TREATMENT BY HIGHLY SIALYLATED IgG COMPOSITION

Abstract

To provide a method for treating a patient by using an IgG pharmaceutical highly added with sialic acid.SOLUTION: A method for treating a disease includes administering an hsIgG pharmaceutical to a subject by 1% to 10% dosage of an effective dosage of IVIG in order to treat the disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Application No. 62 / 744,536, filed October 11, 2018. and the benefit of U.S. Provisional Application No. 62 / 879,930, filed July 29, 2019. The entire contents of the foregoing are incorporated herein by reference. [Background technology]

[0002] Intravenous immunoglobulin (IVIg or IVIG; CAS number: 9007-83-4) , commercially available therapeutic products consisting primarily of human immunoglobulin G (IgG) (e.g., Bivig am®, Carimune®, Cuvitru®, Fl ebogamma (registered trademark), Gammagard (registered trademark), GamaSTAN ( Registered trademark), Gammaked (registered trademark), Gammaplex (registered trademark), Gam unex-C®, Hizentra®, Hyqvia® , Octagam® and Privigen®). IVIG is , prepared from pooled plasma of several thousand healthy donors and therefore This ensures that the diversity of the donor's blood exceeds that of the individual donor. , are used to treat a wide variety of chronic autoimmune and systemic inflammatory conditions. As a sex indicator, idiopathic thrombocytopenic purpura (idiopathic thrombocytopenic purpura, ITP), Kawasaki disease, Guillain-Barré syndrome and other autoimmune neuropathies, myasthenia gravis, These include dermatomyositis, as well as several rare diseases. The exact mechanism of action of IVIg is unclear. Various studies have demonstrated that components of the innate and adaptive immune systems (4, 6) have documented a series of non-mutually exclusive mechanisms that regulate dendritic cells, natural killer cells, regulatory T cells, B cells, and the monocyte / macrophage system and by its action on the immune system, including inflammatory cytokines, chemokines, and pathogenic autoantibodies. Inhibition or neutralization of which soluble factors have been shown to mediate anti-inflammatory responses. Summary of the Invention [Means for solving the problem]

[0003] The present application provides highly sialylated I, which is approximately 1% to 10% of the effective dose of IVIG. The dose of IgG (hsIgG) preparation is effective in treating diseases treated with IVIG. The highly sialylated IgG preparations are based in part on the surprising discovery that they can Therefore, similar IVIG can be prepared from IgG subclasses. It contains a heterogeneous mixture and a wide array of antibodies expected to be present in human serum. When hsIgG is prepared from IVIG, a large number of donors have a high Ig repertoire Ensure diversity.

[0004] Highly sialylated IgG (hsIgG) preparations are characterized by the branched glycans on IgG antibodies. At least 60% of the amino acids were linked via NeuAc-α2,6-Gal terminal bonds. It has sialic acid in both the alpha 1,3 branched and alpha 1,6 branched (i.e., sialic acid In other words, at least 60% of the branched glycans However, it has sialic acid on each branch, and this sialic acid is NeuAc, which is bonded by α2,6 linkage. IgG antibodies have highly sialylated N297 and N3 positions in the Fc domain. The branched structure on the Fc domain of the IgG antibody has glycosylation sites in the IgG preparation. At least 60% of the glycans are linked via terminal NeuAc-α2,6-Gal bonds. IgG antibodies also contain sialic acid in both the α1,3 and α1,6 branches of the Fab region. The glycans may have branched glycans on the glycan region, and at least 50% of these branched glycans may be Neu Alpha 1,3 and alpha 1,6 linked via Ac-α2,6-Gal terminal bonds It has sialic acid on both branches.

[0005] hsIgG preparations also contain highly sialylated IVIG when prepared from IVIG. It may also be referred to as IG or hypersialylated IVG (hsIVIG).

[0006] In some embodiments, the hsIgG preparations used in the methods described herein are I Contains IgG, and at least 60% (70%, 75%, 80%, 85%, 90%, or 95%) of the α1,3- and α1,6-branched sialic acids In some embodiments, at least 65%, 70%, or 80% of the Fc glycans on the IgG are 75%, 80%, 85%, 90% or 95% of the α1,3 and α1,6 branches In some embodiments, at least one of the Fab branched glycans on an IgG has sialic acid. In both cases, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the It has both triantennary and α1,6 branched sialic acids. In some cases, the branched structures on IgG At least 80% of the glycans have sialic acid in both α1,3 and α1,6 branches In some embodiments, at least 85% of the Fc glycans on an IgG are α1,3 In some embodiments, the sialic acid on the IgG is present at both the branched and α1,6 branched sites. At least 60% of the Fab branched glycans are α1,3- and α1,6-branched. Contains alkane.

[0007] In some embodiments, at least 90%, 92% of the protein in the hsIgG preparation , 93%, 94% or 95% w / w is IgG.

[0008] In some embodiments, the present invention relates to a method for treating a disease, the method comprising: A composition containing an hsIgG preparation is administered to a subject at a dose that is 1% to 10% of the effective dose of IVIG. In some embodiments, the effective dose of IVIG is 400 mg / kg g, 500mg / kg, 600mg / kg, 1000mg / kg, or 2000mg / kg In some embodiments, the composition comprising the hsIgG formulation is about 2, 3, 4, 5 , 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 6 0, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 1 20, 125, 130, 135, 140, 145, 150, 155, 160, 165, 1 70, 175, 180, 185, 190, 195, or 200, 250, 300, 350 , 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950, 975, or 1000 mg / kg. In embodiments, the composition comprising the hsIgG formulation is administered daily, weekly, twice weekly, biweekly, monthly, semi-monthly, or semi-monthly. Every, every other month, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 14 days once a day, once every 21 days, once every 28 days, once a day for two consecutive days in a 28-day cycle, or Administered at the same frequency as FDA-approved IVIG doses. In some cases, hsIgG preparations is administered less frequently than the effective or approved frequency of IVIG. In some embodiments, the compositions containing the hsIgG formulations are administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the composition is administered in a single dose. In some cases, the composition is administered in multiple doses.

[0009] In some embodiments, the disease is an inflammatory disease. In some embodiments, the subject suffers from a primary antibody deficiency. In some embodiments, the disease is associated with the presence of autoantibodies.

[0010] In some embodiments, the disease is a neurological disease. The diseases include dermatomyositis, Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy. ammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (mul from the group consisting of unifocal motor neuropathy (MMN), myasthenia gravis, and stiff-person syndrome be selected.

[0011] In some embodiments, the disease is immune cytopenia, parvovirus B19 associated erythropenia Hypogammaglobulinemia secondary to myeloma and chronic lymphocytic leukemia, and after bone marrow transplantation.

[0012] In some embodiments, the disease is vasculitis, systemic lupus erythematosus, erythematosis, SLE), mucous membrane pemphigoid, and uveitis, In science, to treat Kawasaki syndrome, dermatomyositis, toxic epidermal necrolysis, and bullous disease Most commonly used.

