Preparation of tolerizing nanoparticles for the treatment of peanut allergy

Abstract

The present disclosure relates to processes for the preparation of tolerizing, immune-modulating nanoparticles encapsulating peanut proteins, compositions comprising the particles, and uses thereof for the treatment of peanut allergy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 282,889, filed November 24, 2021, which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to a process for the preparation of tolerizing immune-modulating nanoparticles encapsulating peanut proteins for the treatment of peanut allergy.

[0003] Incorporation by Reference of Electronically Submitted Materials Incorporated by reference in its entirety is the computer readable nucleotide / amino acid sequence listing, filed contemporaneously herewith and identified as the file named "57037_Seqlisting.hml", created on November 15, 2022, 27,276 bytes. [Background technology]

[0004] Peanut allergy is one of the most common food allergies in the United States, affecting approximately 1.2% of the total population and 2.5% of the pediatric population, and has been increasing in incidence over the past decade. 1 Peanut allergy is caused by a pathological hyperimmune response, and exposure to peanuts can lead to mild to severe symptoms, including nausea, vomiting, rash, breathing problems, low blood pressure, and even death.

[0005] Allergic immune responses to peanut proteins are mediated by T cell-dependent mechanisms involving upregulation of T helper type 2 responses, B cell class switching leading to the production of peanut protein-specific IgE antibodies, and degranulation of mast cells and basophils. 2 .

[0006] Currently, there is no cure for peanut allergy, and strict avoidance of exposure to peanut antigens and management of anaphylaxis are the only options available to patients. Immune tolerization therapy capable of inducing T cell tolerance to allergenic peanut proteins is considered the gold standard for the treatment of peanut allergy, but such therapy has been elusive.

[0007] Attempts to develop immune tolerization therapies have been made using oral immunotherapy (OIT), subcutaneous immunotherapy (SCIT), epithelial immunotherapy (EPIT), and sublingual immunotherapy (SLIT). 3 The success of these approaches has been highly variable, with only desensitization to peanut proteins reported, which provides protection only against accidental exposure but is not a cure. 4~6 Moreover, these approaches rely on chronic administration of formulations containing free peanut proteins. As a result, these treatments pose a risk of adverse reactions, including anaphylaxis, in peanut-allergic patients due to exposure of free allergenic peanut proteins to an immune system with pre-existing sensitivity to these allergens. 6~7 . Summary of the Invention

[0008] Tolerizing immune-modifying particles (TIMPs) containing one or more antigens have been previously described for the treatment of immune-mediated disorders (e.g., autoimmune diseases and allergies) through the induction of antigen-specific immune tolerance (WO2013 / 192532 and WO2015 / 023796, which are incorporated herein by reference). Encapsulation of peanut proteins within the TIMP core is advantageous because it ensures safe delivery of the encapsulated proteins to APCs without inducing immune activation (e.g., by exposure to IgE), reducing the risk of adverse reactions (e.g., anaphylaxis) associated with administration of free peanut proteins in peanut allergic patients.

[0009] The process for the manufacture of TIMP-PPE involves multiple steps, each of which impacts the physicochemical properties of the resulting composition that are essential for safe and therapeutic administration. Importantly, the process must be optimized to ensure efficient encapsulation of peanut proteins within the particle core.

[0010] The present disclosure provides a process for producing a composition comprising negatively charged particles encapsulating peanut proteins (TIMP-PPE). The process is directed to a process for producing particles optimized for safe and therapeutic administration of TIMP-PPE for the treatment of peanut allergy. In various embodiments, the method includes (a) generating a primary emulsion by mixing an aqueous solution of peanut proteins (PPE) with an oil phase containing a polymer; (b) mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers to form a secondary emulsion; (c) hardening the secondary emulsion by evaporation to remove the solvent, resulting in hardened polymeric nanoparticles encapsulating peanut proteins in their cores; (d) filtering, washing, and concentrating the nanoparticles; and (e) lyophilizing the nanoparticles. In various embodiments, the primary emulsion of step (a) is a water-in-oil emulsion. In various embodiments, the secondary emulsion of step (b) is an oil-in-water emulsion.

[0011] In various embodiments, the aqueous solution of step (a) comprises a solvent. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent. In various embodiments, the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethylsulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in the aqueous solution is at a concentration of 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% (v / v). In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 mM. In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 M.

[0012] In various embodiments, the surfactant and / or stabilizer solution of step (b) comprises a solvent. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent. In various embodiments, the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethylsulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in the aqueous solution is at a concentration of 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% (v / v). In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 mM. In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 M. In various embodiments, the solvent in the solutions of steps (a) and (b) is the same. In various embodiments, the solvent in the solutions of steps (a) and (b) is different.

[0013] In various embodiments, the aqueous solution of step (a) comprises 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / mL of peanut protein. In various embodiments, the peanut protein is dissolved in the aqueous solution by mixing for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours. In various embodiments, the peanut protein used in the process of producing TIMP-PPE is obtained from roasted peanuts. In various embodiments, the peanut protein is obtained from raw peanuts. In various embodiments, the peanut protein used in the process of making TIMP-PPE is obtained using a method comprising: (a) grinding raw peanuts into a paste; (b) defatting the peanut paste; (c) drying the defatted peanut paste; (d) powdering the dried peanut paste; (e) extracting the peanut protein from the peanut powder using ammonium bicarbonate; and (f) concentrating and clarifying the peanut protein to provide a refined peanut extract. In various embodiments, the refined peanut extract is further refined to isolate the allergenic peanut protein. In various embodiments, the isolated allergenic peanut protein is obtained by fractionation. In various embodiments the allergenic peanut proteins are Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18. In various embodiments the aqueous solution of step (a) contains peptides from allergenic peanut proteins.In various embodiments, the peptides comprise allergenic epitopes from allergenic peanut proteins Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18. In various embodiments, the peptides are derived from naturally occurring peanut proteins. In various embodiments, the peptides are synthetically produced. In various embodiments, the peptides are produced by solid phase peptide synthesis or solution phase peptide synthesis.

[0014] In various embodiments, the refined peanut extract used in step (a) is dissolved in a solvent. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent. In various embodiments, the refined peanut extract used in step (a) is dissolved in an inorganic solvent comprising one or more acids and / or one or more bases. In various embodiments, the solvent has a pH of 1.0 to 14.0. In various embodiments, the pH is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, including all values ​​within this range. In various embodiments, the solvent may be 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, fluoroantimonic acid, fluoroboric acid, hexafluorosulfuric acid, fluoroisopropyl ether ... The solvents are fluorophosphoric acid, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, carbonic acid, 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 0.01% and 100% (v / v). In various embodiments, the concentration of the solvent 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%, about 99%, about 100% (v / v), including all values ​​within this range. In various embodiments, the solvent concentration is 0.1M to 36M.In various embodiments, the solvent concentration is about 0.1M, about 0.5M, about 1M, about 2M, about 3M, about 4M, about 5M, about 6M, about 7M, about 8M, about 9M, about 10M, about 11M, about 12M, about 13M, about 14M, about 15M, about 16M, about 17M, about 18M, about 20M, about 30M, about 36M, including all values ​​within this range. In various embodiments, the concentration of the dissolved refined peanut extract is about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg / mL, including all values ​​within this range. In various embodiments, the refined peanut extract is dissolved in the solvent by mixing for 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours.

