Preparation of tolerant nanoparticles for treating primary biliary cholangitis

Abstract

The present disclosure relates to a method for preparing tolerant immunomodified nanoparticles encapsulating antigens associated with primary biliary cholangitis (PBC), compositions comprising said particles and their use for the treatment of PBC.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 503,771, filed May 23, 2023, which is incorporated herein by reference in its entirety.

[0003] By incorporating materials submitted electronically.

[0004] By reference, the computer-readable nucleotide / amino acid sequence listing submitted at the same time and identified as follows is incorporated in its entirety: Filename: 58505_SeqListing.xml; Size: 1,972 bytes; Created: May 20, 2024. Technical Field

[0005] This disclosure relates to a method for preparing tolerable immunomodulated nanoparticles encapsulating primary biliary cholangitis (PBC)-associated antigens. Background Technology

[0006] Primary biliary cholangitis (PBC, formerly known as primary biliary cirrhosis) is a typical autoimmune liver disease characterized by destructive lymphocytic cholangitis and specific antimitochondrial autoantibodies (AMAs) that are primarily targeted at the E2 component (PDC-E2) of the mitochondrial pyruvate dehydrogenase complex. 1 In the United States, the prevalence of PBC is higher in women than in men, and it is estimated to be approximately 580 per million women. 2 The medical needs for this rare disease are significantly unmet, and TIMP-PBC has the potential to have a positive impact on subjects with this disease.

[0007] Tolerable immunomodulated particles (TIMPs) containing one or more antigens have previously been described for the treatment of immune-mediated conditions (e.g., autoimmune diseases and allergies) by inducing antigen-specific immune tolerance (WO2013192532 and WO 2015023796 are incorporated herein by reference). Summary of the Invention

[0008] Encapsulating one or more primary biliary cholangitis (PBC)-associated antigens within a TIMP core (TIMP-PBC) is advantageous because it ensures the safe and efficient delivery of the encapsulated protein to the APC without inducing immune activation.

[0009] The preparation of TIMP-PBC involves numerous steps, each of which affects the physicochemical properties of the resulting composition, which are crucial for safe and therapeutic administration. Importantly, the method must be developed to ensure efficient encapsulation of the PBC antigen within the particle core.

[0010] In various embodiments, the TIMP-PBC prepared by the method described herein encapsulates one or more PBC-related antigens or their epitopes. In various embodiments, the primary biliary cholangitis (PBC)-related antigens comprise intracellular proteins, extracellular proteins, mitochondrial proteins, and / or nuclear proteins. In various embodiments, the PBC antigens are selected from the group consisting of: pyruvate dehydrogenase complex E2 subunit (PDC-E2), gp210, nucleoporin 62, Sp100, PML, CENP A, CENP B, and CENP. C, dsDNA, histones, branched 2-oxyacid dehydrogenase complex (BCOADC), 2-oxyacid dehydrogenase complex, lipoamide acyltransferase component of branched α-ketoacid dehydrogenase complex (BCOADC-E2), dihydrolipoamide lysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex (OGDC-E2), sulfite oxidase, mitochondrial outer membrane, glycogen phosphorylase, sarcosine dehydrogenase, smooth muscle protein, soluble liver antigen, liver / kidney microsomes, centromere protein, tubulin, actin, vimentin, desmin, cytokeratin, F-actin, UDP glucuronyltransferase family 1 member A complex (UGTA1), aminomethylene transferase cyclic deaminase, desialyl glycoprotein receptor (ASGPR), cardiolipin, h-Lamp-2, protease 3, CYP 2C9, CYP 2A6, and CYP P450 2D6. In some embodiments, PBC-associated antigens are selected from the group comprising bacterial epitopes, viral epitopes, or xenobiotics. In some embodiments, the bacterial epitopes, viral epitopes, or xenobiotics comprise Novo 1, Novo 2, Novo 3, Novo 4 proteins from *Novosphingobium aromaticivorans* or β-galactosidase, 2-octamide, 2-nonanoamide, *Escherichia coli* PDC-E2, *Escherichia coli* ATP-dependent ClpX, *Escherichia coli* periplasmic maltose-binding protein, *Escherichia coli* ATP-dependent helicase Hrp, *Escherichia coli* fatty acid oxidation complex α, *Escherichia coli* ppGpp synthase II, *Escherichia coli* nitrate reductase 2, and *Helicobacter pylori*. Helicobacter pylori ) Urease β subunit, Pseudomonas aeruginosa ( Pseudomonas aeruginosa Diaminopimelic acid decarboxylase, human cytomegalovirus capsid assembly protein UL47, Haemophilus influenzae (Haemophilus influenzae t-RNA (uracil-5-)-methyltransferase. In various embodiments, TIMP-PBC particles encapsulate one or more polynucleotides encoding PBC-associated antigens. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0011] In various embodiments, TIMP-PBC encapsulates a pyruvate dehydrogenase complex E2 subunit (PDC-E2) peptide containing an antigenic epitope. In various embodiments, TIMP-PBC encapsulates the PDC-E2 antigenic epitope (PDC-E2) containing amino acids 155-185. 155-185 ), which has the amino acid sequence (KVGEKLSEGDLLAEIETDKATIGFEVQEEGY) (SEQ ID NO: 1).

[0012] This disclosure provides a method for preparing a composition comprising negatively charged particles (TIMP-PBC) encapsulating one or more PBC-associated antigens. The method relates to a method for preparing particles developed for the safe and therapeutic administration of TIMP-PBC for the treatment of PBC. In various embodiments, the method comprises: (a) generating primary emulsion particles by mixing one or more PBC-associated antigens with an oil phase comprising a polymer; (b) mixing the primary emulsion particles with one or more surfactants and / or stabilizers; (c) homogenizing the mixture from (b) to form secondary emulsion particles; and (d) hardening the secondary emulsion particles.

[0013] In various embodiments, the method comprises: (a) generating an aqueous solution of one or more PBC-associated antigens; (b) generating a primary emulsion by mixing the aqueous solution of step (a) with an oil phase comprising a polymer; (c) mixing the primary emulsion of step (b) with a mixture comprising one or more surfactants and / or stabilizers to form a secondary emulsion; and (d) hardening the secondary emulsion by evaporation to produce hardened polymer nanoparticles that encapsulate the PBC-associated antigens within their core.

[0014] In various embodiments, the method further includes step (e) filtering, washing, and concentrating the nanoparticles. In various embodiments, the method further includes (e) filtering, washing, and concentrating the nanoparticles and (f) freeze-drying the nanoparticles. In various embodiments, the primary emulsion in step (b) is a water-in-oil emulsion. In various embodiments, the secondary emulsion in step (c) is an oil-in-water emulsion.

[0015] In various embodiments, the method comprises: (a) generating an aqueous solution of one or more PBC-associated antigens; (b) generating a primary emulsion by mixing the aqueous solution of step (a) with an oil phase comprising a polymer; (c) mixing the primary emulsion of step (b) with a mixture comprising one or more surfactants and / or stabilizers to form a secondary emulsion; (d) hardening the secondary emulsion by evaporation to remove solvent, thereby generating hardened polymer nanoparticles that encapsulate PBC-associated antigens within their cores; (e) filtering, washing, and concentrating the nanoparticles; and (f) freeze-drying the nanoparticles.

[0016] In various embodiments, the one or more PBC-associated antigens in step (a) are dissolved in an aqueous solution containing a solvent. In various embodiments, the one or more PBC-associated antigens in step (a) are dissolved in an aqueous solution containing the same solvent. In various embodiments, the one or more PBC-associated antigens in step (a) are dissolved in an aqueous solution containing a different solvent. In various embodiments, the one or more PBC-associated antigens in step (a) are dissolved in an aqueous solution containing a solvent and additionally containing one or more stabilizers. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent.

[0017] In various embodiments, the solvent comprises acetaldehyde, acetone, acetonitrile, acetic acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, diethanolamine, diethylenetriamine, dimethoxyethane, dimethylformamide, 1,1-dimethylformamide, 1,1-dimethylhydrazine, 1,2-dimethylhydrazine, dimethyl sulfoxide, 1,4-dioxane, formic acid, ethanol, ethyl acetate, ethylamine, ethylene glycol, furanol, glycerol, isopropanol, methanol, methyldiethanolamine, methylisocyanate, N-methyl-2-pyrrolidone, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propylene glycol, pyridine, tetrahydrofuran, triethylene glycol, benzoic acid, and ascorbic acid. Acids, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, oxalic acid, carbonic acid, methylamine, diethylamine, pyridine, sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimonyic acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, boric acid, barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, sodium bicarbonate, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, lithium hydroxide, rubidium hydroxide. In various embodiments, the one or more PBC-related antigens are dissolved in a solvent containing one or more acids and / or one or more bases.

[0018] In various embodiments, the acids include acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimony acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid.

[0019] In various embodiments, the base comprises barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide.

[0020] In various embodiments, the concentration of the acid is between 0.1 N and 36 N, including all ranges and values ​​within this range. In various embodiments, the concentration is about 0.1 N, about 0.5 N, about 1 N, about 2 N, about 3 N, about 4 N, about 5 N, about 6 N, about 7 N, about 8 N, about 9 N, about 10 N, about 11 N, about 12 N, about 13 N, about 14 N, about 15 N, about 16 N, about 17 N, about 18 N, about 20 N, about 30 N, or about 36 N, including all values ​​within this range. In various embodiments, the concentration of the base is between 0.01% and 100% (v / v or wt / v), including all values ​​within this range. In various embodiments, the concentrations are about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 10%, about 25%, about 50%, about 75%, or about 100% (v / v or wt / v).

[0021] In various embodiments, the pH of the acid is between pH 1.0 and pH 7.0, including all ranges and values ​​within this range. In various embodiments, the pH is approximately pH 1.0, approximately pH 1.5, approximately pH 2.0, approximately pH 2.5, approximately pH 3.0, approximately pH 3.5, approximately pH 4.0, approximately pH 4.5, approximately pH 5.0, approximately pH 5.5, approximately pH 6.0, approximately pH 6.5, or approximately pH 7.0. In various embodiments, the pH of the base is between pH 7.0 and pH 14.0, including all ranges and values ​​within this range. In various embodiments, the base pH is approximately pH 7.5, approximately pH 8.0, approximately pH 8.5, approximately pH 9.0, approximately pH 9.5, approximately pH 10.0, approximately pH 10.5, approximately pH 11.0, approximately pH 11.5, approximately pH 12.0, approximately pH 12.5, approximately pH 13.0, approximately pH 13.5, or approximately pH 14.0.

[0022] In various embodiments, one or more PBC-related antigens in step (a) are dissolved in an aqueous solution containing one or more stabilizers. In various embodiments, the stabilizers comprise detergents / surfactants, penetrants, metal complexes, proteins, or amino acids. In various embodiments, the stabilizers are selected from the group consisting of: polyvinyl alcohol, poly(acrylic acid) (PAA), sorbitan monostearate, Triton X, Triton X-100, poloxamer, polyvinylpyrrolidone, Pluronic F68, n-dodecyl-β-D-maltose glycoside (DDM), neopentyl lauryl maltose glycol (LMNG), 1-myristoyl-2-hydroxy- sn-glycerol-3-[phosphate-rac-(1-glycerol)] (LMPG), 1-palmitoyl-2-hydroxy- sn -glycerol-3-[rac-(1-glycerol)] (LPPG), polyethylene glycol 400 dedecyl ether (Thesit), nonylphenyl polyethylene glycol (NP40), polyoxyethylene-(10)-dodecyl-ether (Genapol C-100), dodecyl-phosphocholine (DPC), n-decyl-β-maltodextrin (DM), 1,2-dioctanoyl- sn -Glyceryl-3-phosphatecholine (diC8PC), 1,2-dihexanoyl- sn -Glyceryl-3-phosphocholine (diC6PC), 1,2-diheptanyl- sn - Glyceryl-3-phosphate choline (DHPC), n-octyl-β-D-glucose (B-OG), Brih-35, Brij-56, Brij-58, Brij-72, Brij-78, Brij-97, Brij-98, 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate (CHAPS), detergents containing disulfides, fluorinated diglucose detergents, fluorinated maltose detergents, maltose amphiphiles with 1,3,5-triazine nuclei, steroid-based pentasaccharides, vitamin E-based glycoside amphiphiles, calixarene-based detergents, cyclodextrin, trehalose, mannitol, dextran, carboxymethyl cellulose, ethyl stearate, sodium acetate, monosodium glutamate, polysorbate, albumin, bovine serum albumin, lysine, histidine, arginine, zinc salts, sodium bicarbonate or magnesium hydroxide. In various embodiments, the concentration of the stabilizer in step (a) is between 0.01% and 15% (e.g., about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10% or 15%), including all values ​​within this range.

[0023] In various embodiments, the one or more PBC-associated antigens are dissolved in a solvent by mixing for 0.1 to 96 hours (including all values ​​within this range). In various embodiments, the one or more PBC-associated antigens are dissolved in a solvent by mixing for the following hours: about 0.1 hours, about 0.2 hours, about 0.3 hours, about 0.4 hours, about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours.

[0024] In various embodiments, the one or more PBC-associated antigens are synthetically prepared. In various embodiments, the antigens are prepared by solid-phase peptide synthesis or solution-phase peptide synthesis. In various embodiments, the PBC-associated antigens are prepared using recombinant protein production technologies, such as in mammalian cells, E. coli or other bacterial cells, yeast, insects, or cell-free systems.

[0025] In various embodiments, the pH of the solution containing one or more PBC-related antigens dissolved in step (a) is adjusted with a solvent.