[0013] In some embodiments, the disease is a condition that is FDA-approved for treatment with IVIG. In some embodiments, the dose is 1% to 1% of the FDA-approved IVIG dose for the disease. 0% (e.g., 1%-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1 ~4%, 1~3%, 1~2%, 2~10%, 2~9%, 2~8%, 2~7%, 2~6%, 2-5%, 2-4%, 2-3%, 3-10%, 3-9%, 3-8%, 3-7%, 3-6% , 3-5%, 3-4%, 1-2%, 3%, 2%, or 1%). The FDA approved doses of IVIG are 200 mg / kg, 400 mg / kg, and 500 mg / kg. kg, 600mg / kg, 1000mg / kg, or 2000mg / kg. In some embodiments, the composition comprising the hsIgG formulation comprises about 2, 3, 4, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 1 30, 135, 140, 145, 150, 155, 160, 165, 170, 175, 1 80, 185, 190, 195, or 200, 250, 300, 350, 400, 450 , 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 , 975, or 1000 mg / kg. The composition containing the IgG preparation may be administered daily, weekly, twice weekly, every other week, monthly, semi-monthly, bimonthly, or 3 times a day. Every day, every 4 days, every 5 days, every 6 days, every 7 days, once every 14 days, once every 21 days Once every 28 days, once daily for 2 consecutive days in a 28-day cycle, or FDA-approved IVI In some embodiments, the hsIgG formulation is administered at the same dosing frequency as the IgG dose. The composition is administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the composition comprises: In some embodiments, the composition is administered in multiple doses.

[0014] In some embodiments, the disease is selected from the group consisting of: myocarditis, acute motor axonal neuropathy, painful steatosis, Anti-glomerular basement membrane nephritis; Goodpasture's syndrome, Antiphospholipid syndrome (APS, APLS), Anti-synthetase antibody syndrome; myositis, ILD, ataxic neuropathy (acute and chronic), Autoimmune enteropathy (AIE), autoimmune neutropenia, autoimmune retinopathy, autoimmune thyroiditis, autoimmune urticaria, dermatitis herpetiformis, Acquired epidermolysis bullosa, essential mixed cryoglobulinemia, Granulomatosis with polyangiitis (GPA), Mixed connective tissue disease (MCTD), Neuromyotonia, optic neuritis, paraneoplastic cerebellar degeneration, Anti-N-Methyl-D-Aspartate (anti-NMDA) receptor somatic encephalitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, chronic inflammatory demyelinating polyradiculoneuropathy, dermatomyositis, gestational pemphigoid, Graves' disease, Guillain-Barré syndrome, IgG4-related disease Lambert-Eaton myasthenic syndrome, lupus nephritis, Myositis, multifocal motor neuropathy, myasthenia gravis, Neuromyelitis optica, pemphigus vulgaris, Polymyositis, and Systemic lupus erythematosus (SLE).

[0015] In some embodiments, the disease is selected from the group consisting of: Acute disseminated encephalomyelitis (ADEM), autoimmune angioedema (acquired angioedema type II), Autoimmune hepatitis (types I and II), autoimmune hypophysitis; lymphocytic hypophysitis; Autoimmune inner ear disease (AIED), Evans syndrome, Graves' ophthalmopathy, Hashimoto's encephalopathy, IgA vasculitis (IgAV), latent autoimmune hepatitis, Linear IgA disease (LAD), lupus vasculitis, Membranous glomerulonephritis, Microscopic polyangiitis (MPA), Mooren's ulcer, Morphea, opsoclonus-myoclonus syndrome, Oldo's thyroiditis, relapsing rheumatism, Paraneoplastic opsoclonus-myoclonus ataxia with neuroblastoma, Pediatric Autoimmune Neuropsychiatric Disorders Disorder Associated with Streptococcus, PANDAS) Postpericardiotomy syndrome, Primary biliary cirrhosis (PBC), Rasmussen's encephalitis, rheumatoid vasculitis, Schnitzler syndrome, Sydenham chorea, Undifferentiated connective tissue disease (UCTD), and Miller-Fisher syndrome.

[0016] In some embodiments, the composition comprises an hsIgG preparation, wherein the branched IgG on the Fab domain At least 60% of the glycans are linked via terminal NeuAc-α2,6-Gal bonds. The α1,3 arm and α1,6 arm both have sialic acid, and the branched Fc domain At least 60% of the glycans are linked via terminal NeuAc-α2,6-Gal bonds It has sialic acid in both the α1,3 arm and the α1,6 arm.

[0017] Administering hsIgG preparations at a dose of 10% or less than the effective dose of IVIG Disclosed herein are methods for treating CIDP in a subject with CIDP, including In some embodiments, the effective dose of IVIG is 200-2000 mg / kg. In some embodiments, the hsIgG formulation is administered at a dose that is 10% of the effective dose of IVIG. In some embodiments, the hsIgG formulation is administered at 1% to 1% of the effective dose of IVIG. 10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1 In some embodiments, the hsIgG formulation is administered at a dose of about 2% or 1%. , 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 5 0, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 , 115, 120, 125, 130, 135, 140, 145, 150, 155, 160 , 165, 170, 175, 180, 185, 190, 195, or 200 mg / kg It is administered in doses.

[0018] Administration of hsIgG preparations at a dose of 10% or less than 10% of the effective dose of IVIG is recommended. Disclosed herein are methods for treating ITP in a subject with ITP, including: In some embodiments, the effective dose of IVIG is 1000-2000 mg / kg. In some embodiments, the hsIgG formulation is administered at a dose that is 10% of the effective dose of IVIG. In some embodiments, the hsIgG formulation is 1% to 10%, 1% to 9% of the effective dose of IVIG. , 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 2-10 %, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2-4%, 2-3%, 3-1 0%, 3-9%, 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 1-2%, 2% In some embodiments, the hsIgG formulation is administered at a dose of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 5 5, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 11 5, 120, 125, 130, 135, 140, 145, 150, 155, 160, 16 at doses of 5, 170, 175, 180, 185, 190, 195, or 200 mg / kg It is administered.

[0019] Administration of hsIgG preparations at a dose of 10% or less than 10% of the effective dose of IVIG is recommended. Disclosed herein are methods for treating wAIHA in a subject with wAIHA, including: In some embodiments, the effective dose of IVIG is 1000 mg / kg. In some forms, hsIgG preparations are administered at 1% to 10%, 1% to 9%, or 1% to 8% of the effective dose of IVIG. , 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 2-10%, 2-9 %, 2~8%, 2~7%, 2~6%, 2~5%, 2~4%, 2~3%, 3~10%, 3~ 9%, 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 1-2%, 2% or 1% In some embodiments, the hsIgG formulation is administered at a dose less than or equal to an effective dose of IVIG. In some embodiments, the hsIgG formulation is administered at a dose of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, Administered at doses of 170, 175, 180, 185, 190, 195, or 200 mg / kg will be done.

[0020] As used herein, administration of an hsIgG preparation at a dose that is 10% of the effective dose of IVIG is considered to be effective. Disclosed are methods for treating Guillain-Barré syndrome in a subject with Guillain-Barré syndrome, including: In some embodiments, the effective dose of IVIG is 1000-2000 mg / kg. In some embodiments, the hsIgG formulation has a dose that is less than 10% of the effective dose of IVIG. In some embodiments, the hsIgG formulation is administered at a dose less than or equal to an effective dose of IVIG. 1%-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3 %, 1-2%, 2-10%, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2- 4%, 2-3%, 3-10%, 3-9%, 3-8%, 3-7%, 3-6%, 3-5%, 3 In some embodiments, the hsI is administered at a dose of about 4%, about 1-2%, about 2%, or about 1%. The gG preparation is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 , 155, 160, 165, 170, 175, 180, 185, 190, 195, or 2 It is administered at a dose of 00mg / kg.

[0021] As used herein, hsIgG preparations are administered at a dose that is 10% or less than 10% of the effective dose of IVIG. PID (primary humoral immunodeficiency disease, primary humoral immunodeficiency disease, Disclosed are methods for treating PID in subjects with a genetic immunodeficiency disorder (PID). In some embodiments, an effective dose of IVIG is 200-800 mg / kg. hsIgG preparations are 1% to 10%, 1% to 9%, 1% to 8%, and 1% to 7% of the effective dose of IVIG. %, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 2-10%, 2-9%, 2- 8%, 2-7%, 2-6%, 2-5%, 2-4%, 2-3%, 3-10%, 3-9%, 3 Administered at doses of 1% to 8%, 3% to 7%, 3% to 6%, 3% to 5%, 3% to 4%, 1% to 2%, 2% or 1% In some embodiments, the hsIgG formulation is administered at 1% of the effective dose of IVIG. In some embodiments, the hsIgG formulation is administered in an amount of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6 5, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, It is administered at doses of 175, 180, 185, 190, 195, or 200 mg / kg.