[0015] In various embodiments, the dissolved peanut protein extract has a pH of 1.0 to pH 6, including all values ​​within this range. In various embodiments, the dissolved peanut protein extract has a pH of about pH 1 to about pH 6, about pH 2 to about pH 6, about pH 3 to about pH 6, about pH 2 to about pH 4, or about pH 1, about pH 1.5, about pH 2, about pH 2.5, about pH 3, about pH 3.5, about pH 4, about pH 4.5, about pH 5, about pH 5.5, or about pH 6.

[0016] In various embodiments, the polymer in step (a) is a biodegradable polymer. In various embodiments, the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(lactic-co-glycolic) (PLGA), poly(lactic-co-sebacic) acid (PLSA), poly(glycolic-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 various molar ratios of the constituent polymers. 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.

[0017] In various embodiments, the polymer in step (a) is PLGA. In various embodiments, the molar ratio of the copolymer of PLGA 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. In various embodiments, the PLGA has a high molecular weight. In various embodiments, the PLGA has a low molecular weight. In various embodiments, the PLGA has a molecular weight of 10 to 10,000 kDa (e.g., between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 kDa, including all values ​​within this range). In various embodiments, the amount of PLGA in the solution in step (a) is between 0.05 and 100% by weight (e.g., between 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range).

[0018] In various embodiments, the surfactants and / or stabilizers used in step (b) are anionic, cationic, or non-ionic. In various embodiments, the surfactants and / or stabilizers are poloxamers, 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, didodecyldimethylammonium bromide (DMAB), poly(ethylene-alt-maleic acid) (PEMA), vitamin E. TPGS (Da-tocopheryl 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, hydroxyprolylcellulose, hydroxypropylmethylcellulose, gelatin, sodium cholate, carbomer, or sulfate polymers (e.g., hepar sulfate, chondroitin sulfate, fucoidan, ulvan, and carrageenan). In various embodiments, the amount of surfactant and / or stabilizer present in the solution in step (b) is between 0.05 and 100% (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range) by weight or volume. In various embodiments, the surfactant and / or stabilizer has a molecular weight of 0.1 to 10,000 kDa (e.g., between 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 kDa, including all values ​​within this range).

[0019] In various embodiments, the solution containing one or more surfactants and / or stabilizers forming the oil-in-water secondary emulsion in (b) has a pH of about 4 or less than about 4.0. In various embodiments, the oil-in-water secondary emulsion has a pH of about pH 1 to about pH 4, about pH 2 to about pH 4, about pH 3 to about pH 4, or about pH 1, about pH 1.5, about pH 2, about pH 2.5, about pH 3, about pH 3.5, or about pH 4.

[0020] In various embodiments, the method includes (a) generating a primary emulsion by mixing an aqueous solution of peanut protein with an oil phase containing a polymer to result in a water-in-oil primary emulsion; (b) mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers to form a secondary oil-in-water emulsion; (c) hardening the secondary emulsion to remove the solvent and result in polymeric nanoparticles encapsulating the PPE in their core; (d) filtering, washing, and concentrating the nanoparticles; and (e) freeze-drying the nanoparticles.

[0021] In various embodiments, the water-in-oil primary emulsion of step (a) is obtained by homogenization of an aqueous solution of peanut protein with an oil phase containing a polymer. In various embodiments, homogenization is performed for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 700, 800, 900, or 1000 seconds. In various embodiments, the oil-in-water secondary emulsion of step (b) is obtained by homogenization of the primary emulsion with a solution containing one or more surfactants and / or stabilizers. In various embodiments, homogenization is performed for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 700, 800, 900, or 1000 seconds. In various embodiments, the water-in-oil primary emulsion of step (a) is obtained by ultrasonication of an aqueous solution of peanut proteins with an oil phase comprising the polymer. In various embodiments, the sonication is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds. In various embodiments, the secondary oil-in-water emulsion of step (b) is obtained by sonication of the primary emulsion with a solution comprising one or more surfactants and / or stabilizers. In various embodiments, sonication is performed for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

[0022] In various embodiments, the secondary emulsion is cured by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the evaporation is passive evaporation. In various embodiments, the active evaporation is vacuum driven evaporation. In various embodiments, the evaporation is performed for 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours. In various embodiments, the secondary emulsion is cured by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the evaporation is passive evaporation. In various embodiments, the active evaporation is performed using agitation or under vacuum. In various embodiments, the active evaporation is performed under high pressure vacuum. In various embodiments, the active evaporation is performed under low pressure vacuum. In various embodiments, evaporation is carried out for 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours. In various embodiments, evaporation is performed at a pressure between 0.01 and 1000 mBar (e.g., between 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mBar, including all values ​​within this range).

[0023] In various embodiments, the filtering, washing, and concentrating of the particles in step (d) is performed by gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, or a combination thereof.

[0024] The present disclosure also contemplates a process for producing a composition comprising a negatively charged TIMP encapsulating a peanut protein (TIMP-PPE). In various embodiments, the TIMP-PPE particles have a negative zeta potential. In various embodiments, the negative zeta potential of the TIMP-PPE particles is about -100 mV to about 0 mV. In various embodiments, the zeta potential of the particles is about -100 mV to about -25 mV, about -100 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 to about -35 mV, about -70 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 about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV.

[0025] In various embodiments, the size, or diameter, of the TIMP-PPE particles is from 0.05 μm to about 10 μm. In various embodiments, the diameter of the TIMP-PPE particles is from 0.1 μm to about 10 μm. In various embodiments, the diameter of the TIMP-PPE particles is from 0.1 μm to about 5 μm. In various embodiments, the diameter of the TIMP-PPE particles is from 0.1 μm to about 3 μm. In various embodiments, the diameter of the TIMP-PPE particles is from 0.3 μm to about 5 μm. In various embodiments, the diameter of the TIMP-PPE particles is from about 0.3 μm to about 3 μm. In various embodiments, the diameter of the TIMP-PPE particles is from about 0.3 μm to about 1 μm. In various embodiments, the diameter of the TIMP-PPE particles is from about 0.4 μm to about 1 μm. In various embodiments, the TIMP-PPE particles have a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the TIMP-PPE 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 negatively charged particles have a diameter of 400 nm to 800 nm. In various embodiments, the polydispersity index (PDI) or heterogeneity index for particle size is 0.01 to 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 this range).

[0026] In various embodiments, the particles have a homogeneous size distribution. In various embodiments, the particles have a homogeneous size distribution, with at least 90% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the TIMP-PPE 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 homogenous size distribution, with at least 50% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm. In various embodiments, the particles have a homogenous size distribution, with at least 50% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the TIMP-PPE 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 homogenous size distribution, with at least 10% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm.In various embodiments, the particles have a homogenous size distribution, with at least 10% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the TIMP-PPE 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.

[0027] In various embodiments, the invention of the present disclosure provides a process for producing a composition comprising negatively charged particles encapsulating peanut protein (TIMP-PPE). In various embodiments, the peanut protein content encapsulated within the TIMP-PPE composition is between 0.1 and 100 μg / mg. In various embodiments, the peanut protein content is between 0.1 and 100 μg / mg (e.g., 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg / mg), including all values ​​and ranges therebetween. In various embodiments, the peanut protein encapsulated within the TIMP-PPE composition comprises Ara h protein. In various embodiments, the Ara h protein is Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18. In various embodiments, the content of any one or combination of Ara h proteins in the TIMP-PPE composition is 0.01-100 μg / mg (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg / mg, including all values ​​and ranges therebetween). In various embodiments, the process for making TIMP-PPE described herein results in an encapsulation efficiency of 1-100% (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 therebetween). In various embodiments, the process results in an encapsulation efficiency of at least 20%.The peanut protein content in the TIMP-PPE compositions can be determined by methods described in the literature, including ELISA, mass spectrometry, HPLC, CBQCA, and Western blot.