[0026] In various embodiments, the solvent comprises one or more acids and / or one or more bases. In various embodiments, the pH of the solvent is between pH 1.0 and pH 14.0, including all ranges and values ​​within this range. In various embodiments, the pH of the solvent is between pH 0.5 and pH 14.0, including all ranges and values ​​within this range. In various embodiments, the pH is approximately pH 1.0, approximately pH 1.5, approximately pH 2.0, approximately pH 2.5, approximately pH 3.0, approximately pH 3.5, approximately pH 4.0, approximately pH 4.5, approximately pH 5.0, approximately pH 5.5, approximately pH 6.0, approximately pH 6.5, approximately pH 7.0, approximately pH 7.5, approximately pH 8.0, approximately pH 8.5, approximately pH 9.0, approximately pH 9.5, approximately pH 10.0, approximately pH 10.5, approximately pH 11.0, approximately pH 11.5, approximately pH 12.0, approximately pH 12.5, approximately pH 13.0, approximately pH 13.5, or approximately pH 14.0. In various embodiments, the pH is approximately pH 0.9. In various embodiments, the solvents containing acids are acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimonyic acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid. In various embodiments, the solvents containing bases are barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide. In various embodiments, the solvent concentration is between 0.1 N and 36 N, including all values ​​within this range. In various embodiments, the acid concentration is about 0.1 N, about 0.5 N, about 1 N, about 2 N, about 3 N, about 4 N, about 5 N, about 6 N, about 7 N, about 8 N, about 9 N, about 10 N, about 11 N, about 12 N, about 13 N, about 14 N, about 15 N, about 16 N, about 17 N, about 18 N, about 20 N, about 30 N, about 36 N, including all values ​​within this range. In various embodiments, the solvent concentration is between 0.01% and 100% (v / v or wt / v), including all values ​​within this range.In various embodiments, the solvent concentration is about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 10%, about 25%, about 50%, about 75%, or about 100% (v / v or wt / v), including all values ​​within this range. In various embodiments, the adjusted pH of the solution containing the dissolved PBC-associated antigen is between pH 1.0 and pH 7.0. In various embodiments, the pH is about pH 1.0, about pH 1.5, about pH 2.0, about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, or about pH 7.0, including all ranges and values ​​within this range. In various embodiments, the adjusted pH of the solution containing dissolved PBC-associated antigens is between pH 6.0 and pH 7.5. In various embodiments, the pH of the solution containing one or more PBC-associated antigens dissolved in step (a) is adjusted by dialysis. In various embodiments, the pH is adjusted before, during, or after the formation of the primary emulsion in step (b).

[0027] In various embodiments, the PDC-E2 antigen from step (a) is dissolved in an aqueous solution containing one or more acids and / or one or more bases.

[0028] In various embodiments, the PDC-E2 antigen from step (a) is dissolved in an aqueous solution containing an alkali, such as ammonium hydroxide, barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide. Once dissolved, the pH can be adjusted with an acid, such as acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, perchloric acid, fluorosulfonic acid, fluoroantimony acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid. In one embodiment, PDC-E2 is dissolved in an aqueous solution of ammonium hydroxide (e.g., a 0.1% ammonium hydroxide solution). Once PDC-E2 is dissolved, the pH can be adjusted with an acid such as acetic acid (e.g., 0.9 N or 1 N acetic acid).

[0029] In various embodiments, the aqueous solution is diluted to a desired concentration using a solvent in step (a). In various embodiments, the solution is diluted before, during, or after the formation of the primary emulsion in step (b). In various embodiments, the solvent used for dilution comprises an organic solvent. In various embodiments, the solvent comprises an inorganic solvent. In some embodiments, the solvent comprises water.

[0030] In various embodiments, the concentration of the one or more PBC-associated antigens dissolved in step (a) is between about 0.1 mg / mL and about 100 mg / mL, including all values ​​within this range. In various embodiments, the concentration of the one or more PBC-associated antigens dissolved is between about 1 mg / mL and about 10 mg / mL. In various embodiments, the concentrations of the dissolved one or more PBC-related antigens are approximately 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, and 90 mg / mL. mg / mL, approximately 95 mg / mL, or approximately 100 mg / mL.

[0031] In various embodiments, the emulsion of step (b) includes a solvent. In various embodiments, the solvent comprises an organic solvent. In various embodiments, the solvent comprises an inorganic solvent. In various embodiments, the organic solvent is selected from the group consisting of acetone, dichloromethane, dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is selected from the group consisting of water, ammonia, aqueous ammonium hydroxide, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the concentration of the solvent in step (b) is between 1% (v / v) and 50% (v / v), including all values ​​within this range. In various embodiments, the concentration of the solvent in step (b) is between 0.1% (v / v) and 50% (v / v), including all values ​​within this range. In various embodiments, the concentration of the solvent in step (b) is about 1%, about 2%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (v / v). In various embodiments, the concentration of the solvent in step (b) is between 0.1 mM and 10 mM, including all values ​​within this range. In various embodiments, the concentration of the solvent in step (b) is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, or about 10.0 mM. In various embodiments, the concentration of the solvent in step (b) is between 0.1 M and 10 M, including all values ​​within this range. In various embodiments, the concentration of the solvent in step (b) is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.5 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, about 5 M, about 5.5 M, about 6 M, about 6.5 M, about 7.0 M, about 7.5 M, about 8.0 M, about 8.5 M, about 9.0 M, or about 10.0 M, including all values ​​within this range.

[0032] In various embodiments, the polymer in step (b) comprises a biodegradable polymer. In various embodiments, the biodegradable polymer comprises polyglycolic acid (PGA), polylactic acid (PLA), polysaccharide (PSA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-sebacic acid) (PLSA), poly(glycolic acid-co-sebacic acid) (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharides, or lipids. In various embodiments, the polymer is a copolymer. In various embodiments, the copolymer has different constitutive polymer molar ratios. In various embodiments, the molar ratio is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0, including all values ​​within this range.

[0033] In various embodiments, the polymer in step (b) comprises PLGA. In various embodiments, the molar ratio of the PLGA copolymer is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0, including all values ​​within this range. In various embodiments, the PLGA has a high molecular weight. In various embodiments, the PLGA has a low molecular weight. In various embodiments, the molecular weight of the PLGA is between 1 kDa and 100 kDa (e.g., between 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, and 100 kDa), including all values ​​within this range. In various embodiments, the amount of PLGA in step (b) is between 0.05% by weight and 100% by weight (e.g., between 0.05% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight, or 100% by weight, including all values ​​within this range). In various embodiments, the polymer in step (b) is dissolved in an organic solvent. In various embodiments, the polymer in step (b) is dissolved in dichloromethane, ethyl acetate, dimethylformamide, tetrahydrofuran, or chloroform. In various embodiments, the PLGA polymer is dissolved in ethyl acetate. In various embodiments, the method comprises a 5% PLGA solution (50:50) with a molecular weight between 10,000 Daltons and 60,000 Daltons (Da).

[0034] In various embodiments, the surfactant and / or stabilizer mixture in step (c) includes a solvent. In various embodiments, the solvent comprises an organic solvent. In various embodiments, the solvent comprises an inorganic solvent. In various embodiments, the organic solvent is selected from the group consisting of acetone, ethanol, dichloromethane, dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is selected from the group consisting of water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the concentration of the solvent in the mixture is between 1% (v / v) and 50% (v / v), including all values ​​within this range. In various embodiments, the concentration of the solvent in the mixture is about 1%, about 2%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (v / v). In various embodiments, the solvent concentration in the mixture is between 0.1 mM and 10 mM, including all values ​​within this range. In various embodiments, the solvent concentration in the mixture is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, or about 10.0 mM. In various embodiments, the solvent concentration in the mixture is between 0.1 M and 10 M, including all values ​​within this range. In various embodiments, the concentration of the solvent in the mixture is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.5 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, about 5 M, about 5.5 M, about 6 M, about 6.5 M, about 7.0 M, about 7.5 M, about 8.0 M, about 8.5 M, about 9.0 M, or about 10.0 M. In various embodiments, the solvent used in steps (b) and (c) is the same. In various embodiments, the solvent used in steps (b) and (c) is different.

[0035] In various embodiments, the emulsion of step (b) includes one or more PBC-associated antigens ranging from 0.001 mg / mL to 10 mg / mL, including all values ​​within this range. In various embodiments, the emulsion of step (b) comprises about 0.0011 mg / mL, about 0.002 mg / mL, about 0.005 mg / mL, about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, or about 10 mg / mL of one or more of the PBC-associated antigens.

[0036] In various embodiments, the emulsion of step (b) includes one or more PBC-associated antigens ranging from 0.1 mg / mL to 100 mg / mL, including all values ​​within this range. In various embodiments, the emulsion in step (b) comprises approximately 0.1 mg / mL, approximately 0.2 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12 mg / mL, approximately 13 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 75 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 95 mg / mL, etc. mg / mL or about 100 mg / mL of one or more PBC-associated antigens.

[0037] In some embodiments, the emulsion of step (b) comprises 0.2 µg PBC-associated antigen / mg polymer to 100 µg PBC-associated antigen / mg polymer, including all values ​​within this range. In some embodiments, the emulsion of step (b) comprises 0.2 µg / mg, 2 µg / mg, 5 µg / mg, 10 µg / mg, 20 µg / mg, 25 µg / mg, 50 µg / mg, 100 µg / mg, 200 µg / mg, including all values ​​within this range.

[0038] In various embodiments, the surfactant and / or stabilizer in step (c) is anionic, cationic, nonionic, or amphoteric. In various embodiments, the surfactants and / or stabilizers are poloxamer, polyamines, polyethylene glycol (PEG), Tween-80, gelatin, dextran, Pluronic L-63, Pluronic F-68, Pluronic 188, Pluronic F-127, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, dodecyl dimethyl ammonium bromide (DMAB), poly(ethylene-alt-maleic acid) (PEMA), vitamin E TPGS (Da-tocopherol polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine ​​or their enantiomers), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gelatin, sodium cholate, carbomer, or sulfate polymers (e.g., heparin sulfate, chondroitin sulfate, fucoidan, ulva polysaccharide, and carrageenan). In various embodiments, the amount of the surfactant and / or stabilizer present in the mixture in step (c) is between 0.05 wt% or vol% and 100 wt% or vol% (e.g., between 0.05 wt% or vol%, 0.1 wt% or vol%, 0.2 wt% or vol%, 0.3 wt% or vol%, 0.4 wt% or vol%, 0.5 wt% or vol%, 0.6 wt% or vol%, 0.7 wt% or vol%, 0.8 wt% or vol%, 0.9 wt% or vol%, 1 wt% or vol%, 2 wt% or vol%, 3 wt% or vol%, 4 wt% or vol%, 5 wt% or vol%, 6 wt% or vol%, 7 wt% or vol%, 8 wt% or vol%, 9 wt% or vol%, 10 wt% or vol%, 20 wt% or vol%, 30 wt% or vol%, 40 wt% or vol%, 50 wt% or vol%, 60 wt% or vol%, 70 wt% or vol%, 80 wt% or vol%, 90 wt% or vol% or 100 wt% or vol%, including all values ​​within this range).In various embodiments, the molecular weight of the surfactant and / or stabilizer is between 0.1 kDa and 10,000 kDa (e.g., between 0.1 kDa, 0.2 kDa, 0.3 kDa, 0.4 kDa, 0.5 kDa, 0.6 kDa, 0.7 kDa, 0.8 kDa, 0.9 kDa, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 500 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa). (The values ​​are between 100 kDa and 10000 kDa, inclusive). In various embodiments, the mixture is prepared by mixing 4% PVA and PAA (100 kDa, 35% wt) with ethyl acetate. In various embodiments, the mixture is prepared by mixing an aqueous solution of 4% PVA and an aqueous solution of PAA (100 kDa, 35% wt) with ethyl acetate.

[0039] In various embodiments, the aqueous solution or mixture of one or more surfactants and / or stabilizers comprising forming the oil-in-water secondary emulsion in (c) has a pH less than pH 4.0. In various embodiments, the pH of the oil-in-water secondary emulsion is between pH 1.0 and less than pH 4.0, including all values ​​within this range. In various embodiments, the pH of the oil-in-water secondary emulsion is about pH 1.0 to pH less than pH 4.0, about pH 2.0 to pH less than pH 4, about pH 3 to pH less than pH 4, about pH 1.0 to about pH 3.5, about pH 2.0 to about pH 3.5, about pH 3 to about pH 3.5, about pH 1, about pH 1.5, about pH 2, about pH 2.5, about pH 3, about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, or about pH 3.9, including all values ​​within this range.

[0040] In various embodiments, the water-in-oil primary emulsion of step (b) is obtained by homogenizing an aqueous solution of one or more PBC-associated antigens with an oil phase comprising a polymer. In various embodiments, homogenization is performed between 5 seconds and 1000 seconds, including all values ​​within this range. In various embodiments, homogenization is performed for approximately 5 seconds, approximately 10 seconds, approximately 15 seconds, approximately 20 seconds, approximately 25 seconds, approximately 30 seconds, approximately 35 seconds, approximately 40 seconds, approximately 45 seconds, approximately 60 seconds, approximately 90 seconds, approximately 120 seconds, approximately 150 seconds, approximately 180 seconds, approximately 210 seconds, approximately 240 seconds, approximately 270 seconds, approximately 300 seconds, approximately 330 seconds, approximately 360 seconds, approximately 390 seconds, approximately 420 seconds, approximately 450 seconds, approximately 480 seconds, approximately 510 seconds, approximately 540 seconds, approximately 570 seconds, approximately 600 seconds, approximately 700 seconds, approximately 800 seconds, approximately 900 seconds, or approximately 1000 seconds. In various embodiments, the oil-in-water secondary emulsion of step (c) is obtained by homogenizing the primary emulsion with a solution comprising one or more surfactants and / or stabilizers. In various embodiments, homogenization is performed between 5 seconds and 1000 seconds, including all values ​​within this range. In various embodiments, homogenization is performed for approximately 5 seconds, approximately 10 seconds, approximately 15 seconds, approximately 20 seconds, approximately 25 seconds, approximately 30 seconds, approximately 35 seconds, approximately 40 seconds, approximately 45 seconds, approximately 60 seconds, approximately 90 seconds, approximately 120 seconds, approximately 150 seconds, approximately 180 seconds, approximately 210 seconds, approximately 240 seconds, approximately 270 seconds, approximately 300 seconds, approximately 330 seconds, approximately 360 seconds, approximately 390 seconds, approximately 420 seconds, approximately 450 seconds, approximately 480 seconds, approximately 510 seconds, approximately 540 seconds, approximately 570 seconds, approximately 600 seconds, approximately 700 seconds, approximately 800 seconds, approximately 900 seconds, or approximately 1000 seconds. In various embodiments, the water-in-oil primary emulsion of step (b) is obtained by sonicating the aqueous solution of the PBC-associated antigen with the oil phase comprising the polymer. In various embodiments, the sonication is performed between 5 seconds and 1000 seconds, including all values ​​within this range. In various embodiments, the ultrasonic treatment is performed for approximately 5 seconds, approximately 10 seconds, approximately 15 seconds, approximately 20 seconds, approximately 25 seconds, approximately 30 seconds, approximately 35 seconds, approximately 40 seconds, approximately 45 seconds, approximately 60 seconds, approximately 90 seconds, approximately 120 seconds, approximately 150 seconds, approximately 180 seconds, approximately 210 seconds, approximately 240 seconds, approximately 270 seconds, approximately 300 seconds, approximately 330 seconds, approximately 360 seconds, approximately 390 seconds, approximately 420 seconds, approximately 450 seconds, approximately 480 seconds, approximately 510 seconds, approximately 540 seconds, approximately 570 seconds, approximately 600 seconds, approximately 700 seconds, approximately 800 seconds, approximately 900 seconds, or approximately 1000 seconds. In various embodiments, the oil-in-water secondary emulsion of step (c) is obtained by ultrasonically treating the primary emulsion of step (b) with a mixture comprising one or more surfactants and / or stabilizers. In various embodiments, the ultrasonic treatment is performed between 5 seconds and 1000 seconds, including all values ​​within this range.In various embodiments, the ultrasonic treatment is performed for approximately 5 seconds, approximately 10 seconds, approximately 15 seconds, approximately 20 seconds, approximately 25 seconds, approximately 30 seconds, approximately 35 seconds, approximately 40 seconds, approximately 45 seconds, approximately 60 seconds, approximately 90 seconds, approximately 120 seconds, approximately 150 seconds, approximately 180 seconds, approximately 210 seconds, approximately 240 seconds, approximately 270 seconds, approximately 300 seconds, approximately 330 seconds, approximately 360 seconds, approximately 390 seconds, approximately 420 seconds, approximately 450 seconds, approximately 480 seconds, approximately 510 seconds, approximately 540 seconds, approximately 570 seconds, approximately 600 seconds, approximately 700 seconds, approximately 800 seconds, approximately 900 seconds, or approximately 1000 seconds.

