors such as rotenone; P38 inhibitors; 6Bio, dexamethasone, TCPA-1, IKK) NF-κβ inhibitors such as VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2) such as misoprostol; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors (PDE4) such as rolipram; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB inhibitors such as TGX-221; autophagy inhibitors such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATP such as P2X receptor blockers.Immunosuppressants include IDO, vitamin D3, cyclosporine A and other cyclosporines, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, triptolide; O PN-305, OPN-401; Eritran (E5564); TAK-242; Cpn10; NI-0101; 1A6; AV411; IRS-954 (DV-1079); IMO-3100; CPG-52363; CPG-52364; OPN-305; ATNC05; NI-0101; IMO-8400; Hydroxychloroquine; CU-CPT22; C29; Orthovanillin; SSL3 protein; OPN-305; 5 SsnB; Byzantine; (+)-N-Phenethylnoroxymorphone; VB3323; Monosaccharide 3; (+)-Naltrexone and (+)-Naloxone; HT52; HTB2; Compound 4a; CNTO2424; TH1020; INH-ODN; E6446; AT791; CpG ODN 2088; ODN TTAGGG;COV08-0064;2R9;GpG oligonucleotide;2-aminopurine;Amlexanox;Bay11-7082;BX795;CH-223191;Chloroquine;CLI-095;CU-CPT9a;Cyclosporine A;CTY387;Gefitinib;Glibenclamide;H-89;H-131;Isoliquitigenin;MCC950;MRT67307;OxPAPC;Pal This also includes, but is not limited to, AHR-specific ligands such as tenoride; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU.521; VX-765; YM201636; Z-VAD-FMK; and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); tryptamine (TA); and 6-formylindoro[3,2b]carbazole (FICZ).In certain embodiments, the immunosuppressant is fingolimod, rapamycin, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE) or its related ligand, trichostatin A, and / or suberoylanilide hydroxamic acid (SAHA).

[0085] The present invention is not limited to any particular type or variety of nanoparticles associated with (e.g., complexed, bound, encapsulated, absorbed, adsorbed, mixed with) the peptides described herein, or not associated with them.

[0086] Examples of nanoparticles include, but are not limited to, fullerene (C 60 , C 70 , C 76 , C 80 , C 84 Examples include encapsulated metallic fullerenes (EMI: buckyballs containing other atoms, ions, or clusters within a fullerene cage), ternary metal nitride template encapsulated metallic fullerenes (TNT EME: highly symmetrical tetraatomic molecular cluster encapsulations formed in a ternary metal nitride template within a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods (nanotubes containing metallic fullerenes and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Embodiments of the particles may also include microparticles having properties that enhance efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, and gold, silver, carbon, and iron nanoparticles.

[0087] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise a polyol polymer modified to include a hydrophobic polymer block. As used in this disclosure, the term “hydrophobic polymer block” refers to a segment of the polymer that is hydrophobic by itself. As used herein, the term “micelle” refers to an aggregate of molecules dispersed in a liquid. A typical micelle in an aqueous solution forms an aggregate in which a hydrophilic “head” region is in contact with the surrounding solvent and a hydrophobic single tail region is isolated at the micelle center. In some embodiments, the head region may be, for example, a surface region of the polyol polymer, and the tail region may be, for example, a hydrophobic polymer block region of the polyol polymer.

[0088] The present invention further encompasses the use of micrometer-scale particles in addition to nanometer-scale particles. When microparticles are used, they are preferably relatively small, on the order of 1 to 50 micrometers. For ease of explanation, when "nanoparticles" is used herein, it encompasses true nanoparticles (particle size about 1 nm to about 1000 nm), microparticles (e.g., about 1 micrometer to about 50 micrometers), or both.

[0089] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers, dendrimers having covalently bonded metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some embodiments, the nanoparticles are metallic nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or two or more alloys thereof). The nanoparticles may consist of a core, or a core and a shell, as in core-shell nanoparticles.

[0090] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles consist of a mixture of HDL apolipoprotein and amphiphilic lipids.

[0091] The present invention is not limited to the use of a specific type or category of HDL apolipoprotein. Examples of HDL apolipoproteins include apolipoprotein AI (apoAI), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apoA4), apolipoprotein Cs (apoCs), apolipoprotein M (apoM), and apolipoprotein E (apoE). In some embodiments, HDL apolipoproteins are selected from preproapolipoproteins, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, apolipoprotein A-II xxx (ApoA-II-xxx), preproApoA-IV, proApoA-IV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-I Milano, proApoA-I Milano, ApoA-I Milano, preproApoA-I Paris, proApoA-I Paris, ApoA-I Paris, and peptide mimes of these proteins, as well as mixtures thereof. The carrier particles are preferably composed of ApoA-I or ApoA-II, but other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate a carrier particle mixture for delivering the therapeutic agent. In some embodiments, mimics of such HDL apolipoproteins are used.

[0092] ApoA-I is synthesized in the liver and small intestine as a preproapolipoprotein, secreted as a proprotein, and rapidly cleaved to produce a mature polypeptide with 243 amino acid residues. ApoA-I is mainly composed of 6-8 distinct repeat sequences of 22 amino acids and 2 distinct repeat sequences of 11 amino acids, each of which has an amphiphilic α-helix spiral wheel signature separated by a linker moiety consisting of a stretch, often proline, and sometimes several residues. ApoA-I forms three stable complexes with lipids: a small, low-lipid complex called pre-β1HDL; a flat, disc-shaped particle containing polar lipids (phospholipids and cholesterol), called pre-β2HDL; and a spherical particle containing both polar and non-polar lipids, called spherical HDL or mature HDL (HDL3 and HDL2). The majority of HDL circulating in the blood contains both ApoA-I and ApoA-II (the second major HDL protein).

[0093] In some embodiments, agonists or mimetic compounds of ApoA-I are provided. In some embodiments, such ApoA-I mimetic compounds can mimic the activity of ApoA-I to form amphiphilic α-helices having a specific activity approaching or exceeding that of the native molecule. In some embodiments, the ApoA-I mimetic compounds are peptides or peptide analogs that form amphiphilic helices (in the presence of lipids), bind to lipids to form pre-β-like or HDL-like complexes, activate lecithin:cholesterol acyltransferase (LCAT), increase serum HDL fraction levels, and promote cholesterol excretion.

[0094] The present invention is not limited to the use of a specific ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetic described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 is used. In some embodiments, any of the ApoA-I mimetic described in U.S. Patent Application Publication No. 20110046056 and No. 20130231459 is used.

[0095] In some embodiments, the ApoA-I mimetic "22A" (PVLDLFRELLNELLEALKQKLK) (Sequence ID 4) (see, for example, U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1, as described in U.S. Patent No. 7,566,695, are used.

[0096] [Table 1] JPEG2026522663000002.jpg227169JPEG2026522663000003.jpg227169JPEG2026522663000004.jpg227169JPEG2026522663 000005.jpg226169JPEG2026522663000006.jpg227169JPEG2026522663000007.jpg227169JPEG2026522663000008.jpg27169

[0097] * indicates an N-terminally acetylated and C-terminally amidated peptide, indicates an N-terminally dansylated peptide, sp indicates a peptide that exhibited solubility problems under experimental conditions, X is Aib, Z is Na, O is Orn, and ~ indicates a deleted amino acid.

[0098] In some embodiments, an ApoA-I mimetic having the following sequence described in U.S. Patent No. 6,743,778 is used: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 255).

[0099] In some embodiments, one of the following ApoA-I mimics shown in Table 2, as described in U.S. Patent Application Publication No. 2003 / 0171277, is used.

[0100] [Table 2] JPEG2026522663000010.jpg220169JPEG2026522663000011.jpg144169

[0101] In some embodiments, an ApoAI mimetic having the following sequence described in U.S. Patent Application Publication No. 2006 / 0069030 is used: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333).

