Modified Fc polypeptides with enhanced sialylation

Abstract

The present disclosure relates to compositions containing highly sialylated immunoglobulin Fc polypeptides having an amino acid substitution at amino acid residue 241 of the Fc heavy chain from phenylalanine to an aliphatic amino acid residue, such as alanine. Methods of producing the highly sialylated Fc polypeptides in recombinant expression systems are further disclosed. Also disclosed are methods of using the disclosed compositions for the treatment of inflammatory diseases or disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 375,490 (filed September 13, 2022) and 63 / 512,013 (filed July 5, 2023), the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This specification refers to a Sequence Listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file was created on September 12, 2023, is named NVG_003WO_ST26.xml, and is 8kb in size. [Background technology]

[0003] Inflammatory disorders, including autoimmune diseases, involve the abnormal activation and subsequent migration of leukocytes to affected areas of the body. These conditions encompass a wide range of illnesses that affect the lives of millions of people worldwide. Although various treatments are currently available, many have significant side effects or are only insufficiently effective in alleviating symptoms.

[0004] Immunoglobulin G (IgG) has long been understood to mediate both pro- and anti-inflammatory activities through interactions mediated by its fragment crystallizable (Fc) region. While Fc-FcγR interactions are responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies, intravenous gamma globulin (IVIG) and its component Fc fragments are anti-inflammatory and are widely used to suppress inflammation in disease states.

[0005] Sialylation of recombinant therapeutic glycoproteins, eg, Fc polypeptides, is generally performed in mammalian or mammalian cell lines capable of reproducing a mammalian-like glycosylation profile. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides compositions comprising Fc polypeptides with high levels of sialylation on their N-glycans and methods of making and using the same.

[0007] In certain embodiments, a population of modified Fc polypeptides is disclosed herein, each modified Fc polypeptide having (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2; and (ii) an aliphatic amino acid residue (e.g., alanine, glycine, isoform, methylamino acid ... leucine, leucine, proline, valine, and methionine), and at least 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides have sialic acid (SA) moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, the aliphatic amino acid at position 241 is alanine (Ala; F241A). In some embodiments, the aliphatic amino acid at position 241 is glycine (Gly; F241G). In some embodiments, the aliphatic amino acid at position 241 is isoleucine (Ile; F241I). In some embodiments, the aliphatic amino acid at position 241 is leucine (Leu; F241L). In some embodiments, the aliphatic amino acid at position 241 is proline (Pro; F241P). In some embodiments, the aliphatic amino acid at position 241 is valine (Val; F241V). In some embodiments, the aliphatic amino acid at position 241 is methionine (Met; F241M). In some embodiments, the N-glycan is attached to an asparagine (Asn) at amino acid residue 297 of the polypeptide (Asn297; numbered according to Kabat and corresponding to amino acid residue 88 of SEQ ID NO: 2).In some embodiments, at least 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, the N-glycans of the Fc polypeptide are monosialylated or disialylated. In some embodiments, at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the Fc polypeptides comprise monosialylated N-glycans comprising an SA moiety attached via an α(2,6) linkage. In some embodiments, at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage. In some embodiments, about 90% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage.In some embodiments, at least about 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the Fc polypeptides comprise a galactose moiety. In some embodiments, about 100% of the Fc polypeptides comprise a galactose moiety. In some embodiments, the galactose moiety is attached to the α(1,3) arm and / or the α(1,6) arm of the N-glycan. In some embodiments, the galactose moiety is a branched galactose moiety.

[0008] In certain embodiments, disclosed herein is a population of modified Fc polypeptides, each modified Fc polypeptide having (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2; and (ii) a sequence at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2), and about 40% (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%) of the modified Fc polypeptides have an SA moiety attached to the N-glycan of the Fc polypeptide via an α(2,3) linkage. In some embodiments, the aliphatic amino acid at position 241 is alanine (Ala; F241A). In some embodiments, the aliphatic amino acid at position 241 is glycine (Gly; F241G). In some embodiments, the aliphatic amino acid at position 241 is isoleucine (Ile; F241I). In some embodiments, the aliphatic amino acid at position 241 is leucine (Leu; F241L). In some embodiments, the aliphatic amino acid at position 241 is proline (Pro; F241P). In some embodiments, the aliphatic amino acid at position 241 is valine (Val; F241V). In some embodiments, the aliphatic amino acid at position 241 is methionine (Met; F241M). In some embodiments, the N-glycan is attached to an asparagine (Asn) at amino acid residue 297 (Asn297; numbered according to Kabat and corresponding to amino acid residue 88 of SEQ ID NO: 2) of the polypeptide. In some embodiments, the Fc polypeptide is an IgG1. In some embodiments, the Fc polypeptide is an IgG3.

