Peptide-related nanoparticle compositions

Abstract

The present invention relates to a composition comprising sHDL nanoparticles. In particular, the present invention relates to sHDL nanoparticles comprising a phospholipid; an apolipoprotein mimetic; a thiol reactive lipid; and a peptide comprising a linker moiety linked to a payload moiety wherein the linker comprises cysteine (C) and one to five amino acids independently selected from aspartic acid (D), glutamic acid (E) and serine (S), the payload comprising a polypeptide having a length of 5 to 35 amino acids and having a net positive charge at pH 7 to 12, and the peptide comprises a net negative charge at pH 7 and an isoelectric point from 0.4 to 12; and wherein the peptide is covalently linked to the thiol-reactive lipid via the cysteine (C).

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 522,816, filed June 23, 2023, which is incorporated herein by reference in its entirety.

[0002] Statement of government funding This invention was carried out with government support under DE030691 granted by the National Institutes of Health. The government owns certain rights to this invention.

[0003] sequence list The text of the computer-readable sequence list, titled "UM_41715_601_SequenceListing.xml", created on June 18, 2024 and with a file size of 671,386 bytes, which was submitted along with this file, is hereby incorporated in its entirety by reference. Technical Field

[0004] This invention relates to compositions comprising sHDL nanoparticles. Specifically, this invention relates to sHDL nanoparticles comprising phospholipids; apolipoprotein mimics; thiol-reactive lipids; and a peptide comprising a linker portion connected to a payload portion, wherein the linker comprises cysteine ​​(C) and one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), wherein the payload comprises a polypeptide of 5 to 35 amino acids in length and having a net positive charge at pH 7 to 12, and wherein the peptide contains a net negative charge at pH 7 and an isoelectric point of 0.4 to 12; and wherein the peptide is covalently linked to the thiol-reactive lipid via the cysteine ​​(C). Background Technology

[0005] Peptide-based therapeutics have gained significant 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 therapeutics is their ability to generate diverse specific sequences, resulting in a vast functional diversity. To date, over 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 trigger intracellular effects by binding to specific cell surface receptors or ion channels. However, the vast majority of peptides cannot autonomously leave lysosomes or penetrate cell membranes [4]. In addition, low plasma stability, short circulation time, low oral bioavailability, and high cost of large-scale manufacturing significantly limit the use of peptide drugs [5].

[0006] These drawbacks of peptides can be overcome by developing novel drug delivery systems. To this end, a large number of peptide / protein delivery systems have been proposed, including those using lipid nanocarriers [6, 7], polymers [8, 9], mesoporous silica nanoparticles

[10] , cell-penetrating peptides [11, 12], and various other methods [13–16]. Notably, 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 even surpass them, as they exhibit long circulation, good tolerability, and high stability

[19] . Furthermore, the receptor-mediated internalization capacity and unique ultrasmall size of approximately 10 nm make sHDLs a promising carrier for tissue penetration and high intracellular accumulation, which is particularly useful for applications such as drug delivery to tumors [20–22].

[0007] A number of sHDL-based ND delivery systems have been previously developed, consisting of phospholipids and apolipoprotein A1 mimic peptides [23-28]. In conventional approaches, a “Cys-Ser-Ser” linker is added to the N-terminus of the peptide, and thiol chemistry is used to conjugate the peptide to the lipid. The resulting peptide-lipid conjugate is then loaded onto a pre-formed ND. While this strategy works for many peptides, it has proven to be not universally applicable. For example, hydrophobic peptides and those with isoelectric points close to the pH of the solution tend to form some aggregates when incorporated into ND [29, 30]. Furthermore, while peptides with neutral charges exhibit an aggregation tendency, peptides with high positive isoelectric points at pH 7 also form aggregates [31, 32]. In this case, arginine aggregates more readily than lysine, due to the greater tendency of the Arg side chain to form protein-protein interactions

[33] .

[0008] There is a need for improved compositions and methods for delivering peptides.

[0009] This invention satisfies these needs. Summary of the Invention

[0010] Experiments conducted during the development of embodiments of the present invention led to the development of a novel strategy for attaching negatively or positively charged amino acids at the C or N terminus to increase the water solubility of peptides and improve the manufacturability of sHDL NDs loaded with peptides. Such experiments demonstrated that, for peptides with high isoelectric points (pI), adding a negatively charged amino acid (such as aspartic acid or glutamic acid) to the peptide sequence results in a net charge conversion from positive to negative at pH 7. This charge conversion allows peptide-lipid conjugates to be efficiently and robustly loaded onto NDs, resulting in homogeneous and uniform NDs loaded with peptides. Therefore, adding negatively or positively charged amino acids at the C or N terminus of a peptide provides a general strategy for improving peptide incorporation into NDs and for improving the manufacture of peptide-ND products. Furthermore, our method demonstrates versatility in using peptides with cysteine ​​residues not only at the N-terminus but also at the C-terminus or non-terminal positions to obtain sHDL NDs loaded with peptides. In summary, the embodiments described herein relate to a novel composition that improves the incorporation of various peptides into NDs and the manufacture of peptide-ND products. Furthermore, this method is generally applicable to incorporating various peptides into other nanoparticles and polymers.

[0011] Therefore, in some embodiments, the present invention provides compositions comprising sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise phospholipids; apolipoprotein mimics; thiol-reactive lipids; and a peptide comprising a linker portion connected to a payload portion, wherein the linker comprises: [cysteine ​​(C) and optionally one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S)] or [DD or DDD, if the payload comprises a C amino acid], the payload comprising a polypeptide of 5 to 35 amino acids in length and having a net positive charge at pH 7 to 12, and the peptide comprising a net negative charge at pH 7 and an isoelectric point of 0.4 to 12; and wherein the peptide is covalently linked to the thiol-reactive lipid via the linker.

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

[0013] In some embodiments, if the payload contains C amino acids, the peptide is covalently linked to a thiol-reactive lipid via a linker, wherein the linker contains DD or DDD.

[0014] In some implementations, the peptide comprises the following formula: [linker]-[payload] or [payload]-[linker].

[0015] In some embodiments, the payload has a net positive charge at pH 7 to 12. In some embodiments, the payload has a net zero charge at pH 7 to 12. In some embodiments, the payload has a net negative charge at pH 7 to 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 DDDDD. In some embodiments, if the payload contains a C amino acid, the linker sequence is DD or DDD.

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

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

[0021] In some embodiments, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute the 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 of 12 to 35 amino acids in length.

[0023] In some implementations, 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 mimic is an ApoA-I mimic having any of the following sequences: SEQ ID NO: 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). 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: 353) TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354) 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: 361) NO:363), AQAWGERLRARMEEMGSRTRDR (SEQ IDNO:364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO:365), DWLKAFYDKVAEKLKEAF (SEQ ID NO:236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQID NO:366), PVLDLFRELLNELLEALKQKL (SEQ ID NO:367),PVLDLFRELLNELLEALKQKLA (SEQID NO:368),PVLDLFRELLNELLEALKQKLA (SEQ ID NO:4),PVLDLFRELLNELLEALKQKLA (SEQID NO:369),PVLDLFRELLNELLEALKKLLK (SEQ ID NO:370),PVLDLFRELLNELLEALKKLLA (SEQID NO:371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO:372) and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

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

[0026] In some embodiments, the thiol-reactive lipid is selected from dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridinedithio)propionate]. (DOPE-PDP), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide], 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-maleimide (DOPE-Mal) and 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide].

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

[0028] Based on the teachings contained herein, alternative implementation methods will be readily apparent to those skilled in the art. Attached Figure Description

[0029] Figure 1 Size distribution of ND-SIINFEKL (with CSS, CSE, or CEE connectors) and ND-Ea (with CSS or CSE connectors) as measured by DLS in 10 mM phosphate buffer (pH = 7.4).

[0030] Figure 2 Size distribution of ND-MOG38-50 (with CSS, CDD, or CDDD connectors) and ND-PLP178-191 (with CSS or CDD connectors) as measured by DLS in 10 mM phosphate buffer (pH = 7.4).

[0031] Figure 3 Size distribution of ND-KV11 (with CSS, CSE, or CEE connectors) and ND-NRPA7 (with CSS or CEE connectors) as measured by DLS in 10 mM phosphate buffer (pH = 7.4).

[0032] Figure 4 Size distribution of ND-mIns2 B:9-23 (no connector) and ND-HMOG186-200 (no connector or with DDD connector) as measured by DLS in 10 mM phosphate buffer (pH = 7.4).

[0033] Figure 5 : such as the size distribution of ND-gliadin-C1 (linkerless) measured by DLS in 10 mM phosphate buffer (pH = 7.4).

[0034] definition The term “about” is used in this document to mean a value of ±10% of the listed values.

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

[0036] As used herein, the term "association with" refers to the state of two or more entities (e.g., nanoparticles and one or more peptides) connected by direct or indirect covalent or non-covalent interactions. In some embodiments, association is covalent. In some embodiments, covalent association is mediated by a junctional portion. In some embodiments, association is non-covalent (e.g., charge interactions, affinity interactions, metal coordination, physisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.). For example, in some embodiments, peptides are co-mixed with nanoparticles. In some embodiments, peptides are conjugated with nanoparticles. In some embodiments, peptides are encapsulated within nanoparticles. In some embodiments, peptides are adsorbed into nanoparticles. In some embodiments, peptides are adsorbed onto nanoparticles. In some embodiments, peptides are co-mixed with nanoparticles.

[0037] As used herein, the term “absorption” refers to the absorption of peptides and their stable retention within nanoparticles and / or microparticles (i.e., within the outer surface).

[0038] As used herein, the term "blending" refers to the dissolution, dispersion, or suspension of peptides within nanoparticles and / or microparticles. In some cases, biomacromolecules can be uniformly blended within nanoparticles and / or microparticles.

[0039] As used herein, the term "adsorption" refers to the attachment of a peptide to the outer surface of nanoparticles and / or microparticles. This adsorption preferably occurs via electrostatic attraction. Electrostatic attraction is an attraction or bond between two or more chemical groups with opposite charges or ions. Generally, adsorption is usually reversible.

[0040] As used herein, the term “protrusion protein” is intended to include proteins and polypeptides that have altered amino acid sequences and are produced due to mutation or recombination DNA programs.

[0041] As used herein, "combination therapy" or "combination administration" means administering two (or more) different agents or treatments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) to a subject as part of a defined treatment regimen for a specific disease or ailment. The treatment regimen defines the dosage and administration period of each agent to allow the effects of the individual agents to overlap with those of the subject. In some embodiments, the two or more agents are delivered simultaneously or in parallel, and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated and are administered sequentially as part of a prescription regimen. In some embodiments, the combination administration of two or more agents or treatments results in a reduction in symptoms or other parameters related to the condition that is greater than the reduction observed when a single agent or treatment is delivered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic effect). The order or substantially simultaneous administration of each therapeutic agent can be affected by any suitable route, including but not limited to inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, local, buccal, or direct absorption through mucosal tissue. Therapeutic agents can be administered via the same or different routes. For example, the first therapeutic agent in a combination can be administered intravenously, while the second therapeutic agent in a combination can be administered orally.

[0042] As used herein, the terms "drug" or "therapeutic agent" are intended to include any molecule, molecular complex, or substance administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraception, cosmetic, nutritional health, pharmaceutical, and preventative applications. The term "drug" further means any such molecule, molecular complex, or substance that is chemically modified and / or operatively linked to a biological or biocompatible structure.

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

[0044] As used in this article, the term “amplification” refers to the increase in the number of cells in a cell population or sample due to cell replication.

