Tolerance-inducing immunomodulatory nanoparticles for the treatment of myasthenia gravis

Abstract

Currently, there is no cure for myasthenia gravis (MG), and standard treatment focuses on symptom relief with the use of steroids or immunosuppressants, which have only temporary effects and are associated with serious side effects such as an increased risk of infection and death. [Solution] This application generally relates to compositions comprising tolerance-immunomodified particles encapsulating myasthenia gravis (MG)-related antigens, methods for treating MG using tolerance-immunomodified nanoparticles encapsulating MG-related antigens, and processes for preparing tolerance-immunomodified nanoparticles encapsulating MG antigens.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 492,165, filed on 24 March 2024, which is incorporated in its entirety by reference herein.

[0002] This application generally relates to tolerant immunomodulatory particles (TIMP-MG) containing antigens associated with myasthenia gravis, methods for treating myasthenia gravis using TIMP-MG, and methods for manufacturing TIMP-MG. [Background technology]

[0003] Myasthenia gravis (MG) is a rare autoimmune disorder of the neuromuscular junction characterized by fluctuating muscle weakness and abnormal fatigability. 1 .

[0004] Myelomyelitis (MG) is a T-cell-driven autoimmune disease primarily directed at acetylcholine (AChR) receptors. Activated T cells drive the production of AChR-specific T-cell-mediated B-cell antibodies (Abs) that prevent AChR from binding to acetylcholine. 2 The combined activation of T cells and B cells leads to IgG Ab and complement deposition at the neuromuscular junction, causing impaired neuromuscular transmission. Rare forms of MG are attributed to autoantibodies targeting muscle-specific kinase (MuSK), lipoprotein receptor-associated protein (LRP4), agrin, cortactin, titin, and / or ryanodine. 3 .

[0005] The progression of the disease can lead to life-threatening complications, including myasthenic crisis, thymic tumors, thyroid disorders, and / or other autoimmune conditions.

[0006] The global prevalence of myasthenia gravis (MG) is between 40 and 180 cases per million people. 4 The estimated annual incidence rate is 1.74 to 12 cases per million people. 4 .

[0007] Currently, there is no cure for MG, and standard treatment focuses on symptom relief with the use of steroids or immunosuppressants, which only have a temporary effect and are associated with serious side effects such as an increased risk of infection and death. [Overview of the Initiative]

[0008] Tolerant immunomodulatory 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 / 1319253 and WO2015 / 023796, incorporated herein by reference). Encapsulation of one or more MG-related antigens within the TIMP core is advantageous because it ensures the safe and effective delivery of the encapsulated protein to the APC without inducing immune activation. In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy in inducing T-cell tolerance. Induction of antigen-specific tolerance to MG autoantigens using TIMPs encapsulating MG-related antigens (TIMP-MG) may potentially cure MG.

[0009] This disclosure describes a TIMP-MG composition, a method for treating MG in a subject, comprising administering TIMP-MG to the subject, and a method for manufacturing TIMP-MG for the treatment of MG.

[0010] Provided herein are TIMP-MG compositions comprising negatively charged TIMP-MG particles for encapsulating an antigen, wherein the antigen comprises one or more MG-related antigens and / or a portion thereof, or a combination of antigens or a portion thereof. In various embodiments, the TIMP-MG particles comprise a polymer and have a negative zeta potential. In various embodiments, the polymer is a biodegradable polymer.

[0011] In various embodiments, TIMP-MG particles contain polymers selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(lactic acid-co-glycol) (PLGA), poly(lactic acid-co-sebacic) acid (PLSA), poly(glycol-co-sebacic) acid (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharides, or lipids, polystyrene, diamond, liposomes, PEG, cyclodextran, lipids, or metals, such as iron (Fe), zinc (Zn), cadmium (Cd), gold, or silver, or combinations thereof.

[0012] In various embodiments, the polymer is a copolymer. In various embodiments, the copolymer has constituent polymers in various molar ratios. In various embodiments, the molar ratios are approximately 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, and include all values ​​and ranges between these values.

[0013] In various embodiments, TIMP-MG particles contain poly(lactic acid-coglycolic acid) (PLGA). In various embodiments, the particles contain polylactic acid:polyglycolic acid in a ratio of about 50:50, about 80:20 to about 100:0, or polyglycolic acid:polylactic acid in a ratio of about 50:50, about 80:20 to about 100:0. In various embodiments, the particles contain 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles contain polylactic acid:polyglycolic acid in a ratio of approximately 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 (including all values ​​and ranges that fall between these values).

[0014] In various embodiments, TIMP-MG particles have a negative zeta potential. In various embodiments, the zeta potential of the particles is approximately -100mV to approximately 0mV, approximately -100mV to approximately -25mV, approximately -100mV to approximately -30mV, approximately -80mV to approximately -30mV, approximately -75mV to approximately -30mV, approximately -70mV to approximately -30mV, approximately -75mV to approximately -35mV, approximately -70mV to approximately -25mV, approximately -60mV to approximately -30mV, approximately -60mV to approximately -35mV, or approximately -50mV to approximately -30mV. In various embodiments, the zeta potential is approximately -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV (including all values ​​and ranges within these). In various embodiments, TIMP-MG particles have a negative zeta potential of -30mV to -80mV. In various embodiments, TIMP-MG particles have a negative zeta potential of -30mV to -60mV. In various embodiments, the negative zeta potential is achieved by surface functionalization of the TIMP-MG particles. In various embodiments, the surface functionalization is carboxylation.

[0015] In various embodiments, the size, or diameter, of the TIMP-MG particles is 0.05 μm to about 10 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 10 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 5 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 3 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.3 μm to about 5 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.3 μm to about 3 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.3 μm to about 1 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.4 μm to about 1 μm. In various embodiments, the TIMP-MG particles have diameters of approximately 100-10000 nm, 100-5000 nm, 100-3000 nm, 100-2000 nm, 100-1500 nm, 300-5000 nm, 300-3000 nm, 300-1000 nm, 300-800 nm, 400-800 nm, or 200-700 nm. In various embodiments, TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges within these). In various embodiments, the diameter of negatively charged particles is 400 nm to 800 nm. In various embodiments, the diameter of negatively charged particles is 350 nm to 800 nm.

[0016] In various embodiments, the TIMP-MG particles have a homogeneous size distribution. In various embodiments, the TIMP-MG particles have a homogeneous size distribution, and at least 90% of the particles have diameters of 0.05 μm to about 10 μm, 0.1 μm to about 10 μm, 0.1 μm to about 5 μm, 0.1 μm to about 3 μm, 0.3 μm to about 5 μm, and 0.3 μm to about 3 μm (including all values ​​and ranges within these). In various embodiments, the TIMP-MG particles have a homogeneous size distribution, with at least 90% of the particles having a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm (including all values ​​and ranges within these ranges). In various embodiments, TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges within these). In various embodiments, TIMP-MG particles have a homogeneous size distribution, with at least 50% of the particles having diameters of approximately 0.05 μm to approximately 10 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm to approximately 3 μm, approximately 0.3 μm to approximately 5 μm, and approximately 0.3 μm to approximately 3 μm (including all values ​​and ranges within these). In various embodiments, the particles have a homogeneous size distribution, and at least 50% of the particles have a diameter of about 100-10000 nm, about 100-5000 nm, about 100-3000 nm, about 100-2000 nm, about 300-5000 nm, about 300-3000 nm, about 300-1000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm (including all values ​​and ranges within these).In various embodiments, TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges within these). In various embodiments, TIMP-MG particles have a homogeneous size distribution, with at least 10% of the particles having diameters of approximately 0.05 μm to approximately 10 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm to approximately 3 μm, approximately 0.3 μm to approximately 5 μm, and approximately 0.3 μm to approximately 3 μm (including all values ​​and ranges within these). In various embodiments, the TIMP-MG particles have a homogeneous size distribution, with at least 10% of the particles having a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm (including all values ​​and ranges within these ranges). In various embodiments, TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges within these).

[0017] In various embodiments, TIMP-MG particles encapsulate one or more MG-related antigens, have a negative zeta potential of -100mV to 0mV, and have a diameter of 100 to 1000nm. In various embodiments, TIMP-MG particles encapsulate one or more MG-related antigens, a portion thereof, or a combination thereof, have a particle size of 400 to 800nm, and have a negative zeta potential of -30mV to -80mV.

[0018] In various embodiments, TIMP-MG particles encapsulate one or more MG-related antigens, parts thereof, or combinations thereof. In various embodiments, the antigen comprises one or more proteins, peptides, antigenic epitopes, or combinations thereof. In various embodiments, TIMP-MG particles encapsulate one or more polynucleotides encoding MG-related antigens. In some embodiments, the polynucleotide comprises DNA, RNA, messenger RNA (mRNA), or circular RNA.

[0019] In various embodiments, MG-related antigens include acetylcholine receptor (AChR) protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), parts thereof, and / or one or more antigenic epitopes thereof. In various embodiments, one or more MG-related antigens are selected from the list in Table 1. In various embodiments, the antigen is an AChR protein subunit and / or a part thereof. In various embodiments, the antigen is an AChR antigen (Table 2) and / or a part thereof selected from the group consisting of SEQ ID NOs: 1 to 21, or one or more antigenic epitopes thereof. In various embodiments, one or more antigens encapsulated within a particle include combinations of SEQ ID NOs: 3, 5, 8, 13, 19, 20, and / or 21 listed in Table 2.

[0020] In various embodiments, TIMP-MG particles encapsulate one or more MG-related antigens, have a negative zeta potential of -100mV to 0mV, and have a diameter of 100 to 1000nm. In various embodiments, TIMP-MG particles encapsulate antigens selected from the group listed in Table 1, have a particle size of 400 to 800nm, and have a negative zeta potential of -30mV to -80mV. In various embodiments, TIMP-MG particles encapsulate antigens (Table 2), parts thereof, or combinations thereof selected from the group consisting of SEQ ID NOs: 1 to 21, have a particle size of 400 to 800nm, and have a negative zeta potential of -30mV to -80mV. In various embodiments, the TIMP-MG particles encapsulate one or more MG-related antigens (Table 2), including SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and / or SEQ ID NO: 21, a portion thereof, or a combination thereof, the particle size being 400-800 nm, and the TIMP-MG particles having a negative zeta potential of -30 mV to -80 mV.

[0021] A method for treating MG in a subject is provided herein, comprising administering TIMP-MG to the subject, wherein TIMP-MG is administered in a dose of 0.001 mg / kg to 12 mg / kg. In various embodiments, TIMP-MG is administered in a dose determined based on the weight of the subject. In various embodiments, TIMP-MG is administered in a fixed dose of 0.1 mg to 800 mg. In various embodiments, TIMP-MG is administered in doses of approximately 0.001 to 10 mg / kg, approximately 0.005 to 12 mg / kg, approximately 0.01 to 12 mg / kg, approximately 0.05 to 12 mg / kg, approximately 0.1 to 12 mg / kg, approximately 0.5 to 10 mg / kg, approximately 1 mg / kg to 8 mg / kg, approximately 1.5 to 10 mg / kg, approximately 2 mg / kg to 12 mg / kg, approximately 2 mg / kg to 10 mg / kg, approximately 3 mg / kg to 10 mg / kg, approximately 4 to 10 mg / kg, approximately 4 to 12 mg / kg, or approximately 5 to 12 mg / kg. In various embodiments, TIMP-MG is administered in doses of approximately 0.001 mg / kg, approximately 0.0025 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.025 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 0.25 mg / kg, approximately 0.5 mg / kg, approximately 1.0 mg / kg, approximately 2.0 mg / kg, approximately 3.0 mg / kg, approximately 4.0 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 8.0 mg / kg, approximately 10 mg / kg, or approximately 12 mg / kg. In various embodiments, TIMP-MG is administered in a fixed dose of 0.1 mg to 800 mg. The Specified Provision also provides a method for reducing the immune response to MG-related antigens in subjects suffering from MG, comprising administering TIMP-MG to the subjects, wherein TIMP-MG is administered in a fixed dose of 0.1 mg to 800 mg.In various embodiments, TIMP-MG is administered in fixed doses of approximately 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.

[0022] In various embodiments, TIMP-MG is administered in single or multiple doses. In various embodiments, TIMP-MG is administered in two doses at one-week intervals. In various embodiments, TIMP-MG 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 four months, once every five months, once every six months, or once a year.

[0023] In various embodiments, the TIMP-MG booster dose is administered as a single dose or multiple doses after the loading phase of TIMP-MG administration. In various embodiments, the booster dose 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 four months, once every five months, once every six months, or once per year. In various embodiments, TIMP-MG is administered in two doses one week apart, followed by a booster dose administered as a single dose every three months.

[0024] In various embodiments, the booster dose of TIMP-MG is administered at a dose of 0.001 mg / kg to 12 mg / kg. In various embodiments, the booster dose of TIMP-MG is about 0.001 to 10 mg / kg, about 0.005 to 12 mg / kg, about 0.01 to 12 mg / kg, about 0.05 to 12 mg / kg, about 0.1 to 12 mg / kg, about 0.5 to 10 mg / kg, about 1 to 8 mg / kg, about 1.5 to 10 mg / kg, about 2 to 12 mg / kg, about 2 to 10 mg / kg, about 3 to 10 mg / kg, about 4 to 10 mg / kg, about 4 to 12 mg / kg, or about 5 to 12 mg / kg, or at a dose of 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. In various embodiments, the booster dose of TIMP-MG is administered at 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.

[0025] In various embodiments, TIMP-MG is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

[0026] In various embodiments, the Disclosure provides a method for treating MG or MG-related symptoms in a subject, comprising administering to the subject a composition comprising TIMP-MG alone or in combination with a therapeutic agent. In various embodiments, the therapeutic agent is a cholinesterase inhibitor, steroid, corticosteroid, nonsteroidal immunosuppressant, immunomodulator, therapeutic plasma exchange (plasma replacement), intravenous immunoglobulin preparation (IVIG), chloride ion channel inhibitor, monoclonal antibody, proteasome inhibitor, cytokine and chemokine targeted therapy, microRNA inhibitor, siRNA, JAK inhibitor, BTK inhibitor, complement inhibitor, IgG degrader, chimeric antigen receptor (CAR) T-cell therapy, regulatory T-cell (Treg) therapy, hematopoietic stem cell transplantation, B-cell targeted therapy, or surgical treatment. In various embodiments, the treatment includes azathioprine, sirolimus, edrophonium, neostigmine, pyridostigmine, cyclosporine, mycophenolate mofetil, calcineurin inhibitors, levamisole, chlorambucil, cyclophosphamide, tacrolimus, methotrexate, eculizumab, ravulizumab, efgaltidimod alfa, rozanolixizumab, zircoplan, and CD20 targeted therapy. Therapies are selected from the group including CD19-targeted therapy, CD40 / CD40L-targeted therapy, B-cell targeting factor (BAFF)-targeted therapy, B-cell maturation antigen (BCMA)-targeted therapy, anti-IL6 therapy, anti-IFN therapy, anti-timocytoglobulin, factor D inhibitors, amifampridine, batoclimab, inbilizumab, nipokalimab, pozelimab, rituximab, satralizumab, tocilizumab, tofacitin, and trebulimin. In various embodiments, the steroid or corticosteroid is selected from the group including beclomethasone, cyclusonide, fluticasone floritol, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, oxymetazoline hydrochloride, or hydrocortisone. In various embodiments, the surgical treatment is thymectomy. In various embodiments, the therapeutic agent is administered before, simultaneously with, or after the administration of TIMP-MG.In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days before administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks before administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks before administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks after administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-MG.

[0027] In various embodiments, TIMP-MG is administered via an intravenous infusion that lasts about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours (including all values within this range).

[0028] In various embodiments, TIMP-MG consists of poly(coglycolic acid lactate) (PLGA) particles encapsulating one or more MG antigens and a suitable buffer or excipient. In various embodiments, TIMP-MG particles are surface-functionalized. In various embodiments, TIMP-MG particles are surface-functionalized by carboxylation. In various embodiments, TIMP-MG particles have a negative zeta potential. In various embodiments, the negative zeta potential of TIMP-MG particles is approximately -100mV to approximately 0mV. In various embodiments, the zeta potential of the particles is approximately -100mV to approximately -25mV, approximately -100 to approximately -30mV, approximately -80mV to approximately -30mV, approximately -75mV to approximately -30mV, approximately -70mV to approximately -30mV, approximately -75 to approximately -35mV, approximately -70 to approximately -25mV, approximately -60mV to approximately -30mV, approximately -60mV to approximately -35mV, or approximately -50mV to approximately -30mV. In various embodiments, the zeta potential is approximately -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV.

[0029] In various embodiments, TIMP-MG is administered at concentrations ranging from approximately 0.0005 mg / mL to approximately 50 mg / mL. In various embodiments, TIMP-MG is administered at concentrations of approximately 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, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. In various embodiments, TIMP-MG is administered via intravenous infusion lasting approximately 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0030] In various embodiments, administering TIMP-MG to a subject in need improves or alleviates one or more symptoms of MG. In various embodiments, the symptoms of MG are selected from the group consisting of drooping eyelids (ptosis), diplopia (double vision), difficulty making facial expressions, chewing problems, dysphagia, dysarthria (slurred speech), shortness of breath, dyspnea, muscle fatigue, skeletal weakness, neck weakness, limb weakness, dysphagia, dysphonia, neuromuscular junction weakness, synaptic signaling disorders, muscle damage, anti-Musk antibodies, anti-AChR antibodies, increased activated CD4+ cells compared to healthy subjects, increased activated CD8+ T cells compared to healthy subjects, increased Myasthenia Gravis Activities of Daily Living score, quantitative MG score, MG Quality of Life 15 revised score, and increased MG composite score.

[0031] In various embodiments, administering TIMP-MG to subjects in need improves or reduces the duration and severity of the inflammatory immune response to one or more MG antigens. In various embodiments, the inflammatory immune response is a T-cell response, a B-cell response, a Th1 / Th17 response, a myeloid cell response, a complement response, and / or an antibody response. In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to one or more MG antigens is determined by assays of one or more biological samples from the subject. In various embodiments, the biological samples are selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

[0032] Compositions comprising TIMP-MG as described herein for use in the treatment of myasthenia gravis are also intended. In various embodiments, this disclosure provides the use of compositions comprising TIMP-MG as described herein in the preparation of pharmaceuticals for the treatment of myasthenia gravis.

[0033] The process for manufacturing TIMP-MG involves numerous steps, each of which affects the physicochemical properties of the resulting composition, which are essential for safe and therapeutic administration. Importantly, the process must be optimized to ensure efficient encapsulation of the MG antigen within the particle core.

[0034] In various embodiments, TIMP-MG encapsulates one or more MG-related antigens or antigenic epitopes. In various embodiments, the antigen is selected from Table 1 and is an AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-associated protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or a portion thereof, or one or more antigenic epitopes thereof. In various embodiments, the antigen is an AChR protein subunit and / or a portion thereof. In various embodiments, the antigen is an AChR antigen (Table 2) and / or a portion thereof selected from the group consisting of SEQ ID NOs: 1 to 21, or one or more antigenic epitopes thereof. In various embodiments, TIMP-MG encapsulates an antigen (Table 2) including a combination of SEQ ID NOs: 3, 5, 8, 13, 19, 20, and / or 21.