[0041] In various embodiments, the secondary emulsion is hardened by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the active evaporation is carried out using stirring or under vacuum (i.e., vacuum-driven evaporation). In various embodiments, the active evaporation is carried out under high-pressure vacuum. In various embodiments, the active evaporation is carried out under low-pressure vacuum. In various embodiments, the evaporation is passive evaporation. In various embodiments, the evaporation is carried out between 0.25 hours and 96 hours, including all values ​​within this range. In various embodiments, the evaporation is carried out for approximately 0.5 hours, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 48 hours, approximately 72 hours, or approximately 96 hours.

[0042] In various embodiments, the evaporation is between 0.01 mBar and 1000 mBar (e.g., between 0.01 mBar, 0.02 mBar, 0.03 mBar, 0.04 mBar, 0.05 mBar, 0.1 mBar, 0.2 mBar, 0.3 mBar, 0.4 mBar, 0.5 mBar, 0.6 mBar, 0.7 mBar, 0.8 mBar, 0.9 mBar, 1 mBar, 2 mBar, 3 mBar, 4 mBar, 5 mBar, 6 mBar, 7 mBar, 8 mBar, 9 mBar, 10 mBar, 20 mBar, 30 mBar, 40 mBar, 50 mBar, 60 mBar, 70 mBar, 80 mBar, 90 mBar, 100 mBar, 150 mBar, 200 mBar, 250 mBar, 300 mBar, 350 mBar, 400 mBar, 450 mBar). The pressure is between mBar, 500 mBar, 550 mBar, 600 mBar, 650 mBar, 700 mBar, 750 mBar, 800 mBar, 850 mBar, 900 mBar, 950 mBar, or 1000 mBar (including all values ​​within this range).

[0043] In various embodiments, the filtration, washing, and concentration of particles in step (e) are performed by gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, tangential flow filtration (TFF), or a combination thereof.

[0044] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) generating PDC-E2 particles of a negatively charged peptide, the peptide comprising the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1); 155-185 (a) an aqueous solution of a peptide; (b) generating a primary emulsion by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) homogenizing the primary emulsion of step (b) with a mixture comprising ethyl acetate, polyacrylic acid (PAA) and polyvinyl alcohol (PVA) to form a secondary emulsion; (d) hardening the secondary emulsion by evaporation to produce hardened polymer nanoparticles, the hardened polymer nanoparticles being PDC-E2 155-185 The peptide is encapsulated within its nucleus.

[0045] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) generating PDC-E2 particles of a negatively charged peptide, the peptide comprising the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1); 155-185 (a) an aqueous solution of the peptide; (b) generating a primary emulsion by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) homogenizing the primary emulsion of step (b) with a mixture comprising ethyl acetate, polyacrylic acid (PAA) and polyvinyl alcohol (PVA) and water to form a secondary emulsion; (d) hardening the secondary emulsion by evaporation to produce hardened polymer nanoparticles, the hardened polymer nanoparticles being PDC-E2 155-185 The peptide is encapsulated within its nucleus.

[0046] In various embodiments, the method further includes (e) filtering, washing, and concentrating the nanoparticles. In various embodiments, the method further includes (e) filtering, washing, and concentrating the nanoparticles, and (f) adding sodium citrate, mannitol, and sucrose, and freeze-drying the nanoparticles.

[0047] In various embodiments, this disclosure provides a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) generating PDC-E2 particles of negatively charged peptides. 155-185 (a) An aqueous solution of a peptide with a pH between 1.0 and 7.0; (b) A primary emulsion is generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) The primary emulsion is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; (d) The secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, the hardened polymer nanoparticles being PDC-E2 155-185 The peptides are encapsulated within their cores; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, followed by freeze-drying of the nanoparticles. In each embodiment, the pH is between pH 6.0 and 7.0.

[0048] In various embodiments, this disclosure provides a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) generating PDC-E2 particles of negatively charged peptides.155-185 (a) An aqueous solution of the peptide with a pH between 1.0 and 7.0; (b) A primary emulsion is generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) The primary emulsion is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), polyvinyl alcohol (PVA), and water to form a secondary emulsion; (d) The secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, the hardened polymer nanoparticles being PDC-E2 155-185 The peptides are encapsulated within their cores; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, followed by freeze-drying of the nanoparticles. In each embodiment, the pH is between pH 6.0 and 7.0.

[0049] In various embodiments, this disclosure provides a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) negatively charged particles of a peptide, said peptide containing the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1), the method comprising: (a) PDC-E2 155-185 The peptide is dissolved in ammonium hydroxide, the pH is adjusted to between about pH 1.0 and about pH 7.0 with acetic acid, and diluted with water (such as sterile water for injection (WFI)) to a peptide concentration of about 5 mg / mL to 10 mg / mL; (b) a primary emulsion is produced by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion of step (b) is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA) and polyvinyl alcohol (PVA) and optionally water to form a secondary emulsion; (d) the secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, which will carry PDC-E2 155-185 The peptide is encapsulated within its nucleus.

[0050] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) negatively charged particles of a peptide, said peptide containing the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1), the method comprising: (a) PDC-E2 155-185The peptide is dissolved in ammonium hydroxide, the pH is adjusted to between approximately pH 1.0 and approximately pH 7.0 with acetic acid, and diluted with water (such as sterile water for injection (WFI)) to a peptide concentration of approximately 8.25 mg / mL; (b) a primary emulsion is produced by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion of step (b) is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; (d) the secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, which will carry PDC-E2 155-185 The peptide is encapsulated within its nucleus.

[0051] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) negatively charged particles of a peptide, said peptide containing the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1), the method comprising: (a) PDC-E2 155-185 The peptide was dissolved in ammonium hydroxide, the pH was adjusted to between approximately pH 1.0 and approximately pH 7.0 with acetic acid, and diluted with water for injection (WFI) to a peptide concentration of approximately 8.25 mg / mL; (b) a primary emulsion was generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion of step (b) was homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) and water to form a secondary emulsion; (d) the secondary emulsion was hardened by evaporation to produce hardened polymer nanoparticles, which will carry PDC-E2 155-185 The peptide is encapsulated within its nucleus.

[0052] In various embodiments, the method further includes (e) filtering, washing, and concentrating the nanoparticles. In various embodiments, the method further includes (e) filtering, washing, and concentrating the nanoparticles, and (f) adding sodium citrate, mannitol, and sucrose, and freeze-drying the nanoparticles.

[0053] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) negatively charged particles of a peptide, said peptide containing the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1), the method comprising: (a) PDC-E2155-185 The peptide was dissolved in ammonium hydroxide, the pH was adjusted to between approximately pH 1.0 and approximately pH 7.0 with acetic acid, and diluted with water (e.g., sterile water for injection (WFI)) to a peptide concentration of approximately 8.25 mg / mL; (b) a primary emulsion was generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion was homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; and (d) the secondary emulsion was hardened by evaporation to produce hardened polymer nanoparticles, which would carry PDC-E2 155-185 The peptides are encapsulated within their cores; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, and the nanoparticles are freeze-dried.

[0054] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) negatively charged particles of a peptide, said peptide containing the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO:1), the method comprising: (a) PDC-E2 155-185 The peptide is dissolved in ammonium hydroxide, the pH is adjusted to between approximately pH 1.0 and approximately pH 7.0 with acetic acid, and diluted with water (e.g., WFI) to a peptide concentration of approximately 8.25 mg / mL; (b) a primary emulsion is generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) and water to form a secondary emulsion; (d) the secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, which will PDC-E2 155-185 The peptides are encapsulated within their cores; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, and the nanoparticles are freeze-dried.

[0055] In various embodiments, the ammonium hydroxide content is between about 0.05% and about 0.5%. In various embodiments, the ammonium hydroxide content is 0.1%.

[0056] In various embodiments, the acetic acid is between about 0.5 N and about 2 N. In various embodiments, the acetic acid is 1 N acetic acid. In various embodiments, the acetic acid is between about 0.5 N and about 2 N. In various embodiments, the acetic acid is 0.9 N acetic acid.

[0057] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) displacing PDC-E2 into negatively charged particles of peptides. 155-185 (a) The peptide is dissolved in ammonium hydroxide, and the pH is adjusted to between approximately pH 6.0 and approximately pH 7.0 by dialysis; (b) a primary emulsion is generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; (d) the secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, which will PDC-E2 155-185 The peptide is encapsulated within its core; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, and the nanoparticles are freeze-dried. In various embodiments, the PDC-E2 in step (a) is... 155-185 The peptide was diluted to between approximately 5 mg / ml and approximately 10 mg / ml, for example, approximately 8.25 mg / mL.

[0058] In various embodiments, this disclosure further contemplates a method for preparing a composition comprising encapsulated PDC-E2. 155-185 The method comprises: (a) displacing PDC-E2 into negatively charged particles of peptides. 155-185 (a) The peptide is dissolved in ammonium hydroxide, and the pH is adjusted to between approximately pH 6.0 and approximately pH 7.0 by dialysis; (b) a primary emulsion is generated by homogenizing the aqueous solution of (a) with an oil phase comprising poly(lactic-co-glycolic acid) (PLGA) dissolved in ethyl acetate; (c) the primary emulsion is homogenized with a mixture comprising ethyl acetate, polyacrylic acid (PAA), polyvinyl alcohol (PVA), and water to form a secondary emulsion; (d) the secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, which will PDC-E2 155-185 The peptide is encapsulated within its core; (e) the nanoparticles are filtered, washed, and concentrated; and (f) sodium citrate, mannitol, and sucrose are added, and the nanoparticles are freeze-dried. In various embodiments, the PDC-E2 in step (a) is... 155-185 The peptide was diluted to between approximately 5 mg / ml and approximately 10 mg / ml, for example, approximately 8.25 mg / mL.

[0059] In various embodiments, the method comprises a 5% PLGA solution containing 50:50 PLGA with a molecular weight between 10,000 Da and 60,000 Da.

[0060] In various embodiments, (c) the mixture comprising ethyl acetate, PAA, and PVA was prepared by adding 4% PVA and PAA (100 kDa, 35% wt) to ethyl acetate. In various embodiments, the pH of the PVA / PAA / ethyl acetate mixture was maintained below pH 4.0, below pH 3.9, below pH 3.8, below pH 3.7, below pH 3.6, or below pH 3.5.

[0061] In various embodiments, (c) the mixture comprising ethyl acetate, PAA, and PVA, and water, was prepared by adding 4% PVA and PAA (100 kDa, 35% wt) to ethyl acetate. In various embodiments, the pH of the PVA / PAA / ethyl acetate / water mixture was maintained below pH 4.0, pH 3.9, pH 3.8, pH 3.7, pH 3.6, or pH 3.5.

[0062] This disclosure also contemplates a method for preparing a composition comprising a negatively charged TIMP (TIMP-PBC) encapsulating one or more PBC-associated antigens. In various embodiments, the TIMP-PBC particles have a negative zeta potential. In various embodiments, the negative zeta potential of the TIMP-PBC particles is between about -100 mV and about 0 mV. In various embodiments, the zeta potential of the particles is about -100 mV to about -25 mV, about -100 mV to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 mV to about -35 mV, about -70 mV to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is approximately -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV, or -100 mV, including all values ​​and ranges within this range.

[0063] In various embodiments, the size or diameter of the TIMP-PBC particles is between 0.05 µm and about 10 µm. In various embodiments, the diameter of the TIMP-PBC particles is between 0.1 µm and about 10 µm. In various embodiments, the diameter of the TIMP-PBC particles is between 0.1 µm and about 5 µm. In various embodiments, the diameter of the TIMP-PBC particles is between 0.1 µm and about 3 µm. In various embodiments, the diameter of the TIMP-PBC particles is between 0.3 µm and about 5 µm. In various embodiments, the diameter of the TIMP-PBC particles is between about 0.3 µm and about 3 µm. In various embodiments, the diameter of the TIMP-PBC particles is between about 0.3 µm and about 1 µm. In various embodiments, the diameter of the TIMP-PBC particles is between about 0.4 µm and about 1 µm. In various embodiments, the diameter of the TIMP-PBC particles is about 100 to 10,000 nm, about 100 to 5,000 nm, about 100 to 3,000 nm, about 100 to 2,000 nm, about 300 to 5,000 nm, about 300 to 3,000 nm, about 300 to 1,000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, the diameter of the TIMP-PBC particles is about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1,000 nm, about 1,100 nm, about 1,200 nm, about 1,300 nm, about 1,400 nm, about 1,500 nm, or about 2,000 nm. In various embodiments, the diameter of the negatively charged particles is between 400 nm and 800 nm. In various embodiments, the polydispersity index (PDI) or heterogeneity index of the particle size is between 0.01 and 1.0 (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1, including all values ​​within the range). In various embodiments, the size of the TIMP-PBC particles is between about 400 nm and about 800 nm, and the zeta potential is between -30 mV and -80 mV.