[0102] In some embodiments, an ApoAI mimetic having the following sequence described in U.S. Patent Application Publication No. 2009 / 0081293 is used: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335), PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0103] In some embodiments, an ApoA-I mimetic having one of the following sequences is used: WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347) PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 35 4) QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASLL (SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0104] Amphiphilic lipids include, for example, any lipid molecule that has both a hydrophobic and a hydrophilic portion. Examples include phospholipids and glycolipids. Examples of phospholipids that can be used in sHDL peptide nanoparticles are not limited to these, but include 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dimiristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, and 1,2 -Dilignoceroyl-sn-glycero-3-phosphocholine, 1,2-dimyristreoyl-sn-glycero-3-phosphocholine, 1,2-dimyristeridoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitreoyl-sn-glycero-3-phosphocholine, 1,2-dipalmiteridoyl-sn-glycero-3-phosphocholine, 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dieride Il-sn-glycero-3-phosphocholine, 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, 1,2-dinervonoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-di Stearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitreoyl-sn-glycero-3-phosphoethanolamine, 1,2-dieleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine, N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine, N-(3-maleimido-1-oxopropyl)-L-α-phosphatidyl Ethanolamine (distearoyl), N-[(3-maleimido-1-oxopropyl)aminopropyl polyethylene glycol-carbamyl]distearoylphosphatidylethanolamine, N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (dimyristoyl), N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (dioleoyl), N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (dipalmitoyl), N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (dipalmitoyl), N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (dipalmitoyl), Mido-1-oxopropyl)-L-α-phosphatidylethanolamine (1-palmitoyl-2-oleoyl), phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine (distearoyl), N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine (dioleoyl), N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine (1 Examples include N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine (dipalmitoyl), N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine (dimyristoyl), 3-(N-succinimidyloxyglutaryl)aminopropyl and polyethylene glycol-carbamyl distearoylphosphatidylethanolamine, and N-(3-oxopropoxypolyethylene glycol)carbamyl-distearoylethanolamine.

[0105] In some embodiments, the phospholipid / HDL apolipoprotein molar ratio of sHDL nanoparticles is 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0106] Generally, the sHDL nanoparticles formed in this way are spherical or disc-shaped and have diameters of approximately 5 nm to 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, a more uniform preparation can be obtained by subjecting the sHDL nanoparticles to size exclusion chromatography.

[0107] Such compositions are not limited to a specific type or variety of peptide. In some embodiments, the peptide comprises a linker portion connected to a payload portion, the linker comprising cysteine ​​(C) and 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), the payload comprising a polypeptide having a net positive charge at pH 7 to 12 and a length of 5 to 35 amino acids, the peptide having a net negative charge at pH 7 and an isoelectric point of 0.4 to 12, and the peptide being covalently bonded to a thiol-reactive lipid via cysteine ​​(C).

[0108] In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the carboxyl terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the amino terminus of Cys. In some embodiments, the peptide is covalently bonded to the thiol-reactive lipid via the non-terminal position of Cys.

[0109] In some embodiments, the peptide comprises the following formula: [linker]-[payload] or [payload]-[linker].

[0110] In some embodiments, the linker array is DDCDD.

[0111] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide dimer. In some embodiments, the peptide dimer is selected from DD, SE, SD, and EE.

[0112] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide trimer. In some embodiments, the peptide trimer is selected from DDD, EEE, and KEE.

[0113] In some embodiments, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide tetramer. In some embodiments, the peptide tetramer is selected from DDDD (SEQ ID NO: 762) and EEEE (SEQ ID NO: 763).

[0114] In some embodiments, the payload has a charge greater than 0.1 at pH 7. In some embodiments, the payload has a charge in the range of 0.1 to 5.0. In some embodiments, the payload is a polypeptide with a length of 12 to 35 amino acids.

[0115] In some embodiments, the payload is selected from GWYRSPFSRVVHL (SEQ ID NO: 764), NTWTTSQSIAFPSK (SEQ ID NO: 765), KVAPVWVRMME (SEQ ID NO: 766), KYNKANAFL (SEQ ID NO: 767), and ASFEAQGALANIAVDKA (SEQ ID NO: 768).

[0116] In some embodiments, the peptide is an antigen and / or a tolerogenic antigen.

[0117] In some embodiments, the antigen associated with the nanoparticles comprises a gliadin polypeptide, such as a full-length gliadin polypeptide or any epitope of a gliadin polypeptide. In some embodiments, the antigen associated with the nanoparticles comprises a 33-mer polypeptide derived from α2-gliadin. In some embodiments, the 33-mer gliadin polypeptide has at least 90% (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the polypeptide sequence LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 374). In some embodiments, the antigen associated with the nanoparticles comprises an epitope of the 33-mer gliadin polypeptide. The epitopes of 33-mer gliadin polypeptides may be polypeptides of any length shorter than the 33-mer polypeptide. For example, epitopes may have lengths of 25-23 amino acid residues (e.g., 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3), 20-25 amino acid residues (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5), 12-6 amino acid residues (e.g., 12, 11, 10, 9, 8, 7, or 6), or 9 amino acid residues. Further examples of 33-gliadin epitopes that can be associated with nanoparticles include any one of the epitopes listed in Table 3, including SEQ ID NOs. 375-405. In some embodiments, the tolerogenic antigen associated with the nanoparticle may include any one of the antigens listed in Table 4, including SEQ ID NOs. 406-580. In some embodiments, the antigen associated with the nanoparticle includes a polypeptide sequence having at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, or 100%) sequence identity with any one of SEQ ID NOs. 375-580.In some embodiments, the tolerogenic antigen associated with the nanoparticles may include antigens comprising two or more polypeptides (e.g., 2, 3, 4, 5, or 6) having any two polypeptide sequences from SEQ ID NOs.375–580. In some embodiments, multiple tolerogenic antigens (e.g., 1–30 (e.g., 6–30, or 8–30 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)) per nanoparticle have the same identity as all other tolerogenic antigens associated with the nanoparticles. In some embodiments, multiple immunotolerogenic antigens associated with the nanoparticles comprise a population of 2–10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different antigen sequences involved in the same disease. For example, nanoparticles may be associated with 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8), 4 to 6 (e.g., 4, 5, or 6), or 3 to 4 different polypeptide antigen sequences. In some embodiments, nanoparticles may be associated with (i) a first polypeptide population containing any of the amino acid sequences of SEQ ID NOs. 406-580, or a biologically active fragment or variant thereof; (ii) a second polypeptide population containing any of the amino acid sequences of SEQ ID NOs. 406-580, or a biologically active fragment or variant thereof; and (iii) a third polypeptide population containing any of the amino acid sequences of SEQ ID NOs. 406-580, or a biologically active fragment or variant thereof. In some cases, the first, second, and third polypeptide populations have different amino acid sequences. In some embodiments, the nanoparticles can be associated with (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476) or a biologically active fragment or variant thereof.In some embodiments, the nanoparticles can be associated with (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478) or a biologically active fragment or variant thereof. In some embodiments, nanoparticles can be associated with (i) a first polypeptide comprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 506) or a bioactive fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 507) or a bioactive fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508) or a bioactive fragment or variant thereof. In some embodiments, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include an N-terminal pyroglutamic acid (pyroE). In some embodiments, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include a C-terminal amide group. In some embodiments, tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 506, 507, and 508 include an N-terminal pyroE residue and a C-terminal amide group. In some embodiments, the nanoparticles can be associated with (i) a first polypeptide comprising the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509) or a biologically active fragment or variant thereof, (ii) a second polypeptide comprising the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510) or a biologically active fragment or variant thereof, and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511) or a biologically active fragment or variant thereof.In some embodiments, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include an N-terminal acetyl group. In some embodiments, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include a C-terminal amide group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID NOs: 509, 510, and 511 include an N-terminal acetyl group and a C-terminal amide group. In any of the embodiments described herein, the group of antigens associated with nanoparticles may be fully or partially deamidated. In some embodiments described herein, the tolerogenic antigens associated with nanoparticles may include an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigens associated with nanoparticles may include an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigens associated with nanoparticles may include an N-terminal amide group. In some embodiments described herein, the tolerogenic antigens associated with nanoparticles may include a C-terminal amide group.

[0118] [Table 3]

[0119] [Table 4] JPEG2026522663000014.jpg212169JPEG2026522663000015.jpg64169

[0120] In some embodiments, the immunotolerogenic antigen is the bioactive fragment of SEQ ID NO: 474. In some cases, the bioactive fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 512. In some cases, the bioactive fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 580.

[0121] In some cases, the immunotolerogenic antigen is the bioactive fragment of SEQ ID NO: 475. In some cases, the bioactive fragment of SEQ ID NO: 475 contains a polypeptide that includes the sequence of SEQ ID NO: 542.