[0009] In certain embodiments, disclosed herein are pharmaceutical compositions comprising (a) a population of modified Fc polypeptides of any of the preceding embodiments, and (b) a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutically acceptable carrier, diluent, or excipient is selected from the group consisting of stabilizers, buffers, surfactants, bulking agents, solvents, tonicity or osmolality adjusters, antioxidants, adjuvants, and antimicrobial agents.

[0010] In certain embodiments, disclosed herein are methods of treating an inflammatory disease or condition in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of a population of modified Fc polypeptides of any of the preceding embodiments. In some embodiments, the inflammatory disease or condition is an autoimmune disease or condition. In some embodiments, the inflammatory disease or condition is arthritis. In some embodiments, the inflammatory disease or condition is immune thrombocytopenia (ITP). In some embodiments, the population or pharmaceutical composition has a half-life of at least 3.5 days (e.g., at least 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, or more days) after administration of the population or pharmaceutical composition to the subject. In some embodiments, the population or pharmaceutical composition has a half-life of at least 4 days (e.g., at least 4, 4.5, 5, 6, 7, 8, 9, 10, or more days) after administration of the population or pharmaceutical composition to the subject. In some embodiments, the population or pharmaceutical composition is cleared from the circulation of a subject at a rate of 45 mL / day / kg or less (e.g., 45, 40, 35, 30, 25, 20 mL / day / kg or less). In some embodiments, the population or pharmaceutical composition is cleared from the circulation of a subject at a rate of 40 mL / day / kg or less (e.g., 40, 35, 30, 25, 20 mL / day / kg or less). In some embodiments, the population or pharmaceutical composition is cleared from the circulation of a subject at a rate of 35 mL / day / kg or less (e.g., 35, 30, 25, 20 mL / day / kg or less). In some embodiments, the population or pharmaceutical composition is cleared from the circulation of a subject at a rate of 30 mL / day / kg or less (e.g., 30, 25, 20 mL / day / kg or less). In some embodiments, the population or pharmaceutical composition is cleared from the subject's circulation at a rate of 25 mL / day / kg or less (eg, 25, 25, 20 mL / day / kg or less, or less).In some embodiments, the concentration of the Fc polypeptide over time (AUC) is at least 480 days × mg / mL between 1 and 35 days (e.g., 1, 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, 32, 33, 34, or 35 days) after administration of the population or pharmaceutical composition to the subject. In some embodiments, the AUC is at least 500 day x mg / mL, at least 550 day x mg / mL, at least 600 day x mg / mL, at least 650 day x mg / mL, at least 700 day x mg / mL, at least 750 day x mg / mL, at least 800 day x mg / mL, or at least 850 day x mg / mL for 1 to 35 days (e.g., 1, 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, 32, 33, 34, or 35 days) after administration of the population or pharmaceutical composition to the subject. In some embodiments, the method further comprises administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is administered to the subject prior to, simultaneously with, or after administration of the composition or pharmaceutical composition. In some embodiments, the additional therapeutic agent is a second modified Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the second modified Fc polypeptide comprises the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the second modified Fc polypeptide has the amino acid sequence set forth as SEQ ID NO:5. In some embodiments, the second modified Fc polypeptide is administered at a dose of 1 mg / kg to 20 mg / kg (e.g., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg).In some embodiments, the second modified Fc polypeptide is administered at a dose of 10 mg / kg. In some embodiments, the second modified Fc polypeptide is administered once a week for four weeks. In some embodiments, the additional therapeutic agent is selected from the group consisting of an anti-inflammatory drug, an immunosuppressant, an analgesic, a disease-modifying antirheumatic drug (DMARD), a counterirritant, a platelet-boosting drug, a thrombopoietin receptor (TPOR) agonist, physical therapy, and surgery. In some embodiments, the anti-inflammatory agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, an anti-inflammatory antibody or antigen-binding fragment thereof, an anti-inflammatory cytokine, a kinase inhibitor, and intravenous immunoglobulin (IVIG).