[0064] In various embodiments, the particles have a uniform size distribution. In various embodiments, the particles have a uniform size distribution, wherein at least 90% of the particles have a diameter between 0.05 µm and about 10 µm, between 0.1 µm and about 10 µm, between 0.1 µm and about 5 µm, between 0.1 µm and about 3 µm, between 0.3 µm and about 5 µm, or between 0.3 µm and about 3 µm. In various embodiments, the particles have a uniform size distribution, wherein at least 90% of the particles have a diameter of about 100 nm to 10000 nm, about 100 nm to 5000 nm, about 100 nm to 3000 nm, about 100 nm to 2000 nm, about 300 nm to 5000 nm, about 300 nm to 3000 nm, about 300 nm to 1000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm. In various embodiments, the TIMP-PBC particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the particles have a uniform size distribution, wherein at least 50% of the particles have diameters between approximately 0.05 µm and approximately 10 µm, approximately 0.1 µm and approximately 10 µm, approximately 0.1 µm and approximately 5 µm, approximately 0.1 µm and approximately 3 µm, approximately 0.3 µm and approximately 5 µm, and approximately 0.3 µm and approximately 3 µm. In various embodiments, the particles have a uniform size distribution, wherein at least 50% of the particles have a diameter of about 100 nm to 10000 nm, about 100 nm to 5000 nm, about 100 nm to 3000 nm, about 100 nm to 2000 nm, about 300 nm to 5000 nm, about 300 nm to 3000 nm, about 300 nm to 1000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm. In various embodiments, the TIMP-PBC particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm.In various embodiments, the particles have a uniform size distribution, wherein at least 10% of the particles have a diameter between about 0.05 µm and about 10 µm, about 0.1 µm and about 10 µm, about 0.1 µm and about 5 µm, about 0.1 µm and about 3 µm, about 0.3 µm and about 5 µm, and about 0.3 µm and about 3 µm. In various embodiments, the particles have a uniform size distribution, wherein at least 10% of the particles have a diameter of about 100 nm to 10000 nm, about 100 nm to 5000 nm, about 100 nm to 3000 nm, about 100 nm to 2000 nm, about 300 nm to 5000 nm, about 300 nm to 3000 nm, about 300 nm to 1000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm. In various embodiments, the diameter of the TIMP-PBC particles is approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm.

[0065] In some embodiments, the particle's D90 is less than about 1000 nm. In some embodiments, the particle's D50 is from about 400 nm to about 800 nm, including about 400 nm, about 420 nm, about 440 nm, about 460 nm, about 480 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm, including any value or range therebetween. In some embodiments, the particle's D10 is less than about 600 nm. In some embodiments, the particle's D90 is from about 600 nm to about 700 nm. In some embodiments, the particle's D50 is from about 550 nm to about 600 nm. In some embodiments, the particle's D10 is from about 500 nm to about 550 nm. In some embodiments, the particle's D[4,3] is less than about 1000 nm. In some embodiments, the particle's D[4,3] is between 400 nm and 800 nm.

[0066] In some embodiments, the particles have a spherical or circular shape.

[0067] In various embodiments, this disclosure provides a method for preparing a composition comprising negatively charged particles (TIMP-PBC) encapsulating one or more PBC-associated antigens. In various embodiments, the amount of one or more PBC-associated antigens encapsulated within the TIMP-PBC composition is from 0.1 µg / mg to 100 µg / mg. In various embodiments, the concentration of one or more PBC-related antigens is between 0.1 µg / mg and 100 µg / mg (e.g., 0.1 µg / mg, 0.5 µg / mg, 1 µg / mg, 1.5 µg / mg, 2 µg / mg, 2.5 µg / mg, 3 µg / mg, 3.5 µg / mg, 4 µg / mg, 4.5 µg / mg, 5 µg / mg, 10 µg / mg, 15 µg / mg, 20 µg / mg, 25 µg / mg, 30 µg / mg, 35 µg / mg, 40 µg / mg, 45 µg / mg, 50 µg / mg, 55 µg / mg, 60 µg / mg, 65 µg / mg, 70 µg / mg, 75 µg / mg, 80 µg / mg, 85 µg / mg, 90 µg / mg, 95 µg / mg, or 100 µg / mg). (µg / mg), including all values ​​and ranges between these values. In various embodiments, the methods for preparing TIMP-PBC as described herein produce encapsulation efficiencies between 1 and 100% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, including all values ​​and ranges between these values). The content of the one or more PBC-related antigens in the TIMP-PBC composition can be determined by methods described in the literature, including ELISA, mass spectrometry, NMR, HPLC, CBQCA, and Western blotting.

[0068] In various embodiments, this disclosure provides a method for preparing a composition comprising negatively charged particles (TIMP-PBC) encapsulating one or more PBC-associated antigens, wherein the particle surface contains low levels of one or more PBC-associated antigens. In various embodiments, the particle surface is substantially free of one or more PBC-associated antigens. In various embodiments, the amount of one or more PBC-associated antigens present on the surface of the particles is between 0% and 30% of the total antigen content of the TIMP-PBC composition (e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 5%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, including all values ​​and ranges between these values). In various embodiments, the frequency of particles containing one or more PBC-associated antigens on their surface is less than 30% or between 0 and 30% higher than the frequency of particles containing negative controls (e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 5%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, including all values ​​and ranges between these values). In various embodiments, the frequency of particles containing one or more PBC-associated antigens on their surface is 0-100% lower than the frequency of particles containing positive controls (e.g., 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, including all values ​​and ranges between these values). In various embodiments, the amount of one or more PBC-associated antigens on the surface of the particle is 0 to 10 times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, including all values ​​and ranges between these) of the amount of one or more PBC-associated antigens on the surface of the negative control. In various embodiments, the amount of one or more PBC-associated antigens on the surface of the particle is 0 to 1 / 100 times (e.g., 0, 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, or 1 / 100, including all values ​​and ranges between these).In various embodiments, the number of TIMP-PBC particles with one or more PBC-associated antigens on their surface was determined using previously described methods such as flow cytometry, mass spectrometry, NMR, ELISA, CBQCA, and Western blotting.

[0069] In various embodiments, this disclosure provides a method for preparing a composition comprising negatively charged particles (TIMP-PBC) encapsulating one or more PBC-associated antigens, wherein the particles exhibit low burst release. In various embodiments, the particles do not exhibit burst release. In various embodiments, the particle burst release is between 0% and 75% (e.g., 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 75%, including all values ​​and ranges between these values).

[0070] In various embodiments, an excipient is added to the nanoparticle composition prior to freeze-drying in step (f). In various embodiments, the excipient is a buffer and / or a cryoprotectant. In various embodiments, the excipient is selected from the group consisting of: sucrose, mannitol, trehalose, sorbitol, dextran, Ficoll, dextran 70k, sodium citrate, lactose, L-arginine, or glycine. In various embodiments, the amount of excipient added to the nanoparticle composition prior to freeze-drying is between 0.05 wt% or vol% and 100 wt% or vol% (e.g., between 0.05 wt% or vol%, 0.1 wt% or vol%, 0.2 wt% or vol%, 0.3 wt% or vol%, 0.4 wt% or vol%, 0.5 wt% or vol%, 0.6 wt% or vol%, 0.7 wt% or vol%, 0.8 wt% or vol%, 0.9 wt% or vol%, 1 wt% or vol%, 2 wt% or vol%, 3 wt% or vol%, 4 wt% or vol%, 5 wt% or vol%, 6 wt% or vol%, 7 wt% or vol%, 8 wt% or vol%, 9 wt% or vol%, 10 wt% or vol%, 20 wt% or vol%, 30 wt% or vol%, 40 wt% or vol%, 50 wt% or vol%, 60 wt% or vol%, 70 wt% or vol%, 80 wt% or vol%, 90 wt% or vol%, or 100 wt% or vol%, including all values ​​within this range). In various embodiments, the amount of excipient added to the nanoparticle composition prior to freeze-drying is between 0.01 g and 500 g per gram of nanoparticles (e.g., between 0.01 g, 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 150 g, 200 g, 250 g, 300 g, 350 g, 400 g, 450 g, or 500 g).

[0071] In various embodiments, the batch size of the TIMP-PBC preparation can be scaled up or down proportionally. In various embodiments, the batch size is between 0.01 g and 100 kg. In various embodiments, the batch size is 0.01 g, 0.1 g, 10 g, 20 g, 40 g, 60 g, 80 g, 100 g, 160 g, 240 g, 320 g, 400 g, 480 g, 560 g, 640 g, 720 g, 800 g, 1000 g, 5 kg, 10 kg, 50 kg, or 100 kg, including all values ​​and ranges between these values.

[0072] A composition is also provided comprising particles encapsulated with PBC-associated antigen prepared by the methods described herein. In various embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0073] In various embodiments, the TIMP-PBC formulation or pharmaceutical composition contains negatively charged particles encapsulating PBC-associated antigens and excipients. In various embodiments, the excipients are selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, dextran, Ficoll, dextran 70k, sodium citrate, lactose, L-arginine, or glycine. In various embodiments, the TIMP-PBC formulation contains one to eleven excipients. In various embodiments, the TIMP-PBC formulation contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more excipients.

[0074] In various embodiments, the TIMP-PBC formulation contains negatively charged particles encapsulating PBC-associated antigen, sucrose, mannitol, and sodium citrate. In various embodiments, the concentration of the negatively charged particles in the TIMP-PBC formulation is between 1% and 100%, including all ranges and values ​​between these values. In various embodiments, the concentration of the negatively charged particles in the TIMP-PBC formulation is between 20% and 50%, including all ranges and values ​​between these values. In various embodiments, the concentration of the negatively charged particles in the TIMP-PBC formulation is between 30% and 40%, including all ranges and values ​​between these values. In various embodiments, the concentration of the negatively charged particles in the TIMP-PBC formulation is about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 35.6%, about 36%, about 37%, about 38%, about 39%, or about 40%.

[0075] In various embodiments, the sucrose concentration in the TIMP-PBC formulation is between 1% and 100%, including all ranges and values ​​between these values. In various embodiments, the sucrose concentration in the TIMP-PBC formulation is between 20% and 50%, including all ranges and values ​​between these values. In various embodiments, the sucrose concentration in the TIMP-PBC formulation is between 30% and 40%, including all ranges and values ​​between these values. In various embodiments, the sucrose concentration in the TIMP-PBC formulation is about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 35.6%, about 36%, about 37%, about 38%, about 39%, or about 40%.

[0076] In various embodiments, the mannitol concentration in the TIMP-PBC formulation is between 1% and 100%, including all ranges and values ​​between these. In various embodiments, the mannitol concentration in the TIMP-PBC formulation is between 15% and 35%, including all ranges and values ​​between these. In various embodiments, the mannitol concentration in the TIMP-PBC formulation is between 20% and 30%, including all ranges and values ​​between these. In various embodiments, the mannitol concentration in the TIMP-PBC formulation is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 26%, about 26.7%, about 27%, about 28%, about 29%, or about 30%.

[0077] In various embodiments, the sodium citrate concentration is between 0.01% and 25%, including all ranges and values ​​between these ranges. In various embodiments, the sodium citrate concentration is between 0.5% and 3.5%, including all ranges and values ​​between these ranges. In various embodiments, the sodium citrate concentration is about 0.5%, about 1%, about 1.5%, about 2%, about 2.1%, about 2.5%, about 3%, or about 3.5%.

[0078] The amount or concentration of PBC-associated antigen in the TIMP-PBC formulation can be expressed as the amount of PBC-associated antigen per mg PLGA (e.g., in µg). In various embodiments, the amount of PBC-associated antigen in the TIMP-PBC formulation is between 0.1 µg / mg PLGA and 100 µg / mg PLGA, including all ranges and values ​​between these ranges. In various embodiments, the amount of PBC-associated antigen in the TIMP-PBC formulation is between 0.1 µg / mg PLGA and 60 µg / mg PLGA, including all ranges and values ​​between these ranges. In various embodiments, the amount of PBC-associated antigen in the TIMP-PBC formulation is between 0.5 µg / mg PLGA and 10 µg / mg PLGA, including all ranges and values ​​between these ranges. In some embodiments, the amount of PBC-associated antigen in the TIMP-PBC formulation is between 1 µg / mg PLGA and 12 µg / mg PLGA, including all ranges and values ​​between these ranges. In some embodiments, the amount of PBC-associated antigen in the TIMP-PBC formulation is between 1 µg / mg PLGA and 5.8 µg / mg PLGA, including all ranges and values ​​between these ranges. In various embodiments, the PBC-associated antigen in the TIMP-PBC formulation is about 1 µg / mg PLGA, about 2 µg / mg PLGA, about 3 µg / mg PLGA, about 4 µg / mg PLGA, about 5 µg / mg PLGA, about 5.8 µg / mg PLGA, about 6 µg / mg PLGA, about 7 µg / mg PLGA, about 8 µg / mg PLGA, about 9 µg / mg PLGA, about 10 µg / mg PLGA, about 11 µg / mg PLGA, or about 12 µg / mg PLGA.

[0079] In various embodiments, the TIMP-PBC formulation contains approximately 10 8 10 granules / mg PLGA 10 Particles / mg PLGA, including all ranges and values ​​within this range. In various embodiments, the TIMP-PBC formulation contains approximately 10 9 Particles / mg PLGA

[0080] In some embodiments, the amount of PBC-associated antigen encapsulated in the particles is between 0.01 femtograms / particle and 100 femtograms / particle, including all ranges and values ​​between these ranges. In various embodiments, the amount is between 0.01 femtograms / particle and 10 femtograms / particle, including all ranges and values ​​between these ranges. In various embodiments, the amount is between 0.05 femtograms / particle and 1.5 femtograms / particle, including all ranges and values ​​between these ranges. In some embodiments, the amount is between 0.1 femtograms / particle and 0.75 femtograms / particle, including all ranges and values ​​between these ranges. In various embodiments, the PBC-associated antigen in the TIMP-PBC formulation is about 0.1 femtogram / particle, about 0.25 femtogram / particle, about 0.3 femtogram / particle, about 0.4 femtogram / particle, about 0.5 femtogram / particle, about 0.6 femtogram / particle, about 0.7 femtogram / particle, about 0.8 femtogram / particle, about 0.9 femtogram / particle, about 1 femtogram / particle, about 2 femtogram / particle, about 3 femtogram / particle, about 4 femtogram / particle, about 5 femtogram / particle, or about 6 femtogram / particle, including all ranges and values ​​between these ranges.

[0081] This disclosure provides a method for treating PBC in a subject, the method comprising administering to the subject particles encapsulated with PBC-associated antigens as described herein. Compositions for treating PBC comprising TIMP-PBC as described herein are also contemplated. In various embodiments, this disclosure provides the use of compositions comprising TIMP-PBC as described herein in the preparation of medicaments for treating PBC.