[0028] In various embodiments, the present disclosure provides a process for producing a composition comprising negatively charged particles encapsulating peanut protein (TIMP-PPE), wherein the particle surface contains low levels of peanut protein. In various embodiments, the particle surface is essentially free of peanut protein. In various embodiments, the amount of peanut protein present on the surface of the particle is 0-30% (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 therebetween) of the total protein content of the TIMP-PPE composition. In various embodiments, the frequency of particles containing peanut protein on their surface is 0-30% higher (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), compared to the negative control. In various embodiments, the frequency of particles containing peanut protein on their surface is 0-100% lower (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), compared to the positive control. In various embodiments, the amount of peanut protein on the surface of the particles is 0-10 times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, including all values ​​and ranges between these values) higher than the negative control. In various embodiments, the amount of peanut protein on the surface of the particles is 0-100 times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times, including all values ​​and ranges between these values) lower than the positive control. In various embodiments, the number of TIMP-PPE particles having peanut protein on their surface is determined using the methods previously described, such as flow cytometry, mass spectrometry, ELISA, CBQCA, and Western blot.

[0029] In various embodiments, the present disclosure provides a process for producing a composition comprising negatively charged particles encapsulating peanut proteins (TIMP-PPE), wherein the particles exhibit a low burst release. In various embodiments, the particles do not exhibit a burst release. In various embodiments, the particle burst release is 0-75% (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 therebetween).

[0030] In various embodiments, an excipient is added to the nanoparticle composition prior to lyophilization in step (e). 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 lyophilization is between 0.05 and 100% (e.g., between 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range), by weight or volume. In various embodiments, the amount of excipient added to the nanoparticle composition prior to lyophilization is between 0.01 and 500 g (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 g) per gram of nanoparticles.

[0031] In various embodiments, the manufacturing batch size of the TIMP-PPE can be scaled up or down. In various embodiments, the manufacturing 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 therebetween.

[0032] Contemplated herein is a particle that encapsulates peanut protein produced by the method described herein. Also provided is a composition that includes a particle that encapsulates peanut protein produced by the method described herein. In various embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient. In various embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0033] Also provided is a formulation comprising particles comprising peanut protein extract. In various embodiments, the TIMP-PPE formulation or pharmaceutical composition comprises negatively charged particles encapsulating the purified protein extract, and an excipient. 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 TIMP-PPE formulation contains 1-11 excipients. In various embodiments, the TIMP-PPE formulation contains 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more excipients.

[0034] In various embodiments, the TIMP-PPE formulation contains negatively charged particles encapsulating purified peanut protein, sucrose, mannitol, and sodium citrate. In various embodiments, the concentration of negatively charged particles in the TIMP-PPE formulation is 1-100%, 20-50%, or 30-40%, including all ranges and values ​​therebetween. In various embodiments, the concentration of negatively charged particles in the TIMP-PPE 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%.

[0035] In various embodiments, the sucrose concentration in the TIMP-PPE formulation is 1-100%, 20-50%, or 30-40%, including all ranges and values ​​therebetween, hi various embodiments, the sucrose concentration in the TIMP-PPE 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%.

[0036] In various embodiments, the mannitol concentration in the TIMP-PPE formulation is 1-100%, 15-35%, or 20-30%, including all ranges and values ​​therebetween, In various embodiments, the sucrose concentration in the TIMP-PPE 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%.

[0037] In various embodiments, the sodium citrate concentration is 0.01-25% or 0.5-3.5%, including all ranges and values ​​therebetween, 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%.

[0038] In various embodiments, the purified peanut protein in the TIMP-PPE formulation is from 0.3 μg to 30 μg (micrograms) of peanut protein per milligram (mg) of PLGA, or from 1 μg to 10 μg of peanut protein per mg of PLGA, including all ranges and values ​​therebetween. In various embodiments, the purified peanut protein in the TIMP-PPE formulation is about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, or about 10 μg of peanut protein per mg of PLGA.

[0039] The present disclosure provides a method for treating peanut allergy in a subject, comprising administering to the subject a subject particle that encapsulates peanut protein as described herein.Also contemplated is a composition that comprises TIMP-PPE as described herein for use in treating peanut allergy.In various embodiments, the present disclosure provides the use of a composition that comprises TIMP-PPE as described herein in the preparation of a medicament for treating peanut allergy.

[0040] It is understood that each feature or embodiment, or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention, and as such is meant to be combinable with any other feature or embodiment, or combination described herein. For example, when a feature is described in language such as "one embodiment," "some embodiments," "particular embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, and it is not necessary to list all possible combinations. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​that fall within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of the upper and lower endpoints are envisioned. [Brief description of the drawings]

[0041] [Figure 1] Characterization of PPE by SDS-PAGE. PPE was separated by electrophoretic mobility under non-reducing conditions and stained to visualize protein banding. Standard molecular weight size standards were run in parallel. Characterization by SDS-PAGE clearly identified Ara h1, h2, h3, and h6 bands at the expected sizes. [Diagram 2] FIG. 1 is a high level manufacturing process flow diagram showing the major steps involved in the manufacture of tolerizing nanoparticles encapsulating peanut proteins (TIMP-PPE). [Diagram 3] Physicochemical characterization of TIMP-PPE particles produced in 80 g batches using scanning electron microscopy (SEM). Representative SEM images of nanoparticles at 10,000x magnification are shown. [Figure 4]Physicochemical characterization of TIMP-PPE particles produced in a 160 g batch using scanning electron microscopy (SEM). A representative SEM image of nanoparticles at 10,000x magnification is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] TIMPs are surface-functionalized, negatively charged poly(lactide-co-glycolide) particles that encapsulate antigenic protein or peptide epitopes associated with inflammatory conditions such as autoimmune diseases and allergies. TIMPs are designed to target delivery of the encapsulated proteins / peptides to antigen-presenting cells (APCs) of the mononuclear phagocyte lineage, resulting in APC-mediated T cell reprogramming via a non-inflammatory pathway.

[0043] In preclinical models of autoimmune disease and allergy, TIMPs have demonstrated therapeutic efficacy in inducing T cell tolerance to antigenic / allergenic proteins and peptides, resulting in amelioration of disease symptoms. 8~12 TIMPs encapsulating peanut proteins (TIMP-PPE) could potentially treat peanut allergy by reprogramming the immune system and inducing antigen-specific T cell tolerance to peanut proteins. There is a current need for immune tolerization therapies that can induce T cell tolerance to allergenic peanut proteins for long-term therapeutic benefit without putting patients at risk for adverse events.

[0044] The present disclosure provides processes for producing negatively charged particles encapsulating peanut proteins (TIMP-PPEs) and pharmaceutical compositions comprising the particles.

[0045] definition Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below.

[0046] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly dictates otherwise.

[0047] The term "about" or "approximately" refers to a margin of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means 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" or "approximately" precedes the first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each one of the numbers in the series.