[0022] As used herein, hsIgG preparations are administered at a dose that is 10% or less than 10% of the effective dose of IVIG. and a method for treating Kawasaki disease in a subject with Kawasaki disease, the method comprising administering In some embodiments, the effective dose of IVIG is 1000-2000 mg / kg. In some embodiments, the hsIgG formulation is 1% to 10% of the effective dose of IVIG. , 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2% , 2~10%, 2~9%, 2~8%, 2~7%, 2~6%, 2~5%, 2~4%, 2~3 %, 3~10%, 3~9%, 3~8%, 3~7%, 3~6%, 3~5%, 3~4%, 1~ In some embodiments, the hsIgG formulation is administered at a dose of: In some embodiments, hsIgG is administered at a dose of 1% of the effective dose of IVIG. The agent is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 10 5, 110, 115, 120, 125, 130, 135, 140, 145, 150, 15 5, 160, 165, 170, 175, 180, 185, 190, 195, or 200m It is administered at a dose of 1000 mg / kg.

[0023] In some embodiments, 1% to 10%, 1% to 9%, 1% to 8%, or 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 2-10%, 2-9% , 2~8%, 2~7%, 2~6%, 2~5%, 2~4%, 2~3%, 3~10%, 3~9 %, 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 1-2%, 2% or 1% Administering a composition comprising an hsIgG formulation at a dose equivalent to the administration of a composition comprising an effective dose of IVIG. It has the same effectiveness as administering the substance.

[0024] In some embodiments, the at least one side effect resulting from an effective dose of IVIG is: By administering hsIgG preparations at a dose that is 1% to 10% of the effective dose of IVIG. In some embodiments, administration of a composition comprising an hsIgG formulation is Compared to the effective dose, the following side effects were observed: swelling, pain, discoloration of the limbs, shortness of breath, rapid heartbeat / tachycardia, and numbness or weakness in one or both limbs, brown or red urine, yellowing of eyes or skin, 100° Fever above 10°C, muscle cramps, nausea, vomiting, muscle pain, and / or low blood pressure This results in a reduction in the severity or duration of the above symptoms.

[0025] As used herein, a "glycan" is a sugar and has at least three sugar residues, such as sugars. A "glycan" can be a monomer or polymer of a group, and can be linear or branched. Natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid) , galactose, mannose, fucose, hexose, arabinose, ribose, xylose sugars) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, etc.) The term " "Glycan" includes homo- and heteropolymers of sugar residues. The term "glycan" also refers to complex Also includes glycan components of carbohydrates (e.g., glycoproteins, glycolipids, proteoglycans, etc.) The term includes glycans that have been cleaved or otherwise released from glycoconjugates. This also includes free glycans.

[0026] As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides." Each "Fc polypeptide" refers to an antibody that is an Fc polypeptide, excluding the first constant region immunoglobulin domain. In some embodiments, the "Fc region" comprises one or more peptide constant regions. Two Fc polypeptides linked by a sulfide bond, a chemical linker, or a peptide linker "Fc polypeptide" includes the last two polypeptides of IgA, IgD, and IgG. the constant region immunoglobulin domains of IgE and IgM, and the last three constant region immunoglobulin domains of IgE and IgM. refers to immunoglobulin domains, including portions of the flexible hinge N-terminal to these domains or In the case of IgG, the "Fc polypeptide" may include all of the immunoglobulin domains. C gamma 2 (Cγ2) and C gamma 3 (Cγ3), as well as C gamma 1 (Cγ1) and Cγ The boundaries of an Fc polypeptide can vary, but include the lower part of the hinge between the IgG and Fc domains. The Fc polypeptide usually begins at T223 or C226 or P230 and This numbering is defined to include residues up to the carboxyl terminus, and is based on the numbering scheme used by Kabat et al. (1999) 1, NIH Publication 91-3242, National Techn. ical Information Services,Springfield,VA In the case of IgA, the Fc polypeptide is phosphodomains C alpha 2 (Cα2) and C alpha 3 (Cα3), and C alpha 1 The Fc region contains the lower part of the hinge between (Cα1) and Cα2. It can be synthetic, recombinant, or produced from

[0027] As used herein, an "N-glycosylation site in the Fc region" refers to a site where the glycan is N-linked. This refers to the amino acid residues in the Fc region that are bound to each other.

[0028] For any given parameter, in some embodiments, the "proportion" is the percentage of the formulation This refers to the number of moles of a specific glycan (glycan X) relative to the total moles of glycans. Depending on the molar ratio of PNGase F-released Fc glycans to the total moles of PNGase F-released Fc glycans, This can be assessed by determining the number of moles of Fc glycan X.

[0029] "Purified" (or "isolated") means removed or separated from other components present in the natural environment. The nucleic acid sequence (e.g., polynucleotide) or amino acid sequence (e.g., polypeptide) to be isolated For example, an isolated polypeptide may be separated from other components of the cell in which it is produced (e.g., a vesicle). isolated polynucleotides (somatic or cytoplasmic proteins and RNA). The nucleotides are separated from other nucleic components (e.g., histones) and / or upstream or downstream nucleic acid sequences. An isolated nucleic acid or amino acid sequence is one that is isolated from the nucleic acid or amino acid sequence shown. 60%, or at least 75%, or less than 60% of other components present in the natural environment of the amino acid sequence. It may be at least 90% or at least 95% free.

[0030] As used herein, the term "ST6 sialyltransferase" refers to a sialyltransferase having the amino acid sequence The string is attached to the terminal galactose of the glycan via an α2,6 linkage (e.g., ST6Gal-I). Proteins involved in the transfer of sialic acid to the %, 96%, 95%, 94%, 93%, 92%, 91%, 90% identity The term "ST6 sialic acid" refers to a polypeptide that contains and exhibits a single characteristic sequence. The nucleotide transferase sequences are well known in the art, such as those described herein. In some embodiments, the ST6 sialyltransferase is a sialyltransferase as described herein. The ST6 sialyltransferases described (each of which is incorporated herein by reference) are incorporated by reference. It shares at least one characteristic sequence with one of the and / or with one of the ST6 sialyltransferases described herein. In some embodiments, the sequences described herein exhibit a certain degree of overall sequence identity. The ST6 sialyltransferases used herein are those referred to as reference ST6 sialyltransferases. In some such embodiments, the ATP-binding domain shares at least one biological activity with the ATP-binding domain of the ATP-binding domain. In this study, the covalent biological activity was related to the transfer of sialic acid to the glycan. A suitable ST6 sialyltransferase for this purpose is human ST6, which can be expressed in CHO cells. It's Gal.

[0031] N-linked glycosylation N-linked oligosaccharide chains are added to proteins within the lumen of the endoplasmic reticulum (Molecule ar Biology of the Cell,Garland Publishing g, Inc. (see Alberts et al., 1994). A monosaccharide (typically 14 saccharides) is inserted into the target consensus sequence of Asn X Ser / Thr. It is added to the amino group on the side chain of the contained asparagine residue, and X is any group other than proline. This initial oligosaccharide structure is common to most eukaryotes. , containing 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues. This initial oligosaccharide chain is trimmed by specific glycosidase enzymes in the endoplasmic reticulum. It can be seen that the nucleotide sequence of the ribozyme is composed of two N-acetylglucosamine and three mannose residues (Fig. 1). to obtain a short, branched core oligosaccharide consisting of a saccharide bonded to an asparagine residue. One of the branches is known in the art as "α1, The first branch is called the "α1,6 arm" and the second branch is called the "α1,6 arm."