[0082] It should be understood that each feature, embodiment, or combination described herein is a non-limiting illustrative example of any aspect of the invention and is therefore intended to be combined with any other feature, embodiment, or combination described herein. For example, in the case of features being described using language such as “one embodiment,” “some embodiments,” “certain embodiments,” “another embodiment,” “a particular exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any other feature or combination of features described herein, without necessarily listing every possible combination. Such features or combinations of features are applicable to any aspect of the invention. In the case of instances where values ​​falling within the range are disclosed, any one of these instances is covered as possible endpoints of the range, covering any and all numerical values ​​between such endpoints, and covering any and all combinations of higher and lower endpoints. Attached Figure Description

[0083] Figure 1 An exemplary preparation process flowchart illustrates the steps involved in the preparation of tolerance nanoparticles encapsulated with PBC-associated antigens (TIMP-PBC).

[0084] Figure 2 The physicochemical characterization of TIMP-PBC particles was performed using scanning electron microscopy (SEM). Representative SEM images of the nanoparticles at 10,000X magnification are shown. Detailed Implementation

[0085] TIMPs are surface-functionalized, negatively charged particles made of biodegradable materials that encapsulate antigenic proteins or peptide epitopes associated with inflammatory conditions such as autoimmune diseases and allergies. TIMPs are designed to target the delivery of encapsulated proteins / peptides to antigen-presenting cells (APCs) in the mononuclear phagocyte system, thereby leading to APC-mediated T cell reprogramming via non-inflammatory pathways.

[0086] In preclinical models of autoimmune diseases and allergies, TIMP has demonstrated therapeutic efficacy in inducing T cell tolerance to antigens / sensitizing proteins and peptides, thereby improving disease symptoms. 8-12 TIMPs encapsulating one or more primary biliary cholangitis (PBC)-associated antigens (TIMP-PBC) have the potential to treat PBC by reprogramming the immune system and inducing tolerance of antigen-specific T cells to PBC-associated antigens. Immunotolerance therapy is currently needed, which can induce T cell tolerance to autoimmune PBC-associated antigens to achieve long-term therapeutic benefits without exposing patients to the risk of adverse events.

[0087] This disclosure provides a method for preparing negatively charged particles (TIMP-PBC) encapsulating one or more PBC-related antigens, and a pharmaceutical composition comprising said particles.

[0088] definition

[0089] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0090] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the indefinite article “a / an” and the definite article “the” include both plural and singular references.

[0091] The terms "about" or "approximately" mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined. In some embodiments, the terms "about" or "approximately" mean within 1, 2, 3, or 4 standard deviations. In some embodiments, the terms "about" or "approximately" mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" precedes the first value in a series of two or more values, it should be understood that the term "about" applies to each value in the series.

[0092] As used herein, “particle” refers to any non-tissue-derived composition of matter, which may be a sphere or spheroidal entity, a bead, or a liposome. The terms “particle,” “immunomodulated particle,” “carrier particle,” and “bead” may be used interchangeably depending on the context. Additionally, the term “particle” may be used to encompass both beads and spheres.

[0093] As used in this article, “negatively charged particles” refers to particles that have been modified to have a net surface charge of less than zero.

[0094] As used herein, “surface functionalization” refers to particles having one or more functional groups on their surface. In some embodiments, surface functionalization occurs by introducing one or more functional groups onto the surface of the particles. In various embodiments, surface functionalization can be achieved by carboxylation (i.e., adding one or more carboxyl groups to the particle surface) or by adding other chemical groups (e.g., other chemical groups that impart a negative surface charge).

[0095] "Carboxylated particles," "carboxylated beads," or "carboxylated spheres" include any particles that have been modified or surface-functionalized to add one or more carboxyl groups to their surface. Carboxylation of particles can be achieved using any compound with added carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA), poly(acrylic acid), or polyamino acids (e.g., aspartic acid, glutamic acid) consisting of carboxyl side chains. Carboxylation can also be achieved by forming particles using polymers with naturally occurring carboxyl groups (e.g., PLGA), wherein the preparation method produces additional carboxyl groups, i.e., carboxyl groups in addition to those naturally expressed by the polymer, located on the surface of the particles.

[0096] As used herein, the term “D90” refers to a particle size distribution that contains or finds 90% of the total volume of material in the sample. For example, if D90 is 1000 nm, it indicates that 90% of the sample is 1000 nm or smaller. Similarly, D50 or D10 refers to a particle size distribution that contains or finds 50% or 10% of the total volume of material in the sample, respectively. D[4,3] refers to the volume or mass moment average or the de Brouckere mean diameter. Particle size is measured using techniques known in the art. In some embodiments, D90 / D50 / D10 are measurements based on quantity (counts or frequency) or volume, which can be performed by laser diffraction (LD), SPOS, or SPOS with nanoparticle tracking analysis (SPOS-NTA).

[0097] "Polypeptide" and "protein" refer to polymers consisting of amino acid residues, associated naturally occurring structural variants, and synthetic, non-natural analogs linked by peptide bonds or isosteric peptide bonds. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. The terms "peptide" and "protein" are not limited to a minimum length of product. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. Thus, peptides, oligopeptides, dimers, polymers, and their analogs are all included within the definition. The definition covers full-length proteins and their fragments. The terms "peptide" and "protein" also include post-expression modifications of peptides or proteins, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this disclosure, "peptide" can include "modifications" of a natural sequence, such as deletions, additions, substitutions (which may be conserved in nature or may include substitution with any of the 20 amino acids normally present in human proteins, or any other naturally occurring or non-naturally occurring or atypical amino acids), and chemical modifications (e.g., addition of peptide mimics or substitution with peptide mimics). These modifications may be intentional, such as by removing or attaching chemical parts through site-directed mutagenesis or by chemical modification of amino acids, or they may be accidental, such as by mutations caused by the host cell that produces the protein, or by errors caused by PCR amplification before transfection of the host cell.

[0098] As used herein, "antigen moiety" or "antigen" refers to any part that is recognized by the host's immune system, such as a peptide. Examples of antigen moiety include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0099] "Pharmaceutically acceptable carrier" refers to any of the standard drug carriers, buffers, etc., such as phosphate-buffered saline solutions, 5% glucose solutions, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, flow aids, sweeteners, flavoring agents, and coloring agents. Suitable drug carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co., Easton, 1995). Preferred drug carriers depend on the intended route of administration of the active agent. Typical routes of administration include enteral (e.g., oral), parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or local, transdermal, or transmucosal administration), or inhalation.

[0100] The term “pharmaceutical acceptable” or “pharmacologically acceptable” means a material that is not biologically or otherwise undesirable, i.e., that the material can be administered to an individual without causing any undesirable biological effects or interacting in a harmful manner with any component of the composition containing the material or with any component present on or in the body of an individual.

[0101] The term "subject" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. This term does not specify a particular age or sex.

[0102] The term "epitope" refers to a portion of any molecule that can be recognized and bound by a selective binder at one or more sites within an antigen-binding region. Epitopes typically consist of chemically active surface groups of a molecule, such as amino acid or carbohydrate side chains, and possess specific three-dimensional structural features and mass-charge ratio characteristics. As used herein, epitopes can be continuous or discontinuous. Furthermore, an epitope can be a mimic (simulated epitope) because it contains the same three-dimensional structure as an epitope used to generate antibodies, but does not contain or only contains some amino acid residues found in the target that stimulate an antibody immune response. As used herein, a mimic epitope is not considered an antigen distinct from the epitope bound by a selective binder; the selective binder recognizes the same three-dimensional structure as both the epitope and the mimic epitope. As used herein, "epitope" is also called an "antigenic determinant," which is a portion of an antigen recognized by the immune system, particularly by antibodies, B cells, or T cells. For example, an epitope is a specific fragment of an antigen that an antibody binds to. The portion of an antibody that binds to an epitope is called a complementary site. Although epitopes are typically non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes. T-cell epitopes are located on the surface of antigen-presenting cells, where they bind to MHC (major histocompatibility complex) molecules. In humans, specialized antigen-presenting cells specifically present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T-cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, while MHC class II molecules present longer peptides, ranging from 13 to 17 amino acids in length. Non-classical MHC molecules also present non-peptide epitopes, such as glycolipids.

[0103] As used herein with respect to methods, the terms “treat,” “treated,” “treating,” and “treatment” mean the temporary or permanent, partial or complete elimination, reduction, suppression, or improvement of one or more clinical symptoms, manifestations, or progressions of an event, disease, or condition. Such treatment is not necessarily absolutely beneficial. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean prolonged survival compared to expected survival without treatment. Subjects requiring treatment include those who already have the stated condition or symptom, those who are susceptible to the stated condition or symptom, or those who wish to prevent the stated condition or symptom.

[0104] Particles

[0105] Particle size and charge are important for tolerance induction. While particle size and charge will vary based on the antigen encapsulated therein, generally, the particles described herein are effective in inducing tolerance when the particles are between about 100 nm and about 1500 nm in size and have a negative charge between 0 and about -100 mV. In various embodiments, the particle diameter is 400–800 nm and the charge is between about -25 mV and -70 mV. In various embodiments, the particle diameter is 400–1000 nm and the charge is between about -25 mV and -70 mV. The average particle size and charge can change slightly during lyophilization; therefore, both post-synthetic average and post-lyophilized average are described. As used herein, the terms “post-synthetic size” and “post-synthetic charge” refer to the size and charge of the particles before lyophilization. The terms “post-lyophilized size” and “post-lyophilized charge” refer to the size and charge of the particles after lyophilization.

[0106] In some embodiments, the particles are non-metallic. In these embodiments, the particles may be formed from polymers. In a preferred embodiment, the particles are biodegradable in an individual. In this embodiment, the particles can be delivered in multiple doses in an individual without particle accumulation occurring in the individual. Examples of suitable particles include polystyrene particles, PGA particles, PLA particles, PLGA particles, Pluronic-stabilized polypropylene sulfide particles, polypropylene sulfone, poly(ethylene glycol)-block-poly(propylene sulfide) copolymers, liposomes, and diamond particles.

[0107] In some embodiments, the particle surface is composed of materials that minimize nonspecific or undesirable biological interactions. The interaction between the particle surface and the interstitial space may be a factor playing a role in lymphatic uptake. The particle surface may be coated with materials to prevent or reduce nonspecific interactions. Spatial stabilization is achieved by coating the particles with a hydrophilic layer, such as poly(ethylene glycol) (PEG) and its copolymers, such as PLURONICS® (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), which can reduce nonspecific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All these facts indicate the relevance of the particle's physical properties to lymphatic uptake. Biodegradable polymers can be used to prepare all or some of the polymers and / or particles and / or layers. Biodegradable polymers may undergo degradation, for example, due to the reaction of functional groups with water in solution. As used herein, the term "degradation" refers to becoming soluble by a decrease in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers with ester groups typically undergo spontaneous hydrolysis, such as polylactic acid and polyglycolic acid.

[0108] The particles disclosed herein may also contain additional components. For example, the carrier may have an imaging agent bound to or conjugated with the carrier. An example of a carrier nanosphere with a currently commercially available imaging agent is the Kodak X-sight nanosphere. Inorganic quantum-confined luminescent nanocrystals known as quantum dots (QDs) have become ideal donors for FRET applications: their high quantum yield and tunable size-dependent Stokes shifts allow for different sizes to emit from blue to infrared when excited at a single ultraviolet wavelength. (Bruchez et al., *Science*, 1998, 281, 2013; Niemeyer, C. M. Angew. *Chem. Int. Ed.*, 2003, 42, 5796; Waggoner, A. *Methods Enzymol.*, 1995, 246, 362; Brus, LE. *J. Chem. Phys.*, 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers called dendritic structures, can be used for biolabeling, imaging, and optical biosensing systems. (Lemon et al., *J. Am. Chem. Soc.*, 2000, 122, 12886). Unlike traditional inorganic quantum dot synthesis, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic and unstable reagents. (Etienne et al., Appl. Phys. Lett. 87, 181913, 2005).

[0109] The particles can be formed from a variety of materials. The particles are preferably composed of materials suitable for biological applications. For example, the particles can be composed of glass, silica, citrate, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles can be composed of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aryl-alkenyl, heteroaryl, or alkoxyhydroxy acids; or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aryl-alkenyl, heteroaryl, or alkoxydicarboxylic acids. Additionally, the carrier particles can be quantum dots or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles comprising a mixture of ester bonds and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) can also be used. For example, carrier particles may comprise materials including: polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysaccharide polymer (PSA), poly(lactic-co-glycolic acid) copolymer (PLGA or PLG; terms are interchangeable), poly(lactic-co-sebacic acid) copolymer (PLSA), poly(glycolic acid-co-sebacic acid) copolymer (PGSA), polypropylene sulfide polymer, polypropylene sulfone, poly(ethylene glycol)-block-poly(propylene sulfide) copolymer poly(caprolactone), chitosan, etc. Other biocompatible biodegradable polymers useful in this invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable carbamates, as well as copolymers of these with linear or branched, substituted or unsubstituted alkyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy acids or dicarboxylic acids. Additionally, biologically important amino acids with reactive side-chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in the copolymer along with any of the materials mentioned above to provide reactive groups for conjugation with antigenic peptides and proteins or conjugated moieties. Suitable biodegradable materials for this invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the carrier particles of this invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetates, non-biodegradable carbamates, styrene, vinyl chloride, vinyl fluoride, vinylimidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0110] In some embodiments, the particles are copolymers with a molar ratio of about 80:20 to about 100:0 or about 20:80 to 100:0. Suitable copolymer ratios for these immunomodified particles can be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In some embodiments, the particles are Pluronic-stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), poly(lactic acid-co-glycolic acid) particles, carboxylated polyglycolic acid particles (PGA), carboxylated polylactic acid particles (PLA), or carboxylated poly(lactic acid-co-glycolic acid) particles. In some embodiments, the particles have a copolymer ratio of 80:20 polylactic acid / polyglycolic acid, 90:10 polylactic acid / polyglycolic acid, or 50:50 polylactic acid / polyglycolic acid. In various embodiments, the particles are poly(lactic acid-co-glycolic acid) particles and have a copolymer ratio of approximately 50:50 polylactic acid:polyglycolic acid.

[0111] Consideration has been made that the particles may further comprise surfactants and / or stabilizers. Surfactants and / or stabilizers may be anionic, cationic, or nonionic. Surfactants from the poloxamer and poloxamine families are commonly used in particle synthesis. Surfactants and / or stabilizers that can be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pranic L-63, PVA, PAA, methylcellulose, lecithin, DMAB, and PEMA. Additionally, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (D-α-tocopherol polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine ​​or their enantiomers), sodium cholate, and sulfate polymers. In some embodiments, two surfactants are used. In some embodiments, two stabilizers are used. In some embodiments, a combination of two or more surfactants and stabilizers is used. For example, if the particles are produced via a two-emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion. For example, the stabilizer may be a compound as described herein that stabilizes the primary and / or secondary emulsions by providing a physical or energy barrier between adjacent nanoparticle droplets in the emulsion, thereby reducing the probability of aggregation and the formation of larger nanoparticle droplets.