[0122] In some embodiments, the immunotolerogenic antigen is the bioactive fragment of SEQ ID NO: 476. In some cases, the bioactive fragment of SEQ ID NO: 476 comprises a polypeptide containing the sequence of SEQ ID NO: 563.

[0123] In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 375). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPQPELPY (SEQ ID NO: 376). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQ (SEQ ID NO: 377). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FRPEQPYPQ (SEQ ID NO: 378). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQQ (SEQ ID NO: 379). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence IQPEQPAQL (SEQ ID NO: 380). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPYPQ (SEQ ID NO: 381). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence SQPEQEFPQ (SEQ ID NO: 382). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQEFPQ (SEQ ID NO: 383). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPFPQ (SEQ ID NO: 384). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFCQ (SEQ ID NO: 385). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPEQPQ (SEQ ID NO: 386). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPF (SEQ ID NO: 387). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPW (SEQ ID NO: 388). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 389). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FSQQQESPF (SEQ ID NO: 390).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQ (SEQ ID NO: 391). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 392). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPY (SEQ ID NO: 393). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQ (SEQ ID NO: 394). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 395). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEEPF (SEQ ID NO: 396). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEQPF (SEQ ID NO: 397). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 398). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 399). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPFPQ (SEQ ID NO: 400). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPYPE (SEQ ID NO: 401). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 402). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 403). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQE (SEQ ID NO: 404). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 405). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPW (SEQ ID NO: 406).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPV (SEQ ID NO: 407). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPW (SEQ ID NO: 408). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPIPV (SEQ ID NO: 409). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPFPQ (SEQ ID NO: 410). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPQLPYPQ (SEQ ID NO: 411). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPELPYPQ (SEQ ID NO: 412). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYPQ (SEQ ID NO: 413). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQ (SEQ ID NO: 414). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSQ (SEQ ID NO: 415). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSQ (SEQ ID NO: 416). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFCQ (SEQ ID NO: 417). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFCQ (SEQ ID NO: 418). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYSQ (SEQ ID NO: 419). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYSQ (SEQ ID NO: 420). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQQQCSPVAMPQRLAR (SEQ ID NO: 421). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYLQ (SEQ ID NO: 422).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYLQ (SEQ ID NO: 423). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPQQPFPQ (SEQ ID NO: 424). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPEQPFPQ (SEQ ID NO: 425). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQQQPLPP (SEQ ID NO: 426). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQEQPLPP (SEQ ID NO: 427). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPQQPEQPFPL (SEQ ID NO: 428). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQQGYYPISPQQSGQ (SEQ ID NO: 429). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQPGYYPTSPQQIGQ (SEQ ID NO: 430). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PGQGQSGYYPTSPQQS (SEQ ID NO: 431). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPQQPQLP (SEQ ID NO: 432). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPEQPQLP (SEQ ID NO: 433). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence GQGQSGYYPTSPQQSG (SEQ ID NO: 434). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QYEVIRSLVLRTLPNM (SEQ ID NO: 435). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QVDPSGQVQWPQ (SEQ ID NO: 436). In some embodiments, the immunotolerogenic antigen comprises a polypeptide having the amino acid sequence QVDPSGEVQWPQ (SEQ ID NO: 437).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPL (SEQ ID NO: 438). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPL (SEQ ID NO: 439). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPY (SEQ ID NO: 440). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPY (SEQ ID NO: 441). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPQQPQPY (SEQ ID NO: 442). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPQQPQPY (SEQ ID NO: 443). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFPQQPIPQQPQPY (SEQ ID NO: 444). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPQQPQPY (SEQ ID NO: 445). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQPY (SEQ ID NO: 446). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPQQPFPQ (SEQ ID NO: 447). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPEQPFPQ (SEQ ID NO: 448). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPIPQQPQPYPQQP (SEQ ID NO: 449). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQQPFPQQPQPY (SEQ ID NO: 450). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSW (SEQ ID NO: 451). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSW (SEQ ID NO: 452).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQPYPQQP (SEQ ID NO: 453). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPQ (SEQ ID NO: 454). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPQ (SEQ ID NO: 455). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPQ (SEQ ID NO: 456). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPQ (SEQ ID NO: 457). In some embodiments, the immunotolerogenic antigen comprises an amino acid sequence Q. The immunotolerogenic antigen comprises a polypeptide containing PFPQPQQPTPI (SEQ ID NO: 458). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPTPI (SEQ ID NO: 459). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPQQPFPQ (SEQ ID NO: 460). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPEQPFPQ (SEQ ID NO: 461). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPEQI (SEQ ID NO: 462). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPEQI (SEQ ID NO: 463). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQQI (SEQ ID NO: 464). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPQQI (SEQ ID NO: 465). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 466). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 467). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: 468). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: 469). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQQLPL (SEQ ID NO: 470). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQQLPL (SEQ ID NO: 471). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LFPLPQQPFPQ (SEQ ID NO: 472). In some embodiments, the immunotolerogenic antigen comprises a polypeptide having the amino acid sequence LFPLPEQPFPQ (SEQ ID NO: 473).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 479). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFLPQLPYPQ (SEQ ID NO: 480). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QAFPQPQQTFPH (SEQ ID NO: 481). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence TPIQPQQPFPQ (SEQ ID NO: 482). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 483). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFTQPQQPTPI (SEQ ID NO: 484). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQLQQPQQP (SEQ ID NO: 485). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence VAHAIIMHQQQQQQQE (SEQ ID NO: 486). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence SYPVQPQQPFPQ (SEQ ID NO: 487). In some embodiments, the immunotolerogenic antigen comprises a polypeptide having the amino acid sequence PQQPQPFPQQPVPQQP (SEQ ID NO: 488).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPWQPQQPFPQ (SEQ ID NO: 489). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 490). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFQPQQPFPQ (SEQ ID NO: 491). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence NPLQPQQPFPLQPQPP (SEQ ID NO: 492). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PLQPQQPFPLQPQPPQ (SEQ ID NO: 493). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PNPLQPQQPFPLQ (SEQ ID NO: 494). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence TIPQQPQQPFPL (SEQ ID NO: 495). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSQQPQQPFPL (SEQ ID NO: 496). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSEQPQQPFPL (SEQ ID NO: 497). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence YSPYQPQQPFPQ (SEQ ID NO: 498). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QLPLQPQQPFPQ (SEQ ID NO: 499). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPQQPFPLQPQQPVP (SEQ ID NO: 500). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence IIPQQPQQPFPL (SEQ ID NO: 501). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQIIPQQPQQP (SEQ ID NO: 502). In some embodiments, the immunotolerogenic antigen comprises a polypeptide having the amino acid sequence FLLQPQQPFSQ (SEQ ID NO: 503).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence IISQQPQQPFPL (SEQ ID NO: 504). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQRPQQPFPQ (SEQ ID NO: 505). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 506). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 507). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PELP (SEQ ID NO: 512). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYP (SEQ ID NO: 513). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPY (SEQ ID NO: 514). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELP (SEQ ID NO: 515). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQP (SEQ ID NO: 516). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQ (SEQ ID NO: 517). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYP (SEQ ID NO: 518). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPY (SEQ ID NO: 519).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELP (SEQ ID NO: 520). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQPQ (SEQ ID NO: 521). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQP (SEQ ID NO: 522). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYP (SEQ ID NO: 523). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 524). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELP (SEQ ID NO: 525). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQPQ (SEQ ID NO: 526). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQP (SEQ ID NO: 527). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQ (SEQ ID NO: 528). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYP (SEQ ID NO: 529). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPY (SEQ ID NO: 530). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELP (SEQ ID NO: 531). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQPQ (SEQ ID NO: 532). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQP (SEQ ID NO: 533). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQ (SEQ ID NO: 534). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYP (SEQ ID NO: 535).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPY (SEQ ID NO: 536). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQPQ (SEQ ID NO: 537). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQP (SEQ ID NO: 538). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYP (SEQ ID NO: 539). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQPQ (SEQ ID NO: 540). In some embodiments, the immunotolerogenic antigen is... The immunotolerogenic antigen includes a polypeptide containing the amino acid sequence LQPFPQPELPYPQ (SEQ ID NO: 541). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence QPEQPF (SEQ ID NO: 542). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence QPEQPFP (SEQ ID NO: 543). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence PQPEQPF (SEQ ID NO: 544). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence QPEQPFPW (SEQ ID NO: 545). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence PQPEQPFP (SEQ ID NO: 546). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence FPQPEQPF (SEQ ID NO: 547). In some embodiments, the immunotolerogenic antigen includes a polypeptide containing the amino acid sequence QPEQPFPWQ (SEQ ID NO: 548). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFP (SEQ ID NO: 549). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPWQP (SEQ ID NO: 550). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQ (SEQ ID NO: 551). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPW (SEQ ID NO: 552). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFP (SEQ ID NO: 553). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPF (SEQ ID NO: 554). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQP (SEQ ID NO: 555). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQ (SEQ ID NO: 556). In some embodiments, the immunotolerogenic antigen comprises a polypeptide having the amino acid sequence PFPQPEQPFPW (SEQ ID NO: 557).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFP (SEQ ID NO: 558). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQP (SEQ ID NO: 559). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQ (SEQ ID NO: 560). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQP (SEQ ID NO: 561). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPWQ (SEQ ID NO: 562). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQ (SEQ ID NO: 563). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQP (SEQ ID NO: 564). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQ (SEQ ID NO: 565). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQP (SEQ ID NO: 566). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYP (SEQ ID NO: 567). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPY (SEQ ID NO: 568). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQP (SEQ ID NO: 569). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQ (SEQ ID NO: 570). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQQ (SEQ ID NO: 571). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQ (SEQ ID NO: 572). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYP (SEQ ID NO: 573).In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPY (SEQ ID NO: 574). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQQ (SEQ ID NO: 575). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYPQ (SEQ ID NO: 576). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYP (SEQ ID NO: 577). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYPQQ (SEQ ID NO: 578). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQ (SEQ ID NO: 579). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PDLP (SEQ ID NO: 580). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQ (SEQ ID NO: 581). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQP (SEQ ID NO: 582). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQPQ (SEQ ID NO: 583). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQP (SEQ ID NO: 584). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQ (SEQ ID NO: 585). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYP (SEQ ID NO: 586). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPY (SEQ ID NO: 587). In some embodiments, the immunotolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQP (SEQ ID NO: 588).