[0032] N-glycans are referred to as "high mannose type," "hybrid type," and "complex type." They can be subdivided into three distinct groups, each with a common pentasaccharide core (Man(alpha 1,6) -(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta1 ,4)-GlcpNAc(beta1,N)-Asn) occurs in all three groups.

[0033] After initial processing in the endoplasmic reticulum, glycoproteins are transferred to the Golgi for further processing. Glycans can enter the Golgi plex before they are completely trimmed to the core pentasaccharide structure. When transferred to the nucleotide sequence, a "high mannose glycan" is obtained.

[0034] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added. Galactose may be added to amannose subunits to form "complex glycans." Addition of N-acetylglucosamine subunits and galactose subunits Sialic acid subunits were added to the sucrose to form sialic acid, galactose, or N-acetylglucose. This can result in a chain terminating in either a cosamine residue. Each of these additions may be made to the N-acetylglucosamine residue of the oligosaccharide. It is catalyzed by specific glycosyltransferases known in the art.

[0035] Sialic acids are a family of 9-carbon monosaccharides with heterocyclic structures. Contains a negative charge via the attached carboxylic acid group, as well as N-acetyl and N-glycolyl groups Other chemical decorations are found in glycoproteins produced in mammalian expression systems. A common type of sialic acid is N-acetyl-neuraminic acid (Ne uAc) and N-glycolylneuraminic acid (NeuGc) These usually contain galactose at the non-reducing ends of both N- and O-linked glycans. These sialic groups occur as terminal structures attached to (Gal) residues. The bond configuration can be either α2,3 or α2,6.

[0036] "Hybrid glycans" contain characteristics of both high mannose and complex glycans. For example, one branch of a hybrid glycan may contain primarily or exclusively mannose residues. Alternatively, the additional branching may be N-acetylglucosamine, sialic acid, and / or galactose sugars. may include:

[0037] When referring to a specific percentage of the effective dose of IVIG, the specified percentage may be within a range of ±5 mg / kg. Therefore, 10% of a 1,000 mg / kg dose is 1,000 mg / kg ± 5 %, and 5% of a 1,000 mg / kg dose is 50 mg / kg ± 5%.

[0038] Antibodies are glycosylated at conserved N-linked glycosylation sites within the Fc region of immunoglobulin heavy chains. For example, each heavy chain of an IgG antibody is glycosylated at Asn297 in the CH2 domain. It has a single N-linked glycosylation site (Jefferis, Nature Rev (See J.Iews 8:226-234 (2009)). IgA antibodies bind to CH2 receptors. It has N-linked glycosylation sites in the main and CH3 domains, and IgE antibodies IgM antibodies have N-linked glycosylation sites in three domains: CH1, CH2, and C. It has N-linked glycosylation sites within the H3 and CH4 domains (Arnold et al., J. Biol Chem.280:29080-29087(2005), Mattu et al. , J. Biol. Chem. 273:2260-2272 (1998), Nettlet on et al., Int.Arch.Allergy Immunol.107:328-329 (1995)).

[0039] Each antibody isotype has a variety of different N-linked carbohydrate structures in the constant region. For example, IgG has an Fc region in which Asn of the CH2 domain of each Fc polypeptide is located. 297, and has a single N-linked biantennary carbohydrate for C1q and FcγR. It also contains the binding site (Jefferis et al., Immunol. Rev. 163:59-7 6 (1998) and Wright et al., Trends Biotech 15:26-32 (1997)). In the case of human IgG, the core oligosaccharide usually consists of different numbers of The outer residues are GlcNAc2Man3GlcNAc. The movement is due to the galactose and / or galactose at one or both of the terminal GlcNAcs. Sialic acid attachment or attachment of a third GlcNAc arm (bisected GlcNAc) , and / or fucose linkages.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Methods and materials for use in the art are described herein and are well known in the art. Suitable methods and materials may also be used. The materials, methods, and examples are merely illustrative. All publications, patents, and other references mentioned herein are for illustrative purposes only and are not intended to be limiting. Patent applications, patents, and other references are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0041] Other features and advantages of the invention will be apparent from the following detailed description and claims. It would be. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram of an example of a branched glycan sialylated on both the α1,3 and α1,6 arms with a terminal NeuAc-α2,6-Gal linkage. [Figure 2] 1 is a schematic diagram of a method for preparing hsIgG. The starting substrate can be IVIG or a fraction thereof. [Figure 3] FIG. 1 is a schematic diagram of sialylation of branched glycans by ST6Gal1. [Figure 4] Figure 1 shows a graph depicting the time course of IVIg Fc glycoform ratios in the presence of CMP-NANA and ST6Gal1. Galactosylated IVIg was incubated with 20 mM CMP-NANA and 0.3 U / mg ST6Gal1 at 37°C. Aliquots were removed at different time points, and the relative ratios of IVIg glycoforms were determined by glycopeptide LC-MS / MS analysis. [Figure 5A] The results of an analysis of IVIG dose response in a mouse ITP model are shown (ns, not significant; *P<0.05, **P<0.01, ***P<0.001). [Figure 5B] The results of an analysis of IVIG dose response in a mouse ITP model are shown (ns, not significant; *P<0.05, **P<0.01, ***P<0.001). [Figure 6] 1 shows the results of a comparative study of therapeutic doses of 0.1 g / kg hsIgG with 0.1 and 1 g / kg IVIG in a mouse model of ITP. [Figure 7] 1 shows the results of a comparison of hsIgG and IVIG in human patients with ITP. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure relates to treatment methods using hsIgG preparations. HsIgG preparations contain branched IgG. At least 60% of the glycans were desialylated, i.e., the α1,3 arms (e.g. , with NeuAc-α2,6-Gal terminal linkages) and on the α1,6 arm (e.g., IgG antibodies (with NeuAc-α2,6-Gal terminal linkages) bearing sialic acid on the For example, a formulation containing a mixture of IVIG (prepared from IVIG).

[0044] The hsIgG formulations disclosed herein are useful for treating diseases treated with IVIG. Importantly, hsIgG preparations are much more potent than IVIG, This allows for effective treatment with much lower doses and / or less frequent treatments. As shown in the specification, hsIgG preparations are 10 to 100 times more potent than IVIG. This allows treatment with doses that are 1% to 10% of the effective or approved IVIG dose. .

[0045] Current treatments utilizing IVIG have variable efficacy, clinical risks, high cost, and limited supply. The current maximum dose regimen has clear limitations, including but not limited to: In many cases, only partial and poorly sustained responses have been achieved. The long infusion times (4-6 hours) associated with IVIG treatment (IVIG is generally administered in doses ranging from 1,000 to 2,000 mg / kg). 00 mg / kg) is a resource-intensive and inconvenient method for infusion centers. This has a negative impact on patient-reported outcomes such as sex and quality of life.

[0046] hsIgG preparations are much more potent than IVIG and therefore require shorter infusion times and shorter administration times. This allows for much smaller amounts of protein to be administered, and in some cases, less frequently. For this reason, hsIgG preparations are significantly more effective than IVIG treatment. It can provide an improved patient experience and a higher quality of life. Conditions that should be treated with intravenous IVIG are those treated with subcutaneous (injection) hsIgG. or infusion), e.g., by a subcutaneous pump that allows administration by the patient at home. In addition, the effective dose of hsIgG is less than that required for the effective dose of IVIG. This is because IVIG can be prepared from much smaller amounts of IVIG than is needed for This is an important advantage as they are in limited supply and expensive.