[0048] "Particle" as used herein refers to any non-tissue derived composition and may be a sphere or sphere-like entity, a bead, or a liposome. The terms "particle", "immunomodulating particle", "carrier particle", and "bead" may be used interchangeably depending on the context. Furthermore, the term "particle" may be used to encompass beads and spheres.

[0049] As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge that is less than zero.

[0050] As used herein, "surface functionalized" refers to particles having one or more functional groups on the surface. In some embodiments, surface functionalization occurs by introducing one or more functional groups to the surface of the particle. In various embodiments, surface functionalization can be achieved by carboxylation (i.e., the addition of one or more carboxyl groups to the particle surface) or the addition of other chemical groups (e.g., other chemical groups that impart a negative surface charge).

[0051] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified or surface functionalized to add one or more carboxyl groups to the particle surface. Carboxylation of the particle can be achieved using any compound that adds carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA), poly(acrylic acid), or polyamino acids consisting of carboxyl side chains (e.g., aspartic acid, glutamic acid). Carboxylation can also be achieved by forming the particle using a polymer that has native carboxyl groups (e.g., PLGA), where the manufacturing process results in additional carboxyl groups located on the surface of the particle, i.e., in addition to those naturally expressed by the polymer.

[0052] "Polypeptide" and "protein" refer to polymers composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof, linked via peptide bonds or peptide bond isosteres. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms "polypeptide" and "protein" are not limited to a minimum length of the product. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms "polypeptide" and "protein" also include post-expression modifications of a polypeptide or protein, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, a "polypeptide" can include "modifications" to the native sequence, such as deletions, additions, substitutions (which may be conservative in nature or may include substitutions with any of the 20 amino acids commonly present in human proteins, or any other naturally occurring, non-naturally occurring, or atypical amino acids), and chemical modifications (e.g., addition or substitution by peptidomimetics). These modifications may be deliberate, through site-directed mutagenesis or through chemical modification of amino acids to remove or attach chemical moieties, or may be accidental, such as through mutations that occur via the host cell producing the protein or through errors due to PCR amplification prior to host cell transfection.

[0053] As used herein, "antigenic moiety" or "antigen" refers to any moiety, e.g., peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0054] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as phosphate buffered saline, 5% aqueous solution of dextrose, 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, glidants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration) or via inhalation.

[0055] The terms "pharmacologically acceptable" or "pharmacologically acceptable" mean a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, or with any components present on or in the individual's body.

[0056] The term "subject" includes 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 cows, horses, sheep, goats, pigs, and the like; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex.

[0057] The term "epitope" refers to a portion of any molecule that can be recognized and bound by a selective binding agent at one or more of the antigen-binding regions. An epitope usually consists of a chemically active surface grouping of molecules, such as amino acids or carbohydrate side chains, and has specific three-dimensional structural and charge characteristics. An epitope as used herein can be continuous or discontinuous. An epitope can also be a mimetic (mimotope) in that it contains a three-dimensional structure that is identical to the epitope used to generate the antibody, but does not contain any, or only some, of the amino acid residues found in the target used to stimulate the antibody immune response. As used herein, a mimotope is not considered a different antigen than the epitope bound by the selective binding agent, which recognizes the same three-dimensional structure of the epitope and the mimotope. As used herein, an "epitope" is also known as an "antigenic determinant," which is the portion of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. For example, an epitope is the specific part of an antigen that an antibody binds to. The part of an antibody that binds to the epitope is called the paratope. Epitopes are usually non-self proteins, but also sequences derived from the host that can be recognized (as in the case of autoimmune diseases). T cell epitopes are presented on the surface of antigen-presenting cells, where they are bound to MHC (major histocompatibility complex) molecules. In humans, professional antigen-presenting cells are specialized to 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 8-11 amino acids in length, while MHC class II molecules present longer peptides, 13-17 amino acids in length, and non-classical MHC molecules also present non-peptide epitopes such as glycolipids.

[0058] The terms "treat," "treated," "treating," and "treatment" as used with respect to methods herein refer to eliminating, reducing, inhibiting, or ameliorating, either temporarily or permanently, either partially or completely, one or more clinical symptoms, signs, or progression of an event, disease, or condition. Such treating need not be absolute to be useful. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to having the condition or disorder or those in whom the condition or disorder is to be prevented.

[0059] particle The size and charge of the particles are important for tolerance induction. Although particles vary in size and charge based on the antigen encapsulated within the particle, generally, the particles described herein are effective in inducing tolerance when they are about 100 nanometers to about 1500 nanometers and have a negative charge of 0 to about -100 mV. In various embodiments, the particles are 400 to 800 nanometers in diameter and have a charge of about -25 mV to -70 mV. In various embodiments, the particles are 400 to 1000 nanometers in diameter and have a charge of about -25 mV to -70 mV. The average particle size and charge of the particles may be slightly altered in the lyophilization process, and thus both post-synthesis average and post-lyophilization average are described. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to the size and charge of the particles before lyophilization. The terms "post-lyophilization size" and "post-lyophilization charge" refer to the size and charge of the particles after lyophilization.

[0060] In some embodiments, the particles are non-metallic. In these embodiments, the particles can be formed from polymers. In a preferred embodiment, the particles are biodegradable in the individual. In this embodiment, the particles can be provided in the individual over multiple doses without accumulation of the particles in the individual. Examples of suitable particles include polystyrene particles, PGA particles, PLA particles, PLGA particles, PLURONICS stabilized polypropylene sulfide particles, and diamond particles.

[0061] Preferably, the particle surface is composed of a material that minimizes non-specific or undesirable biological interactions. Interactions between the particle surface and the stroma may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material that prevents or reduces non-specific interactions. Steric stabilization by coating the particle with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS® (comprising copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), may reduce non-specific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All of these facts indicate the relevance of the particle's physical properties in terms of lymphatic uptake. Biodegradable polymers may be used to make all or part of the polymer and / or particle and / or layer. Biodegradable polymers may degrade, for example, as a result of functional groups reacting with water in solution. The term "degradation" as used herein refers to becoming soluble, either by a reduction in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers bearing ester groups, such as polylactides and polyglycolides, are generally subject to spontaneous hydrolysis.

[0062] The particles disclosed herein can also contain additional components. For example, the carrier can have a contrast agent incorporated or conjugated to the carrier. An example of a carrier nanosphere with a contrast agent that is currently commercially available is Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and adjustable size-dependent Stokes shift allow them to emit different sizes from blue to infrared when excited with a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0063] The particles can be formed from a wide range of materials. The particles are preferably composed of materials suitable for biological use. 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 cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Furthermore, the carrier particles can be or be composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Carrier particles containing mixtures of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, the carrier particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG; the terms are interchangeable), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, as well as copolymers thereof having linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acids.In addition, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, can be included in copolymers with any of the aforementioned materials to provide reactive groups and conjugate moieties for conjugation to antigenic peptides and proteins. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, acrylates, ethylene-vinyl acetate, non-biodegradable polymers of acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon can be utilized.

[0064] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0 or 20:80 to 100:0. Suitable copolymer ratios for the 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 certain embodiments, the particles are PLURONICS stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic-co-glycolic acid) particles, or carboxylated polyglycolic acid particles (PGA), carboxylated polylactic acid particles (PLA), or carboxylated poly(lactic-co-glycolic acid) particles. In certain embodiments, the particles have a copolymer ratio of polylactic acid / polyglycolic acid 80:20, polylactic acid / polyglycolic acid 90:10, or polylactic acid:polyglycolic acid / 50:50. In various embodiments, the particles are poly(lactic-co-glycolic acid) particles and have a copolymer ratio of polylactic acid:polyglycolic acid of about 50:50.