[0124] In some embodiments, such tolerogenic antigens include human allograft antigens. Examples of such human allograft antigens include subunits of various MHC class I and MHC class II haplotype proteins, as well as single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Diffy, and Ss.

[0125] In some embodiments, a tolerogenic antigen is an autoantigen to which a subject (e.g., a human patient) has previously developed or is likely to develop an autoimmune response. Examples include proinsulin (e.g., in subjects with or at risk of diabetes), collagen (e.g., in subjects with or at risk of rheumatoid arthritis), and myelin basic protein (e.g., in subjects with or at risk of multiple sclerosis). While there are many proteins that are human autoimmune proteins, the term refers to a variety of autoimmune diseases to which the disease-causing protein(s) are known or can be identified by routine testing. Embodiments include testing a patient to identify an autoimmune protein, creating an antigen for use in molecular fusion, and forming immune tolerance to that protein. Embodiments include including or selecting an antigen from one or more of the following proteins: In type 1 diabetes, several major antigens have been identified, including insulin, proinsulin, preproinsulin, glutamate decarboxylase 65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2β (IA-2β). Other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma / intestinal / pancreatic / pancreatic-associated proteins, S100β, glial fibrillary acidic proteins, regenerative gene II, pancreaticoduodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-associated proteins, and somatostatin (SST) G protein-coupled receptors 1-5. In thyroid autoimmune diseases, including thyroiditis and Graves' disease, the major antigens are thyroglobulin (TG), thyroid peroxidase (TPO), and thyrotropin receptor (TSHR), while other antigens include iodine cotransporter (NIS) and megalin.In thyroid-associated ophthalmopathy and dermatitis, in addition to thyroid autoantigens including TSHR, the insulin-like growth factor 1 receptor is an antigen. In hypoparathyroidism, the main antigen is the calcium-sensing receptor. In Addison's disease, the main antigens include 21-hydroxylase, 17α-hydroxylase, and P450 side-chain cleavage enzyme (P450scc), and other antigens include the ACTH receptor, P450c21, and P450c17. In premature ovarian failure, the main antigens include the FSH receptor and α-enolase. In autoimmune hypophysitis, or pituitary autoimmune disease, the main antigens include pituitary gland-specific protein factors (PGSF) 1a and 2, and another antigen is type 2 iodothyronine deiodinase. In multiple sclerosis, the main antigens include myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein. In rheumatoid arthritis, the main antigen is collagen II. In immune gastritis, the main antigen is H. + ,K +-ATPase. In pernicious anemia, the major antigen is intrinsic factor. In celiac disease, the major antigens are tissue transglutaminase and gliadin. In vitiligo, the major antigens are tyrosinase and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the major antigen is the acetylcholine receptor. In pemphigus vulgaris and its variants, the major antigens are desmoglein 3, 1, and 4, and other antigens include pemfaxine, desmocolin, placoglobin, perplakin, desmoplakin, and the acetylcholine receptor. In bullous pemphigus, the major antigens are BP180 and BP230, and other antigens include plectin and laminin 5. In dermatitis herpetiformis, the major antigens are endomyceum and tissue transglutaminase. In epidermolysis bullosa, the major antigen is collagen VII. In systemic sclerosis, the major antigens include matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, and the PDGF receptor. Other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nuclear proteins, RNA polymerases I, II, and III, PM-Slc, fibrillarin, and B23. In mixed connective tissue disease, the major antigen is U1snRNP. In Sjögren's syndrome, the major antigens are nuclear antigens SS-A and SS-B, and other antigens include fodrin, poly(ADP-ribose) polymerase, and topoisomerase. In systemic lupus erythematosus, the major antigens include SS-A, high-mobility group box 1 (HMGB1), nucleosomes, nuclear proteins (including histone proteins), and double-stranded DNA. In Goodpasture syndrome, glomerular basement membrane proteins, including collagen IV, are major antigens. In rheumatic heart disease, the major antigen is cardiac myosin.Other autoantigens identified in autoimmune polyendocrine syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochrome P450 1A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor proteins, and type 1 interferons (interferon α, β, ω).

[0126] In some cases, a tolerogenic antigen is an exogenous antigen to which a patient has previously developed an undesirable immune response. Examples include food antigens. Embodiments involve testing the patient to identify the exogenous antigen, creating a molecular fusion containing the antigen, and treating the patient to develop immune tolerance to that antigen or food. Examples of such foods and / or antigens are shown. For example, from peanuts: conalatin (Ara h1), allergen II (Ara h2), peanut agglutinin, conglutin (Ara h6); from apples: 31kDa major allergen / disease resistance protein homolog (Mal d2), lipid transfer protein precursor (Mal d3), major allergen Mal d1.03D (Mal d1); from milk: α-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c1, Act d1), phytocystatin, thaumatin-like protein (Act d2), kiwelin (Act d5); from mustard: 2S albumin (Sin a1), 11S globulin (Sin a2), lipid transfer protein (Sin a3), profilin (Sin a4); from celery: profilin (Api Examples of allergenic antigens include high molecular weight glycoprotein (Api g5), Pen a1 allergen (Pen a1), allergen Pen m2 (Pen m2), and tropomyosin fast-twitch muscle type isoforms from shrimp, high molecular weight glutenin, low molecular weight glutenin, α- and γ-gliadin, hordein, secarin, and avenin from wheat and other grains, the major strawberry allergen Fra a1-E (Fra a1) from strawberries, and profilin (Mus xp1) from bananas. In some embodiments, the tolerogenic antigen is one antigenic peptide from sequence numbers 589 to 742 (Table 5).

[0127] [Table 5] JPEG2026522663000017.jpg200169JPEG2026522663000018.jpg205169JPEG2026522663000019.jpg201169JPEG2026522663 000020.jpg207169JPEG2026522663000021.jpg205169JPEG2026522663000022.jpg201169JPEG2026522663000023.jpg84169

[0128] In certain embodiments, amino acid sequence variants of the tolerogenic antigen of the present invention are conceived. For example, it may be desirable to improve the tolerogenicity and / or other biological properties of the tolerogenic antigen. Amino acid sequence variants of the tolerogenic antigen can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the tolerogenic antigen, or by peptide synthesis. Such modifications include, for example, the deletion of residues from the amino acid sequence of the tolerogenic antigen, and / or the insertion of residues into the sequence, and / or the substitution of residues within the sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired properties (e.g., inducing antigen tolerance).