[0045] As used herein, the term "HDL" or "high-density lipoprotein" refers to high-density lipoprotein. HDL comprises a complex of roughly equal amounts of lipids and proteins that act as a cholesterol transporter in the blood. HDL is primarily synthesized and secreted from the epithelial cells of the liver and small intestine. Immediately after secretion, HDL takes the form of discoid granules containing apolipoprotein AI (also known as apoA-I) and phospholipids as its main components, and is also called neonatal HDL. This neonatal HDL receives free cholesterol from the cell membranes of peripheral cells or from the hydrolysis of other lipoproteins in the blood and forms mature globular HDL, while retaining cholesterol esters converted from said cholesterol by the action of LCAT (lecithin-cholesterol acyltransferase) in its hydrophobic center. HDL plays a crucial role in a lipid metabolism process known as "reverse cholesterol transport," which removes cholesterol from peripheral tissues in the blood and transports it to the liver. High levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD), as reverse cholesterol transport is considered one of the main mechanisms by which HDL plays a preventive role in atherosclerosis.

[0046] As used herein, the term "nucleic acid" can be DNA or RNA, such as mRNA. In embodiments, the composition comprises complement of any nucleic acid provided herein, such as full-length complement or degenerate complement (due to the degeneracy of the genetic code). In embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may comprise plasmids, retroviruses, or adenoviruses, etc. Nucleic acids can be isolated or synthesized using standard molecular biology methods, for example, by using a polymerase chain reaction to generate nucleic acid fragments, which are then purified and cloned into an expression vector. Additional techniques useful in the practice of this invention can be found in John Wiley and Sons, Inc.'s Current Protocols in Molecular Biology 2007; Molecular Cloning: A Laboratory Manual (3rd 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 processes or reactions that occur within that environment. An in vitro environment can consist of, but is not limited to, test tubes and cell cultures.

[0048] The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and the processes or reactions that occur in the natural environment.

[0049] As used herein, the term "lipid" or "lipid molecule" refers to fatty substances that are insoluble in water and includes fats, oils, waxes, and related compounds. They can 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, they must be transported and then released for cellular use. The production, transport, and release of lipids for cellular use is called lipid metabolism. While there are several classes of lipids, the two main classes are cholesterol and triglycerides. Cholesterol can be ingested through diet and is produced by cells in most organs and tissues of the body, primarily in the liver. Cholesterol can exist in its free form or, more commonly, in combination with fatty acids to form so-called cholesterol esters. As used herein, "lipid" or "lipid molecule" refers to any lipophilic compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogues. They are generally classified into at least three categories: (1) “simple lipids”, which include fats and oils as well as waxes; (2) “complex lipids”, which include phospholipids and glycolipids; and (3) “derived lipids”, such as steroids. The lipids or lipid molecules applicable to the present invention include both film-forming lipids and non-film-forming lipids.

[0050] As used herein, the term "lipoprotein" refers to a compound whose structure allows water-insoluble lipids to be contained within a partially water-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, which differ in function and their lipid and apoprotein content, and are classified according to increasing density: (i) chylomicrons and chylomicron remnants, (ii) very low-density lipoproteins ("VLDL"), (iii) intermediate-density lipoproteins ("IDL"), (iv) low-density lipoproteins ("LDL"), and (v) high-density lipoproteins ("HDL"). Cholesterol circulates in the bloodstream as particles associated with lipoproteins.

[0051] As used herein, the term "non-naturally occurring amino acid" refers to an α-amino acid that is not naturally produced or absent in mammals. Examples of non-naturally occurring amino acids include D-amino acids; amino acids having an acetylaminomethyl group linked to a sulfur atom in cysteine; polyethylene glycol-modified amino acids; and amino acids of the formula H2(CH2). nω-amino acids of COOH, where n is 2-6; neutral nonpolar amino acids, such as sarcosine, tert-butylalanine, tert-butylglycine, N-methylisoleucine and ortholeucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; sulfoalanine; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminovaleric acid; 2-aminooctanoic acid, 2-carboxypiperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutyric acid; 3-(2-naphthyl)alanine and hydroxyproline. Other amino acids include α-aminobutyric acid, α-amino-α-methylbutyrate, aminocyclopropane-carboxylic acid ester, aminoisobutyric acid, aminononyl-carboxylic acid ester, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-leucine, α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, and D-aminobutyric acid. 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, D-α-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methyl Valine, DN-methylalanine, DN-methylarginine, DN-methylasparagine, DN-methylaspartate, DN-methylcysteine, DN-methylglutamine, DN-methylglutamate, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, N-methylcyclohexylalanine, DN-methylornithine, N-methylglycine, N-methylaminoisobutyrate, N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyl Tyrosine, DN-methylvaline, γ-aminobutyric acid, L-tert-butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α-methylaspartate, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-methylleucine, L-α-methylmethionine, L-α-methyln-valine, L-α-methylphenylalanine, L-α-methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-Diphenylethyl)carbamoylmethylglycine, 1-carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl (o-aminobenzoyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidin-4-carboxylic acid, L-homotyrosine, L-2-furanylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-serine, m-tyrosine, nor-tyrosine, LN,N′,N″-trimethyllysine, homolysine, norlysine N-Glycan Asparagine, 7-Hydroxy-1,2,3,4-Tetrahydro-4-fluorophenylalanine, 4-Methylphenylalanine, Bis-(2-methylpyridine)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, 3, or 4-Aminocyclohexanecarboxylic acid, 1-Amino-1-cyclopentanecarboxylic acid, 1-Aminoindan-1-carboxylic acid, 4-Aminopyrrolidine-2-carboxylic acid, 2-Aminotetrahydronaphthalene-2-carboxylic acid, Azacyclobutane-3-carboxylic acid 4-Benzyl-pyrrolidine-2-carboxylic acid, tert-butylglycine, β-(benzothiazolyl-2-yl)-alanine, β-cyclopropylalanine, 5,5-dimethyl-1,3-thiazolidin-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, tert-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1-Aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxy-benzoic acid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid, N-methyl-o-aminobenzoic 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-tert-butylglycine, L-norvaline, α-methyl-γ-aminobutyrate, 4,4′-Biphenylalanine, α-methylcyclopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine, N-(2-carbamoylethyl)glycine, N-(carbamoylmethyl)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-aminobutyrate, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN-methylthreonine, N-(1-methylethyl)glycine, N-methyl-naphthylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine, L-α-Methyl-tert-butylglycine, L-methylethylglycine, L-α-methylglutamate, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, LN-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl)carbamoylmethylglycine, L-pyroglutamic acid, D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamate, phenylglycine, L-piperidinic acid (homoproline), L-homoleucine, L-lysine (di-) Methyl), L-2-naphthylalanine, L-dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, O-glycan-threonine, o-tyrosine, LN,N′-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothiophene alanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorhalman-3-carboxylic acid, adamantylalanine, symmetrical dimethylarginine, 3-carboxythiomorpholine, D-1,2,3,4-Tetrahydronorhalman-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethyl-pyridin-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-amino-tetrahydrothiaran-4-carboxylic acid, azacyclobutane-2-carboxylic acid, β-(ligniol-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, β-cyclobutylalanine, allylglycine, diaminopropionic acid, homocyclohexylalanine, (2S,4R)-4-hydroxypiperidine-2 -Carboxylic acids, octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, hexahydronicotinic acid, (2S,4R) and (2S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-triphenylmethylmercapto-pyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, tert-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and selenocysteine. In some embodiments, the amino acid residues may be charged or polar. Charged amino acids include alanine, lysine, aspartic acid, or glutamic acid, or their non-natural analogues. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or their non-natural analogues. Particularly noteworthy is that, in some embodiments, the terminal amino group of the amino acid may be an amide group or a carbamate group.

[0052] The "sequence identity percentage (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage. Alignment used to determine the nucleic acid or amino acid sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, the sequence alignment computer program BLAST can be used to generate sequence identity percentage values. As an illustration, the sequence identity percentage of a given nucleic acid or amino acid sequence A with respect to, and or for, a given nucleic acid or amino acid sequence B (which can be alternatively phrased as a given nucleic acid or amino acid sequence A having a certain sequence identity percentage with respect to, and or for, a given nucleic acid or amino acid sequence B) is calculated as follows: 100 × (fraction X / Y) Where X is the number of nucleotides or amino acids that are scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It should be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percentage of sequence identity of A with respect to B will not be equal to the percentage of sequence identity of B with respect to A.

[0053] The term "protein" refers to a polymer of amino acids of any length (e.g., naturally occurring and non-natural amino acids). The term also covers polymers of amino acids that have been modified; for example, by disulfide bond formation, glycosylation, acetylation, phosphorylation, esterification, or conjugation with labeled components.

[0054] As used herein, the term "peptide" refers to a polymer in which monomers are covalently linked together by amide bonds. A peptide is a polymer of two or more amino acid monomers.

[0055] “Pharmaceutical composition” means any composition containing a peptide suitable for administration to a subject. Any formulation may be prepared by methods well known and accepted in the art. See, for example, Remington: The Science and Practice of Pharmacy (21st edition), edited by AR Gennaro, Lippincott Williams & Wilkins, 2005; and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.

[0056] "Pharmaceutically acceptable diluents, excipients, carriers or adjuvants" means diluents, excipients, carriers or adjuvants that are physiologically acceptable to the subject and that retain the therapeutic properties of the pharmaceutical composition administered with them.

[0057] As used herein, the term "sample" is used in its broadest sense. In one sense, it is intended to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, etc. Environmental samples include environmental materials such as surface substances, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to this invention.

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

[0059] As used herein, the terms “synthetic HDL,” “sHDL,” “reconstructed HDL,” and “rHDL” refer to particles that are structurally similar to natural HDL and consist of one or more lipids associated with at least one protein of HDL (preferably ApoA-I) or its mimicry. Typically, the components of sHDL can be derived from blood or produced through recombinant technologies.

[0060] "Therapeutic effective amount" refers to the amount of composition administered in a clinically relevant manner to improve, inhibit, or improve symptoms of a subject's ailment, condition, or disease (e.g., celiac disease). Any improvement in the subject is considered sufficient to achieve therapeutic effect. Preferably, a therapeutically effective amount is an amount that reduces, inhibits, or prevents the occurrence of a disease or condition or one or more symptoms, or reduces the severity of one or more symptoms of a disease or condition or the duration for which the subject has had one or more symptoms of a disease or condition (e.g., a reduction of at least about 10%, about 20%, or about 30%, more preferably at least about 50%, about 60%, or about 70%, and most preferably at least about 80%, about 90%, about 95%, about 99%, or more, relative to a control subject not treated with the composition described herein). The effective amount of the pharmaceutical composition used to practice the methods described herein varies depending on the manner of administration and the age, weight, and general health condition of the subject being treated. A physician or researcher may determine the appropriate amount and dosage regimen.