[0065] It is contemplated that the particles may further comprise a surfactant and / or stabilizer. The surfactant and / or stabilizer may be anionic, cationic, or nonionic. Poloxamer and poloxamine family surfactants are commonly used in particle synthesis. Surfactants and / or stabilizers that may be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, pluronic 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-tocopheryl 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 certain embodiments, two surfactants are used. In certain embodiments, two stabilizers are used. In certain embodiments, a combination of two or more surfactants and stabilizers is used.For example, when particles are produced by double emulsion method, the two surfactants can include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.For example, the stabilizer can be a compound that stabilizes the primary emulsion and / or the secondary emulsion described herein by providing a physical or energy barrier between adjacent nanoparticle droplets in the emulsion, thereby reducing the probability of combining and forming larger nanoparticle droplets.

[0066] In certain embodiments, the polypeptide antigen is encapsulated in the particles by a single emulsion process. In further embodiments, the polypeptide antigen is more hydrophobic. Sometimes, the double emulsion process leads to the formation of large particles that can lead to leakage of the hydrophilic active ingredient and low entrapment efficiency. Coalescence and Ostwald ripening are two mechanisms that can destabilize double emulsion droplets, while diffusion of the hydrophilic active ingredient through the organic phase is the main mechanism responsible for low levels of entrapped active ingredient. In some embodiments, it can be beneficial to reduce the nanoparticle size. One strategy to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by using high polymer concentration and high polymer molecular weight, with an increase in the viscosity of the internal aqueous phase and an increase in the surfactant molecular weight. In certain embodiments, the particles encapsulating the antigen are produced by nanoprecipitation, co-precipitation, inert gas condensation, sputtering, microemulsion, sol-gel method, layer-by-layer technique, or ionic gelation method. Several methods for producing nanoparticles have been described in the literature and are incorporated herein by reference (Sanchez et al., Molecules 25(16):3760, 2020; Zielinska et al. Molecules. 25(16):3731, 2020).

[0067] antigen An antigen refers to a discrete portion of a molecule, such as a polypeptide or peptide sequence, a 3D structural formation of a polypeptide or peptide, a polysaccharide or a polynucleotide that can be recognized by a host immune cell. Antigen specificity refers to the ability of a subject's host cells to recognize and generate an immune response against the antigen alone or against a molecule that closely resembles the antigen, such as an epitope or mimotope.

[0068] "Anergy", "tolerance", or "antigen-specific tolerance" refers to the insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen-specific and persists after exposure to antigenic peptides ceases. For example, anergy in T cells is characterized by a lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of costimulatory molecules) results in failure to produce cytokines and subsequent failure to proliferate. Thus, failure to produce cytokines prevents proliferation. Anergy T cells, however, can proliferate when cultured with cytokines (e.g., IL-2).

[0069] It is contemplated that the tolerization therapy described herein is antigen-specific.For example, the TIMP administered as a tolerization therapy encapsulates one or more antigens related to the tolerization therapy and the associated disease or condition to be treated.It is contemplated that the TIMP used in the tolerization therapy comprises one or more peanut antigens.The one or more peanut antigens can be derived from peanut protein extracts or can be peptides derived from known peanut proteins, for example, isolated from proteins or synthetically produced.

[0070] To date, more than 15 peanut allergens have been recognized by the WHO / IUIS Allergen Nomenclature Sub-Committee (database maintained at www.allergen.org), Ara h1 to Ara h18. Peanut allergens can be classified into different groups based on their architecture (e.g., trimer, monomer, cupin, albumin, prolamin, profilin, oleosin, defensin, vincilin, nonspecific lipid transfer protein (nsLTP)) based on Ara h1, h2, h3, h5, h6, and h8, each of which has a different degree of allergenic potency (Ozias-Akins et al., Allergy 74:888-898, 2019). Known peanut allergens include those from Arachis hypogaea Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, and Ara h8.For example, UNIPROT E5G076, showing the Ara h1 polypeptide sequence (SEQ ID NO:1), UNIPROT A0A445BYI5 for the Ara h2 polypeptide (SEQ ID NO:2), UNIPROT E5G077 for the Ara h3 polypeptide (SEQ ID NO:3) (see also UNIPROT database numbers O82580 (SEQ ID NO:4) and Q9SQH7 (SEQ ID NO:5) for Ara h3 isoallergens 1 and 2 (formerly Ara h4), respectively), UNIPROT L7QH52 for the Ara h5 polypeptide (SEQ ID NO:6), UNIPROT A5Z1R0 for the Ara h6 polypeptide (SEQ ID NO:7), UNIPROT B4XID4 for the Ara h7 polypeptide (SEQ ID NO:8), or UNIPROT Q6VT83 for the Ara h8 polypeptide sequence (SEQ ID NO:9), Ara h9, isoallergens 1 and 2, UNIPROT database numbers B6CEX8 and B6CG41, respectively (SEQ ID NOs:10 and 11); h10, isoallergen 1 and 2, UNIPROT database numbers Q647G5 and Q647G4, respectively (SEQ ID NOs: 12 and 13); Ara h11, isoallergen 1 and 2, UNIPROT database numbers Q45W87 and Q45W86, respectively (SEQ ID NOs: 14 and 15); Ara h12 UNIPROT database number B3EWP3 (SEQ ID NO: 16); Ara h13, isoallergen 1 and 2, UNIPROT database numbers B3EWP4 and C0HJZ1, respectively (SEQ ID NOs: 17 and 18); Ara h14, isoallergen 1, 2, and 3, UNIPROT database numbers Q9AXI1, Q9AXI0, and Q6J1J8, respectively (SEQ ID NOs: 19-21); Ara h15, UNIPROT database number Q647G3 (SEQ ID NO: 22); Ara h16, UNIPROT database number A0A509ZX51 (SEQ ID NO: 23); Ara See h17, UNIPROT A Database No. 0A510A9S3 (SEQ ID NO: 24); and Ara h18, UNIPROT Database No. A0A444XS96 (SEQ ID NO: 25).

[0071] In certain embodiments, one, two, three, or more antigens or antigenic peptides are used in TIMPs. In certain embodiments, one or more peanut antigens are encapsulated in TIMPs by covalent attachment to the inner surface of the particle (see, for example, US Patent Publication No. 2019 / 0282707, which is incorporated herein by reference). In certain embodiments, it is contemplated that the sequences of two or more peanut proteins, for example, from Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, and / or Ara h8, are combined in a fusion protein and encapsulated in the TIMPs described herein. Methods for making TIMPs with linked epitopes are described in US Patent Publication No. 2019 / 0365656, which is incorporated herein by reference.

[0072] Emulsions occur in many processed forms and are widely used by food, cosmetics, and drug delivery. Oil-water (single) or water-oil-water (double) emulsions are methods by which PLGA can be used to encapsulate hydrophobic and hydrophilic drugs in micro- or nanoscale forms. In summary, PLGA is dissolved in an organic phase (oil) that is emulsified with a surfactant or stabilizer (water). Hydrophobic drugs and / or other agents are added directly to the oil phase, while hydrophilic drugs and / or other agents (water) can be first emulsified with a polymer solution before the formation of particles. High intensity homogenization (e.g., sonication bursts) promotes the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours, which allows the solvent to evaporate. The hardened nanoparticles are collected and washed by centrifugation. In certain embodiments, hardened emulsion particles can be obtained by evaporation of the oil phase.