[0035] This disclosure provides a process for producing a composition comprising negatively charged particles (TIMP-MG) encapsulating one or more MG-related antigens. The process relates to a process for producing particles optimized for the safe and therapeutic administration of TIMP-MG for the treatment of MG. In various embodiments, the method involves (a) generating an aqueous solution of one or more MG-related antigens, (b) producing a primary emulsion by mixing an aqueous solution of one or more MG-related antigens with an oil phase containing a polymer; (c) forming a secondary emulsion by mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers; and (d) curing the secondary emulsion by evaporation to obtain cured polymer nanoparticles that encapsulate MG-related antigens within a core.

[0036] In various embodiments, the method further comprises step (e) filtering, washing, and concentrating the nanoparticles. In various embodiments, the method further comprises step (e) filtering, washing, and concentrating the nanoparticles and (f) freeze-drying the nanoparticles. In various embodiments, the primary emulsion of step (b) is a water-in-oil emulsion. In various embodiments, the secondary emulsion of step (c) is an oil-in-water emulsion.

[0037] In various embodiments, one or more MG-related antigens in step (a) are dissolved in an aqueous solution containing a solvent. In various embodiments, the solvent includes an organic solvent. In various embodiments, the solvent includes an inorganic solvent. In various embodiments, one or more MG-related antigens are dissolved in a solvent containing 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 approximately 1.0, approximately 1.5, approximately 2.0, approximately 2.5, approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, approximately 9.0, approximately 9.5, approximately 10.0, approximately 10.5, approximately 11.0, approximately 11.5, approximately 12.0, approximately 12.5, approximately 13.0, approximately 13.5, or approximately 14.0, and includes all values ​​within this range.

[0038] In various embodiments, the acid-containing solvent is selected from the group including 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, hexafluorophosphate, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, and carbonic acid. In various embodiments, the solvent containing the base is selected from the group including 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, iron hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide. In various embodiments, the acid concentration is 0.1N to 36N. In various embodiments, the acid solvent concentration is approximately 0.1 N, approximately 0.5 N, approximately 1 N, approximately 2 N, approximately 3 N, approximately 4 N, approximately 5 N, approximately 6 N, approximately 7 N, approximately 8 N, approximately 9 N, approximately 10 N, approximately 11 N, approximately 12 N, approximately 13 N, approximately 14 N, approximately 15 N, approximately 16 N, approximately 17 N, approximately 18 N, approximately 20 N, approximately 30 N, or approximately 36 N, and includes all values ​​within this range. In various embodiments, the concentration of the base solvent is approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 10%, approximately 25%, approximately 50%, approximately 75%, and approximately 100% (v / v or wt / v), and includes all values ​​within this range.

[0039] In various embodiments, one or more MG-related antigens of step (a) are dissolved in an aqueous solution containing one or more stabilizers. In various embodiments, the stabilizers include detergents / surfactants, osmolites, metal complexes, proteins, or amino acids. In various embodiments, the stabilizers include polyvinyl alcohol, sorbitan monostearate, Triton X, Triton X-100, poloxamer, polyvinylpyrrolidone, Pluronics F68, n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG), polyethylene glycol 400 decyl ether (Thesit), nonylphenyl polyethylene glycol (NP40), and polyoxyethylene-(10)-dodecyl ether (Genapol C-100), dodecylphosphocholine (DPC), n-decyl-β-maltoside (DM), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC6PC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-octyl-β-D-glucose (B-OG), Brih-35, Brij-56, Brij-58, Brij-72, Brij-78, Brij-97, Brij-98, 3-((3-colamidopropyl)dimethylammonium)-1-propanesulfate The following are selected from the group containing fonate (CHAPS), disulfyl-containing detergents, fluorinated diglucose detergents, fluorinated maltose detergents, maltoside amphiphilic substances with 1,3,5-triazine as the core, steroid pentasaccharides, vitamin E glycoside amphiphilic substances, calixarene detergents, cyclodextrin, trehalose, mannitol, dextran, carboxymethylcellulose, ethyl stearate, sodium acetate, sodium glutamate, polysorbate, albumin, bovine serum albumin, lysine, histidine, arginine, zinc salts, sodium bicarbonate, or magnesium hydroxide.In various embodiments, the concentration of the stabilizer in step (a) is 0.01% to 15% (for example, about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, or 15%), and includes all values ​​within this range.

[0040] In various embodiments, the concentration of one or more MG-related antigens dissolved in step (a) is 0.1 mg / mL to 100 mg / mL. In various embodiments, the concentration of one or more MG-related antigens dissolved in step (a) is approximately 0.1 mg / mL, approximately 0.2 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12 mg / mL, approximately 13 mg / mL, approximately 14 mg / mL, Approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 75 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 95 mg / mL, or approximately 100 mg / mL, and including all values ​​within this range.

[0041] In various embodiments, one or more MG-related antigens are approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 48, approximately 72, or approximately 96, and include all values ​​within this range.

[0042] In various embodiments, one or more MG-related antigens are produced synthetically. In various embodiments, the antigens are produced by solid-phase peptide synthesis or solution-phase peptide synthesis. In various embodiments, one or more MG-related antigens are produced using recombinant protein production.

[0043] In various embodiments, one or more MG-related antigens in step (a) are dissolved in an aqueous solution containing a solvent. In various embodiments, one or more MG-related antigens in step (a) are dissolved in the same solvent. In various embodiments, one or more MG-related antigens in step (a) are dissolved in different solvents. One or more MG-related antigens in step (a) are dissolved in a solvent additionally containing one or more stabilizers. Depending on their solubility and / or stability in acids or bases, one or more antigens contained in TIMP-MG are dissolved in an acid, and other antigens are dissolved in a base. In various embodiments, the antigens dissolved in the acid and the antigens dissolved in the base are mixed before, during, or after the formation of the primary emulsion in step (b). In various embodiments, the antigens are mixed in ratios of 100:1, 50:1, 25:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 (including all values ​​within this range). In some embodiments, MG-related antigens (Table 2), including SEQ ID NOs. 3, 8, 13, 19, 20, and 21, are dissolved in acid. In some embodiments, MG-related antigens (Table 2), including SEQ ID NOs. 5, are dissolved in base. In some embodiments, acid-soluble MG-related antigens (Table 2), including SEQ ID NOs. 3, 8, 13, 19, 20, and 21, are mixed with base-soluble MG-related antigens (Table 2), including SEQ ID NOs. 5, in a 4:1 ratio.

[0044] In various embodiments, the MG-related antigen is the AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or parts thereof, and / or one or more antigenic epitopes thereof. In various embodiments, the MG-related antigen is the AChR protein subunit and / or a part thereof, or one or more antigenic epitopes thereof. In various embodiments, the MG-related antigen is the AChR antigen and / or a part thereof from Table 1, or one or more antigenic epitopes thereof. In various embodiments, the MG-related antigen is the AChR antigen and / or a part thereof selected from the group consisting of the AChR antigens of SEQ ID NOs. 1 to 21 (Table 2), or one or more antigenic epitopes thereof. In various embodiments, the MG-related antigen includes an antigen (Table 2) comprising a combination of SEQ ID NOs. 3, 5, 8, 13, 19, 20, and / or 21.

[0045] In various embodiments, the emulsion in step (b) contains 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 solvents are acetone, ethanol, methylene chloride (dichloromethane), dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is selected from the group including water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in step (b) has a concentration of 1% (v / v) to 50% (v / v), and includes all values ​​within this range. In various embodiments, the solvent in step (b) is at a concentration of about 1%, about 2%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (v / v). In various embodiments, the solvent in step (b) is between 0.1 mM and 10.0 mM, and includes all values ​​within this range. In various embodiments, the solvent in step (b) is concentrated at concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, or 10.0 mM. In various embodiments, the solvent in step (b) is concentrated at concentrations between 0.1 M and 10.0 M, encompassing all values ​​within this range.

[0046] In various embodiments, the surfactant and / or stabilizer solution of step (c) contains 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 solvents are acetone, ethanol, methylene chloride (dichloromethane), dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is selected from the group including water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in the mixture is at a concentration of 1% to 50% (v / v), including all values ​​within this range. In various embodiments, the solvent in the mixture 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 mixture is concentrated from 0.1 mM to 10.0 mM, encompassing all values ​​within this range. In various embodiments, the solvent in the mixture has concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, or 10.0 mM. In various embodiments, the solvent in the mixture has concentrations from 0.1 M to 10.0 M, encompassing all values ​​within this range. In various embodiments, the solvent in the mixture is concentrated at concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M, 5 M, 5.5 M, 6 M, 6.5 M, 7.0 M, 7.5 M, 8.0 M, 8.5 M, 9.0 M, or 10.0 M. In various embodiments, the solvent in step (b) and step (c) is the same. In various embodiments, the solvents in step (b) and step (c) are different.

[0047] In various embodiments, the polymer in step (b) is a biodegradable polymer. In various embodiments, the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(milk-co-glycol) (PLGA), poly(milk-co-sebacic) acid (PLSA), poly(glycol-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 constituent polymers in various molar ratios. In various embodiments, the molar ratios are 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, and include all values ​​within this range.

[0048] In various embodiments, the polymer in step (b) is PLGA. In various embodiments, the molar ratio of the PLGA copolymer is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0, and includes all values ​​within this range. In various embodiments, PLGA has a high molecular weight. In various embodiments, PLGA has a low molecular weight. In various embodiments, PLGA has a molecular weight of 1 kDa to 100 kDa (e.g., 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa (including all values ​​within this range)). In various embodiments, the amount of PLGA in the solution of step (b) 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)). In various embodiments, the polymer of step (b) is dissolved in a solvent. In various embodiments, the polymer of step (b) is dissolved in a solvent selected from the group including acetone, ethyl formate, methylene chloride (dichloromethane), ethyl acetate, dimethylformamide, tetrahydrofuran, or chloroform. In various embodiments, the PLGA polymer of step (b) is dissolved in ethyl acetate. In various embodiments, step (b) includes a 5% PLGA solution (50:50) having a molecular weight of 10,000 to 60,000 Daltons (Da).

[0049] In various embodiments, the surfactant and / or stabilizer used in step (c) is anionic, cationic, or nonionic. In various embodiments, the surfactant and / or stabilizer is poloxamer, polyamine, 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-alto-maleic acid) (PEMA), vitamin E Selected from 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., hepulsulfate, chondroitin sulfate, fucoidan, urvan, and carrageenan). In various embodiments, the amount of surfactant and / or stabilizer present in step (c) is 0.05 to 100% by weight or volume (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)). In various embodiments, the surfactant and / or stabilizer has a molecular weight of 0.1 to 10,000 kDa (e.g., 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)).In various embodiments, the surfactant and stabilizer mixture is prepared by mixing 4% PVA and PAA (100 kDa, 35% wt) in water with ethyl acetate.

[0050] In various embodiments, the mixture comprising one or more surfactants and / or stabilizers forming the oil-in-water secondary emulsion of (c) has a pH less than 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, and about pH 4, and includes all ranges and values ​​that exist between these ranges.

[0051] In various embodiments, the method involves (a) generating an aqueous solution of one or more MG-related antigens, (b) producing a primary emulsion by mixing an aqueous solution of one or more MG-related antigens with an oil phase containing a polymer; (c) forming a secondary emulsion by mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers; (d) curing the secondary emulsion by evaporation to obtain cured polymer nanoparticles encapsulating MG-related antigens in a core; (e) filtering, washing, and concentrating the nanoparticles; and (f) freeze-drying the nanoparticles.

[0052] In various embodiments, the water-in-oil primary emulsion of step (b) is obtained by homogenizing an aqueous solution of one or more MG-related antigens with an oil phase containing a polymer. In various embodiments, the homogenization of step (b) is carried out for 5 to 1000 seconds, including all values ​​within this range. In various embodiments, the homogenization is carried out for 5, 10, 15, 20, 25, 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 (c) is obtained by homogenizing the primary emulsion with a solution containing one or more surfactants and / or stabilizers. In various embodiments, the homogenization in step (c) is performed for 5 to 1000 seconds (including all values ​​within this range). In various embodiments, the 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 in step (b) is obtained by sonication of an aqueous solution of MG-related antigen with an oil phase containing a polymer. In various embodiments, the sonication in step (b) is performed for 5 to 1000 seconds, including all values ​​within this range. In various embodiments, the sonication in step (b) 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 in step (c) is obtained by sonication of the primary emulsion with a solution containing one or more surfactants and / or stabilizers. In various embodiments, the sonication in step (c) is performed for 5 to 1000 seconds, including all values ​​within this range.In various embodiments, the ultrasonic treatment in step (c) 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.

[0053] In various embodiments, the secondary emulsion is cured by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the active evaporation is carried out using stirring or under vacuum. In various embodiments, the active evaporation is carried out under high-pressure vacuum. In various embodiments, the active evaporation is carried out under low-pressure vacuum. In various embodiments, the evaporation is passive evaporation. In various embodiments, the evaporation is carried out for 0.25 to 96 hours, including all values ​​within this range. In various embodiments, the 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 carried out at a pressure of 0.01 to 1000 mBar (for example, 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).

[0054] In various embodiments, the filtration, washing, and concentration of the particles in step (e) are carried out by gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, or a combination thereof.

[0055] In various embodiments, the present disclosure further envisions a method for producing a composition comprising negatively charged particles encapsulating a combination of MG-related antigens, including SEQ ID NOs: 3, 5, 8, 13, 19, 20, and 21, as listed in Table 2, comprising: (a) dissolving the peptides of SEQ ID NOs: 3, 8, 13, 19, 20, and 21 in acetic acid, dissolving the peptide of SEQ ID NOs: 5 in ammonium hydroxide, and subsequently mixing the acid-soluble peptides with the base-soluble peptides in a 4:1 ratio; (b) homogenizing the aqueous solution of (a) with an oil phase containing PLGA dissolved in ethyl acetate to produce a primary emulsion; (c) homogenizing the primary emulsion with a mixture containing ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; and (d) curing the secondary emulsion by evaporation to obtain cured polymer nanoparticles encapsulating the peptides of SEQ ID NOs: 3, 5, 8, 13, 19, 20, and 21 within a core.

[0056] In various embodiments, the method further comprises (e) filtering, washing, and concentrating the nanoparticles. In various embodiments, the method further comprises (e) filtering, washing, and concentrating the nanoparticles and (f) adding sodium citrate, mannitol, and sucrose, and freeze-drying the particles.

[0057] In various embodiments, the Disclosure also relates to a method for producing a composition comprising negatively charged particles encapsulating a combination of MG-related antigens, including SEQ ID NOs: 3, 5, 8, 13, 19, 20, and 21, as listed in Table 2, comprising: (a) dissolving the peptides of SEQ ID NOs: 3, 8, 13, 19, 20, and 21 in acetic acid, dissolving the peptide of SEQ ID NOs: 5 in ammonium hydroxide, and subsequently mixing the acid-soluble peptides with the base-soluble peptides in a 4:1 ratio; and (b) homogenizing the aqueous solution of (a) with an oil phase containing PLGA dissolved in ethyl acetate. The method also envisions a method comprising: (c) generating a secondary emulsion; (d) homogenizing the primary emulsion with a mixture containing ethyl acetate, polyacrylic acid (PAA), and polyvinyl alcohol (PVA) to form a secondary emulsion; (e) curing the secondary emulsion by evaporation to obtain cured polymer nanoparticles encapsulating the peptides of SEQ ID NOs. 3, 5, 8, 13, 19, 20, and 21 within the core; (f) filtering, washing, and concentrating the nanoparticles; and (g) adding sodium citrate, mannitol, and sucrose, and freeze-drying the particles.

[0058] In various embodiments, the acetic acid concentration in step (a) is 0.1N to 1N, encompassing all values ​​within this range. In various embodiments, the acetic acid concentration in step (a) is 0.5N.

[0059] In various embodiments, the ammonium hydroxide concentration in step (b) is between 0.01% and 1%, encompassing all values ​​within this range. In various embodiments, the ammonium hydroxide concentration in step (b) is 0.1%.

[0060] In various embodiments, the method includes a 5% PLGA solution (50:50) having a molecular weight of 10,000 to 60,000 Da in step (b).

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

[0062] The disclosure also relates to a process for producing a composition comprising negatively charged TIMP (TIMP-MG) encapsulating one or more MG-related antigens, wherein the process intends the TIMP-MG particles to have a negative zeta potential. In various embodiments, the negative zeta potential of the TIMP-MG particles produced by the process is about -100mV to about 0mV. In various embodiments, the zeta potential of the particles is about -100mV to about -25mV, about -100 to about -30mV, about -80mV to about -30mV, about -75mV to about -30mV, about -70mV to about -30mV, about -75 to about -35mV, about -70 to about -35mV, about -70 to about -25mV, about -60mV to about -30mV, about -60mV to about -35mV, or about -50mV to about -30mV. In various embodiments, the zeta potential is approximately -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV.

[0063] This disclosure describes a method for producing a composition comprising negatively charged TIMP (TIMP-MG) encapsulating one or more MG-related antigens, wherein the process envisions a particle size or diameter of 0.05 μm to about 10 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 10 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 5 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.1 μm to about 3 μm. In various embodiments, the diameter of the TIMP-MG particles is 0.3 μm to about 5 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.3 μm to about 3 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.3 μm to about 1 μm. In various embodiments, the diameter of the TIMP-MG particles is about 0.4 μm to about 1 μm. In various embodiments, TIMP-MG particles have diameters of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the diameter of the negatively charged particles is 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).

[0064] Furthermore, a process is envisioned for producing a composition comprising negatively charged TIMP (TIMP-MG) encapsulating one or more MG-related antigens, wherein 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 0.05 μm to about 10 μm, 0.1 μm to about 10 μm, 0.1 μm to about 5 μm, 0.1 μm to about 3 μm, 0.3 μm to about 2 μm, 0.3 μm to about 5 μm, 0.3 μm to about 3 μm, 0.3 μm to about 1 μm, or 0.4 μm to about 1 μm. In various embodiments, the particles have a homogeneous size distribution, and at least 90% of the particles have a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, the TIMP-MG particles have a diameter of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the particles have a homogeneous size distribution, with at least 50% of the particles having diameters of approximately 0.05 μm to 10 μm, 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 3 μm, 0.3 μm to 2 μm, 0.3 μm to 5 μm, and 0.3 μm to 3 μm, 0.3 μm to 1 μm, or 0.4 μm to 1 μm. In various embodiments, the particles have a homogeneous size distribution, and at least 50% of the particles have a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm.In various embodiments, the TIMP-MG particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the particles have a homogeneous size distribution, with at least 10% of the particles having diameters of approximately 0.05 μm to approximately 10 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm to approximately 3 μm, approximately 0.3 μm to approximately 2 μm, approximately 0.3 μm to approximately 5 μm, and approximately 0.3 μm to approximately 3 μm, 0.3 μm to approximately 1 μm, or 0.4 μm to approximately 1 μm. In various embodiments, the particles have a homogeneous size distribution, and at least 10% of the particles have a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, the TIMP-MG particles have a diameter of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In the embodiment, negatively charged particles have a D90 of less than about 1000 nm. In the embodiment, negatively charged particles have a D50 of about 400 nm to about 800 nm, including about 400 nm, about 420 nm, about 440 nm, about 460 nm, about 480 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm (including all values ​​and ranges in between). In the embodiment, negatively charged particles have a D10 of less than about 600 nm. In the embodiment, negatively charged particles have a D90 of about 600 nm to about 700 nm. In the embodiment, the negatively charged particles have a D50 of approximately 550 nm to approximately 600 nm. In the embodiment, the negatively charged particles have a D10 of approximately 500 nm to approximately 550 nm.In various embodiments, the negatively charged particles have a D[4,3] to less than 1000 nm.