[0061] As used herein, the term "solvent" refers to the medium in which a reaction takes place. Solvents can be liquids, but are not limited to liquids. Solvent categories include, but are not limited to, nonpolar, polar, proton, and aproton. Detailed Implementation

[0062] Experiments conducted during the development of embodiments of the present invention led to the development of a novel strategy for attaching negatively or positively charged amino acids at the C or N terminus to increase the water solubility of peptides and improve the manufacturability of sHDL NDs loaded with peptides. Such experiments demonstrated that, for peptides with high isoelectric points (pI), adding a negatively charged amino acid (such as aspartic acid or glutamic acid) to the peptide sequence results in a net charge conversion from positive to negative at pH 7. This charge conversion allows peptide-lipid conjugates to be efficiently and robustly loaded onto NDs, resulting in homogeneous and uniform NDs loaded with peptides. Therefore, adding negatively or positively charged amino acids at the C or N terminus of a peptide provides a general strategy for improving peptide incorporation into NDs and for improving the manufacture of peptide-ND products. Furthermore, our method demonstrates versatility in using peptides with cysteine ​​residues not only at the N-terminus but also at the C-terminus or non-terminal positions to obtain sHDL NDs loaded with peptides. In summary, the embodiments described herein relate to a novel composition that improves the incorporation of various peptides into NDs and the manufacture of peptide-ND products. Furthermore, this method is generally applicable to incorporating various peptides into other nanoparticles and polymers.

[0063] Therefore, in some embodiments, the present invention provides compositions comprising sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise phospholipids; apolipoprotein mimics; thiol-reactive lipids; and a peptide comprising a linker portion connected to a payload portion, wherein the linker comprises: [cysteine ​​(C) and optionally one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S)] or [DD or DDD, if the payload comprises a C amino acid], the payload comprising a polypeptide of 5 to 35 amino acids in length and having a net positive charge at pH 7 to 12, and the peptide comprising a net negative charge at pH 7 and an isoelectric point of 0.4 to 12; and wherein the peptide is covalently linked to the thiol-reactive lipid via the linker.

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

[0065] In some embodiments, if the payload contains C amino acids, the peptide is covalently linked to a thiol-reactive lipid via a linker, wherein the linker contains DD or DDD.

[0066] In some implementations, the peptide comprises the following formula: [linker]-[payload] or [payload]-[linker].

[0067] In some embodiments, the payload has a net positive charge at pH 7 to 12. In some embodiments, the payload has a net zero charge at pH 7 to 12. In some embodiments, the payload has a net negative charge at pH 7 to 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 DDDDD. In some embodiments, if the payload contains a C amino acid, the linker sequence is DD or DDD.

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

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

[0073] In some embodiments, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute the 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 of 12 to 35 amino acids in length.

[0075] In some implementations, 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 mimic is an ApoA-I mimic having any of the following sequences: SEQ ID NO: 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). 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: 353) TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354) 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: 361) NO:363), AQAWGERLRARMEEMGSRTRDR (SEQ IDNO:364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO:365), DWLKAFYDKVAEKLKEAF (SEQ ID NO:236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQID NO:366), PVLDLFRELLNELLEALKQKL (SEQ ID NO:367),PVLDLFRELLNELLEALKQKLA (SEQID NO:368),PVLDLFRELLNELLEALKQKLA (SEQ ID NO:4),PVLDLFRELLNELLEALKQKLA (SEQID NO:369),PVLDLFRELLNELLEALKKLLK (SEQ ID NO:370),PVLDLFRELLNELLEALKKLLA (SEQID NO:371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO:372) and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

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

[0078] In some embodiments, the thiol-reactive lipid is selected from dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridinedithio)propionate]. (DOPE-PDP), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide], 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-maleimide (DOPE-Mal) and 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide].

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

[0080] In some embodiments, the present invention provides a method for treating, preventing, and / or alleviating a condition, the method comprising administering to a subject (e.g., a person subject who has a disease or medical condition or is at risk of having a disease or medical condition) a composition as described herein.

[0081] Such methods are not limited to treating specific conditions.

[0082] In some implementations, the condition is an autoimmune disease. Such approaches are not limited to treating specific autoimmune diseases. Examples of autoimmune diseases include, but are not limited to: multiple sclerosis (MS), celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), thyroid autoimmune diseases (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated ophthalmopathy and dermatitis, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypophysitis, pituitary autoimmune diseases, immune gastritis, malignant vasculitis, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and its variants, bullous pemphigoid, Durin's herpesoid dermatitis, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Sjogren's syndrome, systemic lupus erythematosus, and Goodpasture's syndrome. Syndrome, rheumatic heart disease, type 1 autoimmune polyglandular syndrome, Aicardi–Goutières syndrome, acute pancreatitis, age-dependent macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, and inflammatory bowel disease.

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

[0084] In some implementations, such methods for treating or preventing autoimmune diseases also include the co-administration (e.g., simultaneously or at different times) of additional therapeutic agents. Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologics (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anaprostol, abatacept), glucocorticoids (e.g., prednisone, methylprednisolone), TNF-α inhibitors (e.g., adalimumab, pego-sertuzumab, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some implementations, the therapeutic agent includes, but is not limited to, infliximab, adalimumab, etanercept, or parenteral or oral gold formulations. 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 trogopterin A; corticosteroids; mitochondrial function inhibitors, such as rotenone; p38 inhibitors; NF-κβ inhibitors, such as 6Bio, dexamethasone, TCPA-1, IKK). 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; PI3KB inhibitors, such as TGX-221; autophagy inhibitors, such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidative ATP, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cyclosporins such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflufenicol, estriol, triptolide; OPN-305, OPN-401; Eritoran (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; o-vanillin; SSL3 protein; OPN-305; 5 SsnB; Vizantin; (+)-N-phenylethyl norhydroxymorphone; VB3323; Monosaccharide 3; (+)-naltrexone and (+)-naloxone; HT52; HTB2; Compound 4a; CNTO2424; TH1020; INH-ODN; E6446; AT791; CpG ODN 2088; ODNTTAGGG; COV08-0064; 2R9; GpG oligonucleotide; 2-aminopurine; ammoniated; Bay11-7082; BX795; CH-223191; chloroquine; CLI-095; CU-CPT9a; cyclosporine A; CTY387; gefitinib; glibenclamide; H-89; H-131; isoliquiritigenin; MCC950; MRT67307; O xPAPC; ternolactone; Pepinh-MYD; Pepinh-TRIF; polymyxin B; R406; RU.521; VX-765; YM201636; Z-VAD-FMK; and AHR-specific ligands; including but not limited to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD); tryptamine (TA); and 6-formylindolo[3,2-b]carbazole (FICZ). In specific embodiments, the immunosuppressant is fingolimod; rapamycin; methyl 2-(1'H-indole-3'-carbonyl)-thiazol-4-carboxylate (ITE) or a related ligand; trichostatin A; and / or succinylanilide isohydroxamic acid (SAHA).

[0085] This invention is not limited to specific types or kinds of nanoparticles that are associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, mixed) or not associated with peptides as described herein.

[0086] Examples of nanoparticles include, but are not limited to: fullerenes (also known as C464-C ... 60 C70 C 76 C 80 C 84 The nanoparticles include: EMI-embedded metallofullerene (EMI) buckyballs (containing additional atoms, ions, or clusters within their fullerene cages); trimetallic nitride-templated EMI-embedded metallofullerenes (TNT EME, highly symmetric tetraatomic molecular cluster embeddings formed within a trimetallic nitride template in 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 / nitrate nanotubes; carbon nanotube pods (nanotubes with internal metallofullerenes and / or other internal chemical structures); carbon nanohorns; carbon nanohorn pods; liposomes; nanoshells; dendritic polymers; quantum dots; superparamagnetic nanoparticles; nanorods; and cellulose nanoparticles. Particle embodiments may also include microparticles with the ability to enhance effectiveness or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, and iron nanoparticles.

[0087] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise polyol polymers modified to contain hydrophobic polymer blocks. As used herein, the term "hydrophobic polymer block" refers to a segment in the polymer that is itself hydrophobic. As used herein, the term "micelle" refers to an aggregate of molecules dispersed in a liquid. Typical micelles in aqueous solutions form aggregates in which a hydrophilic "head" region is in contact with the surrounding solvent, thereby isolating a hydrophobic single-tail region at the center of the micelle. In some embodiments, the head region may be, for example, a surface region of a polyol polymer, while the tail region may be, for example, a hydrophobic polymer block region of a polyol polymer.

[0088] This invention further encompasses the use of micron-sized particles in addition to nanoscale particles. When using microparticles, they are preferably relatively small, on the order of 1-50 micrometers. For ease of discussion, the term "nanoparticle" herein refers to true nanoparticles (sizes from about 1 nm to 1000 nm), microparticles (e.g., from 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, dendritic polymers, dendritic polymers with covalently linked metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanotethers, and quantum dots. In some embodiments, the nanoparticles are metallic nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). Nanoparticles may include a core or a core and shell, as in core-shell nanoparticles.

[0090] In some implementations, the nanoparticles are sHDL nanoparticles. Typically, sHDL nanoparticles consist of a mixture of HDL apolipoproteins and amphiphilic lipids.

[0091] This invention is not limited to the use of a specific type or species of HDL apolipoprotein. HDL apolipoproteins include, for example, apolipoprotein AI (apo AI), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), apolipoprotein M (apo M), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from proto-apolipoprotein, proto-ApoA-I, proto-ApoA-I, ApoA-I, proto-ApoA-II, proto-ApoA-II, ApoA-II, apolipoprotein A-II xxx (apo A-II-xxx), proto-ApoA-IV, proto-ApoA-IV, ApoA-IV, ApoA-IV, ApoA-V, proto-ApoE, proto-ApoE, ApoE, proto-ApoA-lMilano, proto-ApoA-lmilano, ApoA-lMilano, proto-ApoA-Iparis, proto-ApoA-Iparis, ApoA-Iparis, and peptide mimics of these proteins, and mixtures thereof. Preferably, the carrier particles consist of ApoA-I or ApoA-II; however, the use of other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate a mixture of carrier particles for delivering therapeutic agents. In some implementations, analogues of such HDL apolipoproteins are used.

[0092] ApoA-I is synthesized by the liver and small intestine as a proto-apolipoprotein, which is secreted as a proton. This proton is rapidly cleaved to produce a mature polypeptide with 243 amino acid residues. ApoA-I is primarily composed of 6 to 8 distinct 22-amino acid repeat sequences and 2 distinct 11-amino acid repeat sequences. Each repeat sequence has a helical wheel characteristic of an amphiphilic α-helix, separated by linker portions typically composed of proline residues, and in some cases, by extensions consisting of several residues. ApoA-I forms three types of stable complexes with lipids: small, lipid-averse complexes called preβ-1 HDL; flattened, disc-shaped particles containing polar lipids (phospholipids and cholesterol) called preβ-2 HDL; and globular particles containing both polar and nonpolar lipids, called globular or mature HDL (HDL3 and HDL2). Most HDLs in the circulating population contain both ApoA-I and ApoA-II (the second major HDL protein).

[0093] In some embodiments, an ApoA-I agonist or mimic is provided. In some embodiments, such ApoA-I mimics are capable of forming an amphiphilic α-helix that mimics the activity of ApoA-I and have specific activity close to or exceeding that of the natural molecule. In some embodiments, the ApoA-I mimic is a peptide or peptide analog that: forms an amphiphilic helix (in the presence of lipids), binds to lipids, forms a pre-β-like or HDL-like complex, activates lecithin:cholesterol acyltransferase (LCAT), increases serum levels of the HDL fraction, and promotes cholesterol efflux.

[0094] This invention is not limited to the use of a specific ApoA-I emulator. In some embodiments, any ApoA-I emulator described in Srinivasa et al., 2014 Curr. Opinion Lipidology, Vol. 25(4): 304-308 is used. In some embodiments, any ApoA-I emulator described in U.S. Patent Application Publications Nos. 20110046056 and 20130231459 is used.