[0073] "Water-in-oil-in-water" (W / O / W) emulsions are an example of double emulsions, a dispersion of small water droplets within larger oil droplets that are themselves dispersed in a continuous aqueous phase. Due to their compartmentalized internal structure, double emulsions can offer advantages over simple oil-in-water emulsions for encapsulation, such as the ability to carry both polar and non-polar cargo (pharmaceutical / biological agents, e.g., proteins), as well as improved control over the release of therapeutic molecules. The preparation of double emulsions typically requires a surfactant or mixtures thereof for stability. Surfactants stabilize droplets subjected to extreme flows, leading to straightforward mass production of robust double nanoemulsions that are compatible with nanostructured encapsulation applications in various industries. In one example, the double emulsion process involves generating a primary emulsion by mixing an aqueous solution of the pharmaceutical / biological agent(s) with a solution containing a polymer, resulting in a water-in-oil primary emulsion. The primary emulsion is then mixed with a solution containing one or more surfactants to form an oil-in-water secondary emulsion. The secondary emulsion is then hardened by evaporation to remove the solvent(s), resulting in hardened polymeric nanoparticles that encapsulate the pharmaceutical / biological agent(s).

[0074] "Homogenization" as used herein refers to the operation of using a class of processing equipment called homogenizers, which are adapted to reduce the size of droplets in liquid-liquid dispersions. Factors that affect particle or droplet size include, but are not limited to, emulsifier type, emulsifier concentration, solution conditions, and mechanical devices (homogenization power, pressure, rotation speed, time). Non-limiting examples of homogenizers include high speed blenders, high pressure homogenizers, colloid mills, high shear dispersers, ultrasonic disruptor membrane homogenizers, and sonicators. Mechanical homogenizers, manual homogenizers, ultrasonic disintegrators, mixer mills, vortexers, etc. may be utilized for mechanical and physical disruption within the scope of the present disclosure.

[0075] "Batch size" as used herein refers to the scale of production according to the weight of the final product. The manufacturing process can be modified, scaled up or scaled down. The manufacturing process can be modified, scaled up or scaled down by changing the amount or volume of a solvent, antigen / protein, polymer, surfactant, stabilizer, cryoprotectant or excipient. The manufacturing process can be scaled up or scaled down by changing the time of homogenization, sonication, evaporation, filtration, concentration, washing or lyophilization.

[0076] Methods for determining protein content in particles or in solution include ELISA, mass spectrometry, HPLC, CBQCA, and Western blot.

[0077] The Molecular Probes CBQCA Protein Quantitation Kit provides a rapid and sensitive method for the quantitation of proteins in solution. The kit utilizes the ATTO-TAG CBQCA reagent (3-(4-carboxybenzoyl)quinoline-2-carboxyaldehyde), which was 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. The ATTO-TAG CBQCA reagent is virtually non-fluorescent in aqueous solution, but in the presence of cyanide, it reacts with primary amines, such as those found in proteins, to form highly fluorescent derivatives.

[0078] Pharmaceutical preparations The pharmaceutical composition of the present disclosure containing the TIMP-PPE described herein as an active ingredient may contain a pharma- ceutically acceptable carrier or additive depending on the route of administration. Examples of such carriers or additives include water, pharma- ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacryl, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharma- ceutically acceptable surfactants, etc. The additives used are selected from the above or combinations thereof as necessary depending on the dosage form of the present disclosure, but are not limited thereto.

[0079] The formulation of the pharmaceutical composition will vary according to the route of administration (e.g., solution, emulsion) selected. The appropriate composition containing the therapeutic agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient, or electrolyte replenishers.

[0080] Various aqueous carriers, e.g., sterile phosphate buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, may contain other proteins for enhanced stability, such as albumin, lipoproteins, globulins, and the like, that have been subjected to minor chemical modifications, and the like.

[0081] Therapeutic formulations of the inhibitor are prepared for storage by mixing the inhibitor having the desired purity, in the form of a lyophilized formulation or aqueous solution, with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, zeolite, sorbitol ... 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 dextrins; 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 non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0082] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0083] Aqueous suspensions may contain the active compound mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyl-enoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene monostearate sorbitol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as polyethylene sorbitan monostearate. Aqueous suspensions may also contain one or more preservatives, such as ethyl, or n-propyl, p-hydroxybenzoate.

[0084] The TIMP-PPEs described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0085] 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, pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium laurate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0086] Formulations are provided that include particles that include peanut protein extract and excipients. Exemplary excipients include sucrose, mannitol, trehalose, sorbitol, dextran, Ficoll, dextran 70k, sodium citrate, lactose, L-arginine, or glycine. In various embodiments, the TIMP-PPE formulation contains 1-11 excipients. In various embodiments, the TIMP-PPE formulation contains 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more excipients.

[0087] In some embodiments, the TIMP-PPE formulation contains negatively charged particles encapsulating purified peanut protein, sucrose, mannitol, and sodium citrate. In various embodiments, the concentration of negatively charged particles in the TIMP-PPE formulation is 1-100%, 20-50%, or 30-40%, including all ranges and values ​​therebetween. In various embodiments, the concentration of negatively charged particles in the TIMP-PPE 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%.

[0088] In various embodiments, the sucrose concentration in the TIMP-PPE formulation is 1-100%, 20-50%, or 30-40%, including all ranges and values ​​therebetween, hi various embodiments, the sucrose concentration in the TIMP-PPE 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%.

[0089] In various embodiments, the mannitol concentration in the TIMP-PPE formulation is 1-100%, 15-35%, or 20-30%, including all ranges and values ​​therebetween, In various embodiments, the sucrose concentration in the TIMP-PPE 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%.

[0090] In various embodiments, the sodium citrate concentration is 0.01-25% or 0.5-3.5%, including all ranges and values ​​therebetween, 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%.

[0091] In various embodiments, the purified peanut protein in the TIMP-PPE formulation is from 0.3 μg to 30 μg (micrograms) of peanut protein per milligram (mg) of PLGA, or from 1 μg to 10 μg of peanut protein per mg of PLGA, including all ranges and values ​​therebetween. In various embodiments, the purified peanut protein in the TIMP-PPE formulation is about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, or about 10 μg of peanut protein per mg of PLGA.

[0092] How to use Provided herein is a method of treating peanut allergy in a subject, comprising administering TIMP-PPE to the subject, wherein the TIMP-PPE is administered at a dose of 0.001-12 mg / kg or 0.1-12 mg / kg. Also provided herein is a method of reducing an allergic immune response to a peanut antigen in a subject suffering from peanut allergy, comprising administering TIMP-PPE to the subject, wherein the TIMP-PPE is administered at a dose of 0.001-12 mg / kg or 0.1-12 mg / kg.