[0065] In various embodiments, the invention of the present disclosure provides a process for producing a composition comprising negatively paired particles (TIMP-MG) encapsulating one or more MG-related antigens. In various embodiments, the amount of one or more MG-related antigens encapsulated in the TIMP-MG composition is between 0.1 μg / mg and 100 μg / mg of polymer, and includes all values ​​within this range. In various embodiments, the MG-related antigen content is between 0.1 μg / mg and 100 μg / mg of PLGA (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), and includes all values ​​and ranges between these values. In various embodiments, the process for preparing TIMP-MG described herein results in an inclusion efficiency of 1 to 100% (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​and ranges between these values). The content of one or more MG-related antigens in the TIMP-MG composition can be determined by methods described in the literature, including ELISA, mass spectrometry, HPLC, CBQCA, and Western blotting.

[0066] In various embodiments, the Disclosure provides a process for producing a composition comprising negatively charged particles (TIMP-MG) encapsulating one or more MG-related antigens, wherein the particle surface contains low levels of one or more MG-related antigens. In various embodiments, the particle surface is essentially free of one or more MG-related antigens. In various embodiments, the amount of one or more MG-related antigens present on the particle surface is 0 to 30% of the total antigen content of the TIMP-MG composition (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (including all values ​​and ranges between these values)). In various embodiments, the frequency of particles containing one or more MG-related antigens on their surface 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 between these values)) higher than the negative control. In various embodiments, the frequency of particles containing one or more MG-related antigens on their surface is 0–100% (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)) lower than the positive control. In various embodiments, the amount of one or more MG-related antigens on the surface of the particles is 0 to 10 times higher (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (including all values ​​and ranges between these values)) than that of the negative control. In various embodiments, the amount of one or more MG-related antigens is 0 to 1 / 100th (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 1 / 100th (including all values ​​and ranges between these values)) than that of the positive control. In various embodiments, the number of TIMP-MG particles having one or more MG-related antigens is determined using the aforementioned methods such as flow cytometry, mass spectrometry, ELISA, CBQCA, and Western blotting.

[0067] In various embodiments, the disclosure provides a process for producing a composition comprising negatively charged particles (TIMP-MG) encapsulating one or more MG-related antigens, wherein the particles exhibit low burst release. In various embodiments, the particles do not exhibit burst release. In various embodiments, the particle burst release is 0 to 85% (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 75, 80, or 85% (including all values ​​and ranges between these values)).

[0068] In various embodiments, the excipient is added to the nanoparticle composition before freeze-drying in step (f). In various embodiments, the excipient is a buffer and / or cryoprotectant. In various embodiments, the excipient is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, dextran, Ficoll, dextran 70k, sodium citrate, lactose, glycol, glycerol, cyclodextrin, L-arginine, or glycine. In various embodiments, the amount of excipient added to the nanoparticle composition before freeze-drying is 0.05 to 100% by weight or volume (for example, all values ​​within this range 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%). In various embodiments, the amount of excipient added to the nanoparticle composition before freeze-drying is 0.01 to 500 g per gram of nanoparticles (for example, 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).

[0069] In various embodiments, the manufacturing batch size of TIMP-MG can be increased or decreased. In various embodiments, the manufacturing batch size is 0.01g to 100kg. In various embodiments, the batch size is 0.01g, 0.1g, 10g, 20g, 40g, 60g, 80g, 100g, 160g, 240g, 320g, 400g, 480g, 560g, 640g, 720g, 800g, 1000g, 5kg, 10kg, 50kg, or 100kg, and includes all values ​​and ranges between these values.

[0070] Furthermore, compositions comprising particles encapsulating MG-related antigens produced by the methods described herein are also provided. In various embodiments, the compositions further comprise pharmaceutically acceptable carriers, diluents, or excipients. In various embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0071] In various embodiments, the TIMP-MG formulation or pharmaceutical composition contains negatively charged particles encapsulating MG-related antigens and excipients. In various embodiments, the excipients are selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, dextran, Ficoll, dextran 70k, sodium citrate, lactose, L-arginine, or glycine. In various embodiments, the TIMP-MG formulation contains 1 to 11 excipients. In various embodiments, the TIMP-MG 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.

[0072] In various embodiments, the TIMP-MG formulation contains negatively charged particles encapsulating MG-related antigen, sucrose, mannitol, and sodium citrate. In various embodiments, the concentration of negatively charged particles in the TIMP-MG formulation is 1 to 100%, encompassing all ranges and values ​​between these ranges. In various embodiments, the concentration of negatively charged particles in the TIMP-MG formulation is 20 to 50%, encompassing all ranges and values ​​between these ranges. In various embodiments, the concentration of negatively charged particles in the TIMP-MG formulation is 30 to 40%, encompassing all ranges and values ​​between these ranges. In various embodiments, the concentration of negatively charged particles in the TIMP-MG formulation is approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 35.6%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, or approximately 40%.

[0073] In various embodiments, the sucrose concentration in the TIMP-MG formulation is 1 to 100%, encompassing all ranges and values ​​between these ranges. In various embodiments, the sucrose concentration in the TIMP-MG formulation is 20 to 50%, encompassing all ranges and values ​​between these ranges. In various embodiments, the sucrose concentration in the TIMP-MG formulation is 30 to 40%, encompassing all ranges and values ​​between these ranges. In various embodiments, the sucrose concentration in the TIMP-MG formulation is approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 35.6%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, or approximately 40%.

[0074] In various embodiments, the mannitol concentration in the TIMP-MG formulation is 1 to 100%, encompassing all ranges and values ​​between these ranges. In various embodiments, the mannitol concentration in the TIMP-MG formulation is 15 to 35%, encompassing all ranges and values ​​between these ranges. In various embodiments, the mannitol concentration in the TIMP-MG formulation is 20 to 30%, encompassing all ranges and values ​​between these ranges. In various embodiments, the mannitol concentration in the TIMP-MG formulation is approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 25%, approximately 26%, approximately 26.7%, approximately 27%, approximately 28%, approximately 29%, or approximately 30%.

[0075] In various embodiments, the sodium citrate concentration is 0.01 to 25%, encompassing all ranges and values ​​between these ranges. In various embodiments, the sodium citrate concentration is 0.5% to 3.5%, encompassing all ranges and values ​​between these ranges. In various embodiments, the sodium citrate concentration is about 0.5%, about 1%, about 1.5%, about 2%, about 2.1%, about 2.5%, about 3%, or about 3.5%.

[0076] In various embodiments, the total MG-related antigen content in the TIMP-MG formulation ranges from 0.1 μg to 200 μg per mg of PLGA, encompassing all ranges and values ​​between these limits. In various embodiments, the total MG-related antigen or individual MG-related antigen in the TIMP-MG formulation ranges from 2.1 μg to 15.7 μg per mg of PLGA, encompassing all ranges and values ​​between these limits. In various embodiments, the individual MG-related antigen in the TIMP-MG formulation ranges from 0.01 μg to 50 μg per mg of PLGA, encompassing all ranges and values ​​between these limits. In various embodiments, the individual MG-related antigen in the TIMP-MG formulation ranges from 0.1 μg to 5 μg per mg of PLGA, encompassing all ranges and values ​​between these limits. In various embodiments, the individual MG-related antigens in the TIMP-MG formulation are approximately 0.5 μg of antigen per 1 mg of PLGA, approximately 0.7 μg of antigen per 1 mg of PLGA, approximately 1 μg of antigen per 1 mg of PLGA, approximately 1.2 μg of antigen per 1 mg of PLGA, approximately 1.4 μg of MG-related antigen per 1 mg of PLGA, or approximately 1.8 μg of MG-related antigen per 1 mg of PLGA.

[0077] In various embodiments, the AChRα isoform α (324-357) (SEQ ID NO: 3) antigen in the TIMP-MG formulation is 0.05 μg / mg PLGA to 25 μg / mg PLGA. In various embodiments, the AChRε (116-130) (SEQ ID NO: 5) antigen in the TIMP-MG formulation is 0.03 μg / mg PLGA to 23 μg / mg PLGA. In various embodiments, the AChRε (414-435) (SEQ ID NO: 8) antigen in the TIMP-MG formulation is 0.01 μg / mg PLGA to 17 μg / mg PLGA. In various embodiments, the AChRα (43-58) (SEQ ID NO: 13) antigen in the TIMP-MG formulation is 0.02 μg / mg PLGA to 22 μg / mg PLGA. In various embodiments, the AChRα(68~113) (SEQ ID NO: 19) antigen in the TIMP-MG formulation is 0.06 μg / mg PLGA to 26 μg / mg PLGA. In various embodiments, the AChRα(121~158) (SEQ ID NO: 20) antigen in the TIMP-MG formulation is 0.01 μg / mg PLGA to 21 μg / mg PLGA. In various embodiments, the AChRε(201~250) (SEQ ID NO: 21) antigen in the TIMP-MG formulation is 0.03 μg / mg PLGA to 23 μg / mg PLGA.

[0078] In various embodiments, the AChRα isoform α (324-357) (SEQ ID NO: 3) antigen in the TIMP-MG formulation is 0.5 μg / mg PLGA to 2.5 μg / mg PLGA. In various embodiments, the AChRε (116-130) (SEQ ID NO: 5) antigen in the TIMP-MG formulation is 0.3 μg / mg PLGA to 2.3 μg / mg PLGA. In various embodiments, the AChRε (414-435) (SEQ ID NO: 8) antigen in the TIMP-MG formulation is 0.1 μg / mg PLGA to 1.7 μg / mg PLGA. In various embodiments, the AChRα (43-58) (SEQ ID NO: 13) antigen in the TIMP-MG formulation is 0.2 μg / mg PLGA to 2.2 μg / mg PLGA. In various embodiments, the AChRα(68-113) (SEQ ID NO: 19) antigen in the TIMP-MG formulation is 0.6 μg / mg PLGA to 2.6 μg / mg PLGA. In various embodiments, the AChRα(121-158) (SEQ ID NO: 20) antigen in the TIMP-MG formulation is 0.1 μg / mg PLGA to 2.1 μg / mg PLGA. In various embodiments, the AChRε(201-250) (SEQ ID NO: 21) antigen in the TIMP-MG formulation is 0.3 μg / mg PLGA to 2.3 μg / mg PLGA.

[0079] This disclosure provides a method for treating MG in a subject, comprising administering to the subject particles encapsulating the MG-related antigen described herein. Compositions comprising TIMP-MG described herein for use in treating MG are also contemplated. In various embodiments, this disclosure provides the use of compositions comprising TIMP-MG described herein in the preparation of pharmaceuticals for treating MG.

[0080] Each feature or embodiment or combination described herein is a non-limiting, exemplary example of any aspect of the Invention, and is understood to mean that it can be combined with any other feature or embodiment or combination described herein as such. For example, where a feature is described in terms such as “one embodiment,” “several embodiments,” “a particular embodiment,” “further embodiment,” “a particular exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any other feature or combination of features described herein, and it is not necessary to enumerate all possible combinations. Such features or combinations of features are applicable to any aspect of the Invention. Where examples of values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are assumed.

[0081] The headings in this specification are for the convenience of the reader and are not intended to limit the scope of the invention. Additional aspects, embodiments, and variations of the invention will be apparent from the detailed description and / or the drawings and / or claims. [Brief explanation of the drawing]

[0082] [Figure 1A-1D]CNP-106 inhibits antigen-specific T cell responses in animal models. The efficacy of TIMP-MG (CNP-106) in inducing tolerance was tested in a mouse model for treating MG. TIMP-MG consists of negatively charged PLGA particles with a size and diameter of 400 nm to 800 nm and a zeta potential of -30 to -80 mV, encapsulating AChR antigens with SEQ ID NOs. 3, 5, 8, 13, 19, 20, and 21 selected from Table 2. CNP-106 treatment (1.25 mg / dose) significantly improved grip strength compared to the saline-treated group (Figure 1A). CNP-106-treated mice also showed a trend toward improvement in overall clinical scores (Figure 1B) and electromyography (EMG) scores (Figure 1C) compared to immunized saline-controlled mice, with no change in body weight between the different treatment groups (Figure 1D). (*p<0.05, ***p<0.0005) [Figure 2A-2C] CNP-106 treatment inhibits the AChR-specific T cell response. The AChR-specific T cell response was evaluated via the exvivoricol response from the spleen. Treatment of mice immunized with 1.25 mg / dose of CNP-106 reduced levels of IL-2 (Figure 2A), IL-10 (Figure 2B), and IFN-gamma (Figure 2C). (*p<0.05) [Figure 3]Delayed-type hypersensitivity. C57BL / 6 mice were primed on day 0 with 100 μg (200 μL injection) of AChR-peptide emulsified in CFA. On days 0 and 7 after priming, mice were treated with CNP-106 administered intravenously at doses of 0.1 mg / house (0.4 mg / kg HED), 0.5 mg / mouse (2 mg / kg HED), 1.25 mg / mouse (5 mg / kg HED), and 2.5 mg / mouse (10 mg / kg HED). On day 14 after priming, mice were challenged intradermally with 10 μg of MG peptide (right ear, 10 μL injection) or ovalbumin (left ear, 10 μL injection). The thickness of the auricle of each ear was measured immediately after induction and 24 hours after induction using MG peptide and OVA. The DTH response was evaluated by calculating the change in auricle thickness (ΔT) in both ears of each mouse. CNP-106 significantly inhibited the DTH response compared to unloaded control particles at doses of 0.5 mg / mouse (2 mg / kg HED), 1.25 mg / mouse (5 mg / kg HED), and 2.5 mg / mouse (10 mg / kg HED) (Figure 3). (****p<0.00005) [Figure 4] Event schedule for Phase IA / IIB MG clinical trial. The primary objective of this study is to evaluate the safety and tolerability of CNP-106. Participants will be evaluated for safety, tolerability, disease progression (PD), and efficacy according to the event schedule (Figure 4). During post-administration and follow-up periods, participants will return to the clinic for immunosafety lab visits, PD measurements, QMG and MGC evaluations, adverse event (AE) evaluations, and medication changes for each event schedule (Figure 4). [Figure 5]Patient selection criteria for CNP-106 treatment. PBMCs were obtained from 10 MG patients to determine if there was a correlation between HLA-positive restrictions for AChR. Since CNP-106 encapsulates AChR antigens, the presence of HLA-DRB1*03, HLA-DRB3*01, and HLA-DQB1*02 in subjects can be a criterion for inclusion in the CNP-106 clinical trial. 80% of patients in this study expressed HLA restrictions for AChR, specifically HLA-DRB1*03, HLA-DRB3*01, and HLA-DQB1*02 (Figure 5). The selection criteria allow for the identification of patients likely to achieve efficacy with CNP-105. MG patients whose disease is triggered by autoreactivity to AChR antigens are eligible for treatment with CNP-106 and can be identified based on HLA haplotypes in their blood. [Figure 6-7] Process for producing CNP-106. CNP-106 was produced using a double emulsion solvent evaporation process. A high-level production process flow chart is shown in Figure 6. The final TIMP-MG formulation was characterized to determine its physicochemical properties, including particle size, zeta potential, and total peptide content. The results of the TIMP-MG characterization are shown in Table 3. TIMP-MG particles were examined by scanning electron microscopy, revealing a homogeneous composition of intact particles with smooth surfaces (Figure 7). [Modes for carrying out the invention]

[0083] There is a need for therapeutic agents to address the immune imbalance in MG, leading to improved disease symptoms and outcomes without the risk of toxic side effects. TIMPs are surface-functionalized, negatively charged particles made from biodegradable polymers that encapsulate antigenic proteins or peptide epitopes associated with inflammatory conditions such as autoimmune diseases and allergies. TIMPs are designed to target and deliver the encapsulated proteins / peptides to antigen-presenting cells (APCs) of mononuclear phagocytic cell lines, resulting in APC-mediated T cell reprogramming via non-inflammatory pathways.

[0084] In preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated therapeutic efficacy in inducing T-cell tolerance to antigenic / allergenic proteins and peptides, resulting in improved disease symptoms. TIMPs encapsulating one or more antigens involved in or related to myasthenia gravis (TIMP-MG) can potentially treat MG by reprogramming the immune system and inducing antigen-specific T-cell tolerance to MG-related antigens. There is a current need for immunotolerance therapies that can induce tolerance to autoimmune MG-related antigens for long-term therapeutic benefits without exposing patients to the risk of adverse events.

[0085] This disclosure provides compositions of negatively charged particles encapsulating one or more MG-related antigens, portions thereof, or combinations thereof. It also includes methods for inducing antigen-specific tolerance using the TIMP-MG particles described herein, processes for producing TIMP-MG particles encapsulating MG antigens, and processes for producing TIMP-MG particles encapsulating a pharmaceutical composition containing MG antigens and particles. This disclosure provides methodologies for monitoring the induction and maintenance of immune tolerance in subjects with MG after immunotherapy.

[0086] definition Unless otherwise stated, the following terms used in this application, including in this specification and the claims, have the definitions given below.

[0087] As used herein and in the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly indicates otherwise.

[0088] The terms “approximately” or “about” mean a tolerance for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the terms “approximately” or “about” mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms “approximately” or “about” mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the terms “approximately” or “about” precede the first number in a series of two or more numbers, it is understood that the terms “approximately” or “about” apply to each of the numbers in that series.

[0089] As used herein, “particles” refers to any non-tissue-derived composition, which may be spheres or sphere-like entities, beads, or liposomes. The terms “particles,” “tolerant immunomodulated particles,” “carrier particles,” and “beads” may be used interchangeably depending on the context. Additionally, the term “particles” may be used to encompass beads and spheres.

[0090] As used herein, “negatively charged particles” refers to particles that have been modified to have an effective surface charge of less than zero.

[0091] As used herein, "surface-functionalized" refers to particles having one or more functional groups on their surface. In some embodiments, surface functionalization is achieved by introducing one or more functional groups to the surface of a particle. In various embodiments, surface functionalization may be achieved by carboxylation (i.e., addition of one or more carboxyl groups to the particle surface) or by addition of other chemical groups (e.g., other chemical groups that impart a negative surface charge).

[0092] "Carboxylated particles," "carboxylated beads," or "carboxylated spheres" include any particles modified to contain carboxyl groups on their surface. In some embodiments, the addition of carboxyl groups enhances phagocytic / monocyte uptake of particles from circulation through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be achieved using any compound that adds carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA).

[0093] As used herein, the terms “Th cell” or “helper T cell” refer to CD4 + Refers to a cell. CD4 + T cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

[0094] As used herein, the term “Th1 cells” refers to a subset of Th cells that produce pro-inflammatory mediators. Th1 cells play a role in host defense against pathogens by secreting cytokines that stimulate the immune response and, in part, mediating the recruitment of neutrophils and macrophages to infected tissue. Th1 cells secrete cytokines including IFN-gamma, IL-2, IL-10, and TNF-alpha / beta to coordinate defense against intracellular pathogens such as viruses and certain bacteria.

[0095] As used herein, the term “Th2 cells” refers to a subset of Th cells that mediate the activation and maintenance of antibody-mediated immune responses against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines, such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25), which are involved in antibody production, eosinophil activation, and inhibition of several macrophage functions, thereby providing a phagocytic-independent protective response.