[0011] In certain embodiments, the present invention provides (a) a sequence comprising (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2 and (ii) an aliphatic amino acid residue ( For example, disclosed herein are nucleic acid expression vectors comprising (a) a first expression cassette comprising a first mammalian promoter operably linked to a polynucleotide encoding a modified Fc polypeptide having the amino acids alanine, glycine, isoleucine, leucine, proline, valine, and methionine; and (b) a second expression cassette comprising a second mammalian promoter operably linked to a polynucleotide encoding a beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) enzyme. In some embodiments, the aliphatic amino acid at position 241 is Ala (F241A). In some embodiments, the aliphatic amino acid at position 241 is alanine (Ala; F241A). In some embodiments, the aliphatic amino acid at position 241 is glycine (Gly; F241G). In some embodiments, the aliphatic amino acid at position 241 is isoleucine (Ile; F241I). In some embodiments, the aliphatic amino acid at position 241 is leucine (Leu; F241L). In some embodiments, the aliphatic amino acid at position 241 is proline (Pro; F241P). In some embodiments, the aliphatic amino acid at position 241 is valine (Val; F241V). In some embodiments, the aliphatic amino acid at position 241 is methionine (Met; F241M). In some embodiments, the second expression cassette further comprises a polynucleotide encoding a beta-1,4-galactosyltransferase 1 (B4GALT1) enzyme. In some embodiments, the polynucleotide encoding the B4GALT1 enzyme is operably linked to a second promoter.In some embodiments, the second expression cassette further comprises an internal ribosome entry site (IRES) sequence located between the polynucleotide encoding the ST6GAL1 enzyme and the polynucleotide encoding the B4GALT1 enzyme. In some embodiments, the first promoter and the second promoter are each independently selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0012] In certain embodiments, disclosed herein are mammalian host cells comprising the expression vectors of the above embodiments. In some embodiments, the mammalian host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the mammalian host cells are human embryonic kidney 293 (HEK293) cells.

[0013] In certain embodiments, disclosed herein are methods for producing a population of modified Fc polypeptides, each modified Fc polypeptide comprising (i) an amino acid sequence having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:2, and (ii) 241 and (b) purifying the population of modified Fc polypeptides. In some embodiments, the N-glycan is attached to an asparagine (Asn) at amino acid residue 297 of the polypeptide (Asn297; numbered according to Kabat and corresponding to amino acid residue 88 of SEQ ID NO:2). In some embodiments, at least 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages.In some embodiments, at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the Fc polypeptides comprise an SA moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage. In some embodiments, the first promoter and the second promoter are each independently selected from the group consisting of a mouse CMV promoter, an EF1α promoter, an EEF2 promoter, a GAPDH promoter, a PGK promoter, an actin promoter, and a ubiquitin promoter. In some embodiments, the first expression cassette and / or the second expression cassette each independently comprise one or more regulatory sequences selected from the group consisting of a 5' untranslated region (UTR), a 3' UTR, an enhancer, an insulator, an intron, an RNA transport element, a polyadenylation signal, an internal ribosome entry site (IRES), and a transcription terminator. In some embodiments, the N-glycans of the Fc polypeptides are monosialylated or disialylated. In some embodiments, at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the Fc polypeptides comprise monosialylated N-glycans comprising an SA moiety attached via an α(2,6) linkage. In some embodiments, at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage. In some embodiments, about 90% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage.In some embodiments, at least about 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the Fc polypeptides comprise a galactose moiety. In some embodiments, about 100% of the Fc polypeptides comprise a galactose moiety. In some embodiments, the galactose moiety is attached to the α(1,3) arm and / or the α(1,6) arm of the N-glycan. In some embodiments, the galactose moiety is a branched galactose moiety.In some embodiments, the mammalian host cells comprise ST6GAL1 and B4GALT1 in a ratio of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20 (mol:mol). In some embodiments, the mammalian host cells are in a population of mammalian host cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the mammalian host cells remain viable for between 10 and 20 days (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days) after the initiation of step (a). In some embodiments, the method further comprises contacting the mammalian host cells with an additive that enhances sialylation of the Fc polypeptide. In some embodiments, the additive is selected from the group consisting of uridine, manganese, copper, dexamethasone, hydrocortisone, N-acetylmannosamine, tetraacetylated ManNAc, N-azidoacetyl D-mannosamine, 1,3,4-O-Bu3ManNAc, α(2,3)-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), siastatin B, fetuin, and glycerol. In some embodiments, the glycosylation of N-glycans is determined using HPLC, MS, or a combination thereof. In some embodiments, the HPLC is hydrophilic interaction liquid chromatography (HILIC).

[0014] In certain embodiments, disclosed herein are populations or pharmaceutical compositions (as disclosed herein) for use as medicaments.

[0015] In certain embodiments, disclosed herein is a collection or pharmaceutical composition (as disclosed herein) for treating an inflammatory disease or condition, such as an autoimmune disease or condition, in a subject in need of such treatment.For example, the inflammatory disease or condition can be arthritis or immune thrombocytopenia (ITP).

[0016] In some embodiments, the population or pharmaceutical composition has a half-life of at least 3.5 or at least 4 days following administration of the population or pharmaceutical composition to a subject.

[0017] In some embodiments, the population or pharmaceutical composition, after administration to a subject, is cleared from the subject's circulation at a rate of 45 mL / day / kg or less, 40 mL / day / kg or less, 35 mL / day / kg or less, 30 mL / day / kg or less, or 25 mL / day / kg or less.

[0018] In some embodiments, the concentration of the Fc polypeptide over time (AUC) is at least 480 days×mg / mL between 1 and 35 days after administration of the population or pharmaceutical composition to the subject, e.g., at least 500 days×mg / mL, at least 550 days×mg / mL, at least 600 days×mg / mL, at least 650 days×mg / mL, at least 700 days×mg / mL, at least 750 days×mg / mL, at least 800 days×mg / mL, or at least 850 days×mg / mL between 1 and 35 days after administration of the population or pharmaceutical composition to the subject.