[0095] In some embodiments, the “22A” ApoA-I emulator (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) is used (see, for example, U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I emulators shown in Table 1 as described in U.S. Patent No. 7,566,695 are used: Table 1. ApoA-I simulants

[0096] * indicates an N-terminal acetylated and C-terminal amidated peptide; indicates an N-terminal dansyl-substituted peptide; sp indicates a peptide exhibiting solubility problems under experimental conditions; X is Aib; Z is Na1; O is Orn; and ˜ indicates a missing amino acid.

[0097] In some embodiments, an ApoA-I analogue having the following sequence as 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 AlaPhe (SEQ ID NO: 255).

[0098] In some embodiments, any of the following ApoA-I emulators as shown in Table 2 as described in U.S. Patent Application Publication No. 2003 / 0171277 are used: Table 2. ApoA-I simulants

[0099] In some embodiments, an Apo AI analogue with the following sequence, as described in U.S. Patent Application Publication No. 2006 / 0069030, is used: FAEKFKEAVKDYFAKFWD (SEQ ID NO:333).

[0100] In some embodiments, Apo AI analogs having the following sequences, as described in U.S. Patent Application Publication No. 2009 / 0081293, are used: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0101] In some embodiments, an Apo A-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 IDNO: 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 IDNO:359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO:360), ALDKLKEFGNTLEDKARELIS (SEQID NO: 361), VVALLALLASARASEAEDASLL (SEQ ID NO:362), HLRKLRKRLLRDADDLQKRLAVYOA(SEQ ID NO:363), AQAWGERLRARMEEMGSRTRDR (SEQ IDNO: 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).

[0102] Amphiphilic lipids include, for example, any lipid molecule having both hydrophobic and hydrophilic moieties. Examples include phospholipids or glycolipids. Examples of phospholipids that can be used in sHDL-peptide nanoparticles include, but are not limited to, 1,2-dilauroyl-sn-glycerol-3-phosphate choline; 1,2-dimyristoyl-sn-glycerol-3-phosphate choline; 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline; 1,2-distearatel-sn-glycerol-3-phosphate choline; 1,2-distearatel-sn-glycerol-3-phosphate choline; 1,2-diarachidoyl-sn-glycerol-3-phosphate choline; 1,2-dibenzeneyl-sn-glycerol-3-phosphate choline. Choline; 1,2-diliginyl-sn-glycerol-3-phosphate choline; 1,2-dimyristoleoyl-sn-glycerol-3-phosphate choline; 1,2-ditransmyristoleoyl-sn-glycerol-3-phosphate choline; 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline; 1,2-ditranspalmitoyl-sn-glycerol-3-phosphate choline; 1,2-dipetroleum selenyl-sn-glycerol-3-phosphate choline; 1,2-dioleoyl-sn -Glyceryl-3-phosphocholine; 1,2-Ditransoleoyl-sn-glyceryl-3-phosphocholine; 1,2-Diicosenoyl-sn-glyceryl-3-phosphocholine; 1,2-Diceryl-sn-glyceryl-3-phosphocholine; 1,2-Dilauroyl-sn-glyceryl-3-phosphoethanolamine; 1,2-Dimyristoyl-sn-glyceryl-3-phosphoethanolamine; 1,2-Dipentadecanoyl-sn-glyceryl-3-phosphoethanolamine; 1,2-Dibrown Palmitoyl-sn-glycerol-3-phosphate ethanolamine; 1,2-distearate-sn-glycerol-3-phosphate ethanolamine; 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine; 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine; 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine; dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridinyl dithio)propionate]; 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate thioethanol; 1,2-bis-(9Z-octadecenoyl)- sn -Glyceryl-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butyramide]; 1,2-hexadecanoyl- sn -Glyceryl-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butyramide]; 1,2-hexadecanoyl- sn -Glyceryl-3-phosphate ethanolamine-N-[4-(p-maleimidemethyl)cyclohexane-formamide]; 1,2-bis-(9Z-octadecenoyl)- sn-Glyceryl-3-phosphoethanolamine-N-[4-(p-maleimidemethyl)cyclohexane-formamide]; N-[(3-maleimide-1-oxopropyl)aminopropylpolyethylene glycol-carbamoyl]distearylphosphatidyl-ethanolamine; N-[(3-maleimide-1-oxopropyl)aminopropylpolyethylene glycol-carbamoyl]distearylphosphatidyl-ethanolamine; N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, distearylphosphatidyl-3-phosphatidyl-ethanolamine, distearylphosphatidyl-3-phospho ... Acyl group; N-[(3-maleimide-1-oxopropyl)aminopropylpolyethylene glycol-carbamoyl]distearate phosphatidyl-ethanolamine; N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, myristoyl group; N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, dioleoyl group; N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, dipalmitoyl group; N-(3 -maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl; phosphatidylcholine; phosphatidylinositol; phosphatidylserine; phosphatidylethanolamine; N-(succinimide-oxyglutaryl)-L-α-phosphatidylethanolamine, distearate; N-(succinimide-oxyglutaryl)-L-α-phosphatidylethanolamine, dioleoyl; N-(succinimide-oxyglutaryl)-L-α-phosphatidylethanol Amines, 1-palmitoyl-2-oleoyl; N-(succiniminooxyglutarate)-L-α-phosphatidylethanolamine, dipalmitoyl; N-(succiniminooxyglutarate)-L-α-phosphatidylethanolamine, dimyristoyl; 3-(N-succiniminooxyglutarate)aminopropyl and polyethylene glycol-carbamoyl distearate phosphatidyl-ethanolamine; N-(3-oxopropoxy polyethylene glycol)carbamoyl distearate ethanolamine.

[0103] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0104] Typically, the sHDL nanoparticles thus formed are spherical or disc-shaped and have a diameter of about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to produce a more homogeneous formulation.

[0105] 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, wherein the linker comprises cysteine ​​(C) and one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S), wherein the payload comprises a polypeptide of 5 to 35 amino acids in length and having a net positive charge at pH 7 to 12, and wherein the peptide comprises a net negative charge at pH 7 and an isoelectric point of 0.4 to 12; and wherein the peptide is covalently linked to the thiol-reactive lipid via the cysteine ​​(C).

[0106] In some embodiments, the peptide is covalently linked to the thiol-reactive lipid via a Cys group. In some embodiments, the peptide is covalently linked to the thiol-reactive lipid via the carboxyl terminus of the Cys group. In some embodiments, the peptide is covalently linked to the thiol-reactive lipid via the amino terminus of the Cys group. In some embodiments, the peptide is covalently linked to the thiol-reactive lipid via a non-terminal end of the Cys group.

[0107] In some implementations, the peptide comprises the following formula: [linker]-[payload] or [payload]-[linker].

[0108] In some implementations, the connector sequence is DDDDD.

[0109] In some embodiments, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute the peptide dimer. In some embodiments, the peptide dimer is selected from DD, SE, SD, and EE.

[0110] In some embodiments, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute the peptide trimer. In some embodiments, the peptide trimer is selected from DDD, EEE, and KEE.

[0111] In some embodiments, one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S) constitute the peptide tetramer. In some embodiments, the peptide tetramer is selected from DDDD (SEQ ID NO:762) and EEEE (SEQ ID NO:763).

[0112] 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 of 12 to 35 amino acids in length.

[0113] In some implementations, 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).

[0114] In some implementations, the peptide is an antigen and / or a tolerogenic antigen.

[0115] In some embodiments, the antigen associated with the nanoparticles comprises gliadin polypeptides, such as full-length gliadin polypeptides or any epitope of gliadin polypeptides. In some embodiments, the antigen associated with the nanoparticles comprises a 33-meric polypeptide derived from α2-gliadin. In some embodiments, the 33-meric 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 of LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 374). In some embodiments, the antigen associated with the nanoparticles comprises an epitope of the 33-meric gliadin polypeptide. The epitope of the 33-meric gliadin polypeptide can be any length shorter than the 33-meric polypeptide. For example, the epitope can contain 25 to 3 (e.g., 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3) amino acid residues, 20 to 5 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5) amino acid residues, 12 to 6 (e.g., 12, 11, 10, 9, 8, 7, or 6) amino acid residues, or 9 amino acid residues in length. Other examples of 33-gliadin epitopes that can associate with nanoparticles include any of the epitopes described in Table 3, including SEQ ID No: 375-405. In some embodiments, the tolerogenic antigen associated with the nanoparticles may include any one of the antigens described in Table 4, including SEQ ID No: 406-580. In some embodiments, the antigen associated with the nanoparticles 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 No: 375-580. In some embodiments, the tolerogenic antigen associated with the nanoparticles may include an antigen comprising two or more (e.g., 2, 3, 4, 5, or 6) polypeptide sequences having any two of the polypeptide sequences in SEQ ID No: 375-580. In some embodiments, the multiple tolerogenic antigens associated with the nanoparticles (e.g., 1-30 (e.g., 6-30 or 8-30 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) per nanoparticle) have the same identity as each other tolerogenic antigen associated with the nanoparticles.In some embodiments, the plurality of tolerogenic antigens associated with the nanoparticles comprise a group of 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different antigen sequences relating to the same disease; for example, the nanoparticles may associate with 3-8 (e.g., 3, 4, 5, 6, 7, or 8), 4-6 (e.g., 4, 5, or 6), or 3-4 different polypeptide antigen sequences. In some embodiments, the nanoparticles may associate with: (i) a first polypeptide group comprising an amino acid sequence of any one of SEQ ID No: 406-580 or a biologically active fragment or variant thereof; (ii) a second polypeptide group comprising an amino acid sequence of any one of SEQ ID No: 406-580 or a biologically active fragment or variant thereof; and (iii) a third polypeptide group comprising an amino acid sequence of any one of SEQ ID No: 406-580 or a biologically active fragment or variant thereof. In some cases, the first, second, and third polypeptide groups have different amino acid sequences. In some embodiments, the nanoparticles may be associated with: (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a bioactive fragment or variant thereof; (ii) a second polypeptide comprising the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475) or a bioactive fragment or variant thereof; and (iii) a third polypeptide comprising the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476) or a bioactive fragment or variant thereof. In some embodiments, the nanoparticles may be associated with: (i) a first polypeptide comprising the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474) or a bioactive fragment or variant thereof; (ii) a second polypeptide comprising the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477) or a bioactive fragment or variant thereof; and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478) or a bioactive fragment or variant thereof.In some embodiments, the nanoparticles may associate 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, the tolerogenic antigen having the polypeptide sequences of SEQ ID No: 506, 507, and 508 comprises an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigen having the polypeptide sequences of SEQ ID No: 506, 507, and 508 comprises a C-terminal amide group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID No: 506, 507, and 508 comprise an N-terminal pyroE residue and a C-terminal amide group. In some embodiments, the nanoparticles may associate with: (i) a first polypeptide comprising the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509) or a bioactive fragment or variant thereof; (ii) a second polypeptide comprising the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510) or a bioactive fragment or variant thereof; and (iii) a third polypeptide comprising the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511) or a bioactive fragment or variant thereof. In some embodiments, the tolerogenic antigens having the polypeptide sequences of SEQ ID No: 509, 510, and 511 comprise an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID No: 509, 510, and 511 comprise a C-terminal amide group. In some embodiments described herein, the tolerogenic antigens having the polypeptide sequences of SEQ ID No: 509, 510, and 511 comprise an N-terminal acetyl group and a C-terminal amide group. In any embodiment described herein, the antigen population associated with the nanoparticles may be completely or partially deamidated. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal pyroglutamic acid (pyroE). In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal acetyl group. In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may comprise an N-terminal amide group.In some embodiments described herein, the tolerogenic antigen associated with the nanoparticles may contain a C-terminal amide group.