[0093] It is also contemplated that the TIMP-PPE is administered at a dose of about 0.001-10 mg / kg, about 0.005-12 mg / kg, about 0.01-12 mg / kg, about 0.05-12 mg / kg, about 0.1-12 mg / kg, about 0.5-10 mg / kg, about 1-8 mg / kg, about 1.5-10 mg / kg, about 2-12 mg / kg, about 2-10 mg / kg, about 3-10 mg / kg, about 4-10 mg / kg, about 4-12 mg / kg, or about 5-12 mg / kg. Optionally, the TIMP-PPE is administered at a dose of about 0.001 mg / kg, 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. Alternatively, the TIMP-PPE is administered in a dose of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In another embodiment, the TIMP-PPE is administered at a concentration of about 0.0005 mg / mL to about 50 mg / mL or about 0.05 mg / mL to about 50 mg / mL, optionally about 0.0005 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 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, 12.5 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.

[0094] It is contemplated that TIMP-PPE is administered in a single dose or multiple doses.In various embodiments, TIMP-PPE is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.In certain embodiments, TIMP-PPE is administered in two doses, one week apart.

[0095] In various embodiments, the TIMP-PPE is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. It is contemplated that when the TIMP-PPE is given intravenously, it may be via an intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, or 20 hours.

[0096] It is further contemplated that TIMP-PPE may be administered alone or in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents include, but are not limited to, inhibitors of IgE, inhibitors of basophil activation, inhibitors of mast cell activation, antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), or small molecule or biological therapeutic agents.

[0097] Administration of TIMP-PPE, alone or in combination with one or more additional therapeutic agents, to a subject in need thereof provides relief from one or more symptoms of peanut allergy, including skin reactions, hives, skin redness, skin swelling, itching, throat tightness, difficulty breathing, shortness of breath, and anaphylaxis.

[0098] It is also contemplated that administering TIMP-PPE, alone or in combination with one or more additional therapeutic agents, to a subject in need thereof reduces the duration and severity of an allergic immune response to or after exposure to peanut protein. Allergic immune responses contemplated herein include Th2 T cell responses, B cell activation, basophil activation, eosinophil activation, mast cell activation, and / or IgE induction.

[0099] kit In an additional aspect, the present disclosure includes a kit that includes one or more compounds or compositions packaged in a manner that facilitates their use to practice the methods of the present disclosure. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition that includes a TIMP alone or in combination with a second agent) packaged in a container such as a sealed bottle or vessel, with a label attached to the container or included in the package that describes the use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a suitable device for administering the composition according to a specific route of administration or for performing a screening assay. Preferably, the kit includes a label that describes the use of the inhibitor composition.

[0100] In a further embodiment, the disclosure provides an article of manufacture or unit dose form comprising: (a) a composition comprising a TIMP-PPE as described herein; (b) a container comprising the composition; and (c) a label affixed to, or package insert included with, the container regarding use of the TIMP-PPE in treating peanut allergy as described herein.

[0101] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. EXAMPLES

[0102] Example 1. Process for preparing purified peanut extract for the manufacture of tolerizing nanoparticles encapsulating peanut proteins (TIMP-PPE) One process for PPE manufacturing utilizes raw peanuts (Arachis hypogaea), which can be procured from commercial vendors. Briefly, the process begins with grinding raw peanuts into a fine paste. The ground peanuts are then defatted via acetone extraction. The defatted peanut material is air-dried for 1-5 days and then heat-dried for 12-24 hours. The dried material is powdered by forcing it through a sieve with a mesh of 2.0 mm or less. Proteins are then extracted from the powdered peanut raw material with ammonium bicarbonate, separated by centrifugation, and clarified by 1 μm filtration. The final solution is concentrated / dialyzed using hollow fiber cartridges, recentrifuged, and clarified.

[0103] Prior to use in the production of TIMP-PPE, the peanut protein extract was characterized to confirm the presence of antigenic peanut proteins (e.g., Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18) by SDS-PAGE and total protein content by CBQCA. The results of the SDS-PAGE assay are shown in Figure 1. The protein content in the peanut protein extract was determined to be 0.73 mg / mL.

[0104] Example 2. Process for producing tolerizing nanoparticles encapsulating peanut proteins (TIMP-PPE) TIMP-PPE (CNP-201) was manufactured using a double emulsion solvent evaporation process. A high-level manufacturing process flow diagram is shown in Figure 2. Briefly, purified peanut extract dissolved in 1 M acetic acid (10 mg / mL) was rapidly mixed with a 5% PLGA solution (50:50; molecular weight of 10,000-60,000 Da) in ethyl acetate to generate a primary water-in-oil emulsion. The primary emulsion was then rapidly mixed with a surfactant and stabilizer solution containing 4% PVA and PAA (Sigma Aldrich, 100 KDa, 35 wt%) dissolved in ethyl acetate to form an oil-in-water secondary emulsion. The composition of the PVA / PAA / ethyl acetate blend is maintained at a pH below 4.0. Mixing of the primary and secondary emulsions was performed by homogenization.

[0105] The solvent was removed from the secondary emulsion by evaporation under pressure for a total of at least 3-4 hours. The hardened nanoparticles were then washed with sterile water and concentrated by filtration using a 20 μm filter. The cryoprotectants sucrose and mannitol, and the buffer sodium citrate dihydrate were added to the hardened nanoparticles. The formulation was then lyophilized.

[0106] The final CNP-201 formulation was characterized to determine physicochemical properties such as particle diameter, zeta potential, total protein content, presence of Ara h protein, burst release, and presence of protein on the surface of the particles. The results of CNP-201 characterization produced in 80 g or 160 g batch sizes are provided in Tables 1 and 2. CNP-201 particles were examined by scanning electron microscopy and show a homogenous composition of intact particles with smooth surfaces produced in the 80 g batch size (Figure 3) and 160 g batch sizes (Figure 4). [Table 1] [Table 2]

[0107] Example 3. Determining the frequency of TIMP-PPE particles (CNP-201) with surface-present proteins One vial of CNP-201 was reconstituted in water to form a stable suspension. Similarly, one vial of control particles containing no other proteins or peptides (negative control) and one vial of control particles with known high surface protein levels (positive control) were reconstituted in water. The particles were mixed to ensure that they were uniformly dispersed in the suspension.

[0108] CNP-201, positive control, and negative control particles were incubated with staining buffer (bovine serum albumin in PBS) containing a primary polyclonal anti-peanut protein antibody. A separate set of particles was incubated with staining buffer alone to serve as an internal negative control for the experiment. The stained particles were washed three times in staining buffer by centrifugation and incubated with staining buffer containing a fluorophore-conjugated secondary antibody. The particles were then washed three times in staining buffer.

[0109] The stained particles were acquired on a flow cytometer and the frequency of CNP-201 particles that stained positive for peanut proteins was compared to negative and positive control samples. Results are expressed as the percentage of particles positive for surface proteins compared to the negative control (Tables 1 and 2).

[0110] Example 4. Pharmaceutical Compositions of TIMP-PPE Exemplary pharmaceutical formulations or compositions of TIMP-PPE include the individual components listed in Table 3. [Table 3]

[0111] It is understood that all embodiments of the present disclosure described herein may be optionally combined with any one or more of the other embodiments described herein. All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0112] It is to be understood, therefore, that the present invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which come within the spirit and scope of the present invention as defined by the appended claims, the above description, and / or shown in the accompanying drawings. Consequently, only such limitations as appear in the appended claims should be placed on this disclosure.

[0113] References 1.Cannon HE.The economic impact of peanut allergies.Am J Manag Care.2018;24(19 Suppl):S428-s433.

[0114] 2.Sampath V, Nadeau KC.Newly identified T cell subsets in mechanistic studies of food immunotherapy.J Clin Invest.2019;129(4):1431-1440.