[0096] As used herein, the term “Th17 cells” refers to a subset of Th cells that produce pro-inflammatory responses to extracellular parasites and bacteria. Th17 cells mediate these functions by producing cytokines such as IFN-gamma, IL-17A, IL-17F, IL-21, IL-22, TNF-alpha, and GM-CSF, which are involved in the recruitment of neutrophils, myeloid cells, and B cells.

[0097] "Polypeptide" and "protein" refer to polymers composed of amino acid residues, associated naturally occurring structural variants, and naturally non-natural analogs of their synthesis, linked via peptide bonds or peptide bond isostears. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. The terms "polypeptide" and "protein" are not limited to the 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, etc., are included in the definition. Both full-length proteins and their fragments are included by definition. The terms "polypeptide" and "protein" also include post-expression modifications of polypeptides or proteins, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this disclosure, "polypeptide" may include "modifications" to the native sequence, such as deletions, additions, substitutions (which may include substitutions with any of the 20 amino acids that may be essentially conserved or commonly present in human proteins, or any other naturally occurring, non-natural, or atypical amino acids), and chemical modifications (e.g., addition or substitution with peptide mimes). These modifications may be intentional, through site-directed mutagenesis, or through chemical modification of amino acids to remove or add chemical moieties, or they may be accidental, such as through mutations occurring via protein-producing host cells, or through errors resulting from PCR amplification prior to host cell transfection.

[0098] As used herein, “antigenic moiety” or “antigen” refers to any portion recognized by the host’s immune system, such as a peptide. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0099] As used herein, "MG-related antigen" refers to an antigen or a part or fragment thereof that elicits an immune response to a protein or a part or fragment thereof. MG-related antigens may include the whole protein or other proteins or parts related to the protein that can induce an immune response in a subject.

[0100] "Pharmacologically acceptable carriers" refer to any of the standard pharmaceutical carriers, buffers, etc., such as phosphate-buffered saline, a 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, flow enhancers, sweeteners, flavoring agents, and colorants. Preferred 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 activator. Typical modes of administration include enteral (e.g., oral), parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration), or inhalation.

[0101] "Pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, that is, a material that can be administered to an individual without causing any undesirable biological effect, or without harmful interaction with any of the components of a composition containing it, or with any components present on or within the individual's body.

[0102] As used herein, the term “subject” encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals such as rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. The term does not indicate a specific age or sex.

[0103] The term "epitope" refers to any portion of a molecule that can be recognized and bound by a selective binder at one or more antigen-binding regions. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate side chains, and possess specific three-dimensional structural and charge characteristics. Epitopes as used herein may be continuous or discontinuous. Epitopes may also be mimotopes, meaning they are identical to the epitope used to generate an antibody, but contain none or only some of the amino acid residues found in the target used to stimulate the antibody immune response, and they contain a three-dimensional structure. As used herein, a mimotope is not considered a different antigen from the epitope bound by the selective binder, and the selective binder recognizes the same three-dimensional structure of the epitope and the mimotope.

[0104] The term "therapeutic dose" is used herein to indicate the amount of the antigen-specific composition of this disclosure that is effective in improving or alleviating one or more symptoms or signs of a disease to be treated.

[0105] The term “symptom” is used herein to mean any physical or observable sign of a disorder, whether or not the disorder is a general characteristic of that disorder. The term “symptom” can mean all such signs or any subset thereof.

[0106] As used herein in relation to methods, the terms “to treat,” “to be treated,” “to treat,” and “treatment” mean eliminating, reducing, inhibiting, or improving, whether temporary or permanent, partial or complete, the clinical symptoms, signs, or progression of an event, disease, or condition. Such treatment does not need to be absolute in order to be useful.

[0107] particle Tolerant immunomodulatory particles (TIMPs) containing one or more antigens have been previously described for inducing antigen-specific tolerance to treat inflammatory conditions (e.g., autoimmune diseases and allergies) (WO20131319253 and WO2015023796, incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy in inducing antigen-specific tolerance and inhibiting pathological inflammatory immune responses.

[0108] Particle size and charge are important for tolerance induction. While particles vary in size and charge based on the antigen encapsulated within them, generally, the particles described herein are effective in inducing tolerance when they are approximately 100 nanometers to approximately 1500 nanometers in diameter and have a charge of 0 to approximately -100 mV. In various embodiments, particles have a diameter of 400 to 800 nanometers and a charge of approximately -25 mV to -70 mV. In various embodiments, particles have a diameter of 400 to 800 nanometers and a charge of approximately -30 mV to -80 mV. In various embodiments, particles have a diameter of 400 to 800 nanometers and a charge of approximately -30 mV to -60 mV. Average particle size and particle charge may be slightly altered during the freeze-drying process; therefore, both post-synthesis average and post-freeze-drying average are described. As used herein, the terms “post-synthesis size” and “post-synthesis charge” refer to the size and charge of particles before freeze-drying. The terms "freeze-dried size" and "freeze-dried charge" refer to the size and charge of particles after freeze-drying.

[0109] In some embodiments, the particles are nonmetallic. In these embodiments, the particles may be formed from polymers. In preferred embodiments, the particles are biodegradable in solids. In this embodiment, the particles can be supplied in solids in multiple doses without particle accumulation in the solid. Examples of preferred particles include polystyrene particles, PLGA particles, PLURONICS-stabilized polypropylene sulfide particles, and diamond particles.

[0110] In some embodiments, the particle surface is composed of a material that minimizes nonspecific or undesirable biological interactions. Interactions between the particle surface and the interstitial space can be factors that play a role in lymphatic uptake. The particle surface may be coated with a material that prevents or reduces nonspecific interactions. Steric stabilization by coating particles with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS® (containing the copolymer of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) can reduce nonspecific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All of these facts demonstrate the relevance of the physical properties of the particles from the perspective of lymphatic uptake. Biodegradable polymers may be used to produce all or part of the polymer and / or particles and / or layers. Biodegradable polymers may be degraded, for example, as a result of the reaction of functional groups with water in solution. As used herein, the term “degradation” refers to becoming soluble, either by a reduction in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers containing ester groups, such as polylactides and polyglycolides, are generally subjected to spontaneous hydrolysis.

[0111] The particles disclosed herein may also contain additional components. For example, the carrier may have a contrast agent incorporated into or conjugated to the carrier. An example of a currently commercially available carrier nanosphere with a contrast agent is the Kodak X-sight nanosphere. Inorganic quantum confinement luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors for FRET applications: their high quantum yield and tunable size-dependent Stokes shift allow them to emit different sizes from blue to infrared when excited by a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Chem. Int. 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 the conventional 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).

[0112] The particles can be formed from a wide range of materials. Preferably, the particles are composed of materials suitable for biological use. For example, the particles may consist of citrates, glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, TIMP-MG particles may consist of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, polyesters of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxyhydroxy acids, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, polyanhydrides of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxydicarboxylic acids. Additionally, TIMP-MG particles can be quantum dots or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). TIMP-MG particles containing mixtures of ester and anhydride bonds (e.g., glycolic acid and sebaciate copolymers) may also be used. For example, TIMP-MG particles may include materials containing polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysebacate polymer (PSA), poly(lactic-co-glycol) acid copolymer (PLGA or PLG, terms are interchangeable), poly(lactic-co-sebacin) acid copolymer (PLSA), poly(glycol-co-sebacin) acid copolymer (PGSA), polypropylene sulfide polymer, poly(caprolactone), chitosan, etc. Other biocompatible and biodegradable polymers useful in the present invention may include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and biodegradable urethanes, as well as copolymers thereof having linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acid chains.In addition, biologically important amino acids having reactive side chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups and conjugate moieties for conjugation to antigen peptides and proteins. Suitable biodegradable materials for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the TIMP-MG particles of the present invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinylimidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0113] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. Preferred copolymer ratios for these immunomodulated particles may 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 various embodiments, the particles are PLURONICS-stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic acid-coglycolic acid) particles. In various embodiments, the particles are carboxylated PLGA particles. In various 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 acid-coglycolic acid) particles with a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles contain polylactic acid:polyglycolic acid in a ratio of about 50:50, about 80:20 to about 100:0, or polyglycolic acid:polylactic acid in a ratio of about 50:50, about 80:20 to about 100:0. In various embodiments, the particles contain polylactic acid:polyglycolic acid in a ratio of 50:50. In various embodiments, the particles contain polylactic acid:polyglycolic acid in a ratio of approximately 99:1 to approximately 1:99, for example, approximately 99:1, approximately 95:5, approximately 90:10, approximately 85:15, approximately 80:20, approximately 75:25, approximately 70:30, approximately 65:35, approximately 60:40, approximately 55:45, approximately 50:50, approximately 45:55, approximately 40:60, approximately 35:65, approximately 30:70, approximately 25:75, approximately 20:80, approximately 15:85, approximately 10:90, approximately 5:95, and approximately 1:99 (including all values ​​and ranges that fall between these values).

[0114] It is intended that the particles may further contain surfactants and / or stabilizers. The surfactants may be anionic, cationic, or nonionic. Surfactants of the poloxamer and poloxamine families are commonly used in particle synthesis. Surfactants 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. In addition, 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 some 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, if the particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion. For example, the stabilizer may be a compound that stabilizes the primary and / or secondary emulsions described herein by providing a physical or energy barrier between adjacent nanoparticle droplets within the emulsion, thereby reducing the probability of larger nanoparticle droplets binding and forming.

[0115] In various embodiments, polypeptide antigens are encapsulated in particles by a single emulsion process. In further embodiments, polypeptide antigens are more hydrophobic. Sometimes, a double emulsion process results in the formation of larger particles, which can lead to leakage of hydrophilic active components and low capture efficiency. Coalitioning and Ostwald maturation are two mechanisms that can destabilize double emulsion droplets, and diffusion of hydrophilic active components through the organic phase is the main mechanism causing low levels of captured active components. In some embodiments, reducing nanoparticle size may be beneficial. One strategy to achieve this is to apply a second strong shear rate. Leakage effects can be reduced by using high polymer concentrations and high polymer molecular weights, which involves increasing the viscosity of the internal aqueous phase and increasing the surfactant molecular weight. In certain embodiments, particles encapsulating the antigen are produced by nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsion, sol-gel methods, layer-by-layer techniques, or ion gelation methods. Several methods for producing nanoparticles are described in the literature and are incorporated herein by reference. 14、15 .

[0116] In some embodiments, the particles are liposomes. Liposomes can be prepared from a variety of lipid substances, including but not limited to phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearyl aluminium, ovolecithin, and cholesterol lipids, as well as mixtures thereof in various stoichiometric systems. As used herein, liposomes can also be formed from non-lipid amphiphilic molecules such as block copolymers of poly(oxyethylene-β-isoprene-β-oxyethylene). In preferred embodiments, liposomes are prepared from lipids or incorporate lipids that form negatively charged liposomes, such as those produced from phosphatidylserine, dicetyl phosphate, and dimyristoylphosphatidic acid. In various embodiments, negatively charged liposomes have zeta potentials of approximately -100mV to approximately 0mV, approximately -100mV to approximately -25mV, approximately -100mV to approximately -30mV, approximately -80mV to approximately -30mV, approximately -75mV to approximately -30mV, approximately -70mV to approximately -30mV, approximately -75mV to approximately -35mV, approximately -70mV to approximately -25mV, approximately -60mV to approximately -30mV, approximately -60mV to approximately -35mV, or approximately -50mV to approximately -30mV. In various embodiments, the zeta potential is approximately -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV (including all values ​​and ranges within these). In various embodiments, the liposome has a negative zeta potential of -30mV to -80mV. In various embodiments, the liposome has a negative zeta potential of -30mV to -60mV. In various embodiments, the liposome encapsulates one or more MG-related antigens, parts thereof, or combinations thereof. In various embodiments, the antigen comprises one or more proteins, peptides, antigenic epitopes, or combinations thereof. In various embodiments, TIMP-MG particles encapsulate one or more polynucleotides encoding MG-related antigens.In some embodiments, the polynucleotide includes DNA, RNA, messenger RNA (mRNA), or circular RNA. In various embodiments, the antigen is acetylcholine receptor (AChR) protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-associated protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), parts thereof, and / or one or more antigenic epitopes thereof. In various embodiments, one or more MG-associated antigens are selected from the list in Table 1. In various embodiments, the antigen is an AChR protein subunit and / or a part thereof. In various embodiments, the antigen is an AChR antigen (Table 2) and / or a part thereof selected from the group consisting of SEQ ID NOs: 1 to 21, or one or more antigenic epitopes thereof. In various embodiments, one or more antigens encapsulated within the liposome include combinations of SEQ ID NOs. 3, 5, 8, 13, 19, 20, and / or 21 listed in Table 2.

[0117] In various embodiments, the liposomes encapsulate one or more MG-related antigens, have a negative zeta potential of -100mV to 0mV, and have a diameter of 100 to 1000nm. In various embodiments, the liposomes encapsulate one or more MG-related antigens, a portion thereof, or a combination thereof, have a size of 400 to 800nm, and have a negative zeta potential of -30mV to -80mV.

[0118] In various embodiments, the liposomes encapsulate an antigen selected from the group consisting of SEQ ID NOs: 1-21 (Table 2), a portion thereof, or a combination thereof, the size of the liposomes being 100-1000 nm, and the liposomes having a negative zeta potential of -100 mV-0 mV. In various embodiments, the liposomes are used to induce tolerance in subjects having MG. In various embodiments, the administration of the liposomes is intravenous.

[0119] antigen An antigen refers to an individual part of a molecule, such as a polypeptide or peptide sequence, the formation of a polypeptide or peptide's 3D structure, or a polysaccharide or polynucleotide that can be recognized by host immune cells. Antigen-specificity refers to the ability of the target host cell to recognize and generate an immune response to the antigen alone or to a molecule very similar to the antigen, as well as to an epitope or mimotope.

[0120] "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 cessation of exposure to the antigenic peptide. 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 cells to the same antigen (even if re-exposure occurs in the presence of a costimulatory molecule) results in failure of cytokine production and subsequent failure of proliferation. Thus, failure of cytokine production prevents proliferation. However, anergistic T cells can proliferate when cultured with cytokines (e.g., IL-2).

[0121] The tolerance therapies described herein are intended to be antigen-specific. For example, a TIMP administered as a tolerance therapy encapsulates one or more antigens related to the tolerance therapy and the associated disease or condition being treated. A TIMP used in a tolerance therapy comprises one or more MG antigens, portions thereof, or combinations thereof, and / or MG proteins, portions thereof, or combinations thereof.

[0122] In certain embodiments, one, two, three, or more antigens or antigenic peptides are used in the TIMP. In certain embodiments, one or more MG antigens are encapsulated in the TIMP by covalent bonding to the inner surface of a particle (see, for example, U.S. Patent Publication 2019 / 0282707, incorporated herein by reference). In certain embodiments, sequences of two or more MG antigens are linked within a fusion protein and intended to be encapsulated within a TIMP as described herein. In certain embodiments, sequences of two or more MG antigens are linked within a fusion protein and intended to be encapsulated within a TIMP selected from Table 2. In certain embodiments, the TIMP is intended to encapsulate a fusion protein containing linked antigens and individual antigens. In certain embodiments, two or more MG-related antigens are linked together by one or more linkers that are easily cleaved by a specific protease. A method for constructing a TIMP having linked epitopes is described in U.S. Patent Publication 2019 / 0365656, incorporated herein by reference. Linking MG-related antigens improves their solubility and handling for inclusion within TIMP.

[0123] Examples of MG-related proteins (Table 1) include acetylcholine receptor subunit alpha (UNIPROT ID E7ENE5), acetylcholine receptor subunit alpha (UNIPROT ID P02708), nicotinic cholinergic receptor alpha 1 (UNIPROT ID Q53SH4), acetylcholine receptor subunit epsilon (UNIPROT ID Q04844), acetylcholine receptor subunit gamma (UNIPROT ID 07001), acetylcholine receptor subunit beta (UNIPROT ID P11230), acetylcholine receptor subunit gamma (UNIPROT ID P07510), agrin (UNIPROT ID O00468), musculoskeletal receptor tyrosine-protein kinase (UNIPROT ID O15146), cortactin (UNIPROT ID Q14247), titin (UNIPROT ID Q8WZ42), and liodin receptor 1 (UNIPROT ID P21817), liodin receptor 2 (Q92736), liodin receptor 3 (Q15413), Frizzled-9 (UNIPROT ID O00144), segment polar protein dishevelled homolog DVL-1 (UNIPROT ID DO14640), low-density lipoprotein receptor-associated protein 4 (UNIPROT ID O75096), neuronal acetylcholine receptor subunit beta-2 (UNIPROT ID P17787), choline O-acetyltransferase (UNIPROT ID P28329), neuronal acetylcholine receptor subunit alpha-5 (UNIPROT ID P30532), neuronal acetylcholine receptor subunit beta-4 (UNIPROT ID P30926), acetylcholine receptor subunit alpha-3 (UNIPROT ID P32297), acetylcholine receptor subunit alpha-7 (UNIPROT ID P36544), acetylcholine receptor subunit alpha-4 (UNIPROT ID P43681), neuronal acetylcholine subunit-beta-3 (UNIPROT ID Q05901), acetylcholine receptor subunit-delta (UNIPROT IDExamples include Q07001), serine / threonine protein kinase (UNIPROT ID Q13153), 43kDa synaptic receptor-associated protein (RAPsyn) (UNIPROT ID Q13702), acetylcholine receptor subunit alpha-2 (UNIPROT ID Q15822), neuronal acetylcholine receptor subunit alpha-6 (UNIPROT ID Q15825), protein Dok-7 (downstream of tyrosine kinase 7) (UNIPROT ID Q18PE1), protein RIC-3 (resistant to cholinesterase 3 inhibitors) (UNIPROT ID Q7Z5B4), plextrin-like domain family B member 2 (protein LL5-beta) (UNIPROT ID Q86SQ0), AchR epsilon subunit (UNIPROT ID Q8N731), and neuronal acetylcholine receptor subunit alpha-10 (nicotinic acetylcholine receptor subunit-10) (UNIPROT ID Q9GZZ6). [Table 1-1] [Table 1-2]

[0124] Examples of MG-related antigens include AChR antigens having amino acid sequences selected from Table 2. In various embodiments, TIMP-MG encapsulates antigens including combinations of SEQ ID NOs. 3, 5, 8, 13, 19, 20, and 21 shown in Table 2. [Table 2]

[0125] Emulsions are generated in many processing forms and are widely used in food, cosmetics, and drug delivery. Oil-water (single) and water-oil-water (double) emulsions are methods that use PLGA to encapsulate hydrophobic and hydrophilic drugs in micro or nanoscale forms. In summary, PLGA is dissolved in an organic phase (oil) which is emulsified with a surfactant or stabilizer (water). Hydrophobic drugs and / or other drugs are added directly to the oil phase, while hydrophilic drugs and / or other drugs (water) may be first emulsified in a polymer solution before particle formation. High-intensity homogenization (e.g., sonication burst) promotes the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours, thereby evaporating the solvent. The cured nanoparticles are collected and washed by centrifugation. In certain embodiments, cured emulsion particles can be obtained by evaporation of the oil phase.