[0116] Table 3: From CD4 + Celiah-associated T cell epitopes recognized by T cells

[0117] Table 4: Tolerogenic Antigens

[0118] In some embodiments, the tolerogenic antigen is the biologically active fragment of SEQ ID NO: 474. In some cases, the biologically active fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 512. In some cases, the biologically active fragment of SEQ ID NO: 474 comprises a polypeptide containing the sequence of SEQ ID NO: 580.

[0119] In some cases, the tolerogenic antigen is the biologically active fragment of SEQ ID NO: 475. In some cases, the biologically active fragment of SEQ ID NO: 475 comprises a polypeptide containing the sequence of SEQ ID NO: 542.

[0120] In some embodiments, the tolerogenic 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.

[0121] In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 375). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPQPELPY (SEQ ID NO: 376). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQ (SEQ ID NO: 377). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FRPEQPYPQ (SEQ ID NO: 378). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQQ (SEQ ID NO: 379). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IQPEQPAQL (SEQ ID NO: 380). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPYPQ (SEQ ID NO: 381). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SQPEQEFPQ (SEQ ID NO: 382). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQEFPQ (SEQ ID NO: 383). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPEQPFPQ (SEQ ID NO: 384). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFCQ (SEQ ID NO: 385). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPEQPQ (SEQ ID NO: 386). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPF (SEQ ID NO: 387). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPW (SEQ ID NO: 388). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 389). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FSQQQESPF (SEQ ID NO: 390). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQ (SEQ ID NO: 391).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 392). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPY (SEQ ID NO: 393). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQ (SEQ ID NO: 394). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPQ (SEQ ID NO: 395). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEEPF (SEQ ID NO: 396). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PYPEQEQPF (SEQ ID NO: 397). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFSEQEQPV (SEQ ID NO: 398). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 399). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPFPQ (SEQ ID NO: 400). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPQQPYPE (SEQ ID NO: 401). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 402). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EGSFQPSQE (SEQ ID NO: 403). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQSFPEQE (SEQ ID NO: 404). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGYYPTSPQ (SEQ ID NO: 405). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPW (SEQ ID NO: 406). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPV (SEQ ID NO: 407). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPW (SEQ ID NO: 408).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPIPV (SEQ ID NO: 409). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPFPQ (SEQ ID NO: 410). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPQLPYPQ (SEQ ID NO: 411). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LPYPQPELPYPQ (SEQ ID NO: 412). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYPQ (SEQ ID NO: 413). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQ (SEQ ID NO: 414). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSQ (SEQ ID NO: 415). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSQ (SEQ ID NO: 416). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFCQ (SEQ ID NO: 417). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFCQ (SEQ ID NO: 418). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYSQ (SEQ ID NO: 419). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYSQ (SEQ ID NO: 420). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQQQCSPVAMPQRLAR (SEQ ID NO: 421). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQLPYLQ (SEQ ID NO: 422). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYLQ (SEQ ID NO: 423). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPQQPFPQ (SEQ ID NO: 424).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFIQPEQPFPQ (SEQ ID NO: 425). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQQQPLPP (SEQ ID NO: 426). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LERPWQEQPLPP (SEQ ID NO: 427). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPQQPEQPFPL (SEQ ID NO: 428). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQQGYYPISPQQSGQ (SEQ ID NO: 429). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QGQPGYYPTSPQQIGQ (SEQ ID NO: 430). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PGQGQSGYYPTSPQQS (SEQ ID NO: 431). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPQQPQLP (SEQ ID NO: 432). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQTFPEQPQLP (SEQ ID NO: 433). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence GQGQSGYYPTSPQQSG (SEQ ID NO: 434). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QYEVIRSLVLRTLPNM (SEQ ID NO: 435). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QVDPSGQVQWPQ (SEQ ID NO: 436). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QVDPSGEVQWPQ (SEQ ID NO: 437). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPL (SEQ ID NO: 438). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPL (SEQ ID NO: 439). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPY (SEQ ID NO: 440).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPY (SEQ ID NO: 441). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPQQPQPY (SEQ ID NO: 442). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPVPEQPQPY (SEQ ID NO: 443). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFPQQPIPQQPQPY (SEQ ID NO: 444). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPQQPQPY (SEQ ID NO: 445). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPIPEQPQPY (SEQ ID NO: 446). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPQQPFPQ (SEQ ID NO: 447). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQFPQPEQPFPQ (SEQ ID NO: 448). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPIPQQPQPYPQQP (SEQ ID NO: 449). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQQPFPQQPQPY (SEQ ID NO: 450). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFSW (SEQ ID NO: 451). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFSW (SEQ ID NO: 452). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQPYPQQP (SEQ ID NO: 453). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPIPQ (SEQ ID NO: 454). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPIPQ (SEQ ID NO: 455). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPFPQ (SEQ ID NO: 456).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPQ (SEQ ID NO: 457). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQPTPI (SEQ ID NO: 458). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPTPI (SEQ ID NO: 459). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPQQPFPQ (SEQ ID NO: 460). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PAPIQPEQPFPQ (SEQ ID NO: 461). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPEQI (SEQ ID NO: 462). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPEQI (SEQ ID NO: 463). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQQPQQI (SEQ ID NO: 464). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPEQPQQI (SEQ ID NO: 465). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 466). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIISQ (SEQ ID NO: 467). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: 468). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQQPEQIIPQ (SEQ ID NO: 469). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPQQQLPL (SEQ ID NO: 470). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQQLPL (SEQ ID NO: 471). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LFPLPQQPFPQ (SEQ ID NO: 472).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LFPLPEQPFPQ (SEQ ID NO: 473). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 474). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 475). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 476). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPFPQPEQPFPWQP (SEQ ID NO: 477). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 478). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 479). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPFLPQLPYPQ (SEQ ID NO: 480). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QAFPQPQQTFPH (SEQ ID NO: 481). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence TPIQPQQPFPQ (SEQ ID NO: 482). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 483). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFTQPQQPTPI (SEQ ID NO: 484). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQLQQPQQP (SEQ ID NO: 485). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence VAHAIIMHQQQQQQQE (SEQ ID NO: 486). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SYPVQPQQPFPQ (SEQ ID NO: 487). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQQPQPFPQQPVPQQP (SEQ ID NO: 488).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPWQPQQPFPQ (SEQ ID NO: 489). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPLQPQQPFPQ (SEQ ID NO: 490). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFQPQQPFPQ (SEQ ID NO: 491). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence NPLQPQQPFPLQPQPP (SEQ ID NO: 492). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PLQPQQPFPLQPQPPQ (SEQ ID NO: 493). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PNPLQPQQPFPLQ (SEQ ID NO: 494). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence TIPQQPQQPFPL (SEQ ID NO: 495). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSQQPQQPFPL (SEQ ID NO: 496). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence SFSEQPQQPFPL (SEQ ID NO: 497). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence YSPYQPQQPFPQ (SEQ ID NO: 498). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QLPLQPQQPFPQ (SEQ ID NO: 499). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPQQPFPLQPQQPVP (SEQ ID NO: 500). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IIPQQPQQPFPL (SEQ ID NO: 501). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQIIPQQPQQP (SEQ ID NO: 502). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FLLQPQQPFSQ (SEQ ID NO: 503). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence IIQQPQQPFPL (SEQ ID NO: 504).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQRPQQPFPQ (SEQ ID NO: 505). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 506). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 507). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 508). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QLQPFPQPELPYPQPQ (SEQ ID NO: 509). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QQPFPQPEQPFPWQP (SEQ ID NO: 510). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPEQPIPEQPQPYPQQ (SEQ ID NO: 511). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELP (SEQ ID NO: 512). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYP (SEQ ID NO: 513). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPY (SEQ ID NO: 514). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELP (SEQ ID NO: 515). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPBYPQP (SEQ ID NO: 516). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQ (SEQ ID NO: 517). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYP (SEQ ID NO: 518). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPY (SEQ ID NO: 519). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELP (SEQ ID NO: 520).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPBYPQPQ (SEQ ID NO: 521). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQP (SEQ ID NO: 522). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYP (SEQ ID NO: 523). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPY (SEQ ID NO: 524). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELP (SEQ ID NO: 525). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPELPYPQPQ (SEQ ID NO: 526). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQP (SEQ ID NO: 527). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQ (SEQ ID NO: 528). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYP (SEQ ID NO: 529). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPY (SEQ ID NO: 530). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELP (SEQ ID NO: 531). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPELPYPQPQ (SEQ ID NO: 532). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQP (SEQ ID NO: 533). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQ (SEQ ID NO: 534). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYP (SEQ ID NO: 535). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPY (SEQ ID NO: 536).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPELPYPQPQ (SEQ ID NO: 537). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQP (SEQ ID NO: 538). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYP (SEQ ID NO: 539). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPELPYPQPQ (SEQ ID NO: 540). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQ (SEQ ID NO: 541). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPF (SEQ ID NO: 542). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFP (SEQ ID NO: 543). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPF (SEQ ID NO: 544). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPW (SEQ ID NO: 545). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFP (SEQ ID NO: 546). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPF (SEQ ID NO: 547). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPWQ (SEQ ID NO: 548). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFP (SEQ ID NO: 549). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPEQPFPWQP (SEQ ID NO: 550). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQ (SEQ ID NO: 551). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPW (SEQ ID NO: 552). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFP (SEQ ID NO: 553).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPF (SEQ ID NO: 554). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PQPEQPFPWQP (SEQ ID NO: 555). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQ (SEQ ID NO: 556). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPW (SEQ ID NO: 557). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFP (SEQ ID NO: 558). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence FPQPEQPFPWQP (SEQ ID NO: 559). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQ (SEQ ID NO: 560). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PFPQPEQPFPWQP (SEQ ID NO: 561). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPEQPFPWQ (SEQ ID NO: 562). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQ (SEQ ID NO: 563). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQP (SEQ ID NO: 564). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQ (SEQ ID NO: 565). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQP (SEQ ID NO: 566). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYP (SEQ ID NO: 567). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPY (SEQ ID NO: 568). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQP (SEQ ID NO: 569).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQ (SEQ ID NO: 570). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQQ (SEQ ID NO: 571). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQ (SEQ ID NO: 572). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYP (SEQ ID NO: 573). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPY (SEQ ID NO: 574). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYPQQ (SEQ ID NO: 575). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYPQ (SEQ ID NO: 576). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYP (SEQ ID NO: 577). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPYPQQ (SEQ ID NO: 578). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQPYPQ (SEQ ID NO: 579). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PDLP (SEQ ID NO: 580). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PELPYPQ (SEQ ID NO: 581). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQP (SEQ ID NO: 582). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPFPQPELPYPQPQ (SEQ ID NO: 583). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence LQPFPQPELPYPQP (SEQ ID NO: 584). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PIPEQPQPYPQ (SEQ ID NO: 585).In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence QPIPEQPQPYP (SEQ ID NO: 586). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence EQPIPEQPQPY (SEQ ID NO: 587). In some embodiments, the tolerogenic antigen comprises a polypeptide containing the amino acid sequence PEQPIPEQPQP (SEQ ID NO: 588).

[0122] In some implementations, such tolerogenic antigens include human allogeneic transplantation antigens. Examples of such human allogeneic transplantation antigens include, but are not limited to, subunits of various MHC class I and MHC class II haplotype proteins, as well as single amino acid polymorphisms on minor blood group antigens (including RhCE, Kell, Kidd, Duffy, and Ss).