[0019] In some embodiments, an additional therapeutic agent is administered to the subject. In some embodiments, the additional therapeutic agent is administered to the subject before, simultaneously with, or after administration of the population or pharmaceutical composition.

[0020] In some embodiments, the additional therapeutic agent is a second modified Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the second modified Fc polypeptide comprises the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F. In some embodiments, the second modified Fc polypeptide has the amino acid sequence set forth as SEQ ID NO:5.

[0021] In some embodiments, the second modified Fc polypeptide is administered to the subject at a dose of 1 mg / kg to 20 mg / kg, for example, at a dose of 10 mg / kg, hi some embodiments, the second modified Fc polypeptide is administered to the subject once a week for four weeks.

[0022] In some embodiments, the additional therapeutic agent is selected from the group consisting of anti-inflammatory agents, immunosuppressants, analgesics, disease-modifying antirheumatic drugs (DMARDs), counterirritants, platelet-increasing agents, thrombopoietin receptor (TPOR) agonists, physical therapy, and surgery.

[0023] In some embodiments, the anti-inflammatory agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, an anti-inflammatory antibody or antigen-binding fragment thereof, an anti-inflammatory cytokine, a kinase inhibitor, and intravenous immunoglobulin (IVIG).

[0024] definition 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 the claimed subject matter belongs. Generally, the nomenclatures and techniques used in connection with immunology, oncology, cell and tissue culture, molecular biology and protein chemistry described herein are well known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not binding on any claimed subject matter. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0025] As used herein, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" includes a plurality of polypeptides.

[0026] As used herein, all numerical values ​​or ranges include whole integers within or encompassing such ranges and fractions of values ​​or integers within or encompassing such ranges, unless the context clearly dictates otherwise. Thus, for example, a reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., etc. In another example, a reference to a range of 1 to 5,000 times includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc., etc.

[0027] As used herein, "about" a number refers to a number and a range that includes that number and a range from 10% below that number to 10% above that number. "About" a range refers to one that extends from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0028] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a modified Fc polypeptide of the present disclosure or a composition containing same) to a subject by any effective route. Exemplary routes of administration are described in the following section.

[0029] The term "effective amount," as used herein, refers to the amount of an Fc polypeptide or composition of the present disclosure that, when administered to a subject, is sufficient to induce a disclosed effect, e.g., to provide treatment, prognosis, or diagnosis of a disease (e.g., an inflammatory disease or disorder) as described herein. The therapeutically effective amount of the compositions provided herein will vary depending on the relative activity of the disclosed compositions and combinations (e.g., in treating, reducing, or alleviating a disease or disorder described herein) when used alone or in combination, as well as the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the method of administration, etc.

[0030] As used herein, the terms "Fc polypeptide," "Fc peptide," "Fc fragment," "Fc region," and "Fc domain" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain. An "Fc polypeptide" is, in some embodiments, a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.

[0031] An "isolated" polypeptide or protein or population thereof refers to a polypeptide or protein or population thereof that is free from other proteins, lipids, and nucleic acids with which it is naturally associated. In some embodiments, the polypeptide / protein or population thereof comprises at least 10% (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), or mass spectrometry (MS) analysis. In some embodiments, the isolated polypeptide / protein or population thereof described herein is produced by recombinant DNA technology or by chemical methods. In some embodiments, the isolated polypeptide comprises SEQ ID NO:2.

[0032] A "native" or "parent" Fc region comprises an amino acid sequence identical to that of an Fc region found in nature. A "variant" or "modified" Fc region comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, e.g., an amino acid substitution (e.g., F241A). In some embodiments, a modified Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, compared to a native-sequence Fc region or to the Fc region of a parent polypeptide. The modified Fc regions described herein, in some embodiments, have at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a native sequence Fc region and / or to the Fc region of a parent polypeptide.

[0033] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. Alignment for determining nucleic acid or amino acid sequence identity percentage can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2 or Megalign software. Appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared, can be determined by any suitable means. For example, sequence identity percentage values ​​can be generated using the sequence comparison computer program BLAST. As illustrated, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or relative to a given nucleic acid or amino acid sequence, B (alternatively, it can be expressed as a given nucleic acid or amino acid sequence, A, having a certain percent sequence identity to, with, or relative to a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (ratio X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0034] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, e.g., a mammal (e.g., a human), without excessive toxicity, irritation, allergic response and other problem complications, commensurate with a reasonable benefit / risk ratio.