[0126] Water-in-oil-in-water (W / O / W) emulsions are an example of a double emulsion, in which a dispersion of smaller water droplets within larger oil droplets is 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 nonpolar cargo (pharmaceutical / biological factors, e.g., proteins), and improved control over the release of therapeutic molecules. The preparation of double emulsions typically requires surfactants or mixtures thereof for stability. Surfactants stabilize droplets subjected to extreme flows, leading to the direct mass production of robust double nanoemulsions suitable for nanostructure encapsulation applications in various industries. In one embodiment, the double emulsion process involves generating a primary emulsion by mixing an aqueous solution of a pharmaceutical / biological agent with a polymer-containing solution, 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. Next, the secondary emulsion is cured by evaporation to remove the solvent, resulting in cured polymer nanoparticles that encapsulate pharmaceutical / biological agents.

[0127] As used herein, “homogenization” refers to operations using a class of processing equipment called homogenizers, which are tuned to reduce the size of droplets in a liquid-liquid dispersion. Factors influencing particle or droplet size include, but are not limited to, the type of emulsifier, the concentration of the emulsifier, the solution conditions, and the mechanical devices (homogenization power, pressure, rotation speed, time). Non-limiting examples of homogenizers include high-speed blenders, high-pressure homogenizers, colloidal mills, high-shear dispersers, ultrasonic disruptor membrane homogenizers, and ultrasonic processors. Mechanical homogenizers, manual homogenizers, ultrasonic processors, mixer mills, vortexers, etc., may be used for mechanical and physical destruction within the scope of this disclosure.

[0128] As used herein, “batch size” refers to the manufacturing scale corresponding to the weight of particles in the final product. The manufacturing process may be modified, expanded, or reduced. The manufacturing process may be modified, expanded, or reduced by changing the amount or volume of solvent, antigen / protein, polymer, surfactant, stabilizer, cryoprotectant, or excipient. The manufacturing process may be expanded or reduced by changing the time of homogenization, sonication, evaporation, filtration, concentration, washing, or freeze-drying. The number of vials filled with TIMP-MG varies depending on the batch size. Approximately 1, 5, 50, 500, 5000, 50,000, or 500,000 vials are filled, including all values ​​within this range. The amount of TIMP-MG filled in each vial is approximately 0.01 mg, approximately 0.1 mg, approximately 1 mg, approximately 10 mg, approximately 100 mg, approximately 200 mg, or approximately 250 mg, including all values ​​within this range.

[0129] Methods for determining the protein content in particles or solutions include ELISA, mass spectrometry, HPLC, CBQCA, and Western blotting.

[0130] The Molecular Probe CBQCA Protein Quantification Kit provides a rapid and sensitive method for the quantification of proteins in solution. This kit utilizes the ATTO-TAG CBQCA reagent (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde), which was originally developed as a chromatographic derivatization reagent for amines. This reagent has also been proven to be 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 reacts with primary amines such as those found in proteins in the presence of cyanide to form highly fluorescent derivatives.

[0131] acid An acid is a molecule or ion that can donate a proton (i.e., a hydrogen ion, H + ) or form a covalent bond with an electron pair.

[0132] An aqueous solution of an acid has a pH less than 7. A lower pH means a higher acidity and thus a higher concentration of positive hydrogen ions in the solution. A chemical substance or material that has the properties of an acid is said to be acidic.

[0133] In various embodiments, the acid is acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrenesulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimonic acid, fluoroboric acid, hexafluorophosphoric acid, chromic acid, phosphoric acid, hydrofluoric acid, oxalic acid, boric acid, carbonic acid.

[0134] base A base is a chemical species that donates electrons, accepts protons, or releases hydroxide (OH-) ions in aqueous solution.

[0135] Aqueous solutions of bases have a pH ranging from greater than 7.0 to 14.0. A higher pH indicates higher basicity and therefore a higher concentration of negative hydroxide ions in the solution. Chemical substances or materials that possess basic properties are said to be basic.

[0136] In various embodiments, the bases are barium hydroxide, calcium hydroxide, chromium hydroxide, potassium hydroxide, ammonium hydroxide, zinc hydroxide, barium hydroxide, sodium bicarbonate, methylamine, diethylamine, sodium hydroxide, magnesium hydroxide, ammonium bicarbonate, ammonia, aluminum hydroxide, sodium carbonate, magnesium hydroxide, zinc hydroxide, iron hydroxide, acetone, lithium hydroxide, pyridine, and rubidium hydroxide.

[0137] How to use This specification provides a method for treating MG in a subject, comprising administering TIMP-MG to the subject, wherein TIMP-MG is administered at a dose level determined based on the subject's body weight. It is also intended that TIMP-MG may be administered in a fixed dose regardless of the subject's body weight. In various embodiments, a method for treating MG in a subject is intended, comprising administering TIMP-MG to the subject, wherein TIMP-MG is administered in a dose of 0.001 to 12 mg / kg based on the subject's body weight, or in a fixed dose of 0.1 mg to 800 mg. This specification also provides a method for reducing the inflammatory immune response to the MG antigen in a subject suffering from MG, comprising administering TIMP-MG to the subject, wherein TIMP-MG is administered in a dose of 0.001 to 12 mg / kg based on the subject's body weight, or in a fixed dose of 0.1 mg to 800 mg.

[0138] Furthermore, TIMP-MG may also be administered in doses of approximately 0.001 to 10 mg / kg, approximately 0.005 to 12 mg / kg, approximately 0.01 to 12 mg / kg, approximately 0.05 to 12 mg / kg, approximately 0.1 to 12 mg / kg, approximately 0.5 to 10 mg / kg, approximately 1 mg / kg to 8 mg / kg, approximately 1.5 to 10 mg / kg, approximately 2 mg / kg to 12 mg / kg, approximately 2 mg / kg to 10 mg / kg, approximately 3 mg / kg to 10 mg / kg, approximately 4 to 10 mg / kg, approximately 4 to 12 mg / kg, or approximately 5 to 12 mg / kg. TIMP-MG may be administered at any dose of approximately 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 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. Alternatively, TIMP-MG is administered in fixed doses of approximately 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.

[0139] In another embodiment, TIMP-MG is administered at concentrations ranging from approximately 0.0005 mg / mL to approximately 50 mg / mL, with the optional concentrations being approximately 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, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. In various embodiments, TIMP-MG is administered via intravenous infusion lasting approximately 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0140] TIMP-MG is intended to be administered as a single dose or in multiple doses. In various embodiments, TIMP-MG is administered once a week, once every two weeks, once every three weeks, once every four months, once every three months, once every four months, once every five months, once every six months, once a year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, or once every ten years. In certain embodiments, TIMP-MG is administered in two doses, one week apart.

[0141] In various embodiments, a booster dose of TIMP-MG is administered as a single dose or multiple doses after the initial administration of TIMP-MG. In various embodiments, the booster dose 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 four months, once every five months, once every six months, or once per year. In various embodiments, TIMP-MG is administered in two doses one week apart, followed by a booster dose administered as a single dose every three months. The booster dose is administered as needed, as determined by analytical tests or screening methods used to test for weakening of immune tolerance. Methods for tracking the maintenance or weakening of immune tolerance are described by reference below and / or in Patent Publication WO2022 / 221622 of this Spec.

[0142] In various embodiments, TIMP-MG is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. When TIMP-MG is administered intravenously, it is intended to be administered via intravenous infusion lasting approximately 1, 2, 3, 4, 5, 6, 7, 8, 12, 18, or 20 hours.

[0143] In various embodiments, TIMP-MG administration is based on one or more of the following: the patient's profile / HLA haplotype, the Myasthenia Gravis Foundation (MGFA) clinical classification score (Class I, Class II, Class III, Class IV, Class V), the baseline MG-ADL score (e.g., 1-24, including all values ​​within this range), the baseline QMG score (e.g., 1-39, including all values ​​within this range), the baseline MGC score (e.g., 1-50, including all values ​​within this range), symptoms (e.g., ocular or non-ocular symptoms), concurrently or previously administered medications / therapeutic agents / drugs, comorbid or active diseases, the presence / level of anti-AChR antibodies, the presence / level of anti-MuSK antibodies or other MG-related antibodies. In various embodiments, the HLA haplotype is selected from HLA-DRB1*03, HLA-DRB3*01, or HLA-DQB1*02. In some embodiments, the MGFA clinical classification score is III-IV or II-IV. In various embodiments, the MG-ADL score is ≥6, and ≥50% of the score is based on non-ocular symptoms. In various embodiments, the QMG score is ≥11.

[0144] In various embodiments, TIMP-MG is injected at an increasing rate. In various embodiments, the increasing rate doubles 15 minutes after the initial injection. In various embodiments, the increasing rate doubles 15 minutes after the second injection, for example, four times the initial injection rate. In some embodiments, TIMP-GLIA is injected at approximately 20 mL / hour for the first 15 minutes over approximately 2.5 hours, then at 40 mL / hour for the next 15 minutes, with a total injection duration of 80 mL / hour.

[0145] In various embodiments, the Disclosure provides a method for treating MG or MG-related symptoms in a subject, comprising administering to the subject a composition comprising TIMP-MG alone or in combination with a therapeutic agent. In various embodiments, the therapeutic agent is a cholinesterase inhibitor, steroid, corticosteroid, nonsteroidal immunosuppressant, immunomodulator, therapeutic plasma exchange (plasma replacement), intravenous immunoglobulin preparation (IVIG), chloride ion channel inhibitor, monoclonal antibody, proteasome inhibitor, cytokine and chemokine targeted therapy, microRNA inhibitor, siRNA, JAK inhibitor, BTK inhibitor, complement inhibitor, IgG degrader, neonatal Fc receptor (FcRn) inhibitor, chimeric antigen receptor (CAR) T-cell therapy, regulatory T-cell (Treg) therapy, hematopoietic stem cell transplantation, B-cell targeted therapy, or surgical treatment. In various embodiments, the treatment includes azathioprine, sirolimus, edrophonium, neostigmine, pyridostigmine, cyclosporine, mycophenolate mofetil, calcineurin inhibitors, levamisole, chlorambucil, cyclophosphamide, tacrolimus, methotrexate, eculizumab, ravulizumab, efgaltidimod alfa, rozanolixizumab, zircoplan, and CD20 targeted therapy. Therapies are selected from the group including CD19-targeted therapy, CD40 / CD40L-targeted therapy, B-cell targeting factor (BAFF)-targeted therapy, B-cell maturation antigen (BCMA)-targeted therapy, anti-IL6 therapy, anti-IFN therapy, anti-timocytoglobulin, factor D inhibitors, amifampridine, batoclimab, inbilizumab, nipokalimab, pozelimab, rituximab, satralizumab, tocilizumab, tofacitin, and trebulimin. In various embodiments, the steroid or corticosteroid is selected from the group including beclomethasone, cyclusonide, fluticasone floritol, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, oxymetazoline hydrochloride, or hydrocortisone. In various embodiments, the surgical treatment is thymectomy. In various embodiments, the therapeutic agent is administered before, simultaneously with, or after the administration of TIMP-MG.In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days before the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks before the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks after the administration of TIMP-MG. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the administration of TIMP-MG.

[0146] TIMP-MG therapy is intended to alleviate, reduce, or improve one or more symptoms of myasthenia gravis. Symptoms of MG include, but are not limited to, ptosis (drooping eyelids), diplopia (double vision), difficulty making facial expressions, chewing problems, dysphagia (difficulty swallowing), dysarthria (slurred speech), shortness of breath, dyspnea, muscle fatigue, skeletal weakness, neck weakness, limb weakness, dysphagia, dysphonia, neuromuscular junction weakness, synaptic signaling disorders, muscle injury, rescue therapy with IVIG / PLEX, complement activation, anti-Musk antibodies, anti-AChR antibodies, increased activated CD4+ cells and activated CD8+ T cells in PBMCs, increased myasthenia gravis activity of daily living score, quantitative MG score, MG quality of life 15 revised score, and increased MG composite score.

[0147] TIMP-MG therapy is also intended to reduce, shorten, or improve the duration and severity of the inflammatory immune response to one or more MG antigens in the subject. Inflammatory immune responses include T-cell responses, B-cell responses, Th1 responses, myeloid cell responses, and / or antibody responses. In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to one or more MG antigens is determined by assays of one or more biological samples derived from the subject as described herein.

[0148] A method for inducing tolerance in a subject requiring tolerance induction is provided herein, comprising administering to the subject a composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more MG-related antigens, parts thereof, or combinations thereof.

[0149] In various embodiments, the subjects are subjects with MG, subjects receiving therapy for MG, or subjects receiving therapy for MG.

[0150] In various embodiments, TIMP-MG particles comprise one or more MG-related antigens, portions thereof, combinations thereof, or one or more antigenic epitopes thereof. In various embodiments, TIMP-MG particles comprise one or more MG-related proteins, portions thereof, combinations thereof, or one or more antigenic epitopes thereof.

[0151] When TIMP-MG particles containing a single antigen are administered in combination with another carrier particle containing a different antigen, or with a second agent, the particles and / or the second agent may be administered simultaneously or sequentially. Concomitant or simultaneous administration of two therapeutic agents does not require them to be administered simultaneously or via the same route, as long as there is an overlap in the periods during which the agents exert their therapeutic effects. Simultaneous or sequential administration is intended, as is administration on different days or weeks. The term "therapeutic agent" is further intended to refer to agents administered in separate formulations, simultaneously or in combination, and administered simultaneously to each other within 30 minutes of each other. Pre-administration refers to the administration of therapeutic agents within the period from one week before treatment with TIMP-MG particles to 30 minutes before administration of TIMP-MG particles. Subsequent administration means describing administration from 30 minutes after treatment to one week after administration.

[0152] Screening method The induction and maintenance of immune tolerance are monitored in subjects affected by MG, who are being treated with or are intended to be treated with antigen-specific tolerance therapies, including TIMP-MG, as described herein.

[0153] Methods for screening cell types, cytokines, or other measures of tolerance from subjects receiving tolerance therapy as described herein are known in the art. Methods for assessing tolerance are performed using techniques such as flow cytometry, radioimmunoassays, mass cytometry (CyTOF), ELISA, ELISPOT, in vitro or ex vivo cell stimulation assays (including, but not limited to, cell proliferation assays and macrophage stimulation assays), autoantibody measurements, or Ig serotype measurements, for example, by ImmunoCap assays.

[0154] In various embodiments, the immune tolerance status of a subject is determined by assays of one or more biological samples from the subject. Biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, the assays of biological samples include analyzing the levels, and / or presence or absence, of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof that are associated with disease or disorder.

[0155] Cells assayed from biological samples include immune cells, non-immune cells, and / or combinations thereof. Immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from biological samples are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from biological samples include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells. In certain embodiments, the cells assayed from a biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSEC), and / or Kupffer cells.

[0156] The immune tolerance state of a subject and one aspect of its immune signature are determined by analyzing one or more proteins from one or more biological samples from the subject. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL -26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CC L6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL 23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3(MIP-1α, CXCL4(MIP-1β, CXCL5(RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, The protein is selected from the group consisting of IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, soluble CD14, and / or combinations thereof. In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartate protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease.In various embodiments, the following are selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the apoptosis-related protein is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples are described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or their variants. In various embodiments, the immunoglobulin is antigen-specific. In various embodiments, the immunoglobulin is MG antigen-specific. In various embodiments, the immunoglobulin is AChR antigen-specific. In various embodiments, the immunoglobulin is Musk antigen-specific. Several methods for detecting immunoglobulins from biological samples are described in the literature, including radioimmunoassays, ELISA, and ImmunoCap.

[0157] The immune tolerance state and one aspect of the immune signature of the subject are determined by analyzing one or more metabolites from a biological sample. In various embodiments, the metabolites are inflammatory metabolites. In various embodiments, the metabolites are anti-inflammatory metabolites. Examples of inflammatory metabolites in various embodiments include acids, lipids, sugars, amino acids, lactates, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinates, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyl lactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include kynurenine, 3-hydroxykynurenine, 2-amino-3-carboxymucone-6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine.

[0158] A list of human metabolites that can be assayed from biological samples can be found in literature including (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35 (Database issue): D521-6, 2007), and the Human Metabolome Database (HMDB), which are incorporated herein by reference.

[0159] The immune tolerance state of a target and one aspect of its immune signature are determined by analyzing one or more cell surface proteins from a biological sample. In various embodiments, the cell surface proteins include CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, C D45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD11 2, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR 2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, N Kp44, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL -6Rα, gp130, IL-7Rα, IL-9R, IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL-23R, IL-27Rα, IL-31Rα, OSMR,CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL -28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, L IGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BA FF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 , CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IG SF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 This includes α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains, and / or combinations thereof. Several methods for assaying cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), are described in the literature.

[0160] In certain embodiments, the tolerance status of the subject is determined by analyzing nucleic acids from a biological sample. In various embodiments, nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, mRNA, rRNA, tRNA, siRNA, miRNA, long non-coding RNA (long ncRNA, lncRNA), and mitochondrial RNA, DNA and / or RNA. In various embodiments, miRNAs include miR-146, miR-15a, miR-125a-5p, miR-15-5p, miR-21-5p, miR-139-5p, miR-452-5p, miR-7-5p, miR-548k, miR-653, miR-146, miR-612, miR-3651, miR-3653, miR-15b, mi The group is selected from those including R-15b, miR-145, miR-20b, miR-150-5p, miR-21-5p, miR-20b, miR-30e-5p, miR-145, miR-146, miR-181c, miR320a, miR155, miR-181a, miR-21-5p, miR-27a-3p, or miR-30e-5p. In certain embodiments, treatment with TIMP-MG reduces miRNA levels compared to baseline. In certain embodiments, treatment with TIMP-MG increases miRNA levels compared to baseline.In a particular embodiment, treatment with TIMP-MG reduces miRNA levels from 0.01% to 100% relative to the baseline measurement of the subject (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%). Reduce to 85%, approximately 90%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100). A specific implementation In this state, treatment with TIMP-MG reduces miRNA levels by 0.01% to 100% (for example, approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90%) relative to the baseline measurement of the subject. Increase by approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0161] In various embodiments, the immune tolerance state of a target is determined by assaying gene expression from a biological sample. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immune function, antibodies, xenobiotic response, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, phagocytosis, tight junction regulation, cell adhesion, differentiation, and / or combinations thereof. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immunosuppression. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immune activation. In various embodiments, the immune tolerance state is determined by assaying gene expression related to regulatory function. In various embodiments, nucleic acid analysis is used to generate an immune tolerance signature. Several methodologies for high-throughput gene expression analysis, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis, are described in the literature.

[0162] Biological samples are optionally assayed after in vivo and / or ex vivo stimulation with one or more stimuli, such as antigens, allergens, and one or more activators. The T cells, B cells, and immunoglobulins used in the assay are intended to be antigen-specific. Exemplary T cells include effector memory T cells, antigen-specific T cells, activated antigen-specific T cells, Th1 cells, pathogenic Th2a+ cells, Th17 cells, T follicle helper (TFH) cells, TH0 cells, a proportion of antigen-specific CD4+ T cells (disease-specific T cells / effector memory T cells), a proportion of activated antigen-specific CD4+ T cells (disease-specific T cells / disease-specific T cells), and antigen-specific CD8+ T cells (CD154-CD137+C). Examples include the proportion of D8+ T cells (total CD8+ T cells), the proportion of activated antigen-specific CD8+ T cells (CD38+CD154-CD137+CD8+ T cells / CD154-CD137+CD8+ T cells), the proportion of antigen-specific regulatory T cells (CD154-CD137+CD127-CD25+TIGIT+effector memory CD4+ T cells / CD154-CD137+effector memory CD4+ T cells), or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and regulatory B (Breg) cells. In certain embodiments, T cells are identified based on the expression of proteins listed in Table 3. [Table 3]

[0163] The target immune tolerance signature is generated using one or more of the following parameters, assayed from one or more biological samples obtained from the target and stimulated in vivo and / or ex vivo: A. The proportion of effector T cells in the total T cell population. B. The proportion of Treg cells in the total T cell population, C. The proportion of effector B cells in the total B cell population. D. Levels of specific IgG and / or IgM, E. Levels of inflammatory cytokines and chemokines, F. Levels of anti-inflammatory cytokines and chemokines, G. Liver enzyme levels H. Levels of inflammatory metabolites, and I. Levels of anti-inflammatory metabolites.