[0112] In some embodiments, the peptide antigen is encapsulated in particles via a single emulsion process. In other embodiments, the peptide antigen is more hydrophobic. Sometimes, dual emulsion processes result in the formation of large particles, which can lead to leakage of the hydrophilic active component and low retention efficiency. Agglomeration and Ostwald ripening are two mechanisms that can destabilize dual emulsion droplets, and diffusion of the hydrophilic active component through the organic phase is the primary mechanism leading to low levels of active component retention. In some embodiments, reducing the nanoparticle size may be beneficial. One strategy to achieve this is to apply a second strong shear rate. By using high polymer concentrations and high polymer molecular weights, accompanied by increased internal aqueous phase viscosity and surfactant molecular weights, leakage effects can be reduced. In some embodiments, the particles encapsulating the antigen are prepared via nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsions, sol-gel methods, layer-by-layer techniques, or iontophoresis.

[0113] TIMP-PBC particles can be synthesized via a nanoprecipitation method with or without the aid of a microfluidic device. In the nanoprecipitation method without a microfluidic device, a biodegradable polymer (e.g., PLA, PGA, PLGA) is dissolved in one or more organic solvents or combinations thereof. In some embodiments, the organic solvent is selected from the group consisting of acetone, acetonitrile, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, ethyl formate, or dimethyl carbonate. The polymer is dissolved at concentrations of about 1.0 mg / mL and 100 mg / mL, including all ranges and values ​​within this range. The polymer solution is added to an aqueous solution containing the PBC-associated antigen. In some embodiments, the PBC-associated antigen is PDC-E2. In some embodiments, the addition is performed while stirring the mixture. In some embodiments, the stirring is magnetic. The mixture is then added to a higher volume of another mixture comprising one or more surfactants, in some embodiments of which surfactants are selected from the group consisting of: poloxamer, polyamines, PEG, Tween-80, gelatin, dextran, Pluronic L-63, Pluronic F-68, Pluronic 188, Pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, vitamin E TPGS (Da-tocopherol polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids, polylysine, polyarginine, polyaspartic acid, polyglutamic acid, polyserine, polythreonine, polytyrosine, polycysteine, enantiomers of polyamino acids, sodium cholate, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gelatin, carbomer, and sulfate polymers. Organic solvents can be removed by solvent diffusion or solvent evaporation. In some embodiments, the evaporation is active evaporation. In some embodiments, the evaporation is active evaporation under vacuum, followed by passive overnight evaporation. The particles are washed with TFF. TIMP-PBC particles can be synthesized using one or more microfluidic devices via a nanoprecipitation method. In some embodiments, the devices are selected from the group consisting of: continuous microfluidic mixing, Y-shaped and T-shaped microfluidic devices, hydrodynamic flow focusing (HFF), impingement jet mixers, and multi-inlet vortex mixers. Monodisperse particles with sizes of 400-800 nm can be obtained with low PDI. The method involves adjusting the total flow rate (TFR) and flow rate ratio (FRR) of the three solutions: (i) A biodegradable polymer containing an organic phase. In some embodiments, the polymer is selected from the group consisting of PLA, PGA, and PLGA. (ii) A PBC-associated antigen containing aqueous phase 1 (W1). In some embodiments, the PBC-associated antigen is a PDC-E2 peptide.

[0114] (iii) Surfactants containing the W2 phase. In some embodiments, the surfactant is selected from the group consisting of: poloxamer, polyamines, PEG, Tween-80, gelatin, dextran, Pluronic L-63, Pluronic F-68, Pluronic 188, Pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, vitamin E TPGS (Da-tocopherol polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids, polylysine, polyarginine, polyaspartic acid, polyglutamic acid, polyserine, polythreonine, polytyrosine, polycysteine, enantiomers of polyamino acids, sodium cholate, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gelatin, carbomer, and sulfate polymers.

[0115] Organic solvents can be removed by solvent diffusion or solvent evaporation. In some embodiments, the evaporation is active evaporation. In some embodiments, the evaporation is active evaporation under vacuum, followed by passive overnight evaporation. The particles are washed with TFF.

[0116] Several methods for preparing nanoparticles have been described in the literature and are incorporated herein by reference. 3,4 .

[0117] antigen

[0118] An antigen is a discrete part of a molecule, such as a polypeptide or peptide sequence, a 3D structural formation of a polypeptide or peptide, or a polysaccharide or polynucleotide that can be recognized by the host's immune cells. Antigen specificity refers to the ability of a subject's host cells to recognize and generate an immune response against a single antigen or a molecule that is very similar to an antigen (such as an epitope or mimic epitope).

[0119] "Unresponsiveness," "tolerance," or "antigen-specific tolerance" refers to the insensitivity or reprogramming of T cells to T cell receptor-mediated stimuli. This reprogramming is typically antigen-specific and persists after cessation of exposure to the antigenic peptide. This reprogramming induces unresponsiveness in regulatory T cells (Tregs), Tr1 cells, and other T cells. For example, T cell insensitivity is characterized by a lack of effector cytokine production, lack of proliferation, or lack of activation. Reprogramming occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (co-stimulatory signal) or in the presence of an inhibitory signal (negative co-stimulatory or regulatory cytokines). Under these conditions, re-exposure to the same antigen (even in the presence of co-stimulatory molecules) results in a failure to produce cytokines and subsequently a failure to proliferate. Thus, the failure to produce cytokines prevents proliferation. However, if cultured with certain agents (e.g., IL-2), dysfunctional T cells can proliferate.

[0120] The tolerable therapies described herein are considered to be antigen-specific. For example, the TIMP administered as a tolerable therapy encapsulates one or more antigens associated with the tolerable therapy and the relevant disease or symptom being treated. It is considered that the TIMP used for a tolerable therapy contains one or more PBC-associated antigens.

[0121] In various embodiments, TIMP-PBC encapsulates one or more PBC-associated antigens or their epitopes. In various embodiments, the PBC-associated antigens are intracellular proteins, extracellular proteins, mitochondrial proteins, and / or nuclear proteins. In various embodiments, TIMP-PBC particles encapsulate one or more polynucleotides encoding PBC-associated antigens. In some embodiments, the polynucleotides comprise DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0122] In each embodiment, the PBC-related antigen is selected from the group consisting of: pyruvate dehydrogenase complex E2 subunit (acetyltransferase component of dihydrolipoyl lysine residue of pyruvate dehydrogenase complex) (PDC-E2), gp210, nucleoporin 62, Sp100, PML, CENP A, CENP B, and CENP. C, dsDNA, histones, branched 2-oxoacid dehydrogenase complex (BCOADC), lipoamide acyltransferase component of branched α-ketoacid dehydrogenase complex (BCOADC-E2), dihydrolipoamide lysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex (OGDC-E2), sulfite oxidase, mitochondrial outer membrane, glycogen phosphorylase, sarcosine dehydrogenase, smooth muscle protein, soluble liver antigen, liver / kidney microsomes, centromere protein, tubulin, actin, vimentin, desmin, cytokeratin, F-actin, UDP glucuronyltransferase family 1 member A complex (UGTA1), aminomethylene transferase cyclic deaminase, desialyl glycoprotein receptor (ASGPR), cardiolipin, h-Lamp-2, protease 3, CYP 2C9, CYP 2A6, and CYP P450 2D6. In some embodiments, PBC-associated antigens are selected from the group comprising bacterial epitopes, viral epitopes, or xenobiotics. In some embodiments, bacterial epitopes, viral epitopes, or xenobiotics comprise 2-octamide, 2-nonanoamide, *E. coli* PDC-E2, *E. coli* ATP-dependent ClpX, *E. coli* periplasmic maltose-binding protein, *E. coli* ATP-dependent helicase Hrp, *E. coli* fatty acid oxidation complex α, *E. coli* ppGpp synthase II, *E. coli* nitrate reductase 2, *Helicobacter pylori* urease β subunit, *Pseudomonas aeruginosa* diaminopimelic acid decarboxylase, human cytomegalovirus capsid assembly protein UL47, *Haemophilus influenzae* t-RNA (uracil-5-)-methyltransferase, *Neosphingosine monophosphate* proteins Novo 1, Novo 2, Novo 3, Novo 4, or *Lactobacillus delbrueckii* β-galactosidase.

[0123] In various embodiments, the TIMP-PBC encapsulation comprises one or more PDC-E2 peptides containing an antigenic epitope. In various embodiments, the TIMP-PBC encapsulation comprises PDC-E2... 155-185 The antigenic epitope. In various embodiments, TIMP-PBC encapsulates amino acids 155-185 (PDC-E2) of the PDC-E2 antigenic epitope. 155-185 ), which has the amino acid sequence (KVGEKLSEGDLLAEIETDKATIGFEVQEEGY) (SEQ ID NO: 1).

[0124] In some embodiments, the TIMP uses one, two, three, or more antigens or antigenic peptides. In some embodiments, the one or more PBC-associated antigens are encapsulated in the TIMP by covalently linking to the inner surface of the particle (see, for example, U.S. Patent Publication US20190282707, which is incorporated herein by reference). In some embodiments, sequences of two or more PBC-associated antigens are considered to be linked in a fusion protein and encapsulated within the TIMP described herein. Methods for preparing TIMPs having linked epitopes are described in U.S. Patent Publication US20190365656, which is incorporated herein by reference.

[0125] In some embodiments, chemicals, residues, or other impurities are removed or reduced from the antigen or antigenic peptide before encapsulation in the particles. In some embodiments, chemicals, residues, or other impurities are removed or reduced from the antigen or antigenic peptide before or during the formation of the primary emulsion. In some embodiments, the residue is trifluoroacetic acid (TFA). The residue can be removed or reduced by reverse osmosis (dialysis), lyophilization, ultrafiltration, washing, HPLC, reversed-phase HPLC, ion exchange resins, washing with trifluoroethanol, or any other method known in the art.

[0126] Emulsions come in various processing forms and are widely used in food, cosmetics, and pharmaceutical delivery. Oil-water (single), water-oil-water (bi) emulsions, and / or solid-oil-water emulsions are methods for encapsulating hydrophobic and hydrophilic drugs in micron or nanoscale form using PLGA or other biodegradable polymers. For example, PLGA is dissolved in an organic phase (oil) emulsified with a surfactant or stabilizer (water). The "oil phase" refers to an organic solvent that is insoluble in water. Hydrophobic drugs and / or other agents are added directly to the oil phase, while hydrophilic drugs and / or other agents (water) may first be emulsified with the polymer solution before particle formation. High-intensity homogenization (e.g., ultrasonic bursting) or other high-energy inputs facilitate the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours to allow the solvent to evaporate. The hardened nanoparticles are collected and washed by centrifugation. In some embodiments, hardened emulsion particles can be obtained by evaporation of the oil phase.

[0127] "Water-in-oil-in-water" (W / O / W) emulsions are an example of biemulsions, in which a dispersion of small water droplets within large oil droplets is itself dispersed in a continuous aqueous phase. Due to their compartmentalized internal structure, biemulsions offer advantages for encapsulation compared to simple oil-in-water emulsions, such as the ability to carry polar and non-polar cargoes (drugs / biologics, e.g., proteins), and improved control over the release of therapeutic molecules. The preparation of biemulsions typically requires surfactants or mixtures thereof to maintain stability. Surfactants stabilize droplets subjected to extreme flow, enabling the direct, large-scale production of robust bi-nanoemulsions suitable for nanostructure encapsulation applications across various industries. In one example, a biemulsion method involves generating a primary emulsion, i.e., a water-in-oil primary emulsion, by mixing an aqueous solution of a drug / biologic with a solution comprising a polymer. The primary emulsion is then mixed with a solution comprising one or more surfactants to form an oil-in-water secondary emulsion. The secondary emulsion is then hardened by evaporation to remove the solvent, thereby forming hardened polymer nanoparticles encapsulating the drug / biologic.

[0128] As used herein, “homogenization” refers to the operation of a processing device called a homogenizer, designed to reduce the size of droplets in a liquid-liquid dispersion. Factors affecting particle or droplet size include, but are not limited to, the type of emulsifier, emulsifier concentration, solution conditions, and mechanical means (homogenization power; pressure, rotational speed, time). Non-limiting examples of homogenizers include high-speed stirrers, high-pressure homogenizers, colloid mills, high-shear dispersers, ultrasonic disruptors, membrane homogenizers, and ultrasonic generators. Within the scope of this disclosure, mechanical homogenizers, manual homogenizers, ultrasonic instruments, mixing mills, vortex mixers, etc., can be used for both mechanical and physical disruption.

[0129] As used herein, “batch size” refers to the scale of preparation depending on the weight of the final product particles. Preparation methods can be modified, scaled up, or scaled down. This can be done by changing the amount or volume of solvents, antigens / proteins, polymers, surfactants, stabilizers, cryoprotectants, or excipients. Preparation methods can also be scaled up or scaled down by changing the time for homogenization, sonication, evaporation, filtration, concentration, washing, or lyophilization. The number of vials filled with TIMP-PBC will vary depending on the batch size. Fillings are approximately 1, 5, 50, 500, 5000, 50,000, or 500,000 vials, including all values ​​and ranges between these. The amount of TIMP-PBC filled in each vial is approximately 0.01 mg, approximately 0.1 mg, approximately 1 mg, approximately 10 mg, approximately 100 mg, approximately 200 mg, or approximately 250 mg, including all values ​​and ranges between these.

[0130] Methods for determining the protein content in particles or solutions include ELISA, mass spectrometry, HPLC, CBQCA, and Western blotting.

[0131] The CBQCA Molecular Probe Protein Quantification Kit provides a rapid and highly sensitive method for the quantification of proteins in solution. This kit uses ATTO-TAG CBQCA reagent (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde), originally developed as a chromatographic derivatization reagent for amines. This reagent has also proven very useful for quantifying amines in solution, including accessible amines in proteins. ATTO-TAG CBQCA reagent is virtually non-fluorescent in aqueous solution; however, in the presence of cyanide, it reacts with primary amines (such as those found in proteins) to form highly fluorescent derivatives.

[0132] acid

[0133] An acid is a substance that can donate protons (i.e., hydrogen ions, H+). + (or molecules or ions that form covalent bonds with electron pairs.)