[0123] In some implementations, the tolerogenic antigen is an autoantigen to which the subject (e.g., a human patient) has already generated an autoimmune response or is capable of generating an autoimmune response. Examples include proinsulin (e.g., for subjects with diabetes or at risk of developing diabetes), collagen (e.g., for subjects with rheumatoid arthritis or at risk of developing rheumatoid arthritis), and myelin basic proteins (e.g., for subjects with multiple sclerosis or at risk of developing multiple sclerosis). Many proteins are human autoimmune proteins, a term relating to various autoimmune diseases in which one or more proteins causing said diseases are known or can be identified by routine testing. Implementations include testing the patient to identify autoimmune proteins and generate an antigen for the molecular fusion, and generating immune tolerance to the protein. Implementations include an antigen, or an antigen selected from one or more of the following proteins. In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, proinsulin, 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, Imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripheral proteins, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreas / pancreas-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreaticoduodenal homology box 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-associated protein, and SST G-protein-coupled receptors 1-5. In autoimmune diseases of the thyroid, including Hashimoto's thyroiditis and Graves' disease, the main antigens include thyroglobulin (TG), thyroid peroxidase (TPO), and thyroid-stimulating hormone receptor (TSHR); other antigens include sodium-iodide cotransporter (NIS) and macroprotein. In thyroid-associated ophthalmopathy and dermatopathy, in addition to thyroid autoantigens (including TSHR), the main antigen is also the insulin-like growth factor 1 receptor. In hypoparathyroidism, the main antigen is the calcium-sensitive receptor. In Addison's disease, the main antigens include 21-hydroxylase, 17α-hydroxylase, and P450 side-chain lyase (P450scc); other antigens include ACTH receptor, P450c21, and P450c17. In premature ovarian failure, the main antigens include FSH receptor and α-enolase. In autoimmune hypophysitis or pituitary autoimmune diseases, the main antigens include pituitary gland-specific protein factors (PGSF) 1a and 2; another antigen is iodothyronine deiodinase type 2. In multiple sclerosis, the main antigens include myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipoprotein. In rheumatoid arthritis, the main antigen is collagen II.In immune gastritis, the main antigen is H. + K + -ATPase. In malignant vasculitis, 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-associated proteins 1 and 2. In myasthenia gravis, the major antigen is acetylcholine receptor. In pemphigus vulgaris and its variants, the major antigens are desmosome core proteins 3, 1, and 4; other antigens include pemphaxin, desmosome collagen, globin, desmosomin, desmosomal plaque, and acetylcholine receptor. In bullous pemphigoid, the major antigens include BP180 and BP230; other antigens include reticulin and laminin 5. In dulin-herpetic dermatitis, the major antigens include endothelial membrane and tissue transglutaminase. In acquired epidermolysis bullosa, the major antigen is collagen VII. In systemic sclerosis, major antigens include matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrin-1, and PDGF receptor; other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere, topoisomerase I, nucleolar protein, RNA polymerase I, II, and III, PM-Slc, fibrin, and B23. In mixed connective tissue diseases, the major antigen is U1snRNP. In Scheringer's syndrome, the major antigens are nuclear antigens SS-A and SS-B; other antigens include cytosine, poly(ADP-ribose) polymerase, and topoisomerase. In systemic lupus erythematosus, major antigens include nucleoproteins (including SS-A), high-mobility group box 1 (HMGB1), nucleosomes, histones, and double-stranded DNA. In Goodpassuu syndrome, major antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the primary antigen is cardiac myosin. Other autoantigens found in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylases, histidine decarboxylases, cysteine ​​sulfinate decarboxylases, tryptophan hydroxylases, tyrosine hydroxylases, phenylalanine hydroxylases, liver P450 cytochrome P450 1A2 and 2A6, SOX-9, SOX-10, calcium-sensitive receptor proteins, and type 1 interferons interferon α, β, and ω.

[0124] In some cases, tolerance-inducing antigens are foreign antigens against which a patient has already developed an unwanted immune response. An example is a food antigen. Implementation methods include testing the patient to identify the foreign antigen and generating a molecular fusion containing the antigen, and treating the patient to develop immune tolerance to the antigen or food. Examples of such foods and / or antigens are provided. Examples include: peanuts: conaragenin (Ara h 1), allergen II (Ara h 2), peanut lectin, and blue bean protein (Ara h 6); apples: 31 kDa major allergen / anti-disease protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), and major allergen Mal d 1.03D (Mal d 1); milk: α-lactalbumin (ALA) and lactoferrin; kiwifruit: kiwifruit protease (Act c 1, Act d 1), phytocystatin, kiwifruit sweet protein-like protein (Act d 2), and kiwifruit defense protein (Act d 5); mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), and profilin (Sin a 4); and celery: profilin (Api g). 4) High molecular weight glycoproteins (Api g 5); from shrimp: Pen a 1 allergen (Pen a 1), Pen m 2 allergen (Pen m 2), rapid isoform of tropomyosin; from wheat and / or other grains: high molecular weight glutenin, low molecular weight glutenin, α- and γ-gliadin, barley gliadin, rye gliadin, oat protein; from strawberry: major strawberry allergy reaction Fra a 1-E (Fra a 1); from banana: inhibitory protein (Mus xp 1). In some embodiments, the tolerogenic antigen is an antigenic peptide of any one of SEQ ID No: 589-742 (Table 5).

[0125] Table 5. Tolerogenic antigens

[0126] In some embodiments, amino acid sequence variants of the tolerogenic antigens of the present invention are contemplated. For example, it may be desirable to improve the tolerogenic antigenicity 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, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the tolerogenic antigen. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as inducing antigen tolerance.

[0127] In some embodiments, tolerogenic antigen variants with one or more amino acid substitutions are provided. Conserved substitutions are shown under the heading "Preferred Substitutions" in Table 6. More substantial variations are provided under the heading "Exemplary Substitutions" in Table 6 and are further described below with respect to the amino acid side chain categories. Amino acid substitutions can be introduced into the tolerogenic antigen of interest, and desired activities of the product can be screened, such as retained / improved tolerogenic antigenicity.

[0128] Table 6. Exemplary and preferred amino acid substitutions

[0129] Amino acids can be grouped based on their common side-chain characteristics: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Fang ethnic group: Trp, Tyr, Phe.

[0130] Non-conservative substitution would require replacing members of one of these categories with members of another category.

[0131] A useful method for identifying residues or regions in tolerogenic antigens that can serve as targets for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989). ScienceAs described in 244:1081-1085, in this method, a specific residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Tolerogenic antigen variants may be screened to determine whether they contain the desired properties.

[0132] Amino acid sequence insertions include amino and / or carboxyl terminus fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues.

[0133] In some embodiments, the tolerogenic antigen comprises an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises a pyroglutamic acid residue at the N-terminus. In another embodiment, the tolerogenic antigen comprises an acetyl group at the N-terminus. In some embodiments, the tolerogenic antigen comprises an N-terminal pyroglutamic acid residue and an amide group at the C-terminus. In some embodiments, the tolerogenic antigen comprises an N-terminus or C-terminus modified with a cysteine ​​residue that binds to a linker. In some embodiments, the tolerogenic antigen comprises both an N-terminus and a C-terminus modified with a cysteine ​​residue that binds to a linker.

[0134] In some embodiments of any of the compositions described herein, the tolerogenic antigen population is combined with nanoparticle phospholipids to enable administration to subjects (e.g., subjects with or at risk of developing autoimmune diseases, such as MS, celiac disease, rheumatoid arthritis, primary biliary cholangitis, primary sclerosing cholangitis, MOG antibody disease, diabetes (e.g., type 1 diabetes), thyroid autoimmune diseases (e.g., Hashimoto's thyroiditis, Graves' disease), thyroid-associated ophthalmopathy and dermatitis, hypoparathyroidism, Addison's disease, premature ovarian failure, autoimmune hypophysitis, pituitary autoimmune diseases, immune gastritis) This condition is associated with a combination of conditions that promote strong immune tolerance, including inflammation, malignant vasculitis, celiac disease, vitiligo, myasthenia gravis, pemphigus vulgaris and its variants, bullous pemphigoid, Durin's herpes-like dermatitis, acquired epidermolysis bullosa, systemic sclerosis, mixed connective tissue disease, Schergerian syndrome, systemic lupus erythematosus, Goodpasser syndrome, rheumatic heart disease, type 1 autoimmune polyglandular syndrome, Ekady-Guterres syndrome, acute pancreatitis, age-dependent macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, or inflammatory bowel disease.

[0135] Depending on the nature of the molecule, such peptides can be prepared using many techniques known in the art. Short peptides are conveniently prepared via amino acid synthesis. Longer proteins with known sequences can be prepared by synthesizing the coding sequence or by PCR amplifying the coding sequence from a natural source or vector and then expressing the coding sequence in a suitable bacterial or eukaryotic host cell.

[0136] The nanoparticles of this invention can be characterized in size and uniformity using any suitable analytical technique. These include, but are not limited to, atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, LC-MS / MS, etc. 13 360° nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC) (equipped with multi-angle laser light scattering, dual UV and refractive index detectors), capillary electrophoresis, and gel electrophoresis. These analytical methods ensure the homogeneity of the sHDL nanoparticle population and are important for quality control in the final production for in vivo applications.

[0137] In some embodiments, gel permeation chromatography (GPC) can be used to analyze sHDL nanoparticles by separating them from liposomes and free ApoA-I mimetic peptides. In some embodiments, size distribution and zeta potential are determined by dynamic light scattering (DLS) using an instrument such as the Malven Nanosizer.

[0138] Such compositions comprising peptide-associated nanoparticles as described herein are not limited to specific methods of administering the composition to a subject. In fact, any acceptable method known to those skilled in the art can be used to administer such compositions to a subject. Administration can be local (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic. Such compositions can be administered via a variety of routes, including but not limited to oral, inhalation (nose or lung), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, local, subcutaneous, sublingual, or rectal administration. Injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal injection. In some embodiments, injection can be administered at multiple sites.

[0139] In consideration of clinical applications, in some embodiments of the invention, compositions comprising peptide-associated nanoparticles as described herein are prepared as part of a pharmaceutical composition in a form suitable for the intended application. Generally, this requires the preparation of compositions substantially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the invention, direct compositions comprising peptide-associated nanoparticles as described herein can be administered using one or more of the methods described herein.

[0140] In a preferred embodiment, the composition is used in combination with a suitable salt and buffer to deliver the composition in a stable manner, thereby allowing it to be taken up by target cells. A buffer is also used when either composition is introduced into a patient.

[0141] Aqueous compositions comprise a cell-effective amount of sHDL nanoparticles dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also known as inoculums.

[0142] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce side effects, allergic reactions, or other adverse reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption-delaying agents, etc. Unless any conventional media or agents are incompatible with the carriers or cells of the present invention, their use in therapeutic compositions should be considered. Supplemental active ingredients may also be incorporated into the composition.

[0143] The active compositions can also be administered parenterally, intraperitoneally, or intratumorally. Solutions of the active compound as a free base or a pharmacologically 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. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0144] Suitable drug forms for injectable applications include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The carrier can 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 flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenols, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars or sodium chloride, may be preferred. Prolonged absorption of the injectable composition can be achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0145] Sterile injectable solutions are prepared by incorporating, as needed, any of the required amounts of the composition along with the various other components listed above into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile medium containing a basic dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, thereby producing powders containing the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0146] After formulation, any composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation can be readily administered in various dosage forms, such as injectable solutions, drug-release capsules, etc. Regarding parenteral administration with aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be prepared with adequate physiological saline or glucose for isotonicity. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous perfusion fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences," 15th edition, pp. 1035-1038 and 1570-1580). In some embodiments of the invention, the active particles or agent are formulated in a therapeutic mixture to contain about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 mg, or even about 10 mg per dose. Multiple doses can be administered.