[0115] 3.Feuille E,Nowak-Wegrzyn A.Allergen-Specific Immunotherapies for Food Allergy.Allergy Asthma Immunol Res.2018;10(3):189-206.

[0116] 4. Chinthrajah RS, Purington N, Andorf S, et al. Sustained outcomes in oral immunotherapy for peanut allergy(POISED study): a large, randomised, double-blind, placebo-controlled, phase 2 study. Lancet.2019;394(10207):1437-1449.

[0117] 5.Fleischer DM,Greenhawt M,Sussman G,et al.Effect of Epicutaneous Immunotherapy vs Placebo on Reaction to Peanut Protein Ingestion Among Children With Peanut Allergy:The PEPITES Randomized Clinical Trial.Jama.2019;321(10):946-955.

[0118] 6.Vickery BP,Vereda A,Casale TB,et al.AR101 Oral Immunotherapy for Peanut Allergy.N Engl J Med.2018;379(21):1991-2001.

[0119] 7.Dunlop JH.Oral immunotherapy for treatment of peanut allergy.J Investig Med.2020;68(6):1152-1155.

[0120] 8.Getts DR,Martin AJ,McCarthy DP,et al.Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis.Nature Biotechnology.2012;30(12):1217-1224.

[0121] 9.Hunter Z,McCarthy DP,Yap WT,et al.A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease.ACS Nano.2014;8(3):2148-2160.

[0122] 10.Kelly CP,Murray JA,Leffler DA,et al.TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac Disease:A Randomized,Double-Blind,Placebo-Controlled Study.Gastroenterology.2021;161(1):66-80.e68.

[0123] 11.Prasad S,Neef T,Xu D,et al.Tolerogenic Ag-PLG nanoparticles induce tregs to suppress activated diabetogenic CD4 and CD8 T cells.J Autoimmun.2018;89:112-124.

[0124] 12.Smarr CB,Yap WT,Neef TP,et al.Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated allergic airway inflammation pre-and postsensitization.Proc Natl Acad Sci U S A.2016;113(18):5059-5064.

[0125] 13.Keet CA,Johnson K,Savage JH,Hamilton RG,Wood RA.Evaluation of Ara h2 IgE thresholds in the diagnosis of peanut allergy in a clinical population.J Allergy Clin Immunol Pract.2013;1(1):101-103.

[0126] 14.Ozias-Akins P,Breiteneder H.The functional biology of peanut allergens and possible links to their allergenicity.Allergy.2019;74(5):888-898

Claims

1. 1. A method for preparing a composition comprising particles encapsulating peanut proteins, the method comprising: a. forming a primary emulsion by mixing an aqueous solution of peanut protein with a solution containing a polymer to provide a primary emulsion; b. mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers to form a secondary emulsion; c. hardening the secondary emulsion by evaporation to remove the solvent, resulting in hardened polymeric nanoparticles encapsulating peanut protein within their cores; d. filtering, washing, and concentrating the nanoparticles; e. freeze-drying the nanoparticles to form a composition.

2. 10. The method of claim 1, wherein the solution in step (a) comprises a solvent or the solution in step (b) comprises a solvent.

3. 10. The method of claim 1, wherein the solutions in step (a) and step (b) comprise the same solvent, or the solutions in step (a) and step (b) comprise different solvents, optionally wherein the solvents in step (a) and step (b) are organic or inorganic solvents.

4. 4. The method of claim 3, wherein the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, acetic acid, or a mixture thereof.

5. 2. The method of claim 1, wherein the emulsion obtained from step (a) is a water-in-oil emulsion and / or the emulsion obtained from step (b) is an oil-in-water emulsion.

6. 10. The method of claim 1, wherein the polymer in step (a) is a biodegradable polymer and / or the surfactant or stabilizer in step (b) is anionic, cationic, or nonionic.

7. the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(lactic-co-glycolic) (PLGA), poly(lactic-co-sebacic) acid (PLSA), poly(glycol-co-sebacic) acid (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharides, or lipids, and / or optionally the surfactant and / or stabilizer is poloxamer, polyamine, PEG, Tween-80, gelatin, dextran, pluronic L-63, pluronic F-68, pluronic 188, pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, vitamin E 7. The method of claim 6, wherein the surfactant is TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or enantiomers thereof), methylcellulose, hydroxyethylcellulose, hydroxyprolylcellulose, hydroxypropylmethylcellulose, gelatin, sodium cholate, carbomer, or sulfate polymer.

8. 2. The method of claim 1, wherein the primary emulsion of step (a) is obtained by homogenization or sonication and / or the secondary emulsion of step (b) is obtained by homogenization or sonication, optionally wherein the homogenization or sonication is performed for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

9. 10. The method of claim 1, wherein the pH of the secondary emulsion in step (b) is about pH 4 or less.

10. 2. The method of claim 1, wherein the hardening of the nanoparticles in step (c) is carried out by i) active evaporation of the solvent, optionally wherein the active evaporation is vacuum-driven evaporation, or ii) passive evaporation of the solvent, optionally wherein the passive evaporation is carried out by stirring.

11. 10. The method of claim 1, wherein the particles have a negative zeta potential, optionally the zeta potential is between about 0 and −100 mV, or between about −30 mV and −80 mV.

12. 10. The method of claim 1, wherein the particles have a diameter of about 0.3 μm to 3 μm, or about 0.3 μm to 1 μm, or about 0.4 μm to 1 μm.

13. The method of claim 1, wherein i) at least 90% of the particles have a diameter of about 0.3 μm to 3 μm, or about 0.3 μm to 1 μm, or about 0.4 μm to 1 μm, ii) at least 50% of the particles have a diameter of about 0.3 μm to 3 μm, or about 0.3 μm to 1 μm, or about 0.4 μm to 1 μm, or iii) at least 10% of the particles have a diameter of about 0.3 μm to 3 μm, or about 0.3 μm to 1 μm.

14. 10. The method of claim 1, wherein at least 50% of the particles have a diameter of about 0.3 μm to 3 μm, or have a diameter of about 0.3 μm to 1 μm, or have a diameter of about 0.4 μm to 1 μm.

15. 2. The method of claim 1, wherein the peanut protein content encapsulated in the particulate composition is about 0.1 to 100 μg / mg.

16. 2. The method of claim 1, wherein the peanut protein comprises an Ara h protein, optionally wherein the Ara h protein is Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18.

17. 17. The method of claim 16, wherein the particles encapsulate peanut peptides, optionally the peptides are purified from natural peanut proteins or synthetically produced, and optionally the peptides comprise allergenic epitopes from Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, and Ara h 18.

18. 10. The method of claim 1, wherein the peanut protein is dissolved in an acid, and optionally the pH of the peanut protein solution is from 1.0 to 6.0, or from 1.0 to 4.

0.

19. A particle encapsulating peanut protein made by the method of any one of claims 1 to 18.

20. 19. A composition comprising particles encapsulating peanut protein made by the method of any one of claims 1 to 18, and optionally further comprising a pharmaceutically acceptable carrier, diluent, or excipient, optionally wherein the excipient is sucrose, mannitol, and sodium citrate.

21. A pharmaceutical composition comprising negatively charged particles encapsulating peanut protein, sucrose, mannitol, and sodium citrate.

22. A pharmaceutical composition comprising the particles described in claim 19.

23. The composition of claim 20 for treating a subject with a peanut allergy.

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