[0129] In certain embodiments, tolerogenic antigens having one or more amino acid substitutions are provided. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 6. More significant modifications are shown under the heading "Exemplary Substitutions" in Table 6 and are further described in relation to the classes of amino acid side chains. Amino acid substitutions can be introduced into a tolerogenic antigen of interest, and the product can be screened for desired activity, e.g., retained / improved tolerogenic antigenicity.

[0130] [Table 6]

[0131] Amino acids can be classified according to the following general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0132] Non-conservative substitution involves replacing one member of one of these classes with one of another.

[0133] A useful method for identifying residues or regions of tolerogenic antigens that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaces them with neutral or charged amino acids (e.g., alanine or polyalanine) to examine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition to the above, a crystal structure of the antigen-antibody complex is used to identify the antibody-antigen contact points. Such contact residues and adjacent residues may be targeted or removed as candidate substitutions. Tolerogenic antigen variants can also be screened to determine if they possess desired properties.

[0134] Amino acid sequence insertions include the fusion of one to 100 or more residues to the amino and / or carboxyl termini of polypeptides, as well as the insertion of one or more amino acid residues into the sequence.

[0135] In some embodiments, the tolerogenic antigen contains an amide group at the C-terminus. In certain embodiments, the tolerogenic antigen contains a pyroglutamic acid residue at the N-terminus. In some embodiments, the tolerogenic antigen contains an acetyl group at the C-terminus. In some embodiments, the tolerogenic antigen contains a pyroglutamic acid residue at the N-terminus and an amide group at the C-terminus. In some embodiments, the tolerogenic antigen contains an acetyl group at the N-terminus and an amide group at the C-terminus. In certain embodiments, the tolerogenic antigen contains an N-terminus or C-terminus modified with a linker-bound cysteine ​​residue. In some embodiments, the tolerogenic antigen contains an N-terminus and a C-terminus modified with linker-bound cysteine.

[0136] In some embodiments of any one of the compositions described herein, a population of tolerogenic antigens is conjugated to nanoparticle phospholipids, thereby targeting (e.g., autoimmune diseases, e.g., MS, celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody-associated disease, diabetes (e.g., type 1 diabetes), thyroid autoimmune diseases (e.g., Hashimoto's disease, Graves' disease), thyroid eye disease and thyroid dermatitis, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypophysitis, pituitary autoimmune disease, immunogastritis, pernicious anemia, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and so on) It promotes potent immune tolerance when administered to individuals with or at risk of developing related disorders, bullous pemphigoid, dermatitis herpetiformis of Dueling, acquired epidermolysis bullosa, systemic scleroderma, mixed connective tissue disease, Sjögren's syndrome, systemic lupus erythematosus, Goodpasture syndrome, rheumatic heart disease, autoimmune polyglandular syndrome type 1, Eicardi-Goutier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, or inflammatory bowel disease.

[0137] Such peptides can be prepared by several techniques well known in the art, depending on the properties of the molecule. Short peptides can be easily prepared by amino acid synthesis. Longer proteins with known sequences can be prepared by synthesizing the coding sequence or by PCR amplification of the coding sequence from a natural source or vector, and then by expressing the coding sequence in a suitable bacterial or eukaryotic host cell.

[0138] The nanoparticles of the present invention can be characterized for particle size and uniformity by any suitable analytical method. These analytical methods include, but are not limited to, atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, LC-MS / MS, 13 These analytical methods include 13C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC) (with multi-angle laser light scattering, dual UV and refractive index detectors), capillary electrophoresis, and electrophoresis. These analytical methods ensure the uniformity of the sHDL nanoparticle population and are important for production quality control for final use in in vivo applications.

[0139] In some embodiments, sHDL nanoparticles are analyzed using gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides. In some embodiments, particle size distribution and zeta potential are measured by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0140] Such compositions, comprising nanoparticles associated with the peptides described herein, are not limited to any particular method of administration to a subject. In fact, such compositions can be administered to a subject using any acceptable method known to those skilled in the art. Administration may be topical (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic. Such compositions can be administered by many routes, including but not limited to oral, inhalation (nasal or lung), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, subcutaneous, sublingual, or rectal means. Injections may be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, injections may be administered at multiple locations.

[0141] Where clinical applications are conceived, in some embodiments of the present invention, compositions comprising the peptides associated with nanoparticles described herein are prepared as part of a pharmaceutical composition in a form appropriate for the intended use. Generally, this involves preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the present invention, undiluted compositions comprising the peptides associated with nanoparticles described herein may be administered using one or more of the routes described herein.

[0142] In preferred embodiments, the composition is used in combination with appropriate salts and buffers to ensure stable delivery of the composition and enable uptake by target cells. Buffers are also used when introducing any of the compositions to a patient.

[0143] The aqueous composition contains an effective amount of sHDL nanoparticles dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such a composition is also called an inoculum.

[0144] The phrase "pharmaceutically acceptable" refers to molecules and compositions that, when administered to animals or humans, do not produce adverse reactions, allergic reactions, or other unwanted reactions. As used herein, "pharmaceutically acceptable carriers" include all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. Any conventional media and / or agents are conceivable for use in therapeutic compositions unless they are incompatible with the agents of this disclosure. Auxiliary active ingredients may also be added to the compositions.

[0145] The active composition may also be administered parenterally, intraperitoneally, or intratumorally. Solutions of the active compound as a free base or a pharmacoagulably acceptable salt are prepared in water, appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. These preparations contain preservatives to inhibit microbial growth under normal storage and use conditions.

[0146] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Inhibition of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, the inclusion of isotonic agents, such as sugars or sodium chloride, is considered preferable. Extension of absorption of the injectable composition can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0147] Sterile injectable solutions are prepared by adding one of the compounds, along with various other components mentioned above, in the required amount to a suitable solvent, and then, if necessary, by filtration sterilization. Generally, dispersions can be prepared by adding various sterile active ingredients to a sterile vehicle containing a basic dispersion medium and other required components from the above components. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder combining the active ingredient and any desired additional components from a pre-sterile filtered solution.

[0148] After preparation, one of the compositions is administered in a therapeutically effective amount in a form suitable for the dosage form. The formulations are readily administered in various dosage forms, such as injection solutions and drug-release capsules. For parenteral administration in aqueous solutions, for example, the solution is buffered as needed, and the liquid diluent isotonicized first with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution or injected into the intended injection site (see, for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, the active particles or activator can be incorporated into the therapeutic mixture in amounts such as about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per single dose. Multiple doses can also be administered.

[0149] Further formulations suitable for other modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may also be used. Suppositories are solid dosage forms of various weights and shapes, usually containing a drug, for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the lumen. Generally, in the case of suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides, and such suppositories can be formed from a mixture containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Vaginal suppositories or pessaries are usually spherical or oval in shape, each weighing about 5 g. Vaginal drugs are available in various physical forms that do not fit the classical concept of suppositories, such as creams, gels, or liquids. Compositions can also be formulated as inhalants.

[0150] In some embodiments, the present invention also provides a kit comprising a composition containing one or more nanoparticles associated with the peptides described herein. In some embodiments, the kit comprises one or more reagents and tools necessary for the production of any of the compositions, as well as a method for using any of such compositions.

[0151] Examples The following examples are provided to illustrate and further demonstrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope. The use of pronouns such as "we," "our," and "inventors" refers to the inventors.

[0152] Example I. Synthesis of sHDL ND carrying a peptide with a pI value of less than 7 and a net charge of less than 0 at pH 7. method Preparation of antigen-loaded sHDL ND DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. The ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) (SEQ ID NO: 769) was synthesized by GenScript Biotech. All other peptides used in this study were synthesized by Genemed Synthesis Inc. A list of the antigen peptides used in this study is shown in Table 7.