[0035] The term "polypeptide" refers to a chain of amino acids. A polypeptide is not limited to a product of a particular length. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms are used interchangeably herein unless otherwise indicated. The term also encompasses peptide chains having post-expression modifications, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art, both naturally occurring and non-naturally occurring. In some embodiments, a polypeptide is a whole protein or a fragment thereof.

[0036] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. A "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals and laboratory animals, zoo animals, sport animals, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require the supervision of a medical professional.

[0037] The term "therapeutically effective amount" generally refers to an amount of a disclosed composition effective to "treat" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount effective to produce some desired therapeutic effect by inhibiting a disease or disorder as described herein, at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least a desired therapeutic or prophylactic effect in an organ or tissue. The amount of a therapeutic agent necessary to cause prevention and / or therapeutic treatment of a disease or disorder is not, per se, fixed. In some embodiments, the amount of a therapeutic agent administered varies with the type and extensiveness of the disease and the size of the mammal suffering from the disease or disorder. When used in conjunction with a therapy that involves administration of a therapeutic agent after a subject exhibits symptoms of a disease or disorder, the term "therapeutically effective" means that after treatment, one or more signs or symptoms of the disease or disorder are ameliorated or eliminated.

[0038] An effective response of the present disclosure is achieved when a patient experiences partial or total relief or reduction of signs or symptoms of a disease, and in the case of treating a disease (e.g., an inflammatory disease or disorder), specifically includes, but is not limited to, symptomatic remission, prolonged progression, cure, remission, prolonged survival, or other objective response. In some embodiments, expected progression-free survival is measured in months to years, depending on prognostic factors, including the number of relapses, the stage of the disease, and other factors. Prolonged survival includes, but is not limited to, at least 1 month (month), about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, etc. Overall survival is also measured, for example, in months to years. Alternatively, an effective response, in some embodiments, is when the subject's symptoms remain quiescent. Further indications for treating an indication are described in more detail below.

[0039] In some embodiments, administration of a therapeutic agent in a prophylactic method occurs prior to the manifestation of symptoms of an unwanted disease or disorder, such that the disease or disorder is prevented or, alternatively, its progression is delayed. Thus, when used in conjunction with a prophylactic method, the term "therapeutically effective" means that, following treatment, fewer subjects (on average) will develop the unwanted disease or disorder or progress in the severity of the symptoms.

[0040] As used herein, the terms "treatment," "treating," and the like refer, in some cases, to administering an agent or performing a procedure with the intent to achieve an effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a partial or complete cure of the disease and / or symptoms of the disease is achieved. "Treatment," as used herein, includes treatment of a disease or disorder (e.g., an inflammatory disease or disorder) in a mammal, particularly a human, and includes (a) preventing the disease or symptoms of the disease from occurring in a subject who is predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases associated with or caused by a primary disease), (b) inhibiting the disease, i.e., halting its development, and (c) relieving the disease, i.e., causing regression of the disease. The term "treating" includes any indication of successful treatment, amelioration, or prevention of a disease or disorder, and any objective or subjective parameter, such as alleviation, remission, or reduction of symptoms, or making the disease state more tolerable to the patient, slowing the rate of degeneration or decline, or making the end point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administering an agent or composition of the present disclosure to prevent, delay, reduce, or arrest or inhibit the onset of symptoms or conditions associated with a disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptoms, or disease side effects in a subject. A subject is "treated" for a disease or disorder if, after receiving a therapeutic dose of a therapeutic agent or composition of the present disclosure, the patient exhibits an observable and / or measurable change in a parameter or symptom of the disease or disorder.

[0041] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors, e.g., plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Expression vectors suitable for use with the compositions and methods described herein contain an expression cassette that includes a polynucleotide sequence and, optionally, additional sequence elements used for, e.g., protein expression and, optionally, integration of these polynucleotide sequences into the genome of the host cell. Certain vectors that can be used to express one or more (e.g., one, two, three, or more) recombinant polypeptides as described herein include plasmids containing regulatory sequences that direct gene transcription, e.g., promoter and enhancer elements. Other useful vectors for expressing the disclosed polypeptides contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions (UTRs), internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of the transgene carried in the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. One example of a suitable selection marker is the glutamine synthetase (GS) gene. Additional examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, zeocin, nourseothricin, carbenicillin, tetracycline, streptomycin, and spectinomycin. [Brief explanation of the drawings]

[0042] [Figure 1]