[0047] The level of sialylation can be determined by the individual Fc regions (e.g., the sialic acid of branched glycans within the Fc region). (number of branched glycans with α-1,3 arms, α-1,6 arms, or both) , or the overall composition of the glycoprotein preparation (e.g., F in the glycoprotein preparation Sialic acid in the α1,3 arm, α1,6 arm, or both of the branched glycans in the c region The number or percentage of branched glycans having

[0048] Methods for sialylation of IgG preparations Washburn et al. (Proc Natl Acad Sci 112(11):E1 297-306), ST6GalI sialyltransferase The sialic acid donor (e.g., cytidine 5'-monophospho-N-acetylno) sialyltransferase (sialylamino acid) to the terminal galactose residue of the glycan via an α2,6 bond ST6 sialyltransferase catalyzes the transfer of sialic acid to branched glycans. A second sialic acid is transferred to the α1,3 arm of the α1,6 arm, followed by a second sialic acid being transferred to the α1,6 arm. (producing branched sialylated glycans), followed by further Sialic acid can be removed from the α1,3 arm (with sialic acid on the α1,6 arm) Therefore, the activity of ST6 sialyltransferase By controlling and / or regulating the activity (e.g., kinetics) of specific sialylation patterns, It is possible to produce glycoproteins having the same structure.

[0049] For methods of producing hsIgG, see, for example, Wasserman, 1999, incorporated herein by reference. and U.S. Patent Application Publication No. 2016 / 0108450. T6Gal-1 sialyltransferase (Engel et al., BMC Proceedings ngs 7(Suppl 6):P110,2013) was also used to prepare hsIgG. This can be useful for:

[0050] Regarding a different sialylation method that does not significantly increase Fab sialylation, Hua ng et al. (J Am Chem Soc 134(29):12308-12318,20 12). Despite the lack of Fab sialic acid addition, Sialylated IVIG may be useful.

[0051] hsIgG preparations can be prepared from commercially available IVIG as follows: Bulk I VIG, 10% solution (0.1 g / mL) pooled and 3-(N-morpholino)propanesulfonyl ether 3-(N-morpholino)propanesulfonic acid d, MOPS) pH 7.4 buffer. This solution is diluted with 30 kDa tangential force. Tangential flow filtration (TFF) membrane (polyether Using a membrane made of sulfone, 5 diavolumes ( The diafiltered (DF) solution is then diafiltered with the buffer-exchanged I The VIG solution was concentrated, followed by 0.2 μm depth filtration, to ensure a final IVIG concentration of ≥ 1 The sialic acid addition was performed by adding two sialic acid derivatives dissolved in MOPS pH 7.4 buffer. This is accomplished in two enzymatic steps using the sugar nucleotide: The synthesis was carried out using β-1,4 galactosyltransferase enzyme in MOPS pH 7.4 buffer. B4GalT, uridine diphosphate galactose This occurs through a reaction with ATP (UDP-Gal) and manganese chloride. The reaction solution is adjusted to about 135 mg / mL by adding 100 ml of ethanol and maintained at about 37°C for about 48 hours. Following galactosylation, this material was purified by the human α2,6-sialyltransferase enzyme. Protein (ST6-Gal1) and cytidine 5'-monophospho-N-acetylneuraminic acid (cyti dine 5'monophospho-N-acetyl neuraminic acid (CMP-NANA) was added and the Incubate for another hour and adjust to approximately 120 mg / mL with MOPS buffer pH 7.4. CMP-NANA is charged in small amounts over the course of the reaction at approximately 12 hour intervals.

[0052] Glycan evaluation Glycans on glycoproteins are assessed using any method known in the art. For example, sialylation of the glycan composition (e.g., α1,3 arms and / or or the level of branched glycans with sialic acid on the α1,6 arm) can be determined, for example, by b, Biochemistry 48:9705-9707(2009), Anumul a,J.Immunol.Methods 382:167-176(2012);Gi lar et al., Analytical Biochem. 417:80-88(2011); Wuhrer et al., J. Chromatogr. B. 849:115-128 (2007) In some embodiments, the granules may be characterized using the methods described in In addition to assessing sialic acid addition of the glycan, one or more of the parameters listed in Table 1 may be used. is evaluated.

[0053] In some instances, the glycan structures and compositions described herein may be, for example, one or two More than one enzyme, chromatography, mass spectrometry (MS), MS followed by chromatography, electrophoresis, electrophoresis followed by MS, nuclear magnetic resonance (NMR) The molecules are analyzed by NMR (magnetic resonance) techniques, and combinations of these. Enzymatic methods include removing one or more glycans (e.g., one or more exposed glycans) from the glycans. contacting the glycoprotein preparation with one or more enzymes under conditions and for a time sufficient to release In some instances, the one or more enzymes include PNGase F. Exemplary chromatographic methods include those using pulsed amperometric detection. Strong Anion Exchange Chromatography single Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (li high performance liquid chromatography (LC) id chromatography (HPLC), ultra performance liquid chromatography (ultra performance liquid chromatography (UPLC), thin layer chromatography raphy, TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) methods include, but are not limited to, tandem MS, LC-MS, LC-MS / MS, matrix-assisted laser desorption / ionization mass spectrometry ( matrix assisted laser desorption ionization mass spectrometry, MALDI-MS ), Fourier transform mass spectrometry (FTMS), quality Ion mobility separation with mass spectrometry ry, IMS-MS), electron transfer dissociation (ETD-MS) Exemplary electrophoretic methods include, but are not limited to, electrophoresis, electrophoresis, and combinations thereof. Examples include capillary electrophoresis (CE), CE-MS, Gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, specific glycans SDS-polyacrylamide gel electrophoresis followed by Western blotting using antibodies that recognize the structure SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Exemplary nuclear magnetic resonance (N NMR (NMR) includes one-dimensional NMR (1D-NMR) and two-dimensional NMR (two-dimensional NMR, 2D-NMR), correlation spectroscopy magnetic angle spinning NMR (correla tion spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlation spectroscopy MR (total correlated spectroscopy NMR, TOCSY-NMR), heteronuclear single quantum Heherence NMR (heteronuclear single-quantum coherence NMR, HSQC-NMR) heteronuclear multiple quantum coherence (HMQC) NMR), rotational nuclear overhauser effect spectroscopy NMR effect spectroscopy NMR, ROESY-NMR), nuclear Overhauser effect spectroscopy (nucle ar overhauser effect spectroscopy, NOESY-NMR), and combinations of these These include, but are not limited to:

[0054] In some instances, the techniques described herein include the detection of glycans or glycoproteins; It may be combined with one or more other techniques for analysis and / or isolation. For example, in certain instances, glycans can be synthesized using one or more available methods. Analyses are performed according to the instructions (although in some cases, chem.,350(1):1,2006; Klein et al., Anal.Biochem. , 179:162, 1989, and / or Townsend, R.R. Carbohyd. rate Analysis “High Performance Liquid Ch romatography and capillary electrophores is., Ed. Z. El Rassi, pp181-209, 1995, International Publication No. 20 08 / 128216, 2008 / 128220, 2008 / 128218 , No. 2008 / 130926, No. 2008 / 128225, No. 2008 / 13 No. 0924, No. 2008 / 128221, No. 2008 / 128228, No. 20 08 / 128227, 2008 / 128230, 2008 / 128219 , Same No. 2008 / 128222, Same No. 2010 / 071817, Same No. 2010 / 07 No. 1824, No. 2010 / 085251, No. 2011 / 069056, and No. See US Pat. No. 2011 / 127322, each of which is incorporated herein by reference in its entirety. For example, in some instances, glycans can be purified by chromatography. and one or more of the following methods: electrophoresis, nuclear magnetic resonance, and a combination thereof. In some instances, for example, one or more glycoproteins in a glycoprotein formulation are characterized. The method may further include combining two or more target protein-specific parameters, e.g., the parameters disclosed herein. The method for evaluating one or more of the data may be one or more of the following methods: This can be performed.