[0134] Aqueous solutions of acids have a pH less than 7. A lower pH means higher acidity, and therefore a higher concentration of hydrogen ions in the solution. Chemical components or substances that exhibit acidic properties are called acidic.

[0135] In various embodiments, the acid is acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimony acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid.

[0136] alkali

[0137] A base is a chemical species that, in aqueous solution, donates electrons, accepts protons, or releases hydroxyl (OH-) ions.

[0138] Aqueous solutions of alkaline substances have a pH greater than 7.0 and at most 14.0. A higher pH means higher alkalinity, and therefore a higher concentration of hydroxyl ions in the solution. Chemical components or substances that exhibit alkaline properties are called alkaline.

[0139] In various embodiments, the base is barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, acetone, lithium hydroxide, pyridine, or rubidium hydroxide.

[0140] Drug formulation

[0141] Pharmaceutical compositions containing TIMP-PBC as an active ingredient as disclosed herein may contain pharmaceutically acceptable carriers or additives, depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerides, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin wax, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, etc. The additives used are selected from, but not limited to, the above or combinations thereof, and, where appropriate, depending on the dosage form of this disclosure.

[0142] The formulation of a pharmaceutical composition will vary depending on the chosen route of administration (e.g., solution, emulsion). A suitable composition containing a therapeutic drug to be administered can be prepared in a physiologically acceptable carrier or mediator. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol / aqueous solutions, emulsions or suspensions, including saline and buffer media. Parenteral mediators may include sodium chloride solution, Ringer's dextrose, dextran and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous mediators may include various additives, preservatives or fluids, nutrients, or electrolyte supplements.

[0143] Various aqueous carriers, such as sterile phosphate buffer solutions, antibacterial water, water, buffer water, 0.4% saline, 0.3% glycine, etc., may also include other proteins for enhancing stability, such as albumin, lipoprotein, globulin, etc., subject to mild chemical modification, etc.

[0144] Therapeutic formulations of particles for storage are prepared in the form of lyophilized formulations or aqueous solutions by mixing particles of desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington’s Pharmaceutical Sciences, 16th ed., Osol, A. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzyl methoxyamine chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as blood... Albumin, albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0145] Preparations intended for internal use must be sterile. This can be easily accomplished through filtration using a sterile filter membrane.

[0146] Aqueous suspensions may contain active compounds mixed with excipients suitable for preparing aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents may be naturally occurring phospholipids (e.g., lecithin), or condensation products of olefin oxides and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecanethyl-enoxyhexadecyl alcohol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides (e.g., polyvinyl sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethylparaben or n-propylparaben.

[0147] The TIMP-PBC described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0148] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with: at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or enrichers, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) humectants, such as cetyl alcohol and glyceryl monostearate; h) adsorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffers.

[0149] How to use

[0150] This article provides a method for treating primary biliary cholangitis (PBC) in a subject, the method comprising administering TIMP-PBC as described herein to the subject, wherein TIMP-PBC is administered at a dose from 0.01 mg / kg to 12 mg / kg. This article also provides a method for reducing the inflammatory immune response to PBC antigens in a subject with PBC, the method comprising administering TIMP-PBC to the subject, wherein TIMP-PBC is administered at a dose from 0.01 mg / kg to 12 mg / kg.

[0151] This document provides a method for treating PBC in a subject, the method comprising administering TIMP-PBC to the subject, wherein the TIMP-PBC is administered at a dose determined based on the subject's weight. In various embodiments, the TIMP-PBC is administered at a dose of 0.01 to 12 mg / kg. In various embodiments, the TIMP-PBC is administered at a fixed dose between 1 mg and 800 mg. In various embodiments, the TIMP-PBC is administered at the following doses: about 0.01 to about 12 mg / kg, about 0.05 to about 10 mg / kg, about 0.01 to about 5 mg / kg, about 0.1 to about 10 mg / kg, about 1 to about 8 mg / kg, about 1.5 to about 10 mg / kg, about 2 to about 12 mg / kg, about 2 to about 10 mg / kg, about 3 to about 10 mg / kg, about 4 to about 10 mg / kg, about 4 to 12 mg / kg, or about 5 to about 12 mg / kg. In various embodiments, TIMP-PBC was administered at the following doses: about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5 mg / kg, about 6 mg / kg, about 8.0 mg / kg, about 10 mg / kg, or about 12 mg / kg. In various embodiments, TIMP-PBC is administered at the following fixed doses: about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, or about 800 mg.

[0152] It has also been considered that TIMP-PBC be administered at concentrations between about 0.005 mg / mL and about 50 mg / mL. In various embodiments, TIMP-PBC is administered at concentrations of about 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. In various embodiments, TIMP-PBC is administered by intravenous infusion over a period of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0153] TIMP-PBC can be administered as a single dose or in multiple doses. In various embodiments, TIMP-PBC is administered weekly, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, annually, every two years, every three years, every four years, every five years, every six years, every seven years, every eight years, every nine years, or every ten years. In some embodiments, TIMP-PBC is administered with two doses one week apart.

[0154] In various embodiments, TIMP-PBC is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, by inhalation, or orally. It is considered that if TIMP-PBC is administered intravenously, it can be administered over approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours via intravenous infusion.

[0155] TIMP-PBC therapy is intended to relieve, reduce, or improve one or more symptoms of primary biliary cholangitis (PBC). PBC symptoms include, but are not limited to, hepatitis, cirrhosis, cholestasis, liver dysfunction, liver failure, liver fibrosis, increased hepatic immune infiltration, elevated bile acid levels, elevated liver enzyme levels (ALT, ALP, AST, gamma-glutamyl transferase), elevated bilirubin levels, circulating antimitochondrial antibodies (AMA), circulating antinuclear antibodies (ANA), fatigue, pruritus, itching, dry eyes and mouth, abdominal pain, splenomegaly, musculoskeletal pain, edema, fluid accumulation, xanthoma, jaundice, hyperpigmentation, osteoporosis, high cholesterol, diarrhea, steatorrhea, hypothyroidism, and weight loss. The diagnosis and symptoms of PBC are measured using methods known in the art, such as histological, fiber scan, and / or MR elastography analysis of liver pathology.

[0156] It is also considered that TIMP-PBC therapy reduces, shortens, or improves the duration and severity of the inflammatory immune response to one or more PBC antigens in subjects. Inflammatory immune responses include T-cell responses, B-cell responses, Th1 responses, myeloid cell responses, and / or antibody responses. In various embodiments, as described herein, the efficacy of TIMP-PBC in alleviating one or more PBC symptoms and / or reducing the duration and severity of the inflammatory immune response to one or more PBC antigens is determined by assaying one or more biological samples from subjects.

[0157] This document also provides a method for treating an autoimmune disease, wherein PDC-E2 is considered an antigen. In some embodiments, the method comprises administering tolerable nanoparticles encapsulating the PDC-E2 antigen to treat a PDC-E2-related autoimmune disease. In various embodiments, the PDC-E2-related autoimmune disease is selected from the group comprising: PBC, Sjögren's syndrome, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, or autoimmune hepatitis. This disclosure provides a method for treating a subject with a PDC-E2-related autoimmune disease, the method comprising administering to the subject particles encapsulating PDC-E2 as described herein. Compositions comprising TIMPs encapsulating PDC-E2 as described herein are also contemplated for the treatment of PBC, Sjögren's syndrome, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, or autoimmune hepatitis. In various embodiments, this disclosure provides the use of compositions comprising TIMPs encapsulating PDC-E2 as described herein for the preparation of medicaments for the treatment of PBC, Sjögren's syndrome, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, or autoimmune hepatitis.

[0158] Reagent test kit

[0159] In another aspect, this disclosure includes kits containing one or more compounds or compositions packaged in a manner conducive to their use in practicing the methods of this disclosure. In one embodiment, such kits include compounds or compositions (e.g., containing TIMP alone or in combination with a second agent) packaged in containers such as sealed bottles or containers, wherein a label is affixed to the container or included in the packaging, the label describing the use of the compound or composition to practice the method. Preferably, the compound or composition is packaged in unit dosage forms. The kit may further include a device suitable for administering the composition according to a specific route of administration or suitable for performing screening assays. Preferably, the kit contains a label describing the use of the inhibitory composition.

[0160] In another embodiment, this disclosure provides an article of manufacture or unit dosage form comprising: (a) a composition of substance comprising TIMP-PBC as described herein; (b) a container containing the composition; and (c) a label affixed to the container, or including a packaging insert in the container relating to the use of TIMP-PBC as described herein in the treatment of PBC.

[0161] Further aspects and details of this disclosure will become apparent from the following examples, which are intended to be illustrative and not limiting.

[0162] Example

[0163] Example 1. A method for preparing PDC-E2 peptide solutions to prepare tolerance nanoparticles (TIMP-PBC) encapsulating PBC-associated antigens.

[0164] To prepare the antigen solution, PDC-E2 peptide was added to 0.1% ammonium hydroxide to achieve a provisional concentration of 14 mg / mL PDC-E2 peptide. The solution was stirred to ensure complete dissolution of the peptide. Next, the pH of the PDC-E2 peptide solution was adjusted to a pH between 1.0 and 7.0 using 1 N acetic acid. The adjusted solution was then further diluted to a final concentration of 8.25 mg / mL PDC-E2 peptide by adding water for injection (WFI) and mixing until homogeneous.

[0165] In an alternative method, the PDC-E2 peptide was added to 3% ammonium hydroxide to achieve a provisional concentration of 14 mg / mL PDC-E2 peptide. The peptide dissolved in ammonium hydroxide was dialyzed against ultrapure water using a dialysis system (SpectraFlo dialyzer, Repligen). The pH of the dialyzed peptide solution was maintained between pH 6.0 and pH 7.0. The adjusted solution was then further diluted to a final concentration of 8.25 mg / mL PDC-E2 peptide by adding water for injection (WFI) and mixed until homogenized.

[0166] Example 2. Method for preparing tolerance nanoparticles (TIMP-PBC) encapsulating PBC-associated antigens.

[0167] TIMP-PBC (CNP-104) was prepared using a two-emulsion solvent evaporation method. An exemplary preparation method flowchart is provided. Figure 1 In short, the PDC-E2 peptide was dissolved in 0.1% ammonium hydroxide, and the pH was adjusted with 1 N acetic acid (8.2 mg / mL). It was then rapidly mixed with a 5% PLGA solution in ethyl acetate (50:50; molecular weight between 10,000 Da and 60,000 Da) to produce a primary water-in-oil emulsion. A stock mixture of water, PVA, PAA, and ethyl acetate was prepared to generate a blend by mixing PVA (4% in water), PAA (Sigma-Aldrich, 100 kDa, 35% wt in water), and ethyl acetate. The PVA / PAA / ethyl acetate / water blend was maintained at a pH below 4.0. The primary emulsion was then rapidly mixed with the PVA / PAA / ethyl acetate blend to form a secondary oil-in-water emulsion. The mixing of the primary and secondary emulsions was performed by homogenization.

[0168] Solvents were removed from the secondary emulsion by evaporation under pressure for a total of at least 3–4 hours. The hardened nanoparticles were then washed in sterile water and concentrated by filtration using a 20 µm filter. Cryoprotectants sucrose and mannitol, along with a buffer sodium citrate dihydrate, were added to the hardened nanoparticles. The formulation was then lyophilized.

[0169] The final TIMP-PBC formulation was characterized to determine its physicochemical properties, such as particle size, zeta potential, and total peptide content. Table 1 presents the results of the TIMP-PBC characterization. The TIMP-PBC particles were examined by scanning electron microscopy, revealing a uniform composition of intact particles with smooth surfaces. Figure 2 ).

[0170] Table 1: Physicochemical characterization of CNP-104 particles encapsulating PDC-E2 peptide.

[0171]

[0172] Example 3. Pharmaceutical composition of TIMP-PBC

[0173] The pharmaceutical formulations or compositions of TIMP-PBC contain the individual components listed in Table 2.

[0174] Table 2:

[0175] It should be understood that each embodiment of this disclosure described herein may optionally be combined with any one or more embodiments of the other embodiments described herein. Every patent document and every non-patent document cited herein is incorporated herein by reference in its entirety.

[0176] Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications within the spirit and scope of the invention as defined by the appended claims, the foregoing description, and / or the drawings. Thus, only such limitations appearing in the appended claims should be set forth in this disclosure.

[0177] References

[0178] 1. Lu M, Zhou Y, Haller IV, et al. reported an increasing prevalence of primary bile cholangitis and a reduced mortality rate after treatment. (Clinical Gastroenterology and Hepatology) Clin Gastroenterol Hepatol. )》 2018;16(8):1342-1350.e1341.

[0179] 2. Webb GJ, Siminovitch KA, Hirschfield GM. The immunogenetics of primary biliary cirrhosis: A comprehensive review, *Journal of Autoimmunity*. J Autoimmun. )》 2015;64:42-52.

[0180] 3. Mejía SP, Sánchez A, Vásquez V, Orozco J. Functional Nanocarriers for Delivering Itraconazole Against Fungal Intracellular Infections. Frontiers in Pharmacology (… Front Pharmacol. )》 June 28, 2021; 12:685391.

[0181] 4. Zieli Ska A, Carreiró F, Oliveira AM, Neves A, Pires B, VenkateshDN, Durazzo A, Lucarini M, Eder P, Silva AM, Santini A, Souto EB. Polymeric Nanoparticles: Production, Characterization, Toxicology and Ecotoxicology. (Molecular) Molecules. )》 August 15, 2020; 25(16):3731.

Claims

1. A method for preparing a composition comprising particles encapsulating one or more primary biliary cholangitis (PBC)-associated antigens, the method comprising: a. An aqueous solution that produces one or more PBC-related antigens; b. A primary emulsion is produced by mixing the aqueous solution of step (a) with the polymer. c. Mixing the primary emulsion with a mixture comprising one or more surfactants and / or stabilizers to form a secondary emulsion; and d. The secondary emulsion is hardened by evaporation to produce hardened polymer nanoparticles, wherein the hardened polymer nanoparticles encapsulate one or more PBC-associated antigens in their core.