[0023] Figure 1 shows an illustration and nomenclature of a representative biantennary N-glycan structure found at asparagine 297 (Asn297) of the heavy chain of an Fc polypeptide of the present disclosure. The illustrated glycoforms are attached to the heavy chain constant region 2 (CH2) domain of the Fc polypeptide via a covalent bond at the lower terminus of each glycan structure. All subsequent descriptions of N-glycans are provided as a linear or branched sequence of carbohydrates beginning at Asn297 (e.g., Asn297 → carbohydrate 1 → carbohydrate 2, etc.). The carbohydrate closest to Asn297 is considered proximal, those further from Asn297 are considered distal, and those furthest from Asn297 are considered terminal. The G0F glycoform (i.e., core glycan) contains an N-acetylglucosamine (GlcNAc) and mannose (Man) core, modified by a fucosyl (Fuc) residue at the most proximal GlcNAc moiety. The G0 glycoform is identical to the G0F glycoform except for the absence of the Fuc moiety on the core glycan. One branch of the N-glycan is called the α(1,6) arm, and the other branch is called the α(1,3) arm. The Man5 high-mannose glycoform contains a branched glycan structure with five mannose moieties attached to the core glycan. The G1F glycoform contains a core glycan with a single terminal galactose (Gal) attached to either the α(1,6) or α(1,3) arm of the branched N-glycan. The G1 glycoform is identical to the G1F glycoform except for the absence of the Fuc moiety on the core glycan. The G2F glycoform contains a core glycan with a terminal Gal attached to each of the α(1,6) and α(1,3) arms of the branched N-glycan. The G2 glycoform is identical to the G2F glycoform except for the absence of the Fuc moiety on the core glycan. The G2FSA glycoform contains a core G2F glycan structure with a single terminal sialic acid (SA) moiety attached to one of the Gal moieties, and the G2FSA2 glycoform contains a core G2F glycan structure with a terminal SA moiety attached to each of the Gal moieties. [Figure 2A]Figure 2A shows a bar graph depicting the levels of α(2,3) sialylation after transient transfection of CHO cells with wild-type (WT) and mutant Fc polypeptides (IgG1 isotype) having an alanine (A) substitution for phenylalanine (F) at amino acid position 241 (F241), which corresponds to amino acid residue 32 of SEQ ID NO: 2. The WT Fc contains primarily glycans terminating with GlcNAc (GO or GOF; 49%) or glycans terminating with a GlcNAc containing a single Gal (G1 or GIF; 39%), but little or no SA-containing glycoforms (<1.0%; Figure 2A). [Figure 2B] Figure 2B is a bar graph showing the levels of α(2,3) sialylation after transient transfection of CHO cells with wild-type (WT) and mutant Fc polypeptides (IgG1 isotype) having an alanine (A) substitution with phenylalanine (F) at amino acid position 241 (F241), which corresponds to amino acid residue 32 of SEQ ID NO: 2. The F241A mutation results in a significant enhancement of Gal addition to the Fc domain, with molecules containing 22% mono-2,3 SA (G2FSA) and 19% di-2,3 SA (G2FSA2; Figure 2B). [Figure 3A] Figure 3A shows a bar graph depicting the N-glycan analysis of the F241A Fc mutant recombinantly expressed by stable transfection of Chinese hamster ovary (CHO) cells using 1 L culture pools. Figure 3B shows the N-glycan analysis of the F241A Fc mutant expressed alone (Pool 1; Figure 3A). Pool 1 is 18% mono-α(2,3) sialylated, 5% di-α(2,3) sialylated, and 47% with one or two terminal Gals. [Figure 3B]Figure 3B shows a bar graph depicting the N-glycan analysis of the F241A Fc mutant recombinantly expressed by stable transfection of Chinese hamster ovary (CHO) cells using 1 L culture pools. Bar graph depicting the N-glycan analysis of the F241A Fc mutant expressed in combination with beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1; pool 2; Figure 3B). Pool 2 is 30% mono-α(2,6) sialylated, 27% di-α(2,6) sialylated, and 7% with one or two terminal Gals. [Figure 3C] Figure 3C shows a bar graph depicting the N-glycan analysis of the F241A Fc mutant recombinantly expressed by stable transfection of Chinese hamster ovary (CHO) cells using 1 L culture pools. Figure 3C shows the N-glycan analysis of the F241A Fc mutant expressed in combination with ST6GAL1 and beta-1,4-galactosyltransferase 1 (B4GALT1; pool 3; Figure 3C). Pool 3 is 3% mono-α(2,6)sialylated, 81% di-α(2,6)sialylated, and 0% terminal Gal. [Figure 3D] Figure 3D shows a bar graph depicting the N-glycan analysis of the F241A Fc mutant recombinantly expressed by stable transfection of Chinese hamster ovary (CHO) cells using 1 L culture pools. A bar graph depicts the N-glycan analysis of the F241A Fc mutant expressed in combination with siRNA targeting the SA transporter, solute carrier family 35 member A1 (SLC35A1 KD; Pool 4; Figure 3D). Pool 4 is 0% sialylated and 84% has one or two terminal Gals. [Figure 4A] Figure 4A shows a