[0055] In some instances, for example, one or more target proteins in a glycoprotein formulation may be The protein-specific parameters may be, for example, one or two of the parameters disclosed herein. The method for assessing one or more of the following may be carried out by one or more of the following methods:

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058] The above documents are incorporated herein by reference in their entirety, or alternatively, They may be prepared by one or more of the methods for determining parameters described herein. Incorporated above to the extent relevant.

[0059] Pharmaceutical Compositions and Administration The hsIgG can be incorporated into pharmaceutical compositions. Pharmaceutical compositions for the treatment of rheumatoid arthritis can be formulated by methods known to those skilled in the art. For example, The pharmaceutical composition comprises an hsIgG formulation in a solution of sterile water and saline, vegetable oil, emulsifiers, suspending agents, Pharmaceutically acceptable additives such as surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders, etc. The formulation is then generally prepared by combining the formulation with an acceptable vehicle or medium. They can be compounded in unit dosage forms as required by accepted pharmaceutical practice. The amount of active ingredient contained in the agent is such that a suitable dosage within the specified range is provided.

[0060] Sterile compositions for injection may be prepared in a conventional pharmaceutical manner using distilled water for injection as a vehicle. It can be formulated according to conventional methods. For example, glucose and D-sorbitol, Contains other supplements such as D-mannose, D-mannitol, and sodium chloride Physiological saline or isotonic solution as an aqueous solution for injection, optionally with a suitable soluble a solvent, for example, an alcohol such as ethanol, and propylene glycol or polyethylene glycol; Polyalcohols such as ethylene glycol, and polysorbate 80 (trademark), HCO-50, etc. It may be used in combination with any nonionic surfactant.

[0061] Non-limiting examples of oily liquids include sesame oil and soybean oil, with sorbitol as a solubilizer. It may be combined with benzyl benzoate or benzyl alcohol. The formulation may contain a buffer such as a phosphate buffer or a sodium acetate buffer, or procaine hydrochloride. soothing agents, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injection can be placed in a suitable ampoule.

[0062] In some embodiments, the dosage is FDA approved (or other national or international regulatory agency) The IVIG dose is 1% to 10% of the effective IVIG dose for the disease. In embodiments, an FDA (or other national or international regulatory agency) approved or effective dose of IVIG is used. are 200mg / kg, 400mg / kg, 500mg / kg, 600mg / kg, 10 In some embodiments, the hsIgG is 00 mg / kg, or 2000 mg / kg. The composition containing the formulation may be about 4, 5, 6, 10, 15, 20, 30, 40, 50, 60, 70 ,80,90,100,110,120,130,140,150,160,170,1 80, 190, 200, 250, 300, 350, 400, 450, 500, 550, 6 00, 650, 700, 750, 800, 850, 900, 950, 975, or 100 In some embodiments, the composition comprising the hsIgG formulation is administered at a dose of 0 mg / kg. Items can be daily, weekly, twice a week, every other week, monthly, every two weeks, every other month, every three days, or every four days. , every 5 days, every 6 days, every 7 days, once every 14 days, once every 21 days, once every 28 days, Once daily for 2 consecutive days on a 28-day cycle, or at the same frequency as FDA-approved IVIG doses In some embodiments, the composition is administered in a single dose. In embodiments, the composition is administered in multiple doses. The dose and method of administration will vary depending on the patient's weight, age, and other factors. It will vary depending on age, condition, etc., and can be selected appropriately by those skilled in the art as needed.

[0063] As described by Washburn et al., sialic acid addition by ST6Gal1 Analysis of the CMP-NANA sugar nucleotides revealed that ST6Gal1 catalyzes the synthesis of Fc glycans. It not only catalyzes the transfer of sialic acid to thiol but also the removal of sialic acid from the sialylation product. These reactions are shown in Figure 3. show.

[0064] Washburn et al. reported that under certain reaction conditions, the sialic acid in the α1,3 branch of biantennary glycans Acid addition (to form A1F-1,3) was rapid and essentially complete in 30 min, whereas The doubly sialylated species (A2F) formed at a rate approximately 10 times slower and ceased to accumulate at 24 h. After 24 hours, monosialic acid with sialic acid on the α1,6 branch (A1F-1,6) was detected. Glycosylated species began to form and accumulation continued steadily, reaching approximately 35% of the glycosylated species at 64 hours. %. Concomitantly, A2F reached 71% in 20 hours and 44% in 64 hours. Indeed, the A1F-1,6 glycoform outnumbered the desialylated A2F. This suggests that it is generated from the removal of sialic acid residues on the exposed α1,3 branch. Incubation led to cleavage of the 1,6 sialic acid in the A1F-1,6 glycoform. This results in the generation of a sialylated but fully galactosylated and fucosylated species of G2F. G2F, present in trace amounts at the start of the reaction, appeared in measurable amounts by 40 hours and continued to increase. As A2F and A1F-1,6 levels decreased, reaching 15% at 64 hours. .

[0065] In Washburn et al., these observations demonstrate that the yield of the A2F species can be maximized while the A1F To minimize A1F-1,3, A1F-1,6 and G2F glycoforms, parameters It has further been reported that optimizing the data is important.

[0066]

number

[0067]

number

[0068] Galactosylation and sialylation of IVIG Sialylation of IVIG by sialyltransferase ST6 was analyzed. G was first galactosylated and then sialylated. The reactions were carried out sequentially. There was no purification between the sylation and sialylation reactions. Relative abundance of glycoforms was analyzed after sialylation reaction.

[0069] A. Galactosylation Reactions were set up containing the following components at the concentrations indicated:

[0070] [Table 2]

[0071] The reaction was incubated at 37°C for 72 hours.

[0072] B. Sialylation Aliquot the galactosylation reaction with CMP-NANA, MOPS buffer, and ST6 Gal1 was added. The final volume was adjusted to the final concentrations of the components in the reaction as indicated. Ta.

[0073] [Table 3]

[0074] The reaction was incubated at 37°C. Aliquots were frozen at -20°C for later analysis. Extraction was carried out at various times after thawing.

[0075] C. Results FIG. 4 shows the results of experiments carried out essentially as described above in the presence of CMP-NANA and ST6Gal1. The IVIG Fc domain glycoform ratio during the sialic acid addition reaction A time course is shown. Briefly, galactosylated IVIG was incubated at 37°C for 20 min at 20 mM The cells were incubated with 0.1 U / mg of CMP-NANA and 0.3 U / mg of ST6Gal1. The recoat was removed at different time points and the relative proportions of IVIG glycoforms were analyzed using the glycopeptide As determined by LC-MS / MS analysis, the major glycoforms were: During the reaction process involving competitive addition (forward reaction) and elimination (reverse reaction), G2F~A1F( 1,3) to A2F~A1F(1,6) over time.

[0076] Analysis of hsIgG preparations Commercially available hsIgG preparations made from IVIG contain CMP-NA during the sialic acid addition reaction. The starting IVIG material and the resulting IVIG were prepared essentially as described above, with periodic addition of NA. The resulting hsIgG preparations were extensively analyzed before and after sialylation. For acid loading, the hsIgG preparation was tetrasialylated (i.e., 1-sialylated IgG on each Fc chain). The branched glycans were substantially sialylated on both branches. A comprehensive analysis is required to distinguish between different Fc isotypes and Fab glycosylation. Fc glycosylation was performed in a site-specific and site-specific manner. For IgG isotypes, primarily sialylated species of the starting IVIG material (e.g., G 0F and G1F) to over 90% desialylated species (e.g., A2F, A2, and A2F Analysis of the Fab glycans revealed that the IVIG starting material was , significant presence of monosialylated (approximately 35%) and disialylated (approximately 35%) glycans in Fab. Although the distribution contains a variegated distribution, this distribution is consistent with the higher levels of disialylated IgG in the hsIgG preparation. It was further revealed that there was a shift towards Lycan (approximately 75%).