[0153] [Table 7]

[0154] To prepare blank sHDL ND, DMPC and 22A peptide powders were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC:22A = 2:1, mass ratio) (pH = 7.4). This mixture was then subjected to a heating and cooling cycle to obtain blank sHDL ND, which was then sonicated at room temperature for 1 minute. To load the antigen peptide into the blank ND, the cysteine-terminated antigen peptide was conjugated to DOPE-MAL (antigen peptide:DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank ND (22A:antigen peptide = 4:1, mass ratio) and incubated at room temperature for 1 hour with gentle shaking in an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce).

[0155] Particle size distribution measurement using dynamic light scattering (DLS) The hydrodynamic particle size of peptide load ND was measured by dynamic light scattering (DLS, Malvern Septizer Nano ZSP) in 10 mM phosphate buffer (pH=7.4). The refractive index (RI) and absorbance parameters were set to 1.45 and 0.001, respectively. Dispersant: phosphate buffer, temperature / viscosity = 25°C / 0.89, RI = 1.333. Method: Mark-Houwink method for particle size measurement (default setting). Temperature: 25°C. Equilibrium time: 120 seconds. Cell type: Disposable cuvette ZEN0040. Measurement angle: 173° backscattering (NIBS default setting). Measurement time: Automatically performed 11 times of 10 seconds for 3 measurements. Readings: Particle size distribution based on volume and intensity.

[0156] Measurement of loading efficiency by liquid chromatography-mass spectrometry (LC-MS) The chromatogram of DOPE-peptide was characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) equipped with a biphenyl column (1.7 mm, 50 × 2.1 mm, 100 A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold with methanol. Loading efficiency was calculated by comparing the area under the curve (AUC) of DOPE-peptide in the LC-MS chromatograms before and after purification.

[0157] result The inventors first tested a method for preparing sHDL ND loaded with peptides having a pI value < 7 and a net charge < 0 at pH 7. The inventors selected CSS-SIINFEKL (SEQ ID NO: 743) and CSS-Ea (CSS-ASFEAQGALANIAVDKA) (SEQ ID NO: 746) for these tests, as they had pI values ​​of 6.1 and 3.9, and net charges of -0.1 and -1.1 at pH 7, respectively (Table 7). In previous tests, the inventors demonstrated that peptide-lipid conjugates formed by modifying the N-terminus of an antigen peptide with a "Cys-Ser-Ser" (CSS) linker and then attaching a DOPE-MAL lipid to the Cys thiol of the resulting peptide could be loaded into ND. Using this method, CSS-SIINFEKL (SEQ ID NO: 743) and CSS-Ea were readily loaded into ND, and the resulting ND was stable at pH 7.4 and showed a single peak around 10 nm, as indicated by the DLS volume and intensity profiles (Tables 1 and 2). Therefore, these results indicate that efficient loading and the formation of uniform peptide-loaded sHDL ND are possible by modifying the N-terminus of peptides with pI values ​​< 7 and net charge < 0 at pH 7 (e.g., SIINFEKL (SEQ ID NO: 770) and Ea peptides) with a CSS linker.

[0158] Next, the inventors investigated the effects of changing the CSS linker to a CSE or CEE linker. The inventors synthesized SIINFEKL (SEQ ID NO: 770) or Ea peptides (Peptides 2, 3, and 5, Table 7) having a CSE or CEE linker and investigated whether this affected the loading of peptide-lipids into sHDL ND. CSS-SIINFEKL (SEQ ID NO: 743), CSE-SIINFEKL (SEQ ID NO: 744), and CEE-SIINFEKL (SEQ ID NO: 745) were synthesized, bound to DOPE-MAL, and then peptide-lipids were loaded into sHDL. The obtained peptide-loaded sHDL was stable at pH 7.4, and its DLS volume and intensity profiles showed a particle size distribution similar to sHDL ND loaded with either CSS-SIINFEKL (SEQ ID NO: 743), CSE-SIINFEKL (SEQ ID NO: 744), or CEE-SIINFEKL (SEQ ID NO: 745) (Peptides 1, 2, and 3, Figure 1). Similarly, CSS-Ea and CSE-Ea were synthesized, bound to DOPE-MAL, and then the peptide-lipids were loaded into sHDL. The obtained peptide-loaded sHDL was stable at pH 7.4, and its DLS volume and intensity profiles showed a particle size distribution similar to sHDL ND loaded with either CSS-Ea or CSE-Ea (Peptides 4 and 5, Figure 1). Furthermore, the loading efficiency of SIINFEKL (SEQ ID NO: 770) or Ea peptide onto ND remained similar or slightly improved when the peptide linker was changed from CSS to either CSE or CEE (Peptides 1-5, Table 7).

[0159] These results indicate that, for peptides with a pI value of less than 7 and a net charge of less than 0 at pH 7, the CSS-, CSE-, or CEE- linkers were all suitable linkers for modifying the peptide sequence to form a uniform peptide load sHDL ND.

[0160] Example II. Synthesis of sHDL ND carrying a peptide with a pI value greater than 7 and a net charge greater than 0 at pH 7. method Preparation of antigen-loaded sHDL ND DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. The ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) (SEQ ID NO: 769) was synthesized by GenScript Biotech. All other peptides used in this study were synthesized by Genemed Synthesis Inc. A list of the antigen peptides used in this study is shown in Table 8.

[0161] [Table 8]

[0162] To prepare blank sHDL ND, DMPC and 22A peptide powders were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC:22A = 2:1, mass ratio) (pH = 7.4). This mixture was then subjected to a heating and cooling cycle to obtain blank sHDL ND, which was then sonicated at room temperature for 1 minute. To load the antigen peptide into the blank ND, the cysteine-terminated antigen peptide was conjugated to DOPE-MAL (antigen peptide:DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank ND (22A:antigen peptide = 4:1, mass ratio) and incubated at room temperature for 1 hour with gentle shaking in an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce).

[0163] Particle size distribution measurement using DLS The hydrodynamic particle size of peptide load ND was measured by dynamic light scattering (DLS, Malvern Septizer Nano ZSP) in 10 mM phosphate buffer (pH=7.4). The refractive index (RI) and absorbance parameters were set to 1.45 and 0.001, respectively. Dispersant: phosphate buffer, temperature / viscosity = 25°C / 0.89, RI = 1.333. Method: Mark-Houwink method for particle size measurement (default setting). Temperature: 25°C. Equilibrium time: 120 seconds. Cell type: Disposable cuvette ZEN0040. Measurement angle: 173° backscattering (NIBS default setting). Measurement time: Automatically performed 11 times of 10 seconds for 3 measurements. Readings: Particle size distribution based on volume and intensity.

[0164] Measurement of loading efficiency by liquid chromatography-mass spectrometry (LC-MS) The chromatogram of DOPE-peptide was characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) equipped with a biphenyl column (1.7 mm, 50 × 2.1 mm, 100 A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold with methanol. Loading efficiency was calculated by comparing the area under the curve (AUC) of DOPE-peptide in the LC-MS chromatograms before and after purification.

[0165] result Next, the inventors investigated a method for preparing sHDL ND loaded with peptides having a pI value > 7 and a net charge > 0 at pH 7. The inventors selected CSS-MOG38-50, CSS-PLP178-191, CSS-KV11, and CSS-NRPA7 for these tests because they have a pI value > 7 and a net charge > 0 at pH 7 (Table 8).

[0166] In previous studies, we demonstrated that peptide-lipid conjugates formed by modifying the N-terminus of an antigen peptide with a "Cys-Ser-Ser" (CSS) linker and then attaching a DOPE-MAL lipid to the Cys thiol of the resulting peptide can be loaded into NDs. This approach works well for peptides with pI < 7 and a net charge < 0 at pH 7, as shown in Example I. However, this approach can often lead to aggregation of NDs with peptides that are poorly water-soluble, have a pI > 7, and a net charge > 0 at pH 7. This is the case for the CSS-MOG38-50 peptide, which has a pI = 10.2 and a charge of +2 at pH 7 (peptide 6, Table 8). NDs loaded with CSS-MOG38-50 formed aggregates with an average particle size greater than 1000 nm in both DSL volume and intensity profiles (peptide 6, Figure 2). Therefore, when the inventors changed the CSS linker of the MOG38-50 peptide to either a CDD or CDDD linker, the pI values ​​decreased to 7.1 and 5.1, respectively, and the net charge decreased to 0 and -1 (Peptides 7 and 8, Table 8). Furthermore, when the CSS linker was changed to either a CDD linker or a CDDD linker, the water solubility of the MOG38-50 peptide increased (Peptides 6-8, Table 8). Both CDD-MOG38-50 and CDDD-MOG38-50 formed stable and uniform NDs with an average particle size of 10 mM in 10 mM phosphate buffer at pH 7.4 when loaded with sHDL ND, as shown in the volume and intensity profiles of the DLS (Peptides 7 and 8, Figure 2). Furthermore, the loading efficiency of CSS-MOG38-50 into ND was significantly improved from 70.5% to 99.1% and 99.9% respectively when using CDD-MOG38-50 or CDDD-MOG38-50 (peptides 6-8, Figure 2).