blot providing independent validation of the glycan structures of pools 1-4 (P1-P4) from a 1 L stably transfected CHO culture. Images show Coomassie staining of each of P1-P4 (Figure 4A). [Figure 4B]Figure 4B shows a blot providing independent validation of the glycan structures of pools 1–4 (P1–P4) from a 1 L stably transfected CHO culture. The blot shows each of P1–P4 stained with biotinylated Sambucus nigra (SNA) lectin to detect α(2,6)SA (Figure 4B). [Figure 4C] Figure 4C shows a blot providing independent validation of the glycan structures of pools 1–4 (P1–P4) from a 1 L stably transfected CHO culture stained with Maackia amurensis (MAL I) lectin to detect terminal galactose. [Figure 5A] Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A+SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. A plot (Figure 5A) shows the serum concentration (μg / mL) of the F241A Fc mutant over time in JAX-014565 mice (mouse FcRn homozygous knockout, human FcRn hemizygous, Tg32 strain) after intravenous (IV) bolus administration of Pool 2 (F241A+ST6GAL1), Pool 3 (F241A+ST6GAL1+B4GALT1), and Pool 4 (F241A+SLC35A1 KD) using a logarithmic y-axis. Maximum exposure, as measured by area under the curve (AUC), was achieved with Pool 3 material that was 93% α(2,6)-sialylated. [Figure 5B] Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A+SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. Identical data as in Figure 5A shown with a linear scale y-axis (Figure 5B). Maximum exposure, as measured by area under the curve (AUC), was achieved with 93% α(2,6)-sialylated Pool 3 material. [Figure 5C]Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A+SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. Plot showing serum concentrations (μg / mL) of the F241A Fc mutant over time in JAX-014565 mice following intravenous (IV) administration of Pool 1 (F241A only), Pool 3 (F241A+ST6GAL1+B4GALT1), and an IgG1 Fc domain with ABDEG mutations (M252Y / S254T / T256E / H433K / N434F) to enhance FcRn binding (efgartigimod (EFG); Argenx)), shown with a logarithmic y-axis (Figure 5C). The 93% α(2,6) sialylated Fc-F241A (pool 3) showed the highest exposure in these human FcRn mice. The ERG Fc domain had the lowest AUC, similar to that of nonsialylated F241A (pool 4) as shown in Figures 5A-5B. [Figure 5D]Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A + SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. Identical data as in Figure 5C shown with a linear y-axis (Figure 5D). 93% α(2,6) sialylated Fc-F241A (Pool 3) showed the highest exposure in these human FcRn mice. The ERG Fc domain had the lowest AUC, similar to unsialylated F241A (Pool 4) as shown in Figures 5A-5B. [Figure 5E]Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A + SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. Figure 5E shows a plot depicting the mean concentration (μg / mL) of F241A Fc mutant over time in male and female CD1 mice following intravenous (IV) administration of Pool 1 (F241A 2,3 sialylated) or Pool 3 (F241A 2,6 sialylated). There was no apparent difference in exposure between male and female mice receiving either α(2,3)-sialylated (open triangles and closed triangles) or α(2,6)-sialylated (open circles and closed circles) Fc-F241A. However, the α(2,6) sialylated Fc pool had a higher exposure compared to the α(2,3) sialylated pool, although this difference is likely due to the degree of sialylation rather than the α(2,3) versus α(2,6) linkages. [Figure 5F]Figure 1 shows a plot depicting the effect of sialylation on F241A Fc mutant exposure in mice in vivo. These experiments were performed with F241A Fc material generated by stable transfection of 10 mL CHO cell culture pools. Pool 1 material (F241A alone) contained 15% mono-2,3 sialylation and 20% di-2,3 sialylation. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6 sialylation and 35% di-2,6 sialylation. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6 sialylation and 90% di-2,6 sialylation. Pool 4 material (F241A + SLC35A1 KD) contained 0% mono-sialylation and 0% di-sialylation. Bar graph showing the effect of repeated dosing (100 mg / kg weekly for 4 weeks) of α(2,3)-sialylated and α(2,6)-sialylated F241A Fc mutants in CD1 mice (Figure 5F). Greater exposure was achieved in both male and female CD1 mice after dosing with α(2,6)-sialylated Fc-F241A versus α(2,3)-sialylated Fc-F241A, both at C (pre-dose, day 21) and 24 hours after the fourth dose (24 hours post-dose, day 22). [Figure 6A]Figure 6A shows plots depicting the therapeutic effect of sialylated F241A Fc polypeptide in a mouse model of arthritis. Plots depicting the clinical scores of K / BxN transgenic mice treated with phosphate-buffered saline (PBS), 1 g / kg intravenous immunoglobulin (IVIG), or 50 mg / kg of α(2,3)-sialylated F241A material generated by transient transfection (41%) (Figure 6A). * = difference in clinical score