[0164] The immune tolerance signature indicates maintained immune tolerance if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance. In various embodiments, the immune tolerance signature indicates maintained immune tolerance if at least 2 out of 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance. In various embodiments, if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance, the subject is determined not to require treatment with TIMP. In various embodiments, if at least 3 out of 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance, the subject is determined not to require treatment with TIMP.

[0165] The immune tolerance signature of a subject generated using one or more parameters described herein indicates weakened and / or absent immune tolerance before or after treatment with TIMP if:

[0166] a. The proportion of effector T cells in the total T cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values) relative to baseline measurements of subjects and / or healthy subjects, and / or

[0167] b. The percentage of T cells in the total T cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values), and / or

[0168] c. The proportion of effector B cells in the total B cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values) relative to baseline measurements for subjects and / or healthy subjects, and / or

[0169] d. IgG and / or IgM levels should be approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%) relative to baseline measurements of the subject and / or healthy subjects. , increasing to approximately 90%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values) and / or,

[0170] e. Inflammatory cytokine / chemokine levels should be between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9%) Increases to 0%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or increases by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0171] f. Levels of anti-inflammatory cytokines and chemokines are approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9%) relative to baseline measurements of subjects and / or healthy subjects. Decreases to 0%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or decreases to approximately 2-1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values) and / or

[0172] g. Liver enzyme levels should be approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 90%) compared to baseline measurements for subjects and / or healthy subjects. Increases by 5%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0173] h. Levels of inflammatory metabolites should be approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), Increases to approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or increases to approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0174] i. The levels of anti-inflammatory metabolites should be approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) relative to baseline measurements in the subject and / or in healthy subjects. It decreases to approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or to approximately 2-1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0175] The efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to MG antigens is determined by assays of one or more biological samples from the subject. Biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, assays of biological samples may involve analyzing the levels, and / or presence or absence, of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof.

[0176] In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to MG antigens is determined based on assays of cells derived from one or more subject-derived biological samples before and after treatment with TIMP-MG. In various embodiments, the cells are immune cells, non-immune cells, and / or combinations thereof. In various embodiments, immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from biological samples are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from biological samples include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0177] In certain embodiments, the cells assayed from a biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSEC), and / or Kupffer cells.

[0178] In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to one or more MG antigens is determined based on assays of cell surface proteins derived from one or more subject-derived biological samples before and after treatment with TIMP-MG. In various embodiments, the cell surface proteins are CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, C D141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR 3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI , MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R,IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, P D-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, I COS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TR AF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, F The group is selected from AP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. The integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains and / or combinations thereof. Several methods for assaying cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), are described in the literature.

[0179] In various embodiments, treatment with TIMP-MG increases the expression of inflammatory cell surface proteins to 5%–100% (e.g., about 5%, about 10%, 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%) relative to baseline values ​​of the subject and / or healthy subjects. Reduced to 1 / 100 (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-1 / 100 (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between those values) In various embodiments, treatment with TIMP-MG increases the expression of anti-inflammatory cell surface proteins to 5%–100% (e.g., about 5%, about 10%, 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%) relative to baseline values ​​in the subject and / or healthy subjects. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between those values)).

[0180] In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to the MG antigen is determined based on assays of proteins derived from one or more subject-derived biological samples before and after treatment with TIMP-MG. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL- 21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, I L-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL1 2. Selected from the group consisting of CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, and / or combinations thereof.

[0181] In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartate protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease. In various embodiments, the following are selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the apoptosis-related protein is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples are described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or their variants. In various embodiments, immunoglobulins are antigen-specific. Several methods for detecting immunoglobulins from biological samples are described in the literature, including radioimmunoassays, ELISA, and ImmunoCap.In various embodiments, treatment with TIMP-MG reduces inflammatory protein levels to 5%–100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to baseline levels in the subject and / or healthy subjects. (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100th (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or reduced to 1 / 100th (including all values ​​and ranges between those values) In various embodiments, treatment with TIMP-MG reduces anti-inflammatory protein levels to 5%–100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to baseline levels in the subject and / or healthy subjects. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between those values)).

[0182] In various embodiments, the efficacy of TIMP-MG in alleviating one or more symptoms of MG and / or reducing the duration and severity of the inflammatory immune response to the MG antigen is determined based on assays of metabolites derived from one or more subject-derived biological samples before and after treatment with TIMP-MG. In various embodiments, the metabolites are inflammatory metabolites. In various embodiments, the metabolites are anti-inflammatory metabolites. Examples of inflammatory metabolites in various embodiments include acids, lipids, sugars, amino acids, lactates, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinates, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyl lactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include kynurenine, 3-hydroxykynurenine, 2-amino-3-carboxymucone-6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine. In various embodiments, treatment with TIMP-MG reduces the levels of inflammatory metabolites to 5% to 100% (e.g., about 5%, about 10%, 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% (these values) relative to baseline measurements of the subject and / or healthy subjects. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100th (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100th (including all values ​​and ranges between those values)).In various embodiments, treatment with TIMP-MG raises the levels of anti-inflammatory metabolites to 5%–100% (e.g., about 5%, about 10%, 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%) relative to baseline levels in the subject and / or relative to healthy subjects. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between those values)).

[0183] In various embodiments, the efficacy of TIMP-MG is determined based on the following evaluations before and after administration of TIMP-MG. A. Percentage of antigen-specific CD4+ and CD8+ T cells B. Percentage of activated antigen-specific CD4+ and CD8+ T cells Percentage of CT regulatory (Treg) cells D. Percentage of anti-AChR antibodies in serum. E. Percentage of anti-MuSK antibodies in serum. F. Myasthenia Gravis Composite (MGC) Score G. Quantitative score of muscle weakness (QMG) score. H.MG Activities of Daily Living (MG-ADL) score. I. MG Quality of Life 15 Revised (MG-QOL15r) score. J. Healthcare utilization through hospital visits (Rate of MG exacerbations requiring rescue therapy with IVIG / PLEX) K. Use of alternative mycosmetic therapy

[0184] In various embodiments, the effectiveness of TIMP-MG in improving one or more symptoms of MG is determined from the results of assays of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 parameters listed in (A) to (K) above.

[0185] In various embodiments, the efficacy of TIMP-MG is determined based on an assay of antigen-specific CD4+ and / or CD8+ T cells. In various embodiments, treatment with TIMP-MG raises the levels of antigen-specific CD4+ and / or CD8+ T cells to 1% to 100% (e.g., about 1%, about 2%, about 5%, about 10%, 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 1%) relative to the baseline measurement of the subject and / or a healthy subject. Reduce to 00% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0186] In various embodiments, the efficacy of TIMP-MG is determined based on an assay of activated antigen-specific CD4+ and / or CD8+ T cells. In various embodiments, treatment with TIMP-MG raises the level of activated antigen-specific CD4+ and / or CD8+ T cells by 1% to 100% (e.g., about 1%, about 2%, about 5%, about 10%, 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 Reduce to 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0187] In various embodiments, the efficacy of TIMP-MG is determined based on an assay of regulatory T cells or antigen-specific regulatory T cells. In various embodiments, treatment with TIMP-MG raises the level of regulatory T cells or antigen-specific regulatory T cells to 1% to 100% (e.g., about 1%, about 2%, about 5%, about 10%, 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 Increase to 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0188] In various embodiments, the efficacy of TIMP-MG is determined based on an assay of anti-AChR antibodies. In various embodiments, treatment with TIMP-MG raises the level of anti-AChR antibodies to 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (between these values) relative to the baseline measurement of the subject and / or a healthy subject. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0189] In various embodiments, the efficacy of TIMP-MG is determined based on an assay of anti-MuSK antibodies. In various embodiments, treatment with TIMP-MG raises the level of anti-MuSK antibodies to 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (between these values) relative to the baseline measurement of the subject and / or a healthy subject. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0190] In various embodiments, the effectiveness of TIMP-MG is determined based on the subject's score on a clinical disease scale. In various embodiments, the clinical disease scale is the QMG score, myasthenia gravis score (MMS), Vesta Institute of Neurological Assessment Scale for MG (INCB-MG), MG-ADL, manual muscle testing (MMT), MG composite (MGC), oculomedullary facial respiration score (OBFR), MG disability index (MGII), or MG-QOL-15r score. In various embodiments, TIMP-MG reduces the QMG score, myasthenia gravis score (MMS), Vesta Institute of Neurological Assessment Scale for MG (INCB-MG), MG-ADL, manual muscle testing (MMT), MG composite (MGC), oculomedullary facial respiration score (OBFR), MG disability index (MGII), or MG-QOL-15r score. Several clinical disease scoring systems are described in the literature and are incorporated herein by reference. 7 .

[0191] In various embodiments, the effectiveness of TIMP-MG is determined based on hospital visit healthcare use (MG exacerbation rate requiring rescue therapy with IVIG / PLEX). In various embodiments, treatment with TIMP-MG reduces hospital visit healthcare use and reduces MG exacerbation rate or rescue therapy with IVIG / PLEX. In various embodiments, TIMP-MG reduces hospital visit healthcare use, MG exacerbation rate, or rescue therapy with IVIG / PLEX to 2 to 1 / 100 (e.g., approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)) compared to baseline measurements of the subject and / or compared to a healthy subject.

[0192] In various embodiments, the efficacy of TIMP-MG is determined based on the use of alternative MG therapies. In various embodiments, treatment with TIMP-MG reduces the use of alternative MG therapies. In various embodiments, alternative MG therapies include cholinesterase inhibitors, steroids, corticosteroids, nonsteroidal immunosuppressants, immunomodulators, therapeutic plasma exchange (plasma replacement), intravenous immunoglobulin preparations (IVIG), monoclonal antibodies, proteasome inhibitors, cytokine and chemokine targeted therapies, microRNA inhibitors, siRNA, JAK inhibitors, BTK inhibitors, complement inhibitors, IgG degraders, chimeric antigen receptor (CAR) T-cell therapy, regulatory T-cell (Treg) therapy, hematopoietic stem cell transplantation, B-cell targeted therapy, or surgical treatment. In various embodiments, the treatment includes azathioprine, sirolimus, edrophonium, neostigmine, pyridostigmine, cyclosporine, mycophenolate mofetil, calcineurin inhibitors, levamisole, chlorambucil, cyclophosphamide, tacrolimus, methotrexate, eculizumab, ravulizumab, efgaltidimod alfa, rozanolixizumab, zircoplan, CD20 targeted therapy, The group includes CD19-targeted therapy, CD40 / CD40L-targeted therapy, B-cell targeting factor (BAFF)-targeted therapy, B-cell maturation antigen (BCMA)-targeted therapy, anti-IL6 therapy, anti-IFN therapy, anti-timocytoglobulin, factor D inhibitors, amifamplidin, batoclimab, inbilizumab, nipokalimab, pozerimab, rituximab, satralizumab, tocilizumab, tofacitinib, and trebrutinib. In various embodiments, the steroid or corticosteroid is selected from the group including beclomethasone, cyclusonide, fluticasone floratol, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, oxymetazoline hydrochloride, or hydrocortisone.In various embodiments, TIMP-MG administration is used as an alternative MG therapy to baseline measurements of a subject and / or healthy subjects, ranging from 1% to 100% (e.g., about 1%, about 2%, about 5%, about 10%, 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% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%). Reduce. In various embodiments, the use of alternative MG therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-MG. In various embodiments, the use of alternative MG therapy is reduced or eliminated 1, 2, 3, or 4 weeks after administration of TIMP-MG. In various embodiments, the use of alternative MG therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-MG. In various embodiments, the use of alternative MG therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-MG.

[0193] The following analysis is intended to track the immune status or tolerance induction in subjects receiving TIMP-MG therapy. • The percentage of antigen-specific CD4+ T cells (CD154+CD137+effector memory CD4+ T cells / total effector memory CD4+ T cells). • The proportion of activated antigen-specific CD4+ T cells (CD38+CD154+CD137+effector memory CD4+ T cells / CD154+CD137+effector memory CD4+ T cells). ·Percentage of antigen-specific CD8+ T cells (CD154-CD137+CD8+ T cells / total CD8+ T cells). ·Percentage of activated antigen-specific CD8+ T cells (CD38+CD154-CD137+CD8+ T cells / CD154-CD137+CD8+ T cells). • The percentage of antigen-specific T regulatory cells (CD154-CD137+CD127-CD25+TIGIT+effector memory CD4+T cells / CD154-CD137+effector memory CD4+T cells). • Changes in the percentage of anti-acetylcholine receptor (AChR) antibody levels (a positive result of >0.02 nmol / L indicates autoimmune myasthenia gravis). • Changes in the percentage of anti-muscle-specific kinase (Musk) antibody levels (a positive result of >0.02 nmol / L indicates autoimmune myasthenia gravis).

[0194] In various embodiments, the immune tolerance status of the subject determined before administration of TIMP-MG serves as a baseline. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-MG. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, or 4 weeks prior to administration of TIMP-MG. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-MG.

[0195] In various embodiments, the immune tolerance status of the subject is determined after administration of TIMP-MG. In various embodiments, the immune tolerance status of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-MG. In various embodiments, the baseline status of the subject is determined from assays of one or more biological samples 1, 2, 3, or 4 weeks after administration of TIMP-MG. In various embodiments, the baseline status of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-MG. In various embodiments, the baseline status of the subject determined after administration of TIMP-MG is compared to the baseline. In various embodiments, the immune tolerance status of a subject determined after administration of TIMP-MG is compared to that of a healthy subject.

[0196] Pharmaceutical preparations The pharmaceutical compositions of this disclosure containing TIMP-MG and antigen as described herein may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline®, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and pharmaceutically acceptable surfactants. The additives used are selected, as necessary, from the above or combinations thereof, depending on the dosage form of this disclosure, but are not limited to these.

[0197] The formulation of the pharmaceutical composition will vary depending on the chosen route of administration (e.g., solution, emulsion). A suitable composition containing the therapeutic agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. Regarding solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions containing saline and a buffer medium. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oil. Intravenous vehicles may include various additives, preservatives, or liquids, nutrients, or electrolyte supplements.

[0198] Various aqueous carriers, such as sterile phosphate-buffered saline, bacteriostatic water, water, buffer water, 0.4% saline, and 0.3% glycine, may contain other proteins for enhanced stability, such as albumin, lipoproteins, and globulins, which have undergone mild chemical modifications.

[0199] Therapeutic formulations of inhibitors are prepared for storage by mixing the inhibitor of the desired purity in the form of a lyophilized formulation or aqueous solution with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin, gelatin The material comprises proteins such as tin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, algin, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0200] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0201] The aqueous suspension may contain the active compound mixed with excipients suitable for the preparation of the aqueous suspension. Such excipients may be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, and dispersing or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensate products of alkylene oxide and fatty acids, such as polyoxyethylene stearate, or condensate products of ethylene oxide and long-chain aliphatic alcohols, such as heptadecaethyl-enoxycetanol, or condensate products of ethylene oxide and partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monostearate, or condensate products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydride, such as polyethylene sorbitan monostearate. The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl, p-hydroxybenzoate.

[0202] The TIMP-MG described herein can be freeze-dried for storage and reconstituted in a suitable carrier before use.

[0203] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier, e.g., sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as 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 glycol, sodium lauryl acid, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0204] kit This disclosure also provides kits comprising one or more compounds or compositions packaged in a manner that facilitates their use in practicing the methods of this disclosure. In one embodiment, such a kit comprises a compound or composition described herein (e.g., a composition comprising TIMP alone or in combination with another agent), is packaged in a container such as a sealed bottle or vessel, and has a label affixed to the container or included in the package that describes the use of the compound or composition in practicing the methods. Preferably, the compound or composition is packaged in unit dosage form. The kit may further include a suitable device for administering the composition according to a specific route of administration or for practicing a screening assay. Preferably, the kit includes a label that describes the use of the particulate composition.

[0205] In further embodiments, the Disclosure provides a product or unit dosage form comprising (a) a composition of a substance comprising TIMP-MG as described herein, (b) a container comprising the composition, and (c) a label affixed to the container or a document included in the container, with reference to the use of TIMP-MG in the treatment of MG as described herein.

[0206] Additional aspects and details of this disclosure will be apparent from the following examples, which are intended to be illustrative rather than restrictive. [Examples]

[0207] Example 1: Example CNP-106 inhibits antigen-specific T cell responses in animal models. The efficacy of TIMP-MG (CNP-106) encapsulating AChR antigens with SEQ ID NOs. 3, 5, 8, 13, 19, 20, and 21 (Table 2) during tolerance induction was investigated in a therapeutic model of Mycobacterium Grammasum (MG). 8-11 week old C57BL / 6J mice were subcutaneously injected with California electric ray AChR and human major immunogenicity (MIR) peptide on day 0 (40 μg tAChR + 100 μg MIR peptide in CFA), day 21, and day 56 (20 μg tAChR + 100 μg MIR peptide in IFA). On days 42 and 49, the mice were injected with CNP-106 (1.25 mg / mouse or approximately 5 mg / kg HED and 0.5 mg / mouse or approximately 2 mg HED). CNP-embedded electric ray AChR (CNP-tAChR) was used as a positive control. Mice treated with physiological saline were used as a negative control.

[0208] Anti-AChR antibody titers were measured on days 28 and 56. Grip tests, clinical scores, and EMG were evaluated on days 28 and 70. On day 70, mice from each treatment group were sacrificed, their spleens were collected, and their T-cell response was evaluated.

[0209] CNP-106 treatment (1.25 mg / dose) significantly improved grip strength compared to the saline treatment group (Figure 1A). (*p<0.05, ***p<0.0005)

[0210] CNP-106-treated mice also showed a trend toward overall clinical score improvement compared to immunized saline-controlled mice (Figure 1B), electromyography (EMG) scores of mice from various treatment groups expressed as the percentage change in compound muscle action potentials (CMAP) (Figure 1C), and no change in body weight between different treatment groups (Figure 1D).

[0211] AChR-specific T cell responses were evaluated via ex-vivolicle responses from the spleen. Splenocytes from control and treated mice were cultured in medium alone or in two different concentrations of tAChR (5 μg / mL and 20 μg / mL), and the levels of secreted IFN-γ, IL-2, and IL-10 were assessed. CNP-106 treatment inhibits AChR-specific T cell responses. Treatment of mice immunized with 1.25 mg / dose of CNP-106 reduced levels of IL-2 (Figure 2A) (*p<0.05), IL-10 (Figure 2B) (*p<0.05), and IFN-gamma (Figure 2C).