[0167] Similarly, when CSS-PLP178-191, which has low water solubility, a pI of 9.1, and a net charge of 0.9 at pH 7, was loaded onto sHDL ND (peptide 9, Table 8), a heterogeneous ND particle size distribution was produced (peptide 9, Figure 2). In contrast, CDD-PLP178-191, which has a pI of 3.7 and a net charge of -1.1 at pH 7 (peptide 10, Table 8), showed improved water solubility, was stably loaded into ND, and formed uniform ND with an average particle size of 10 nm in 10 mM phosphate buffer at pH 7.4, as shown in the DLS volume and intensity profiles (peptide 10, Figure 2). The loading efficiency of CDD-PLP178-191 also increased significantly to 96.4% compared to 59.6% for CSS-PLP178-191 (peptides 9-10, Figure 2).

[0168] The inventors also investigated a similar approach for peptides with good water solubility, pI > 7, and net charge > 0 at pH 7. CSS-KV11 has a pI of 9.1 and a net charge of 0.9 at pH 7, and exhibits good water solubility (peptide 11, Table 8). When CSS-KV11 was loaded onto sHDL ND, the resulting ND appeared homogeneous in the DLS volume profile, but aggregation of the ND was shown in the DLS intensity profile (peptide 11, Figure 3). In contrast, CSE-KV11 and CEE-KV11 had pI values ​​of 6.3 and 4.5, and net charges of -0.1 and -1.1 at pH 7, respectively (peptides 12 and 13, Table 8). They were stably loaded into ND, and as shown in both the DLS volume and intensity profiles, homogeneous ND with an average particle size of 10 nm was formed in 10 mM phosphate buffer at pH 7.4 (peptides 12 and 13, Figure 3). The loading efficiencies of CSS-KV11, CSE-KV11, and CEE-KV11 into ND were 72.7% to 79.2% (peptides 11-13, Table 8).

[0169] Similar findings were observed with CSS-NRPA7, which had a pI of 9.9, a net charge of 1.9 at pH 7, and good water solubility (peptide 14, Table 8). When CSS-NRPA7 was loaded onto sHDL ND, the resulting ND appeared homogeneous in the DLS volume profile, but aggregation of the ND was shown in the DLS intensity profile (peptide 14, Figure 3). In contrast, CSS-NRPA7 with a pI of 6.4 and a net charge of -0.1 at pH 7 (peptide 10, Table 8) was stably loaded onto ND, and homogeneous ND with an average particle size of 10 nm was formed in 10 mM phosphate buffer at pH 7.4, as shown in both the DLS volume and intensity profiles (peptide 15, Figure 3). The loading efficiency of CEE-NRPA7 into ND increased to 94.5% compared to 80.5% for CSS-NRPA7 (peptides 14-15, Figure 3).

[0170] Overall, these results indicate that for peptides with a pI value of less than 7 and a net charge of less than 0 at pH 7, the CDD-, CDDD-, CSE-, or CEE- linkers are all suitable linkers for modifying the peptide sequence, increasing the water solubility of the peptide and resulting in a more uniform peptide load in sHDL ND and improved peptide loading efficiency into sHDL ND.

[0171] Example III. Synthesis of sHDL ND carrying a peptide with non-terminal cysteine method Preparation of antigen-loaded sHDL ND DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. The ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) (SEQ ID NO: 769) was synthesized by GenScript Biotech. All other peptides used in this study were synthesized by Genemed Synthesis Inc. A list of the antigen peptides used in this study is shown in Table 9.

[0172] [Table 9]

[0173] To prepare blank sHDL ND, DMPC and 22A peptide powders were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC:22A = 2:1, mass ratio) (pH = 7.4). This mixture was then subjected to a heating and cooling cycle to obtain blank sHDL ND, which was then sonicated at room temperature for 1 minute. To load the antigen peptide into the blank ND, a cysteine-containing antigen peptide was conjugated to DOPE-MAL (antigen peptide:DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank ND (22A:antigen peptide = 4:1, mass ratio) and incubated at room temperature for 1 hour with gentle shaking in an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce).

[0174] Particle size distribution measurement using dynamic light scattering (DLS) The hydrodynamic particle size of peptide load ND was measured by dynamic light scattering (DLS, Malvern Septizer Nano ZSP) in 10 mM phosphate buffer (pH=7.4). The refractive index (RI) and absorbance parameters were set to 1.45 and 0.001, respectively. Dispersant: Phosphate buffer, temperature / viscosity = 25°C / 0.89, RI = 1.333. Method: Mark-Houwink method for particle size measurement (default setting). Temperature: 25°C. Equilibrium time: 120 seconds. Cell type: Disposable cuvette ZEN0040. Measurement angle: 173° backscattering (NIBS default setting). Measurement time: Automatically performed 11 times of 10 seconds for 3 measurements. Readings: Particle size distribution based on volume and intensity.

[0175] Measurement of loading efficiency by liquid chromatography-mass spectrometry (LC-MS).

[0176] The chromatogram of DOPE-peptide was characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) equipped with a biphenyl column (1.7 μm, 50 × 2.1 mm, 100 A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold with methanol. Loading efficiency was calculated by comparing the area under the curve (AUC) of DOPE-peptide in the LC-MS chromatograms before and after purification.

[0177] result The inventors investigated a method for preparing sHDL ND loaded with peptides having non-terminal cysteine ​​residues. For these tests, the inventors selected mIns2 B:9-23 and HMOG186-200, which have pI values ​​<7 and a net charge <0 at pH 7 (peptides 16 and 17, Table 9).

[0178] When mIns2 B:9-23, which has low water solubility, a pI of 5.3, and a net charge of -1.0 at pH 7, was loaded onto sHDL ND (peptide 16, Table 9), a non-uniform particle size distribution of ND was produced (peptide 16, Figure 4). Similarly, when HMOG186-200, which has a pI of 3.0 and a net charge of -0.1 at pH 7 (peptide 17, Table 9), was loaded onto sHDL NDs, a non-uniform particle size distribution was produced (peptide 17, Figure 4). To solve this problem, the inventors modified HMOG186-200 by adding a DDD linker (Asp-Asp-Asp) to its N-terminus, obtaining HMOG186-200-DDD with a pI of 0.5 and a net charge of -3.1 at pH 7. This modification significantly improved the stability and water solubility of the peptide. ND loaded with HMOG186-200-DDD was stable at pH 7.4, and a single peak was observed around 10 nm in the DLS volume and intensity profile (Figure 4). The loading efficiency of HMOG186-200-DDD into ND also improved to 82.4% compared to 68.9% for HMOG186-200 (peptides 17-18, Table 9). These results demonstrate the versatility of using peptides that have cysteine ​​residues not only at the N-terminus but also at non-terminal positions. By incorporating a linker, the water solubility of these peptides is increased, promoting the formation of uniform peptide-loaded sHDL ND and improving the efficiency of peptide loading into sHDL ND.

[0179] Example IV. Synthesis of sHDL ND carrying a peptide with a C-terminal cysteine method Preparation of antigen-loaded sHDL ND DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. The ApoA1 mimetic peptide 22A (PVLDLFRELLBELLEALKQKLK) (SEQ ID NO: 769) was synthesized by GenScript Biotech. All other peptides used in this study were synthesized by Genemed Synthesis Inc. A list of the antigen peptides used in this study is shown in Table 10.