[0077] hsIgG is more effective than IVIG in preventing platelet destruction in a mouse model of ITP is about 10 times more potent Washburn et al. reported that thrombocytopenia induced by a mouse anti-CD41 antibody We compared hsIgG preparations to IVIG in patients with HIV-1 infection. aAntibodies were prepared by transient transfection of 293T cells followed by titration as suggested by the manufacturer. Protein G beads (GE Healthcare) were used to detect tissue fragments from serum-free cell culture supernatants. The IVIG preparation was produced by purification of recombinant antibodies. Chronic ITP was treated with 0.1 μg / g 6A6-IgG2a antiplatelet The mice were thrombocytopenic (3%) until the end of the experiment. Platelet counts were obtained before and 4 hours after daily antibody injections of 1:4 diluted PBS into the blood system. (Advia 120; Bayer HealthCare). Platelet count before antibody infusion was set to 100%. The dose-response profile of IVIG in the ITP model (Figure 5 After maximal platelet depletion by antiplatelet antibodies, mice were inoculated with 0.1–1 g / kg IVIG. The platelet counts were measured on the day of IVIG treatment (day 1) and two days after treatment (days 2 and 3 of the experiment). A clear dose-response was observed in this model within this range (Figure 5). The full activity of VIg was lost at 0.1 g / kg. As shown in Figure 6, a clear improvement in efficacy was observed when compared with IVIG administered at 0.1 Treatment with hsIgG at 100 mg / kg resulted in platelet levels on days 2 and 3. was restored to levels similar to those obtained with IVIg at 1 g / kg.

[0078] Testing in human patients HsIgG preparation (at least 80% of the branched glycans are NeuAc-α2,6-Ga (sialylated on both the α1,3 arm and the α1,6 arm by 1-terminal linkage) It has been compared with IVIG in human patients with ITP. This double-blind, placebo-controlled study was administered to healthy volunteers over a 3.5-month period. In the study, the doses were 3mg / kg, 10mg / kg, 30mg / kg, 60mg / kg, and 120mg Patients will receive a single dose of hsIgG every 14 days in the following order: 250 mg / kg, 250 mg / kg, and 250 mg / kg. Endpoint measurements included safety and tolerability.

[0079] In a separate study, patients with ITP received single ascending doses over a 4-month period. For subjects, 60mg / kg, 120mg / kg, 250mg / kg, 500mg / kg, and A single dose of hsIVIG was administered in a fixed sequence of 1000 mg / kg: hsIgG dose IVIG was administered to the same patient 28 days later at 1000 mg / kg. Levels of serotonin were measured throughout the study. Endpoint measures included safety and tolerability.

[0080] In a randomized trial, patients with ITP were started with either hsIgG or IVIG, In one subsection, five patients were treated with high hsIVIG doses. IVIG was started in 1 patient and another 5 patients were started on 1,000 mg / kg IVIG. Patients who started on a higher hsIVIG dose during the study were given 1,000 mg for the remainder of the study. IVIG was given at 1,000 mg / kg, and patients started on 1,000 mg / kg IVIG were given IVIG for the remainder of the study. High hsIgG doses were given to the mice. Platelet responses were measured throughout. In this study, five patients were started on a low hsIgG dose and another five patients were started on a 1,000m Patients were started on IVIG at 200 mg / kg. Midway through the 2-month trial, patients were started on a lower hsIgG dose. Patients received 1,000 mg / kg IVIG for the remainder of the study and 1,000 mg Patients initiated on g / kg IVIG received a lower hsIG dose for the remainder of the study Platelet responses were measured throughout the study to determine the efficacy of hsIVIG.

[0081] hsIgG preparations are more potent than IVIG in human ITP patients The hsIgG preparation was compared with IVIG from human patients with ITP. In this case, at least 80% of the branched glycans are terminally linked to NeuAc-α2,6-Gal. Both the α1,3 arm and the α1,6 arm were sialylated by 43 mg. As shown in Figure 7, this patient received 43 mg / kg hsIVIG. When administered at 1000 mg / kg, the incidence of reticulitis was similar to that observed with 1000 mg / kg of IVIG. Therefore, hsIgG preparations prevent platelet destruction in this patient. It was approximately 25 times more potent than IVIG in some cases.

[0082] Other embodiments Although the present invention has been described in connection with this detailed description, the foregoing description is not intended to be limiting and should not be construed as limiting the scope of the present invention. The invention is not intended to limit the scope of the invention. It is understood that the appended claims are defined by the appended claims. Other aspects, advantages, and modifications are set forth below. Within the scope of the claims below.

Claims

1. 1. A method for treating a disease, comprising administering an effective dose of IVIG to treat said disease. The method comprises administering to the subject an hsIgG preparation at a dose that is 1% to 10% of the

2. 4. The hsIgG formulation of claim 1, wherein the hsIgG formulation is administered at a dose of 5 mg / kg to 100 mg / kg.

1. The method according to claim 1.

3. 10. The method of claim 1, wherein the disease is an inflammatory disease.

4. The method of claim 1 , wherein the subject suffers from an antibody deficiency.

5. The method of claim 4, wherein the subject is suffering from a primary antibody deficiency.

6. The method of claim 1 , wherein the disease is associated with the presence of autoantibodies.

7. 12. The method of claim 1, wherein the dose of hsIVIG is as effective as an effective dose of IVIG. The method described below.

8. 10. The method of claim 1, wherein the hsIgG preparation is administered at the same frequency as an effective dose of the IVIG.

7. The method according to claim 7.

9. The method of claim 1 , wherein the disease is a neurological disease.

10. The neurological disease is dermatomyositis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy ( CIDP), multifocal motor neuropathy (MMN), myasthenia gravis, and stiff-person syndrome 10. The method of claim 9, wherein the compound is selected from the group consisting of:

11. The disease is immune cytopenia, parvovirus B19-associated erythroid aplasia, myeloma, and Secondary hypogammaglobulinemia in chronic lymphocytic leukemia and after bone marrow transplantation The method of claim 1 , wherein the compound is selected from the group consisting of:

12. The diseases include vasculitis, systemic lupus erythematosus (SLE), mucous membrane pemphigoid, and uveal ulcers. in dermatology, Kawasaki syndrome, dermatomyositis, toxic epidermal necrolysis, 10. The method of claim 1, which is most commonly used to treat gallbladder disease and bullous disease.

13. The disease is FDA approved for treatment with IVIG or IVIG is , the method of claim 1 being indicated for the treatment of said disease.

14. The hsIgG preparation is administered at a dose of 1% to 10% of the FDA-approved IVIG dose for the disease. %. The method of claim 13 .