2. The method of claim 1, further comprising (e) filtering, washing and concentrating the nanoparticles.

3. The method according to claim 1 or 2, further comprising (f) freeze-drying the nanoparticles to form a composition.

4. The method according to any one of claims 1 to 3, wherein the aqueous solution in step (a) comprises a solvent.

5. The method according to any one of claims 1 to 4, wherein the solvent in step (a) is selected from the group consisting of: acetaldehyde, acetone, acetonitrile, acetic acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, diethanolamine, diethylenetriamine, dimethoxyethane, dimethylformamide, 1,1-dimethylformamide, 1,1-dimethylhydrazine, 1,2-dimethylhydrazine, dimethyl sulfoxide, 1,4-dioxane, formic acid, ethanol, ethylamine, ethylene glycol, furanol, glycerol, isopropanol, methanol, methyldiethanolamine, methylisocyanate, N-methyl-2-pyrrolidone, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propylene glycol, pyridine, tetrahydrofuran, Triethylene glycol, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrene sulfonic acid, oxalic acid, carbonic acid, methylamine, diethylamine, pyridine, sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimonyic acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, boric acid, barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, sodium bicarbonate, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, lithium hydroxide, and rubidium hydroxide.

6. The method according to any one of claims 1 to 5, wherein the pH of the solvent in step (a) is between 1.0 and 14.

0.

7. The method according to any one of claims 1 to 6, wherein the initial concentration of the one or more PBC-related antigens dissolved in step (a) is between 0.1 mg / mL and 100 mg / mL.

8. The method according to any one of claims 1 to 7, wherein the pH of the solution in step (a) is adjusted to between 1.0 and 7.

0.

9. The method according to any one of claims 1 to 8, wherein the final concentration of one or more PBC-related antigens in step (a) is between 0.1 mg / mL and 100 mg / mL.

10. The method according to any one of claims 1 to 9, wherein the mixture in steps (b) and (c) comprises one or more solvents.

11. The method according to claim 10, wherein the one or more solvents are organic solvents or inorganic solvents.

12. The method according to any one of claims 10 to 11, wherein the one or more solvents in steps (b) and (c) are the same.

13. The method according to any one of claims 10 to 11, wherein the one or more solvents in step (b) and step (c) are different.

14. The method according to any one of claims 11 to 13, wherein the organic solvent is selected from the group consisting of acetic acid, acetone, ethanol, dichloromethane, dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, and chloroform.

15. The method according to any one of claims 1 to 14, wherein the emulsion produced by step (b) is a water-in-oil emulsion.

16. The method according to any one of claims 1 to 15, wherein the emulsion produced by step (c) is an oil-in-water emulsion.

17. The method according to any one of claims 1 to 16, wherein the polymer in step (b) is a biodegradable polymer.

18. The method of claim 15, wherein the biodegradable polymer is selected from the group consisting of: polyglycolic acid (PGA), polylactic acid (PLA), polysaccharide (PSA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-saccharide) (PLSA), poly(glycolic acid-co-saccharide) (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharides, and lipids.

19. The method according to any one of claims 1 to 18, wherein the surfactant or stabilizer in step (c) is anionic, cationic, amphoteric, or nonionic.

20. The method of claim 19, wherein the surfactant and / or stabilizer is selected from the group consisting of: poloxamer, polyamines, PEG, Tween-80, gelatin, dextran, pluronic L-63, pluronic F-68, pluronic 188, pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, vitamin E TPGS (Da-tocopherol polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids, polylysine, polyarginine, polyaspartic acid, polyglutamic acid, polyserine, polythreonine, polytyrosine, polycysteine, enantiomers of polyamino acids, sodium cholate, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gelatin, carbomer, and sulfate polymers.

21. The method according to any one of claims 1 to 20, wherein the primary emulsion in step (b) is obtained by homogenization.

22. The method according to any one of claims 1 to 20, wherein the primary emulsion in step (b) is obtained by ultrasonic treatment.

23. The method according to any one of claims 1 to 22, wherein the secondary emulsion in step (c) is obtained by homogenization.

24. The method according to any one of claims 1 to 22, wherein the secondary emulsion in step (c) is obtained by ultrasonic treatment.

25. The method according to any one of claims 1 to 24, wherein the pH of the emulsion in step (b) is less than pH 4.

0.

26. The method according to claim 21 or 23, wherein homogenization is performed for 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 30 seconds, 40 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds, 180 seconds, 210 seconds, 240 seconds, 270 seconds, 300 seconds, 330 seconds, 360 seconds, 390 seconds, 420 seconds, 450 seconds, 480 seconds, 510 seconds, 540 seconds, 570 seconds, or 600 seconds.

27. The method according to claim 22 or 24, wherein the ultrasonic treatment is performed for 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 30 seconds, 40 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds, 180 seconds, 210 seconds, 240 seconds, 270 seconds, 300 seconds, 330 seconds, 360 seconds, 390 seconds, 420 seconds, 450 seconds, 480 seconds, 510 seconds, 540 seconds, 570 seconds, or 600 seconds.

28. The method according to any one of claims 1 to 27, wherein the hardening of the nanoparticles in step (d) is carried out by evaporation of the solvent.

29. The method of claim 28, wherein the evaporation is active evaporation or passive evaporation.

30. The method of claim 29, wherein the active evaporation is vacuum-driven evaporation.

31. The method of claim 30, wherein the vacuum-driven evaporation is performed under high or low pressure.

32. The method of claim 29, wherein passive evaporation is carried out by stirring.

33. The method according to any one of claims 28 to 32, wherein evaporation is carried out for 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, or 96 hours.

34. The method according to any one of claims 2 to 33, wherein the filtration, washing and concentration of the nanoparticles in step (e) are performed by filtration, gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation or a combination thereof.

35. The method according to any one of claims 1 to 34, wherein the content of one or more PBC-related antigens encapsulated in the particulate composition is from about 0.1 µg / mg to 100 µg / mg.

36. The method according to any one of claims 1 to 35, wherein the one or more PBC-associated antigens are selected from the group consisting of: pyruvate dehydrogenase complex E2 subunit (PDC-E2), gp210, nucleoporin 62, Sp100, PML, CENP A, CENP B, CENP C, dsDNA, histones, branched 2-oxyacid dehydrogenase complex (BCOADC), lipoamide acyltransferase component of branched α-ketoacid dehydrogenase complex (BCOADC-E2), dihydrolipoamide lysuccinyltransferase component of 2-oxoglutarate dehydrogenase complex (OGDC-E2), sulfite oxidase, mitochondrial outer membrane, glycogen phosphorylase, sarcosine dehydrogenase, smooth muscle protein, soluble liver antigen, liver / kidney microsomes, centromere protein, tubulin, actin, vimentin, myodermin, cytokeratin, F-actin, UDP glucuronyltransferase family 1 member A complex (UGTA1), aminomethylene transferase cyclic deaminase, desialyl glycoprotein receptor (ASGPR), cardiolipin, h-Lamp-2, protease 3, CYP 2C9, CYP 2A6 and CYP P450 2D6, 2-octamide, 2-nonanoamide, Escherichia coli ( Escherichia coli PDC-E2, Aromatic Neosphingosine Bacteria ( Novosphingobium aromaticivorans Proteins Novo 1, Novo 2, Novo 3, Novo 4 or Lactobacillus delbrueckii ( Lactobacillus delbrueckii β-galactosidase, 2-octamide, 2-nonanoamide, *E. coli* PDC-E2, *E. coli* ATP-dependent ClpX, *E. coli* periplasmic maltose-binding protein, *E. coli* ATP-dependent helicase Hrp, *E. coli* fatty acid oxidation complex α, *E. coli* ppGpp synthase II, *E. coli* nitrate reductase 2, *Helicobacter pylori* ( Helicobacter pylori ) Urease β subunit, Pseudomonas aeruginosa ( Pseudomonas aeruginosa Diaminopimelic acid decarboxylase, human cytomegalovirus capsid assembly protein UL47, Haemophilus influenzae ( Haemophilus influenzae t-RNA (uracil-5-)-methyltransferase.

37. The method of claim 36, wherein the PBC-associated antigen comprises PDC-E2. 155-185 .

38. The method of claim 36, wherein the PBC-associated antigen is PDC-E2 as shown in SEQ ID NO:

1. 155-185 .

39. A method for preparing a composition comprising an encapsulated pyruvate dehydrogenase complex E2 subunit (PDC-E2). 155-185 The method comprises: negatively charged peptide particles. (a) Generation of PDC-E2 155-185 Aqueous solutions of peptides with pH values ​​between 1.0 and 7.0; (b) By making the PDC-E2 of step (a) 155-185 A primary emulsion is produced by homogenizing an aqueous solution of peptides with an oil phase containing PLGA dissolved in ethyl acetate. (c) Homogenize the primary emulsion from step (b) with a mixture comprising PAA, PVA, and ethyl acetate to form a secondary emulsion; and (d) The secondary emulsion from step (c) is hardened by evaporation to produce hardened polymer nanoparticles, which will then bond PDC-E2. 155-185 The peptide is encapsulated within its nucleus.

40. The method of claim 39, further comprising (e) filtering, washing and concentrating the nanoparticles.

41. The method of claim 40, further comprising (f) adding sodium citrate, mannitol and sucrose to the nanoparticles and freeze-drying the nanoparticles.

42. The method according to any one of claims 39 to 41, wherein the acetic acid is between about 0.5 N and about 2 N.

43. The method according to any one of claims 39 to 42, wherein the PLGA is a 5% PLGA solution comprising 50:50 PLGA with a molecular weight between 10,000 Da and 60,000 Da.

44. The method according to any one of claims 1 to 43, wherein the particle has a negative zeta potential.

45. The method of claim 44, wherein the zeta potential of the particle is between about 0 mV and -100 mV.

46. ​​The method of claim 44, wherein the zeta potential of the particle is between about -30 mV and -80 mV.

47. The method according to any one of claims 1 to 46, wherein the diameter of said particles is between about 0.3 µm and 3 µm.

48. The method of claim 47, wherein the diameter of the particles is between about 0.3 µm and 1 µm.

49. The method of claim 48, wherein the diameter of the particles is between about 0.4 µm and 1 µm.

50. The method according to any one of claims 1 to 49, wherein at least 90% of the particles have a diameter between about 0.3 µm and 3 µm.

51. The method of claim 50, wherein at least 90% of the particles have a diameter between about 0.3 µm and 1 µm.

52. The method of claim 50, wherein at least 90% of the particles have a diameter between about 0.4 µm and 1 µm.

53. The method according to any one of claims 1 to 52, wherein at least 50% of the particles have a diameter between about 0.3 µm and 3 µm.

54. The method of claim 53, wherein at least 50% of the particles have a diameter between about 0.3 µm and 1 µm.

55. The method of claim 54, wherein at least 50% of the particles have a diameter between about 0.4 µm and 1 µm.

56. The method according to any one of claims 1 to 55, wherein at least 10% of the particles have a diameter between about 0.3 µm and 3 µm.

57. The method of claim 56, wherein at least 10% of the particles have a diameter between about 0.3 µm and 1 µm.

58. The method according to any one of the preceding claims, wherein the batch size is between 0.01 g and 100 kg.

59. The method according to claim 58, wherein the batch size is 0.01 g, 0.1 g, 10 g, 20 g, 40 g, 60 g, 80 g, 100 g, 160 g, 240 g, 320 g, 400 g, 480 g, 560 g, 640 g, 720 g, 800 g, 1000 g, 5 kg, 10 kg, 50 kg or 100 kg.

60. The method according to any one of claims 1 to 59, wherein the aqueous solution in step (a) comprises one or more acids and / or one or more bases.

61. The method according to any one of claims 1 to 60, wherein the aqueous solution in step (a) comprises a base selected from the group consisting of: ammonium hydroxide, barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, ferrous hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide.

62. The method according to any one of claims 1 to 60, wherein the aqueous solution in step (a) comprises an acid selected from the group consisting of: acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrenesulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimony acid, fluoroboric acid, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid.

63. The method of claim 62, wherein the acid comprises acetic acid.

64. A composition comprising particles encapsulating one or more primary biliary cholangitis (PBC)-associated antigens, said composition being prepared by the method according to any one of claims 1 to 63.

65. The composition of claim 64, wherein the PBC-associated antigen comprises the pyruvate dehydrogenase complex E2 subunit (PDC-E2). 155-185 Peptide.

66. The composition according to claim 64 or 65, wherein the PBC-associated antigen is the pyruvate dehydrogenase complex E2 subunit (PDC-E2) shown in SEQ ID NO:

1. 155-185 Peptide.

67. The composition according to any one of claims 64 to 66, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

68. The composition according to claim 67, wherein the excipient is sucrose, mannitol, and sodium citrate.

69. A pharmaceutical composition comprising an encapsulated pyruvate dehydrogenase complex E2 subunit (PDC-E2). 155-185 Particles containing peptides, sucrose, mannitol, and sodium citrate, all with a negative charge.

70. The pharmaceutical composition of claim 69, wherein the negatively charged particles comprise poly(lactic-co-glycolic acid) (PLGA) and PDC-E2155-185 peptide, wherein PDC-E2 155-185 The peptide content ranges from 0.1 µg / mg to 100 µg / mg.

71. The pharmaceutical composition according to claim 69 or 70, wherein the PDC-E2 155-185 The peptide content ranges from 1 µg / mg to 5 µg / mg.

72. The pharmaceutical composition according to any one of claims 69 to 71, wherein the PDC-E2 155-185 The peptide contains the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO: 1).

73. The pharmaceutical composition according to any one of claims 65 to 68, wherein the PLGA is a carboxylated PLGA.

74. The pharmaceutical composition according to claims 69 to 73, wherein the zeta potential of the particles is between about 0 mV and -100 mV.

75. The pharmaceutical composition of claim 74, wherein the zeta potential of the particles is between about -30 mV and -80 mV.

76. The pharmaceutical composition according to any one of claims 69 to 75, wherein the diameter of the particles is between about 0.3 µm and 3 µm.

77. The pharmaceutical composition according to any one of claims 69 to 76, wherein the sucrose concentration is between 30% w / v and 40% w / v.

78. The pharmaceutical composition according to any one of claims 69 to 77, wherein the mannitol concentration is between 20% w / v and 30% w / v.

79. The pharmaceutical composition according to any one of claims 69 to 78, wherein the sodium citrate concentration is between 0.5% w / v and 3.5% w / v.

80. The pharmaceutical composition according to any one of claims 69 to 79, wherein the particles are lyophilized.

81. A method of treating a subject suffering from primary biliary cholangitis (PBC), the method comprising administering a composition according to any one of claims 66 to 68 or a pharmaceutical composition according to any one of claims 69 to 80.

82. The composition according to any one of claims 66 to 68 or the pharmaceutical composition according to any one of claims 69 to 80, for treating a subject with primary biliary cholangitis (PBC).

83. Use of a composition according to any one of claims 66 to 68 or a pharmaceutical composition according to any one of claims 69 to 80 for the preparation of a medicament for treating a subject with primary biliary cholangitis (PBC).

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