[0147] Other formulations suitable for different modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may also be used. Suppositories are solid dosage forms of various weights and shapes, typically containing medication, for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the lumen fluid. Typically, for suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from a mixture containing an active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries are typically spherical or oval and each weighs about 5 g. Vaginal medications are available in various physical forms, such as creams, gels, or liquids, which differ from the classic concept of suppositories. These compositions can also be formulated as inhalers.

[0148] In some embodiments, the present invention also provides a kit comprising a composition comprising one or more nanoparticles associated with a peptide as described herein. In some embodiments, the kit comprises one or more reagents and tools necessary to generate any of the compositions, as well as a method for using any of such compositions.

[0149] Example The following examples are provided to illustrate and further explain certain preferred embodiments and aspects of the invention, and should not be construed as limiting its scope. Pronouns such as "we," "our," and "I" are used to refer to the inventive entity.

[0150] Example I. Synthesis of sHDL ND of peptides carrying pI value <7 and net charge <0 at pH 7.

[0151] method Preparation of antigen-loaded sHDL ND.

[0152] DMPC (1,2-dimyristicoyl-sn-glycerol-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. ApoA1-mimic 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 antigenic peptides used in this study is shown in Table 7.

[0153] Table 7. Peptides used in the experiments of Example I. Their peptide sequences, isoelectric points, net charge at pH = 7, water solubility, stability in 10 mM phosphate buffer (pH = 7.4), and loading efficiency when loaded onto sHDL ND are shown.

[0154]

[0155] To prepare blank sHDL NDs, DMPC and 22A peptide powder were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC: 22A = 2:1, mass ratio) at pH 7.4, and then subjected to heating and cooling cycles to obtain blank sHDL NDs, followed by sonication at room temperature for 1 minute. To load the antigenic peptide into the blank NDs, the cysteine-capped antigenic peptide was first conjugated with DOPE-MAL (antigenic peptide: DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank NDs (22A:antigen peptide = 4:1, mass ratio) and incubated for 1 hour at room temperature with gentle shaking on an orbital oscillator. Unreacted antigenic peptides were removed using a Zeba Spin desalting column (Pierce).

[0156] Measured based on the size distribution of dynamic light scattering (DLS).

[0157] Hydrodynamic dimensions of the ND of the loaded peptide were measured in 10 mM phosphate buffer (pH = 7.4) using dynamic light scattering (DLS, Malvern Zetasizer Nano ZSP). 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 (default settings) was used for size measurements. Temperature: 25°C. Equilibration time: 120 seconds. Cell type: disposable cuvette ZEN0040. Measurement angle: 173° backscatter (NIBS default). Measurement duration: 11 automatic runs, 10 seconds each, for 3 measurements. Readings: Size distribution based on volume and intensity.

[0158] Loading efficiency was measured using liquid chromatography-mass spectrometry (LC-MS).

[0159] The chromatograms of DOPE-peptides were characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) using a biphenyl column (1.7 mm, 50 x 2.1 mm, 100A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold in methanol. The loading efficiency was calculated by comparing the area under the curve (AUC) of the DOPE-peptide in the LC-MS chromatograms before and after purification.

[0160] result We first investigated how to prepare sHDL NDs loaded with peptides having a pI value <7 and a net charge <0 at pH 7. We selected CSS-SIINFEKL (SEQ ID NO:743) and CSS-Ea (CSS-ASFEAQGALANIAVDKA) (SEQ ID NO:746) for these studies because they have pI values ​​of 6.1 and 3.9, respectively, and net charges of -0.1 and -1.1 at pH 7 (Table 7). In previous studies, we have shown that NDs can be loaded with peptide-lipid conjugates formed by modifying the N-terminus of an antigenic peptide with a "Cys-Ser-Ser" (CSS) linker and then conjugating the DOPE-MAL lipid to the thiol of Cys in the resulting peptide. Using this method, CSS-SIINFEKL (SEQ ID NO:743) and CSS-Ea were readily loaded into NDs, and the resulting NDs were stable at pH 7.4, exhibiting a singlet near 10 nm as shown by their DLS volume and intensity curves (Tables 1 and 2). Therefore, these results indicate that peptides with pI values ​​<7 and net charge <0 at pH 7 (such as SIINFEKL (SEQ ID NO:770) and Ea peptides) can be modified at the N-terminus with CSS linkers to efficiently load and form homogeneous sHDL NDs containing loaded peptides.

[0161] Next, we examined the effect of changing the CSS linker to a CSE or CEE linker. We synthesized SIINFEKL (SEQ ID NO:770) or Ea peptides (peptides 2, 3, and 5, Table 7) with CSE or CEE linkers and examined whether this affected peptide-lipid loading onto sHDL ND. CSS-SIINFEKL (SEQ ID NO:743), CSE-SIINFEKL (SEQ ID NO:744), and CEE-SIINFEKL (SEQ ID No:745) were synthesized and conjugated with DOPE-MAL, and then the peptide-lipids were loaded onto sHDL. The resulting sHDLs loaded with peptides were stable at pH 7.4, and their DLS volume and intensity curves showed loading with CSS-SIINFEKL (SEQ ID NO:743), CSE-SIINFEKL (SEQ ID NO:744), or CEE-SIINFEKL (SEQ ID NO:745) (peptides 1, 2, and 3, Table 7) Figure 1The sHDL NDs exhibited similar size distributions. Similarly, CSS-Ea and CSE-Ea were synthesized and conjugated with DOPE-MAL, and then the peptide-lipids were loaded onto sHDL. The resulting sHDLs loaded with peptides were stable at pH 7.4, and their DLS volume and intensity curves showed that CSS-Ea or CSE-Ea (peptides 4 and 5) were loaded onto sHDL. Figure 1 The size distribution of sHDL NDs was similar. Furthermore, when the peptide linker was changed from CSS to CSE or CEE (peptides 1-5, Table 7), the loading efficiency of SIINFEKL (SEQ ID NO:770) or Ea peptide onto NDs remained similar or slightly improved.

[0162] These results indicate that for peptides with a pI value less than 7 and a net charge less than 0 at pH 7, CSS, CSE, or CEE adapters are suitable adapters for modifying peptide sequences to form homogeneous loaded peptides in sHDL ND.

[0163] Example II. Synthesis of sHDL ND of peptides carrying pI value >7 and net charge >0 at pH 7.

[0164] method Preparation of antigen-loaded sHDL ND.

[0165] DMPC (1,2-dimyristic-sn-glycerol-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. ApoA1-mimic 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 antigenic peptides used in this study is shown in Table 8. Table 8. Peptides used in the experiments in Example II. Their peptide sequences, isoelectric points, net charges at pH = 7, water solubility, stability in 10 mM phosphate buffer (pH = 7.4), and loading efficiency when loaded onto sHDL ND are shown.

[0166]

[0167] To prepare blank sHDL NDs, DMPC and 22A peptide powder were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC: 22A = 2:1, mass ratio) at pH 7.4, and then subjected to heating and cooling cycles to obtain blank sHDL NDs, followed by sonication at room temperature for 1 minute. To load the antigenic peptide into the blank NDs, the cysteine-capped antigenic peptide was first conjugated with DOPE-MAL (antigenic peptide: DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank NDs (22A:antigen peptide = 4:1, mass ratio) and incubated for 1 hour at room temperature with gentle shaking on an orbital oscillator. Unreacted antigenic peptides were removed using a Zeba Spin desalting column (Pierce).

[0168] Measured based on the size distribution of the DLS.

[0169] Hydrodynamic dimensions of the ND of the loaded peptide were measured in 10 mM phosphate buffer (pH = 7.4) using dynamic light scattering (DLS, Malvern Zetasizer Nano ZSP). 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 (default settings) was used for size measurements. Temperature: 25°C. Equilibration time: 120 seconds. Cell type: disposable cuvette ZEN0040. Measurement angle: 173° backscatter (NIBS default). Measurement duration: 11 automatic runs, 10 seconds each, for 3 measurements. Readings: Size distribution based on volume and intensity.

[0170] Loading efficiency was measured using liquid chromatography-mass spectrometry (LC-MS).

[0171] The chromatograms of DOPE-peptides were characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) using a biphenyl column (1.7 mm, 50 x 2.1 mm, 100A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold in methanol. The loading efficiency was calculated by comparing the area under the curve (AUC) of the DOPE-peptide in the LC-MS chromatograms before and after purification.

[0172] result Next, we investigated how to prepare sHDL NDs loaded with peptides having a pI value >7 and a net charge >0 at pH 7. We selected CSS-MOG38-50, CSS-PLP178-191, CSS-KV11, and CSS-NRPA7 for these studies because they have a pI value >7 and a net charge >0 at pH 7 (Table 8).

[0173] In previous studies, we have shown that NDs can be loaded with peptide-lipid conjugates formed by modifying the N-terminus of an antigenic peptide with a "Cys-Ser-Ser" (CSS) linker and then conjugating DOPE-MAL lipids to the thiol of Cys in the resulting peptide. As shown in Example I, this method is effective for peptides with pI < 7 and net charge < 0 at pH 7. However, for peptides with poor water solubility, pI > 7, and net charge > 0 at pH 7, this method generally leads to ND aggregation. This was the case for the CSS-MOG38-50 peptide with a pI of 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 size > 1000 nm in both the DSL volume and intensity curves (peptide 6, Table 8). Figure 2 Therefore, we changed the CSS linker in the MOG38-50 peptide to a CDD or CDDD linker, which reduced the pI values ​​to 7.1 and 5.1, respectively, and the net charge to 0 and -1, respectively (peptides 7 and 8, Table 8). Furthermore, changing the CSS linker to a CDD or CDDD linker improved the water solubility of the MOG38-50 peptides (peptides 6-8, Table 8). Both CDD-MOG38-50 and CDDD-MOG38-50 were loaded with sHDL ND, forming stable and homogeneous NDs with an average size of 10 nm in 10 mM phosphate buffer at pH 7.4, as shown by the DLS volume and intensity curves (peptides 7 and 8, Table 8). Figure 2 Furthermore, when using CDD-MOG38-50 or CDDD-MOG38-50, the loading efficiency of CSS-MOG38-50 on ND was significantly improved from 70.5% to 99.1% and 99.9%, respectively (peptide 6-8, Figure 2 ).

[0174] Similarly, when sHDL NDs are loaded with CSS-PLP178-191, which has poor water solubility, a pI of 9.1, and a net charge of 0.9 at pH 7 (peptide 9, Table 8), this leads to a heterogeneous ND size distribution (peptide 9, Table 8). Figure 2On the other hand, CDD-PLP178-191 (peptide 10, Table 8), with a pI value of 3.7 and a net charge of -1.1 at pH 7, exhibited improved water solubility and was stably loaded onto NDs, resulting in the formation of homogeneous NDs with an average size of 10 nm in 10 mM phosphate buffer at pH 7.4, as shown by the DLS volume and intensity curves (peptide 10, ...). Figure 2 Compared to CSS-PLP178-191's 59.6%, CDD-PLP178-191's loading efficiency also significantly increased to 96.4% (peptides 9-10, ...). Figure 2 ).