[0180] [Table 10]

[0181] To prepare blank sHDL ND, DMPC and 22A peptide powders were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC:22A=2:1, mass ratio) (pH=7.4). This mixture was then subjected to a heating and cooling cycle to obtain blank sHDL ND, which was then sonicated at room temperature for 1 minute. To load the antigen peptide into the blank ND, the cysteine ​​C-terminal antigen peptide was conjugated to DOPE-MAL (antigen peptide:DOPE-MAL=1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank ND (22A:antigen peptide=4:1, mass ratio) and incubated at room temperature for 1 hour with gentle shaking in an orbital shaker. Unreacted antigen peptide was removed using a Zeba Spin desalting column (Pierce).

[0182] Particle size distribution measurement using DLS The hydrodynamic particle size of peptide load ND was measured by dynamic light scattering (DLS, Malvern Septizer Nano ZSP) in 10 mM phosphate buffer (pH=7.4). The refractive index (RI) and absorbance parameters were set to 1.45 and 0.001, respectively. Dispersant: Phosphate buffer, temperature / viscosity = 25°C / 0.89, RI = 1.333. Method: Mark-Houwink method for particle size measurement (default setting). Temperature: 25°C. Equilibrium time: 120 seconds. Cell type: Disposable cuvette ZEN0040. Measurement angle: 173° backscattering (NIBS default setting). Measurement time: Automatically performed 11 times of 10 seconds for 3 measurements. Readings: Particle size distribution based on volume and intensity.

[0183] Measurement of loading efficiency using LC-MS The chromatogram of DOPE-peptide was characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) equipped with a biphenyl column (1.7 μm, 50 × 2.1 mm, 100 A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold with methanol. Loading efficiency was calculated by comparing the area under the curve (AUC) of DOPE-peptide in the LC-MS chromatograms before and after purification.

[0184] result Next, the inventors investigated a method for preparing sHDL ND loaded with a peptide having a C-terminal cysteine ​​residue. The inventors selected gliadin-C1, which has a pI value of 6.1 and a net charge of -0.1 at pH 7, for these tests (peptide 19, Table 10).

[0185] When gliadin-C1 was loaded onto sHDL ND, the resulting ND was stable at pH 7.4, and a single peak was observed around 10 nm in the DLS volume and intensity profile (peptide 19, Figure 5). The loading efficiency of gliadin-C1 into NDs was 88.9% (Table 10).

[0186] These results demonstrate that stable peptide loads of sHDL ND can be obtained even with peptides containing a C-terminal cysteine ​​residue. Equal parts The present invention may be implemented in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein should be considered illustrative in all respects rather than limiting the invention as described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all modifications that fall within the meaning and equivalence of the claims are intended to be encompassed therein.

[0187] Reference The entirety of the disclosures of the patent documents and scientific papers referenced herein is incorporated by reference for all purposes. The entirety of the following references is incorporated by reference herein.

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[0189] [Figure 1] The particle size distribution of ND-SIINFEKL (with CSS, CSE, or CEE linker) and ND-Ea (with CSS or CSE linker) measured by DLS in 10 mM phosphate buffer (pH=7.4) is shown. [Figure 2] The particle size distribution of ND-MOG38-50 (with CSS, CDD, or CDDD linker) and ND-PLP178-191 (with CSS or CDD linker) measured by DLS in 10 mM phosphate buffer (pH=7.4) is shown. [Figure 3] The particle size distribution of ND-KV11 (with CSS, CSE, or CEE linker) and ND-NRPA7 (with CSS or CEE linker) measured by DLS in 10 mM phosphate buffer (pH=7.4) is shown. [Figure 4]The particle size distribution of ND-mIns2 B:9-23 (without linker) and ND-HMOG186-200 (without linker or with DDD linker) measured by DLS in 10 mM phosphate buffer (pH=7.4) is shown. [Figure 5] The particle size distribution of ND-gliadin-C1 (without linker) measured by DLS in 10 mM phosphate buffer (pH=7.4) is shown.

Claims

1. A composition comprising sHDL nanoparticles, Phospholipids and Apolipoprotein mimetic, Thiol-reactive lipids, A peptide comprising a linker portion connected to a payload portion, The aforementioned linker, Cysteine ​​(C) and, optionally, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), or If the payload contains C amino acids, it includes either DD or DDD. The payload comprises a polypeptide having a length of 5 to 35 amino acids. The peptide has a negative net charge at pH 7 and an isoelectric point of 0.4 to 12. The composition wherein the peptide is covalently bonded to the thiol-reactive lipid via the linker.

2. The linker comprises cysteine ​​(C) and, optionally, 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S). The composition according to claim 1, wherein the peptide is covalently bonded to the thiol-reactive lipid via the amino terminus of the Cys, the carboxyl terminus of the Cys, or an unterminus of the Cys.

3. The payload contains C amino acid, The linker includes DD or DDD, The composition according to claim 1, wherein the peptide is covalently bonded to the thiol-reactive lipid via the linker's DD or DDD.

4. The composition according to claim 1, wherein the peptide comprises the formula: [linker]-[payload] or [payload]-[linker].

5. The composition according to claim 1, wherein, if the payload contains a C amino acid, the linker sequence is selected from C, DDCDD, DD, and DDD.

6. The composition according to claim 1, wherein the payload has a positive net charge at pH 7 to 12.

7. The composition according to claim 1, wherein the payload has a pH of 7 to 12 and a net charge of 0.

8. The composition according to claim 1, wherein the payload has a negative net charge at pH 7 to 12.

9. The composition according to claim 1, wherein the peptide has a charge of less than -0.

1.

10. The composition according to claim 1, wherein the peptide has a charge in the range of -0.1 to -5.

0.

11. The composition according to claim 1, wherein the peptide has an isoelectric point of 3.7 to 12.

12. The composition according to claim 1, wherein the peptide has an isoelectric point of 0.62 to 9.

78.

13. The composition according to claim 1, wherein the linker comprises a peptide dimer.

14. The composition according to claim 1, wherein the peptide dimer is selected from DD, SE, SD, and EE.

15. The composition according to claim 1, wherein the 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide trimer.

16. The composition according to claim 15, wherein the peptide trimer is selected from DDD, EEE, and KEE.

17. The composition according to claim 1, wherein the 1 to 5 amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute a peptide tetramer.

18. The composition according to claim 17, wherein the peptide tetramer is selected from DDDD (SEQ ID NO: 762) and EEEE (SEQ ID NO: 763).

19. The composition according to claim 1, wherein the payload has a charge greater than 0.1 at pH 7.

20. The composition according to claim 19, wherein the payload has a charge in the range of 0.1 to 5.

0.

21. The composition according to claim 1, wherein the payload is a polypeptide having a length of 12 to 35 amino acids.

22. The composition according to claim 21, wherein the payload is selected from GWYRSPFSRVVHL (SEQ ID NO: 764), NTWTTSQSIAFPSK (SEQ ID NO: 765), KVAPVWVRMME (SEQ ID NO: 766), KYNKANAFL (SEQ ID NO: 767), and ASFEAQGALANIAVDKA (SEQ ID NO: 768).

23. The apolipoprotein mimetic is sequence numbers 1-336, and WDRVKDLATVYVDVLKDSGRDYVSQF (sequence number 341), LKLLDNWDSVTSTSFSKLREOL (sequence number 342), PVTOEFWDNLEKETEGLROEMS (sequence number 343), KDLEEVKAKVQ (sequence number 344), KDLEEVKAKVO (sequence number 345), PYLDDFQKKWQEEEMELYRQKVE (sequence number 346), PLRAELQEGARQKLHELOEKLS (sequence number 347), PLGEEMRDRARAHVD ALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354)QTVTDYGKDLME (SEQ ID NO: 35 5) KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAPQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASLL (SEQ ID NO: 362), HLRKLRRKRLLRDADDL QKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLLEALKQKLA (SEQ ID NO: 368), PVLDLFRELLNELLLEALKQKLK (SEQ ID NO: 4),The composition according to claim 1, which is an ApoA-I mimetic having any of the sequences of PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFFAEEFLARKS (SEQ ID NO: 373).

24. The composition according to claim 23, wherein the apolipoprotein mimetic has the sequence PVLDLFRELLNELLEALKQKLK (Sequence ID 4).

25. The thiol-reactive lipids are dioleyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-di-(9Z-octadecenoyl))-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2- The composition according to claim 1, selected from dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-maleimide (DOPE-Mal), and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide].

26. The composition according to claim 25, wherein the thiol-reactive lipid is DOPE-PDP.

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