15. The disease is myocarditis, acute motor axonal neuropathy, painful steatosis, Anti-glomerular basement membrane nephritis; Goodpasture's syndrome, Antiphospholipid syndrome (APS, APLS), Anti-synthetase antibody syndrome; myositis, ILD, ataxic neuropathy (acute and chronic), autoimmune enteropathy (AIE), autoimmune neutropenia, autoimmune retinopathy, autoimmune thyroiditis, autoimmune urticaria, dermatitis herpetiformis, Acquired epidermolysis bullosa, essential mixed cryoglobulinemia, Granulomatosis with polyangiitis (GPA), mixed connective tissue disease (MCTD), Neuromyotonia, optic neuritis, paraneoplastic cerebellar degeneration, anti-N-methyl D-aspartate (anti-NMDA) receptor encephalitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, chronic inflammatory demyelinating polyradiculoneuropathy, dermatomyositis, gestational pemphigoid, Graves' disease, Guillain-Barré syndrome, IgG4-related diseases, Lambert-Eaton myasthenic syndrome, lupus nephritis, Myositis, multifocal motor neuropathy, myasthenia gravis, Neuromyelitis optica, pemphigus vulgaris, Polymyositis, and Systemic lupus erythematosus (SLE) 2. The method of claim 1, wherein the compound is selected from the group consisting of:

16. The disease is acute disseminated encephalomyelitis (ADEM), autoimmune angioedema (acquired angioedema type II), Autoimmune hepatitis (types I and II), autoimmune hypophysitis; lymphocytic hypophysitis; autoimmune inner ear disease (AIED), Evans syndrome, Graves' ophthalmopathy, Hashimoto's encephalopathy, IgA vasculitis (IgAV), latent autoimmune hepatitis, Linear IgA disease (LAD), lupus vasculitis, Membranous glomerulonephritis, Microscopic polyangiitis (MPA), Mooren's ulcer, Morphea, opsoclonus-myoclonus syndrome, Oldo's thyroiditis, relapsing rheumatism, Paraneoplastic opsoclonus-myoclonus ataxia with neuroblastoma, Pediatric autoimmune streptococcal neuropsychiatric disorder (PANDAS), Postpericardiotomy syndrome, Primary biliary cirrhosis (PBC), Rasmussen's encephalitis, rheumatoid vasculitis, Schnitzler syndrome, Sydenham chorea, Undifferentiated connective tissue disease (UCTD), and Miller Fisher syndrome 2. The method of claim 1, wherein the compound is selected from the group consisting of:

17. 60% of the glycans on the IgG in the hsIgG preparation were α1,3- and α1,6-branched. The method according to any one of claims 1 to 16, wherein both branches have sialic acid.

18. At least 60% of the branched glycans on the Fab domain are NeuAc-α2,6-G The α1,3 arm and the α1,6 arm both have sialic acid linked via an α1,6 terminal bond. and at least 60% of the branched glycans on the Fc domain are NeuAc-α2,6- Both the α1,3 arm and the α1,6 arm are linked via Gal-terminal bonds.

18. The method of claim 17, comprising sialic acid.

19. A method of treating CIDP in a subject with CIDP, comprising administering an effective dose of IVIG. The method comprises administering the hsIgG preparation at an effective dose of 10% or less than 10% of the effective dose.

20. 20. The method of claim 19, wherein the effective dose of IVIG is 200 to 2000 mg / kg. method.

21. The hsIgG preparation has an effective dose of 10% or less than 10% of the effective dose of the IVIG.

21. The method of claim 19 or 20, wherein the amount is administered.

22. 10. The method of claim 1, wherein the hsIgG preparation is administered at a dose of 1% of the effective dose of the IVIG. 9 or 20. The method according to claim 9 or 20.

23. The hsIgG preparation is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 、３０、３５、４０、４５、５０、５５、６０、６５、７０、７５、８０、９０、１００ , 110, 120, 130, 140, 150, 160, 170, 180, 190 or 20 20. The method of claim 19, wherein the compound is administered at a dose of 0 mg / kg.

24. A method of treating ITP in a subject with ITP, comprising administering an effective dose of IVIG to a subject. The method comprises administering the hsIgG preparation at an effective dose that is 0% or less than 10%.

25. 25. The method of claim 24, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. How to do it.

26. The hsIgG preparation has an effective dose of 10% or less than 10% of the effective dose of the IVIG.

25. The method of claim 24, wherein the amount is administered.

27. The hsIgG preparation is administered at a dose of 1% to 5% of the effective dose of the IVIG.

25. The method of claim 24.

28. The hsIgG preparation is about 10, 20, 30, 40, 50, 60, 70, 80, 90, １００、１１０、１２０、１３０、１４０、１５０、１６０、１７０、１８０、１９０、 or 200 mg / kg.

29. A method of treating wAIHA in a subject with wAIHA, comprising administering IVIG to a subject. administering the hsIgG formulation at an effective dose that is 10% or less than 10% of the original dose. How to write.

30. 30. The method of claim 29, wherein the effective dose of IVIG is 1000 mg / kg.

31. The hsIgG preparation is administered at a dose that is less than 10% of the effective dose of the IVIG.

30. The method of claim 29.

32. The hsIgG preparation is administered at a dose of 1% to 5% of the effective dose of the IVIG.

30. The method of claim 29.

33. The hsIgG preparation is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

34. A method of treating Guillain-Barré syndrome in a subject with Guillain-Barré syndrome. Therefore, the hsIgG preparation is administered at an effective dose of 10% or less than that of IVIG. The method, comprising:

35. 35. The method of claim 34, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. How to do it.

36. 10. The method of claim 1, wherein the hsIgG preparation is administered at a dose that is 10% of the effective dose of the IVIG.

34. The method according to claim 34.

37. The hsIgG preparation is administered at a dose of 1% to 5% of the effective dose of the IVIG. The method of claim 34.

38. The hsIgG preparation is about 10, 20, 30, 40, 50, 60, 70, 80, 90, １００、１１０、１２０、１３０、１４０、１５０、１６０、１７０、１８０、１９０、 or 200 mg / kg.

39. A method for treating a primary humoral immunodeficiency disease (PID) in a subject with PID. Therefore, the hsIgG preparation is administered at an effective dose that is 10% or less than 10% of the effective dose of IVIG. The method comprises administering.

40. The method of claim 39, wherein the effective dose of IVIG is 200 to 800 mg / kg. Law.

41. The hsIgG preparation is administered at a dose that is less than 10% of the effective dose of the IVIG.

40. The method of claim 39.

42. The hsIgG preparation is administered at a dose of 1% to 5% of the effective dose of the IVIG.

40. The method of claim 39.

43. The hsIgG preparation is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / kg 40. The method of claim 39, wherein the dose is

44. A method of treating Kawasaki disease in a subject with Kawasaki disease, comprising administering an effective dose of IVIG to a subject. The method comprises administering the hsIgG preparation at an effective dose that is 0% or less than 10%.

45. 45. The method of claim 44, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. How to do it.

46. The hsIgG preparation is administered at a dose that is less than 10% of the effective dose of the IVIG. The method of claim 44.

47. The hsIgG preparation is administered at a dose of 1% to 5% of the effective dose of the IVIG. The method of claim 44.

48. The hsIgG preparation is about 10, 20, 30, 40, 50, 60, 70, 80, 90, １００、１１０、１２０、１３０、１４０、１５０、１６０、１７０、１８０、１９０、 or 200 mg / kg.

49. a dose of said hsIgG preparation having an efficacy similar to an effective dose of said IVIG; Item 49. The method according to any one of items 1 to 48.

50. At least one side effect caused by the effective dose of IVIG is not observed when the hsIgG formulation is administered.

50. The method of any one of claims 1 to 49, wherein the pain is relieved by administering

51. A sterile composition comprising 110, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg of an hsIgG preparation. An article of manufacture, including a container that contains an item.

52. At least 60 of the branched glycans on the Fab domain of the IgG in the hsIgG preparation % of the α1,3 arm and α linked via a NeuAc-α2,6-Gal terminal linkage The hsIgG preparation has sialic acid on both the 1 and 6 arms, and At least 60% of the branched glycans of 52. The method of claim 51, wherein the α1,3 arm and the α1,6 arm are both sialic acid. Manufactured items.

53. 52. The article of manufacture of claim 51, wherein the container is a vial, bottle, or bag.

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