[0212] Delayed-type hypersensitivity: C57BL / 6 mice were primed on day 0 with 100 μg (200 μL injection) of AChR-peptide emulsified in CFA. On days 0 and 7 after priming, mice were treated with CNP-106 administered intravenously at doses of 0.1 mg / house (0.4 mg / kg HED), 0.5 mg / mouse (2 mg / kg HED), 1.25 mg / mouse (5 mg / kg HED), and 2.5 mg / mouse (10 mg / kg HED). On day 14 after priming, mice were challenged intradermally with 10 μg of MG peptide (right ear, 10 μL injection) or ovalbumin (left ear, 10 μL injection). The thickness of the auricle of each ear was measured immediately after induction and 24 hours after induction using MG peptide and OVA. The DTH response was evaluated by calculating the change in auricle thickness (ΔT) in both ears of each mouse. CNP-106 significantly inhibited the DTH response compared to unloaded control particles at doses of 0.5 mg / mouse (2 mg / kg HED), 1.25 mg / mouse (5 mg / kg HED), and 2.5 mg / mouse (10 mg / kg HED) (Figure 3) (****p<0.00005).

[0213] Example 2: Phase IB / IIA trial of TIMP-MG This example describes a Phase 1b / 2a randomized, double-blind, placebo-controlled clinical trial to evaluate the safety, tolerability, pharmacodynamics (PD), and efficacy of CNP-106 in subjects with systemic myositis (MG). The trial consists of an escalation phase and an expansion phase with 42 and 180 trial days for screening.

[0214] CNP-106 consists of PLGA particles encapsulating 7AChRα and AChRε peptides (SEQ ID NOs: 3, 5, 8, 13, 19, 20, 21). CNP-106 particles have an average diameter of 400-800 nm and a negative zeta potential of -30 mV to -60 mV. CNP-106 particles are supplied as a lyophilized formulation. CNP-106 particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride) before administration.

[0215] Participants aged 18–75 years with systemic myasthenia gravis will be screened at least 42 days prior to enrollment in the study. If a participant is currently receiving standard treatment, they may continue their current standard treatment throughout the study, at the discretion of the principal investigator, regardless of the treatment group (TIMP-MG or placebo).

[0216] Screening is completed according to the event schedule (Figure 4). After completing the screening visit, subjects who meet all inclusion criteria and do not meet exclusion criteria will be enrolled in the study.

[0217] In this study, subjects with systemic MG who are eligible for registration will be defined based on the following inclusion criteria. 1. Subjects who are willing and able to provide written informed consent and privacy language approved by the Institutional Review Board (IRB) in accordance with domestic regulations. 2. Men and non-pregnant women aged 18-75. 3. Women of childbearing potential must agree not to become pregnant during the trial, have a negative pregnancy test result at the time of their screening visit, and agree to one of the following: • Start with initial screening and continue using two highly effective forms of contraception throughout the trial period. • Practice abstinence starting with the initial screening and continuing throughout the trial period. 4. Subjects with Myasthenia Gravis Foundation clinical classification classes III-IV (Cohort 1). If preliminary safety data obtained from Cohort 1 up to day 15 are successfully reviewed and approved by the Data Monitoring Committee (DMC), Cohort 2 will enroll subjects with MGFA clinical classification classes II-IV. 5. Subjects who test positive for anti-AChR antibodies by radioimmunoassay (RIA). 6. Patients with an MG-ADL score of ≥6 at screening and baseline visit, where ≥50% of the score is due to non-ocular symptoms. 7. Subjects with a QMG score of ≥ 11 at screening and baseline visit. 8. Participants taking steroids must agree not to change their steroid dosage until day 60 of the trial unless reviewed and approved by the medical monitor and the principal investigator. 9. If a subject is taking any medication used to treat symptoms of MG (e.g., corticosteroids, pyridostigmine), the subject must have been taking a stable dose for at least 90 days prior to enrollment and must agree not to increase their dose throughout the study period unless reviewed and approved by the medical monitor and the principal investigator. 10. Women who agree to not breastfeed at the initial screening stage and throughout the trial period. 11. This program is for women who agree not to donate eggs, starting with the initial screening and throughout the trial period. 12. Men who have a spouse or a partner capable of bearing children, and who have agreed that they and their spouse or partner will practice effective contraception as discussed with their physician by the study physician or study staff at the start of the screening and throughout the study period.

[0218] Participants will be randomized to the current cohort in a 2:1 ratio on day 1 and will receive two separate intravenous doses of either CNP-106 or placebo (0.9% sodium chloride injection, USP) on days 1 and 8. The study product will be administered by IV infusion over approximately 3-4 hours using a stepped infusion rate. Participants will be medically observed in the clinic for 4 hours post-infusion on days 1 and 8 for acute adverse events. Participants will be discharged 4 hours after infusion if all scheduled assessments on the day of visit are completed, vital signs (sitting or supine blood pressure, heart rate, and temperature) measured 4 hours post-infusion are within the expected range for the participant, and no other health concerns are noted by the principal investigator.

[0219] After administration, patients return to the clinic for immunosafety lab tests, PD measurements, QMG and MGC evaluations, AE evaluations, and medication changes according to the event schedule (Figure 4).

[0220] During the follow-up period, participants return to the clinic for PD measurement, QMG and MGC evaluation, AE evaluation, and medication changes according to the event schedule (Figure 4).

[0221] Participants will return to the clinic at the end of their trial visit for safety laboratory data collection (serological chemistry, hematology, coagulation, and urine analysis), PD measurement, QMG and MGC evaluation, and final evaluation of adverse events and medication changes.

[0222] The dose escalation phase will enroll up to three cohorts (approximately six subjects per cohort) at multiple dose levels. Subjects will be randomized in a 2:1 ratio to receive either CNP-106 or placebo (0.9% sodium chloride injection, USP) as a 200 mL intravenous infusion on days 1 and 8. The planned dose levels are as follows: Cohort 1: 150 mg Cohort 2: 350 mg Cohort 3: Additional dose levels recommended by the DMC

[0223] The expansion phase is planned to enroll subjects randomized in a 2:1 ratio to receive either placebo or a safe and tolerable dose level of CNP-106 identified in the escalation phase. An additional cohort will be randomized to receive a third dose of CNP-106 on day 90.

[0224] Participants will receive CNP-106 as a 200 mL intravenous infusion on days 1 and 8. Participants randomized to receive a booster dose will receive CNP-106 as a 200 mL intravenous infusion on days 1, 8, and 90. TIMP-MG will be administered using the following stepped infusion rates: 20 mL / hour for the first 15 minutes, 40 mL / hour for the next 15 minutes, and 80 mL / hour for the remaining infusions.

[0225] The subjects will be evaluated for safety, tolerability, disease progression (PD), and efficacy according to the event schedule (Figure 4). The primary objective of this study includes evaluating the safety and tolerability of CNP-106. The primary endpoint includes the frequency of adverse events (AEs) and serious adverse events (SAEs), as measured by MedDRA v. 25.0 or the latest version, or CTCAE v. 5.0 or the latest version.

[0226] Exemplary endpoints of this study include: change from baseline in Myasthenia Gravis Activities of Daily Living (MG-ADL) score at days 15, 60, 90, 120, and 180 (optional at days 365, 540, and 730); change from baseline in Quantitative Myasthenia Gravis (QMG) score at days 15, 60, 90, 120, and 180 (optional at days 365, 540, and 730); Change from baseline in the revised Quality of Life 15 (MG-QOL15r) score at day 180 (optionally selected at days 365, 540, and 730); change from baseline in healthcare use by hospital visits (MG exacerbation rate requiring rescue therapy with IVIG / PLEX) at days 60, 90, and 180 (optionally selected at days 365, 540, and 730); change from baseline at days 15, 60, 90, 120, and 180 (optionally selected at day 365, Change from baseline in the composite myasthenia gravis (MGC) score at days 540 and 730; change from baseline in antigen-specific (number or level) CD4+ and CD8+ T cells in PBMCs at days 15, 60, 90, 120, and 180 (optionally selected at days 365, 540, and 730); change from baseline in activated antigen-specific (number or level) CD4+ and CD8+ T cells in PBMCs at days 15, 60, 90, 120, and 180 (optionally selected at days 365, 540, and 730) This includes changes from baseline in CD4+ and CD8+ T cells (or levels); changes from baseline in anti-acetylcholine receptor (AChR) antibody levels at days 15, 60, 90, 120, and 180 (optionally at days 365, 540, and 730); and changes from baseline in anti-muscle-specific kinase (Musk) antibody levels at days 15, 60, 90, 120, and 180 (optionally at days 365, 540, and 730).

[0227] Clinical efficacy endpoints include MG-ADL score, QMG score, MG-QOL-15r score, healthcare utilization (MG exacerbation rate requiring rescue therapy with IVIG / PLEX), and MGC score.

[0228] The total duration of the study is a maximum of 222 days for the primary study (42 days for screening and 180 days for the study), plus an optional 508 days for the long-term safety follow-up period.

[0229] Safety assessments include the following: 1. A complete physical examination (PE) consisting of the following systems will be performed on the screening day, day 1, and day 8: cardiovascular, dermatological, ear, nose and throat, limbs, digestive system, musculoskeletal system, ophthalmological, neurological, and respiratory system. Height and weight will be obtained at the time of screening. 2. Vital signs at the time of screening, and on days 1, 8, 60, 90, 120, 180, 270, 365, 540, and 730. 3. Electrocardiograms taken at the time of screening, on day 1, and on day 8. 4. Exposure events (AEs) at the time of screening, followed by AEs on days 1, 8, 15, 60, 90, 120, 180, 270, 365, 540, and 730. 5. Safety laboratory tests (serological chemistry, hematology, coagulation, and urinalysis) at the time of screening, followed by tests at days 1, 8, 15, 60, 90, 120, 180, 180, 270, 365, 540, and 730. 6. During screening, cytokine (IL-1β, TNF-α, IL-6, MCP-1, MIP-1α, IFN-γ, IL-4, IL-10) and tryptase profiling was performed on days 1 and 8.

[0230] Laboratory / PD evaluations include the following: 1. Antigen-specific CD4+ and CD8+ T cells at the time of screening, followed by those at day 1, day 8, day 15, day 60, day 90, day 120, day 180, day 270, day 365, day 540, and day 730. 2. Activated antigen-specific CD4+ and CD8+ T cells at the time of screening, followed by those at day 1, day 8, day 15, day 60, day 90, day 120, day 180, day 270, day 365, day 540, and day 730. 3. Anti-AChR antibodies and anti-MuSK antibodies at the time of screening, followed by those at day 1, day 15, day 60, day 90, day 120, day 180, day 270, day 365, day 540, and day 730.

[0231] Clinical efficacy evaluation includes the following: 1. Symptom-driven physical examination on days 15, 60, 90, 120, 180, 270, 365, 540, and 730. 4. At the time of screening, followed by QMG, MG-ADL, MG-QOL-15r, and MGC scores on days 1, 8, 15, 60, 90, 120, 180, 270, 365, 540, and 730. 2. Steroid use on days 60, 90, 120, 180, 270, 365, 540, and 730 (average daily dose of prednisone). 3. Healthcare use at screening, and on days 60, 90, 120, 180, 365, and 730 was reported as hospital use within one year of the start of the study (MG exacerbation rate requiring rescue therapy with IVIG / PLEX).

[0232] Example 3. Patient selection criteria for CNP-106 treatment. PBMCs were obtained from 10 MG patients to determine if there was a correlation between HLA-positive restriction and correlation with AChR. Since CNP-106 encapsulates the AChR antigen, the presence of HLA-DRB1*03, HLA-DRB3*01, and HLA-DQB1*02 in subjects can be a criterion for inclusion in the CNP-106 clinical trial. 80% of patients in this study expressed HLA restriction for AChR, specifically HLA-DRB1*03, HLA-DRB3*01, and HLA-DQB1*02 (Figure 5). Inclusion criteria can identify patients who are likely to achieve efficacy with CNP-105. MG patients whose disease is triggered by autoreactivity to the AChR antigen are eligible for treatment with CNP-106 and can be identified based on HLA haplotypes in their blood.

[0233] Example 4. Process for preparing an MG peptide solution for the production of tolerant nanoparticles (TIMP-MG) encapsulating MG-related antigens. Acid-soluble AChR antigens ((AChRα(43-58), AChRα(68-113), AChRα(121-158), AChRα isoform α(324-357), AChRe(201-250), and AChRe(414-435)) were dissolved in 0.5N acetic acid containing individual AChR peptide concentrations of 2 mg / mL.

[0234] The base-soluble AChR antigen (AChRe(116~130)) is dissolved in 0.1% ammonium hydroxide with individual peptide concentrations of 8 mg / mL.

[0235] Acid and base-soluble antigens are aseptically filtered using a 0.2 μm nylon syringe filter. The filtered 6-peptide solution in 0.5 N acetic acid and the filtered 1-peptide solution in 0.1% ammonium hydroxide are combined in a 4:1 ratio to achieve a final individual peptide concentration of 1.6 mg / mL.

[0236] Example 5. Process for producing tolerant nanoparticles (TIMP-MG) encapsulating MG-related antigens. TIMP-MG was prepared using a double emulsion solvent evaporation process. A high-level production process flow diagram is shown in Figure 6. Briefly, the filtered peptide mixture was rapidly mixed with a 5% PLGA solution (50:50, molecular weight 10,000-60,000 Da) in ethyl acetate to produce a primary water-in-oil emulsion. A blend was prepared by mixing PVA (4% in water), PAA (Sigma Aldrich, 100 kDa, 35% wt), and ethyl acetate. The composition of the PVA / PAA / ethyl acetate blend was maintained at a pH of less than 4.0. The primary emulsion was then mixed with PVA / PAA / ethyl acetate to form a secondary oil-in-water emulsion. The primary and secondary emulsions were mixed by homogenization.

[0237] The solvent was removed from the secondary emulsion by evaporation under pressure for a total of at least 3-4 hours. The cured nanoparticles were then washed with sterile water and concentrated by filtration using a 20 μm filter. Sucrose and mannitol as cryoprotectants, and sodium citrate dihydrate as a buffer were added to the cured nanoparticles. The formulation was then freeze-dried.

[0238] The final TIMP-MG formulation was characterized, and its physicochemical properties, including particle diameter, zeta potential, size distribution, and total peptide content, were determined. The results of the TIMP-MG characterization are shown in Table 4. The TIMP-MG particles were examined using a scanning electron microscope, revealing a homogeneous composition of intact particles with smooth surfaces (Figure 7). [Table 4]

[0239] Example 6: Pharmaceutical composition of TIMP-MG The TIMP-MG pharmaceutical formulation or composition contains the individual components manufactured in 80g and 160g batch scales as listed in Table 5. [Table 5]

[0240] Example 7. Method for preparing MG antigen for the production of TIMP-MG. Acid-soluble AChR antigens ((AChRα(43~58), AChRα(68~113), AChRα(121~158), AChRα isoform α(324~357), AChRe(201~250), and AChRe(414~435)) are dissolved in 0.5N acetic acid containing 0.1% LMPG detergent.

[0241] The base-soluble AChR antigen (AChRe(116~130)) is dissolved in 0.1% ammonium hydroxide containing 0.1% LMPG detergent.

[0242] The dissolved antigen is aseptically filtered using a 0.2 μm nylon syringe filter. The filtered 6-peptide solution in 0.5 N acetic acid and 0.1% LMPG is combined with the filtered 1-peptide solution in 0.1% ammonium hydroxide containing 0.1% LMPG in a 4:1 ratio. The antigen is then rapidly mixed with a 5% PLGA solution in ethyl acetate (50:50, molecular weight 10,000-60,000 Da) to produce a primary water-in-oil emulsion. PVA (4% in water), PAA (Sigma Aldrich, 100 kDa, 35% wt), and ethyl acetate are mixed to produce a blend. The composition of the PVA / PAA / ethyl acetate blend is maintained at a pH of less than 4.0. The primary emulsion is then mixed with PVA / PAA / ethyl acetate to form a secondary oil-in-water emulsion. The primary and secondary emulsions are mixed by homogenization.

[0243] The solvent is removed from the secondary emulsion by evaporation under pressure for a total of at least 3-4 hours. The cured nanoparticles are then washed with sterile water and concentrated by filtration using a 20 μm filter. Sucrose and mannitol as cryoprotectants, and sodium citrate dihydrate as a buffer are added to the cured nanoparticles. The formulation is freeze-dried.

[0244] The final TIMP-MG formulation is characterized to determine its physicochemical properties, such as particle size, zeta potential, and total peptide content.

[0245] Therefore, it should be understood that the present invention is not limited to any specific embodiment disclosed, and is intended to encompass the spirit and scope of the invention as defined by the appended claims, all modifications within the above description, and / or shown in the appended drawings. As a result, only the limitations that appear in the appended claims should be imposed on this disclosure.

[0246] References 1.Conti-Fine,B.M.,Milani,M.,&Kaminski,H.J.Myasthenia gravis:past,present,and future.The Journal of clinical investigation,116(11),2843-2854(2006).

[0247] 2.Wang,Z.Y.,Karachunski,P.I.,Howard,J.F.,Jr.&Conti-Fine,B.M.Myasthenia in SCID mice grafted with myasthenic patient lymphocytes:role of CD4+ and CD8+ cells.Neurology 52,484-497(1999).

[0248] 3.Gilhus,N.E.,Skeie,G.O.,Romi,F.,Lazaridis,K.,Zisimopoulou,P.,&Tzartos,S.Myasthenia gravis-autoantibody characteristics and their implications for therapy.Nature reviews.Neurology,12(5),259-268(2016).

[0249] 4.Carr,A.S.,Cardwell,C.R.,McCarron,P.O.et al.A systematic review of population based epidemiological studies in Myasthenia Gravis.BMC Neurol 10,46(2010)

[0250] 5.Meliani A,Boisgerault F,Hardet R,et al.Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.Nat Commun,9(1):4098(2018).

[0251] 6.Farmakidis,C.,Pasnoor,M.,Dimachkie,M.M.,&Barohn,R.J.Treatment of Myasthenia Gravis.Neurologic clinics,36(2),311-337(2018).

[0252] 7.Barnett,C.,Herbelin,L.,Dimachkie,M.M.,&Barohn,R.J.(2018).Measuring Clinical Treatment Response in Myasthenia Gravis.Neurologic clinics,36(2),339-353.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 13.Bilati U,Allemann E,Doelker E.Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles.Eur J Pharm Sci.2005 Jan;24(1):67-75.

[0259] 14.Mejia SP,Sanchez A,Vasquez V,Orozco J.Functional Nanocarriers for Delivering Itraconazole Against Fungal Intracellular Infections.Front Pharmacol.2021 Jun 28;12:685391.

[0260] 15.Zielinska A,Carreiro F,Oliveira AM,Neves A,Pires B,Venkatesh DN,Durazzo A,Lucarini M,Eder P,Silva AM,Santini A,Souto EB.Polymeric Nanoparticles:Production,Characterization,Toxicology and Ecotoxicology.Molecules.2020 Aug 15;25(16):3731.

Claims

1. A composition comprising tolerance-immunomodulated particles-myasthenia gravis (TIMP-MG) particles encapsulating one or more myasthenia gravis (MG)-related antigens, portions thereof, or combinations thereof.

2. The composition according to claim 1, wherein the particles include a biodegradable polymer.

3. The composition according to claim 1 or 2, wherein the biodegradable polymer is polyglycolic acid (PGA), poly(lactidocoglycolide) (PLG), polylactic acid (PLA), copolymer of PLG and PLA (PLGA), polycaprolactone (PCL), polystyrene, polysebacic acid (PSA), poly(lacticcosebacic) acid (PLSA), polypropylene sulfide, cyclodextran, polyethylene glycol (PEG), chitosan, polysaccharide, one or more lipids, liposomes, diamond, iron, zinc, cadmium, gold, or silver.