[0175] We also examined similar methods 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 with good water solubility (peptide 11, Table 8). When sHDL NDs were loaded with CSS-KV11, the resulting NDs appeared homogeneous in the DLS volumetric curves; however, the DLS intensity curves showed aggregated NDs (peptide 11, Table 8). Figure 3 On the other hand, CSE-KV11 and CEE-KV11 (peptides 12 and 13, Table 8), with pI values ​​of 6.3 and 4.5 respectively and net charges of -0.1 and -1.1 at pH 7, were stably loaded onto NDs, resulting in the formation of homogeneous NDs with an average size of 10 nm in 10 mM phosphate buffer at pH 7.4, as shown by both DLS volume and intensity curves (peptides 12 and 13). Figure 3 The loading efficiencies of CSS-KV11, CSE-KV11, and CEE-KV11 on ND ranged from 72.7% to 79.2% (peptides 11-13, Table 8).

[0176] Similar findings were observed for CSS-NRPA7 (which has a pI of 9.9 and a net charge of 1.9 at pH 7 with good water solubility) (peptide 14, Table 8). When sHDL NDs were loaded with CSS-NRPA7, the resulting NDs appeared homogeneous in the DLS volumetric curves; however, the DLS intensity curves showed aggregated NDs (peptide 14, Table 8). Figure 3 On the other hand, CSS-NRPA7 (peptide 15, Table 8), with a pI value of 6.4 and a net charge of -0.1 at pH 7, was stably loaded onto ND, resulting in the formation of homogeneous NDs with an average size of 10 nm in 10 mM phosphate buffer at pH 7.4, as shown by both DLS volume and intensity curves (peptide 15, Table 8). Figure 3 Compared to CSS-NRPA7's 80.5%, CEE-NRPA7's loading efficiency on ND increased to 94.5% (peptides 14-15, ...). Figure 3 ).

[0177] Overall, these results indicate that CDD, CDDD, CSE, or CEE linkers are suitable for modifying peptide sequences for peptides with pI values ​​greater than 7 and net charge greater than 0 at pH 7, resulting in increased peptide water solubility, homogeneous sHDL NDs of loaded peptides, and improved peptide loading efficiency on sHDL NDs.

[0178] Example III. Synthesis of sHDL ND carrying a peptide with a non-terminal cysteine ​​residue.

[0179] method Preparation of antigen-loaded sHDL ND.

[0180] DMPC (1,2-dimyristicoyl-sn-glycerol-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. ApoA1-mimic 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 antigenic peptides used in this study is shown in Table 9.

[0181] Table 9. Peptides used in the experiments in Example III. Their peptide sequences, isoelectric points, net charge at pH = 7, water solubility, stability in 10 mM phosphate buffer (pH = 7.4), and loading efficiency when loaded onto sHDLND are shown.

[0182]

[0183] To prepare blank sHDL NDs, DMPC and 22A peptide powder were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC: 22A = 2:1, mass ratio) at pH 7.4, and then subjected to heating and cooling cycles to obtain blank sHDL NDs, followed by sonication at room temperature for 1 minute. To load the antigenic peptide into the blank NDs, the cysteine-containing antigenic peptide was first conjugated with DOPE-MAL (antigenic peptide: DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank NDs (22A:antigen peptide = 4:1, mass ratio) and incubated for 1 hour at room temperature with gentle shaking on an orbital oscillator. Unreacted antigenic peptides were removed using a Zeba Spin desalting column (Pierce).

[0184] Measured based on the size distribution of dynamic light scattering (DLS).

[0185] Hydrodynamic dimensions of the ND of the loaded peptide were measured in 10 mM phosphate buffer (pH = 7.4) using dynamic light scattering (DLS, Malvern Zetasizer Nano ZSP). 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 (default settings) was used for size measurements. Temperature: 25°C. Equilibration time: 120 seconds. Cell type: disposable cuvette ZEN0040. Measurement angle: 173° backscatter (NIBS default). Measurement duration: 11 automatic runs, 10 seconds each, for 3 measurements. Readings: Size distribution based on volume and intensity.

[0186] Loading efficiency was measured using liquid chromatography-mass spectrometry (LC-MS).

[0187] The chromatograms of DOPE-peptides were characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) using a biphenyl column (1.7 µm, 50 x 2.1 mm, 100A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold in methanol. The loading efficiency was calculated by comparing the area under the curve (AUC) of the DOPE-peptide in the LC-MS chromatograms before and after purification.

[0188] result We examined how to prepare sHDL NDs loaded with peptides containing non-terminal cysteine ​​residues. We selected mIns2 B:9-23 and HMOG186-200 for these studies, and they had pI values ​​<7 and net charges <0 at pH 7 (peptides 16 and 17, Table 9).

[0189] When sHDL ND is loaded with mIns2 B:9-23, which has poor water solubility, a pI of 5.3, and a net charge of -1.0 at pH 7 (peptide 16, Table 9), this leads to a non-uniform ND size distribution (peptide 16, Figure 4 Similarly, HMOG186-200 (peptide 17, Table 9), with a pI of 3.0 and a net charge of -0.1 at pH 7, resulted in a non-uniform size distribution when loaded into sHDL ND (peptide 17, Table 9). Figure 4To address this issue, we modified HMOG186-200 by adding a DDD linker (Asp-Asp-Asp) to the N-terminus, yielding HMOG186-200-DDD, which has a pI of 0.5 and a net charge of -3.1 at pH 7 (peptide 18, Figure 2 This modification significantly improved the stability and water solubility of the peptide. The ND loaded with HMOG186-200-DDD was stable at pH 7.4 and exhibited a single peak near 10 nm in its DLS volume and intensity curve. Figure 4 The loading efficiency of HMOG186-200-DDD on ND was 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 with cysteine ​​residues not only at the N-terminus but also at non-terminal positions. The inclusion of linkers enhances the water solubility of these peptides, promotes the formation of homogeneous loaded peptides on sHDL ND, and improves the loading efficiency of peptides on sHDL ND.

[0190] Example IV. Synthesis of sHDL ND carrying a peptide with a C-terminal cysteine ​​residue.

[0191] method Preparation of antigen-loaded sHDL ND.

[0192] DMPC (1,2-dimyristicoyl-sn-glycerol-3-phosphocholine) and DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine) were purchased from NOF AMERICA CORPORATION. ApoA1-mimic 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 antigenic peptides used in this study is shown in Table 10.

[0193] Table 10. Peptides used in the experiments in Example IV. The peptide sequences, isoelectric points, net charges at pH = 7, water solubility, stability in 10 mM phosphate buffer (pH = 7.4), and loading efficiency when loaded onto sHDL ND are shown.

[0194]

[0195] To prepare blank sHDL NDs, DMPC and 22A peptide powder were mixed and hydrated in 10 mM sodium phosphate buffer (DMPC: 22A = 2:1, mass ratio) at pH 7.4, and then subjected to heating and cooling cycles to obtain blank sHDL NDs, followed by sonication at room temperature for 1 minute. To load the antigenic peptide into the blank NDs, the cysteine-C-terminated antigenic peptide was first conjugated with DOPE-MAL (antigenic peptide: DOPE-MAL = 1.5:1, molar ratio). The DOPE-peptide conjugate was then added to the blank NDs (22A:antigen peptide = 4:1, mass ratio) and incubated for 1 hour at room temperature with gentle shaking on an orbital oscillator. Unreacted antigenic peptides were removed using a Zeba Spin desalting column (Pierce).

[0196] Measured based on the size distribution of the DLS.

[0197] Hydrodynamic dimensions of the ND of the loaded peptide were measured in 10 mM phosphate buffer (pH = 7.4) using dynamic light scattering (DLS, Malvern Zetasizer Nano ZSP). 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 (default settings) was used for size measurements. Temperature: 25°C. Equilibration time: 120 seconds. Cell type: disposable cuvette ZEN0040. Measurement angle: 173° backscatter (NIBS default). Measurement duration: 11 automatic runs, 10 seconds each, for 3 measurements. Readings: Size distribution based on volume and intensity.

[0198] Based on load efficiency measurements using LC-MS.

[0199] The chromatograms of DOPE-peptides were characterized by LC-MS (Shimadzu LCMS-2020 + LC-2040C) using a biphenyl column (1.7 µm, 50 x 2.1 mm, 100A, Phenomenex). The mobile phase was a mixture of water, acetonitrile, and methanol. The sample was diluted 100-fold in methanol. The loading efficiency was calculated by comparing the area under the curve (AUC) of the DOPE-peptide in the LC-MS chromatograms before and after purification.

[0200] result Next, we examined how to prepare sHDL ND loaded with peptides containing C-terminal cysteine ​​residues. We chose gliadin-C1 for these studies, which has a pI of 6.1 and a net charge of -0.1 at pH 7 (peptide 19, Table 10).

[0201] When sHDL ND was loaded with gliadin-C1, the resulting ND was stable at pH 7.4 and exhibited a single peak near 10 nm in its DLS volume and intensity curve (peptide 19, Figure 5 The loading efficiency of gliadin-C1 on ND was 88.9% (Table 10).

[0202] These results indicate that stable sHDL NDs of loaded peptides can also be obtained for peptides containing C-terminal cysteine ​​residues.

[0203] Equivalent solution This invention may be practiced in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments should be considered illustrative in all respects, and not limiting of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all variations falling within the equivalent meaning and scope of the claims are intended to be included therein.

[0204] By incorporating via reference For all purposes, the full disclosure of each patent document and scientific article cited in this article is incorporated herein by reference. The following references are incorporated herein by reference in their entirety:

[0205]

[0206]

[0207]

Claims

1. A composition comprising sHDL nanoparticles, wherein the sHDL nanoparticles contain... Phospholipids; apolipoprotein mimics; Thiol-reactive lipids; and A peptide containing a connector portion that connects to the payload portion. The connector described herein includes: Cysteine ​​(C) and one to five amino acids, independently selected from aspartic acid (D), glutamic acid (E), and serine (S), or DD or DDD, if the payload contains C amino acids; The payload comprises a polypeptide of 5 to 35 amino acids in length, and The peptides described herein contain a net negative charge at pH 7 and an isoelectric point ranging from 0.4 to 12; and The peptide is covalently linked to the thiol-reactive lipid via the linker.

2. The composition according to claim 1, The linker comprises cysteine ​​(C) and optionally one to five amino acids independently selected from aspartic acid (D), glutamic acid (E), and serine (S). The peptide is covalently linked to the thiol-reactive lipid via the amino terminus of Cys, the carboxyl terminus of Cys, or a non-terminal position of Cys.

3. The composition according to claim 1, The payload contains C amino acids; The connector mentioned above includes DD or DDD; The peptide is covalently linked to the thiol-reactive lipid via the DD or DDD of the linker.

4. The composition of claim 1, wherein the peptide comprises the following formula: [connector]-[payload] or [payload]-[connector].

5. The composition of claim 1, wherein if the payload comprises a C amino acid, the linker sequence is selected from: C, DDDDD, DD, and DDD.

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

7. The composition of claim 1, wherein the payload has a net zero charge at pH 7 to 12.

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

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

1.

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

0.

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

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

78.

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

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

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

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

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

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

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

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

0.

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

22. The composition of claim 21, wherein the effective 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 composition of claim 1, wherein the apolipoprotein mimic is an ApoA-I mimic having any one of the following sequences: SEQ ID NO: 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). 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: 353) TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354) 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: 361) 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).

24. The composition of claim 23, wherein the apolipoprotein mimic has the sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4).

25. The composition of claim 1, wherein the thiol-reactive lipid is selected from dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[3-(2-pyridinedithio)propionate]. (DOPE-PDP), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide phenyl)butamide], 1,2-hexadecanoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide], 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-maleimide (DOPE-Mal) and 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-maleimide methyl)cyclohexane-formamide].

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

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