4. The composition according to any one of claims 1 to 3, wherein the particles have a negative zeta potential.

5. The composition according to claim 4, wherein the particles have a zeta potential of -1 to -100 mV.

6. The composition according to claim 4 or 5, wherein the particles have a zeta potential of -30 to -80 mV.

7. The composition according to any one of claims 1 to 6, wherein the particle size is 100 nm to 1000 nm.

8. The composition according to claim 7, wherein the particle size is 400 to 800 nm.

9. The composition according to any one of claims 1 to 8, wherein the antigen comprises one or more proteins, peptides, or one or more antigenic epitopes thereof.

10. The composition according to claim 9, wherein the antigen is AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or a portion thereof, or one or more antigenic epitopes thereof.

11. The composition according to claim 9, wherein the antigen is an MG-related antigen shown in Table 1.

12. The composition according to claim 9, wherein the antigen is an AChR protein subunit and / or a portion thereof, or one or more antigenic epitopes thereof.

13. The composition according to claim 12, wherein the AChR antigen epitope is selected from the group consisting of SEQ ID NOs: 1 to 21.

14. The composition according to claim 13, wherein the AChR antigen epitope comprises a combination of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and / or SEQ ID NO:

21.

15. A method for inducing tolerance in a subject requiring tolerance induction, comprising administering to the subject a composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more myasthenia gravis (MG) related antigens, a portion thereof, or a combination thereof.

16. The method according to claim 15, wherein the particles include polyglycolic acid (PGA), poly(lactide-co-glycolide) (PLG), polylactic acid (PLA), PLG and PLA copolymer (PLGA), polycaprolactone (PCL), polystyrene, polysebacic acid (PSA), poly(lactic-co-sebacic) acid (PLSA), polypropylene sulfide, cyclodextran, polyethylene glycol (PEG), chitosan, polysaccharide, one or more lipids, liposomes, diamond, iron, zinc, cadmium, gold, or silver.

17. The method according to claim 15 or 16, wherein the particles have a negative zeta potential.

18. The method according to any one of claims 15 to 17, wherein the particles have a zeta potential of 0 mV to -100 mV.

19. The method according to any one of claims 15 to 18, wherein the particles have a zeta potential of -30 mV to -100 mV.

20. The method according to any one of claims 15 to 19, wherein the size of the particles is 100 nm to 1000 nm.

21. The method according to any one of claims 15 to 20, wherein the size of the particles is 400 nm to 800 nm.

22. The method according to any one of claims 15 to 21, wherein the antigen comprises one or more proteins, peptides, or one or more antigenic epitopes thereof.

23. The method according to claim 22, wherein the antigen is AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or a portion thereof, or one or more antigenic epitopes thereof.

24. The method according to claim 22, wherein the antigen is an MG-related antigen shown in Table 1.

25. The method according to claim 22, wherein the antigen is an AChR protein subunit and / or a portion thereof, or one or more antigenic epitopes thereof.

26. The method according to claim 25, wherein the AChR antigen epitope is selected from the group consisting of SEQ ID NOs: 1 to 21.

27. The method according to claim 26, wherein the AChR antigen epitope includes a combination of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and / or SEQ ID NO:

21.

28. The method according to any one of claims 15 to 27, wherein the subject has an autoimmune disease.

29. The method according to any one of claims 15 to 28, wherein the autoimmune disease is myasthenia gravis.

30. The method according to any one of claims 15 to 29, wherein administration of the composition reduces the MG-related immune response.

31. The method according to claim 30, wherein the immune response is an inflammatory immune response.

32. The method according to claim 30 or 31, wherein the immune response is a humoral immune response.

33. The method according to claim 30 or 31, wherein the immune response is an adaptive immune response.

34. The method according to claim 30 or 31, wherein the immune response is an innate immune response.

35. The method according to any one of claims 30 to 34, wherein the immune response is a T cell, B cell, monocyte, macrophage, neutrophil, basophil, or eosinophil response.

36. The method according to any one of claims 30 to 34, wherein the immune response is an antibody response.

37. The method according to claim 36, wherein the antibody response is the formation of an AChR antibody against the AChR protein, antigen, epitope, or a portion thereof.

38. The method according to claim 36, wherein the antibody response is the formation of a MuSK antibody against the MuSK protein, antigen, epitope, or a portion thereof.

39. The method according to any one of claims 15 to 38, wherein the composition is administered intravenously, intramuscularly, intraocularly, intraperitoneally, transdermally, transnasally, orally, and / or subcutaneously.

40. A method for treating myasthenia gravis (MG) in a subject, comprising administering to the subject a tolerant immunomodulatory particle (TIMP-MG) containing one or more MG-related antigens.

41. The method according to claim 40, wherein the MG-related antigen is AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or a portion thereof, or one or more antigenic epitopes thereof.

42. The method according to claim 40 or 41, wherein the antigen is an MG-related antigen selected from the group including Table 1.

43. The method according to any one of claims 40 to 42, wherein the antigen is an AChR protein subunit and / or a portion thereof, or one or more antigenic epitopes thereof.

44. The method according to any one of claims 40 to 43, wherein the AChR antigen epitope is selected from the group consisting of SEQ ID NOs: 1 to 21.

45. The method according to claim 43 or 44, wherein the AChR antigen epitope includes a combination of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO:

21.

46. The method according to any one of claims 40 to 45, wherein the TIMP-MG particles have an average diameter of 100 nm to 1000 nm.

47. The method according to claim 46, wherein the TIMP-MG particles have an average diameter of 400 nm to 800 nm.

48. The method according to any one of claims 40 to 47, wherein the TIMP-MG particles have a negative zeta potential.

49. The method according to claim 48, wherein the particles have a negative zeta potential of 0 mV to -100 mV.

50. The method according to claim 49, wherein the particles have a negative zeta potential of -30 mV to -80 mV.

51. The method according to any one of claims 40 to 50, wherein TIMP-MG is administered at a concentration of 0.0005 mg / mL to 50 mg / mL.

52. The method according to any one of claims 40 to 50, wherein TIMP-MG is administered at a dose level of 0.1 mg / kg, 0.25 mg / kg, 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.

53. The method according to any one of claims 40 to 50, wherein TIMP-MG is administered in a dose level of 0.1 mg to 800 mg.

54. The method according to claim 53, wherein TIMP-MG is administered in doses of 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.

55. The method according to any one of claims 40 to 54, wherein TIMP-MG is administered in a single dose or multiple doses.

56. The method according to any one of claims 40 to 55, wherein TIMP-MG 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 per year.

57. The method according to any one of claims 40 to 56, wherein TIMP-MG is administered in two doses at one-week intervals.

58. The method according to any one of claims 40 to 56, wherein TIMP-MG is administered in two doses at one-week intervals, followed by a booster dose administered as a single dose once every three months.

59. The method according to claims 40 to 58, wherein TIMP-MG is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

60. The method according to any one of claims 15 to 59, wherein administering TIMP-MG to a subject reduces or improves one or more symptoms of MG.

61. The method according to claim 60, wherein one or more of the symptoms of MG are selected from the group consisting of drooping eyelids (ptosis), double vision, difficulty making facial expressions, chewing problems, difficulty swallowing, dysarthria (slurred speech), shortness of breath, difficulty breathing, muscle fatigue, skeletal muscle weakness, neck weakness, limb weakness, swallowing difficulties, dysphonia, weakening of the neuromuscular junction, synaptic signaling disorders, muscle damage, anti-Musk antibody, anti-AChR antibody, increased activated CD4+ cells in PBMCs and increased activated CD8+ T cells in PBMCs, increased myasthenia gravis activity of daily living score, quantitative MG score, MG quality of life 15 revised score, and increased MG composite score.

62. The method according to any one of claims 40 to 61, wherein administration of TIMP-MG to a subject reduces the duration and / or severity of the inflammatory immune response to the MG antigen.

63. The method according to claim 62, wherein the inflammatory immune response is a T cell, B cell, or myeloid cell response.

64. The method according to claim 62 or 63, wherein the inflammatory immune response is assayed from one or more biological samples obtained from the subject.

65. The method according to claim 64, wherein the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

66. The method according to any one of claims 40 to 65, wherein administration of TIMP-MG to a subject reduces the levels of antigen-specific CD4+ and CD8+ T cells.

67. Administering TIMP-MG raises the aforementioned levels of antigen-specific CD4+ and / or CD8+ T cells from 1% to 100% (for example, about 1%, about 2%, about 5%, about 10%, 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% (including all values ​​and ranges between these values)). The method according to claim 66, reducing to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2 to 1 / 100 (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

68. The method according to any one of claims 40 to 65, wherein administration of TIMP-MG to a subject reduces the levels of activated antigen-specific CD4+ and CD8+ T cells.

69. Treatment with TIMP-MG raises the aforementioned levels of activated antigen-specific CD4+ and / or CD8+ T cells from 1% to 100% (for example, about 1%, about 2%, about 5%, about 10%, 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% (including all values ​​and ranges between these values)). The method according to claim 68, which reduces the amount to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2 to 1 / 100 (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

70. The method according to any one of claims 40 to 65, wherein administration of TIMP-MG to a subject increases the level of antigen-specific T regulatory cells.

71. The method according to any one of claims 40 to 65, wherein administering TIMP-MG to a subject reduces the level of anti-AChR antibody.

72. Treatment with TIMP-MG reduces the aforementioned levels of anti-AChR antibody to 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values)), 10% to 95%, 15% to 9%. The method according to claim 71, which reduces the value to 0%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

73. The method according to any one of claims 40 to 65, wherein administering TIMP-MG to a subject reduces the level of anti-MuSK antibody.

74. Treatment with TIMP-MG reduces the aforementioned levels of anti-MuSK antibody to 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values)), 10% to 95%, 15% to 9%. The method according to claim 73, which reduces the amount to 0%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or about 2 to 1 / 100 (for example, about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

75. The method according to claim 40, wherein treatment with TIMP-MG reduces healthcare use by hospital visits and reduces the rate of MG exacerbations or rescue therapy with IVIG / PLEX.

76. The method according to claim 40, wherein treatment with TIMP-MG reduces the use of alternative MG therapy.

77. The method according to claim 40, wherein improvement in one or more symptoms of MG is determined using the MG-ADL score, QMG score, GM-QOL-15r score, healthcare use (MG exacerbation rate requiring rescue therapy with IVIG / IPLEX), and / or the MGC score.

78. The method according to claim 40, wherein TIMP-MG administration is based on the subject's gene profile / HLA haplotype, Myasthenia Gravis Foundation (MGFA) clinical classification score, baseline MG-ADL score, baseline QMG score, baseline MGC score, symptoms, drugs / therapeutic agents / medicines administered concurrently or previously, comorbidities or active diseases, presence / level of anti-AChR antibodies, presence / level of anti-MuSK antibodies or other MG-related antibodies.

79. A method for preparing a composition comprising particles encapsulating one or more MG-related antigens, wherein the method is (a) To produce an aqueous solution of one or more MG-related antigens, (b) Mixing the aqueous solution from step (a) with the oil phase containing the polymer to produce a primary emulsion, (c) Mixing the primary emulsion with a mixture containing one or more surfactants and / or stabilizers to form a secondary emulsion, A method comprising (d) curing the secondary emulsion of step (c) by evaporation to obtain cured polymer nanoparticles that encapsulate MG-related antigens within a core.

80. (e) The method according to claim 79, further comprising filtering, washing, and concentrating the nanoparticles.

81. (f) The method according to claim 79 or 80, further comprising freeze-drying the nanoparticles.

82. The method according to claim 79, wherein the aqueous solution in step (a) contains a solvent.

83. The solvent in step (a) is acetic acid, sulfuric acid, hydrochloric acid, nitric acid, formic acid, benzoic acid, ascorbic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid, trifluoroacetic acid, fluoroacetic acid, tartaric acid, lactic acid, gluconic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, polystyrenesulfonic acid, hydrobromic acid, hydroiodic acid, hypochlorous acid, chloric acid, chloric acid, perchloric acid, fluorosulfuric acid, fluoroantimonic acid, fluoroboric acid, hexafluorophosphate, chromic acid, li The method according to claim 80 or 81, selected from the group comprising: nic 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.

84. The method according to claim 82 or 83, wherein one or more MG-related antigens of step (a) can be dissolved in the same solvent or different solvents.

85. The method according to claims 82 to 84, wherein the MG-related antigen of step (a) or more is dissolved in an acid or base.

86. The method according to claim 85, wherein one or more MG-related antigens dissolved in an acid and a base are mixed in a ratio of 100:1, 50:1, 25:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:

1.

87. The method according to any one of claims 79 to 86, wherein the mixture of steps (b) and (c) comprises one or more solvents.

88. The method according to claim 87, wherein the solvent is an organic solvent or an inorganic solvent.

89. The method according to claim 87 or 88, wherein the solvent is the same.

90. The method according to claim 87 or 88, wherein the solvent is different.

91. The method according to any one of claims 88 to 90, wherein the organic solvent is selected from the group comprising acetone, ethanol, methylene chloride (dichloromethane), dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid.

92. The method according to claims 79 to 91, wherein the emulsion obtained from step (b) is a water-in-oil emulsion.

93. The method according to claims 79 to 91, wherein the emulsion obtained from step (c) is an oil-in-water emulsion.

94. The method according to claims 79 to 93, wherein the polymer in step (b) is a biodegradable polymer.

95. The method according to claim 94, wherein the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(milk-co-glycol) (PLGA), poly(milk-co-sebacin) acid (PLSA), poly(glycol-co-sebacin) acid (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharide, or lipid.

96. The method according to any one of claims 79 to 95, wherein the surfactant or stabilizer in step (c) is anionic, cationic, or nonionic.

97. The method according to claim 96, wherein 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 TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, sodium cholate, or enantiomers thereof), methylcellulose, hydroxyethylcellulose, hydroxyprolylcellulose, hydroxypropylmethylcellulose, gelatin, carbomer, or sulfate polymer.

98. The method according to any one of claims 79 to 97, wherein the primary emulsion in step (b) is obtained by homogenization.

99. The method according to any one of claims 79 to 97, wherein the primary emulsion in step (b) is obtained by ultrasonic treatment.

100. The method according to any one of claims 79 to 99, wherein the secondary emulsion in step (c) is obtained by homogenization.

101. The method according to any one of claims 79 to 99, wherein the secondary emulsion in step (c) is obtained by ultrasonic treatment.

102. The method according to claim 98 or 100, wherein 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, or 600 seconds.

103. The method according to claim 99 or 101, wherein the ultrasonic treatment 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.

104. The method according to any one of claims 79 to 103, wherein the curing of the nanoparticles in step (d) is performed by evaporation of the solvent.

105. The method according to claim 104, wherein the evaporation is active evaporation or passive evaporation.

106. The method according to claim 105, wherein the active evaporation is vacuum-driven evaporation.

107. The method according to claim 106, wherein the vacuum-driven evaporation is performed under high pressure or low pressure.

108. The method according to claim 105, wherein passive evaporation is performed by stirring.

109. The method according to any one of claims 104 to 108, wherein the 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.

110. The method according to any one of claims 80 to 109, wherein filtering, washing, and concentrating the nanoparticles in step (e) is performed by filtration, gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, or a combination thereof.

111. The method according to any one of claims 79 to 110, wherein the particles have a negative zeta potential.

112. The method according to claim 111, wherein the zeta potential of the particle is about 0 to -100 mV.

113. The method according to claim 112, wherein the zeta potential of the particle is approximately -30 mV to -80 mV.

114. The method according to any one of claims 79 to 113, wherein the particles have a diameter of about 0.3 μm to 3 μm.

115. The method according to claim 114, wherein the particles have a diameter of about 0.3 μm to 1 μm.

116. The method according to claim 115, wherein the particles have a diameter of about 0.4 μm to 1 μm.

117. The method according to any one of claims 79 to 114, wherein at least 90% of the particles have a diameter of about 0.3 μm to 3 μm.

118. The method according to claim 117, wherein at least 90% of the particles have a diameter of about 0.3 μm to 1 μm.

119. The method according to claims 79 to 116, wherein at least 90% of the particles have a diameter of about 0.4 μm to 1 μm.

120. The method according to claims 79 to 114, wherein at least 50% of the particles have a diameter of about 0.3 μm to 3 μm.

121. The method according to claim 120, wherein at least 50% of the particles have a diameter of about 0.3 μm to 1 μm.

122. The method according to claim 121, wherein at least 50% of the particles have a diameter of about 0.4 μm to 1 μm.

123. The method according to any one of claims 79 to 114, wherein at least 10% of the particles have a diameter of about 0.3 μm to 3 μm.

124. The method according to claim 123, wherein at least 10% of the particles have a diameter of about 0.3 μm to 1 μm.

125. The method according to any one of claims 79 to 124, wherein one or more MG-related antigens encapsulated in the particle composition are approximately 0.1 to 100 μg / mg.

126. The method according to any one of claims 79 to 125, wherein one or more MG-related antigens are AChR protein, muscle tyrosine kinase (MuSK) protein, low-density lipoprotein receptor-related protein 4 (LRP4) protein, agrin protein (AGRN), cortactin protein (CTTN), titin protein (TTN), ryanodine receptors (RYR1, RYR2, RYR3), and / or parts thereof, or one or more antigenic epitopes thereof.

127. The method according to any one of claims 79 to 126, wherein the antigen is an MG-related antigen selected from the group including Table 1.

128. The method according to any one of claims 79 to 127, wherein the antigen is the AChR protein subunit and / or a portion thereof, or one or more antigenic epitopes thereof.

129. The method according to any one of claims 79 to 128, wherein the AChR antigen epitope is selected from the group consisting of SEQ ID NOs: 1 to 21.

130. The method according to any one of claims 79 to 129, wherein the AChR antigen epitope includes a combination of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and / or SEQ ID NO:

21.

131. The method according to any one of the prior claims, wherein the manufacturing batch size is 0.01 g to 100 kg.

132. A composition comprising particles encapsulating one or more MG-related antigens, prepared by the method described in any one of claims 79 to 131.

133. The composition according to claim 132, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

134. The composition according to claim 133, wherein the excipients are sucrose, mannitol, and sodium citrate.

135. A pharmaceutical composition comprising negatively charged particles encapsulating myasthenia gravis (MG)-associated antigen, sucrose, mannitol, and sodium citrate.

136. A method for treating a subject having myasthenia gravis (MG), comprising administering the composition according to claim 132 or 135.

137. A composition comprising liposomes encapsulating one or more myasthenia gravis (MG) related antigens, portions thereof, or combinations thereof.

138. The composition according to claim 137, wherein the liposomes are negatively charged.

139. The composition according to claim 137 or 138, wherein the liposomes have a negative zeta potential.

140. The composition according to claim 139, wherein the negative zeta potential is between -100 mV and 0 mV.

141. The composition according to claim 140, wherein the negative zeta potential is -80 mV to -30 mV.

142. The composition according to claims 137 to 141, wherein the MG-related antigen is selected from the group including Table 1.

143. The composition according to claims 137 to 141, wherein the AChR antigen epitope is selected from the group consisting of SEQ ID NOs: 1 to 21.

144. The composition according to claims 137 to 141, wherein the AChR antigen epitope includes a combination of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO:

21.

145. The composition according to claims 137 to 144, wherein the size of the liposomes is 100 to 1000 nm.

146. A method for inducing tolerance in a subject requiring tolerance induction, comprising administering to the subject a composition according to any one of claims 137 to 145.

Images