between IVIG and α(2,3)-sialylated F241A Fc on day 6 (p=0.002); ** = difference in clinical score between IVIG and α(2,3)-sialylated F241A Fc on day 7 (p=0.04); for all other days, there were no significant differences between IVIG and α(2,3)-sialylated F241A Fc polypeptide. Peak inflammation was achieved by day 7 in PBS-treated mice. [Figure 6B] Figures 6B-6C show plots depicting the therapeutic effect of sialylated F241A Fc polypeptide in a mouse model of arthritis. Peak inflammation was achieved by day 7 in PBS-treated mice. Accordingly, clinical scores on study days 7 and 8 are also shown separately in Figures 6B-6C as box plots expressed as the mean and standard error of the mean, respectively. Both 1 g / kg IVIG and 50 mg / kg F241A Fc significantly suppressed inflammation relative to PBS. These data suggest that F241A is approximately 20-fold more potent than IVIG in the K / BxN serum transfer model of arthritis. [Figure 6C]Figures 6B-6C show plots depicting the therapeutic effect of sialylated F241A Fc polypeptide in a mouse model of arthritis. Peak inflammation was achieved by day 7 in PBS-treated mice. Accordingly, clinical scores on study days 7 and 8 are also shown separately in Figures 6B-6C as box plots expressed as the mean and standard error of the mean, respectively. Both 1 g / kg IVIG and 50 mg / kg F241A Fc significantly suppressed inflammation relative to PBS. These data suggest that F241A is approximately 20-fold more potent than IVIG in the K / BxN serum transfer model of arthritis. [Figure 6D] Figure 6D shows plots depicting the therapeutic effect of sialylated F241A Fc polypeptide in a mouse model of arthritis. Plots depicting the clinical scores of K / BxN mice treated with PBS, 1 g / kg intravenous immunoglobulin (IVIG), 50 mg / kg of 23% α(2,3)-sialylated F241A Pool 1 material, or 50 mg / kg of 84% α(2,6)-sialylated F241A Pool 3 material produced from 1 L of stable CHO cultures (Figure 6D; asterisks represent statistically significant differences compared to IVIG; p<0.05). IVIG demonstrated significantly greater anti-inflammatory activity than F241A α(2,3)-sialylated Fc from day 5 onward, as determined by unpaired t-test (p<0.01). By comparison, IVIG was significantly different from F241Aα(2,6) sialylated Fc only from day 8 onwards (paired t-test, p<0.05). [Figure 6E] Figures 6E-6F show plots depicting the therapeutic effect of sialylated F241A Fc polypeptides in a mouse model of arthritis. At days 6 and 7, the peak of inflammation in PBS-treated animals, α(2,6) sialylated F241A Fc resulted in significantly less inflammation compared to α(2,3) sialylated F241A Fc. [Figure 6F]Figures 6E-6F show plots depicting the therapeutic effect of sialylated F241A Fc polypeptides in a mouse model of arthritis. At days 6 and 7, the peak of inflammation in PBS-treated animals, α(2,6) sialylated F241A Fc resulted in significantly less inflammation compared to α(2,3) sialylated F241A Fc. [Figure 7] Figure 1 shows box and whisker plots demonstrating the therapeutic efficacy of α(2,3) sialylated F241A in a mouse model of immune thrombocytopenic purpura (ITP) before and after treatment with 6A6-IgG2a anti-mouse platelet antibody, as indicated by platelet counts over time. The 2,3 sialylated Fc-F241A material was as effective as IVIG at a 10-fold lower concentration. [Figure 8A] Figure 8A shows plots demonstrating the half-life and bioavailability of different F241A Fc glycoforms. (Figure 8A) Female humanized FcRn mice (n = 6 per group) were dosed once with 20 mg / kg wild-type (WT) Fc or one of four preparations: (1) F241A Fc alone, (2) F241A Fc + ST6GAL1, (3) F241A Fc + B4GALT1 + ST6GAL1, or (4) F241A Fc + SLC35A1 siRNA. After dosing, serum concentrations of hIgG Fc were measured by ELISA on days 1, 3, and 7, and at 2, 3, and 5 weeks until serum concentrations fell below the detection threshold of 1 μg / ml. From the data obtained, the half-life, area under the curve from the time of administration to the last measurable concentration (AUClast), and clearance rate of each Fc were calculated (see also Table 4). Data are plotted as means with standard deviations. Statistics were performed using conventional one-way analysis of variance and Tukey's multiple comparison. [Figure 8B]Figure 8 shows plots demonstrating the half-life and bioavailability of different F241A Fc glycoforms. Correlations between percent sialylation at FcF241A and clinical score (Figure 8B), half-life (Figure 8C), AUClast (Figure 8D), and clearance (Figure 8E). Red trendlines were generated by simple linear regression, and the corresponding R2 and p-values ​​are also shown. Day 7 clinical score correlations were generated from data obtained in WT mice, and other correlations were generated from data in Tg32 mice. [Figure 8C] Figure 8 shows plots demonstrating the half-life and bioavailability of different F241A Fc glycoforms. Correlations between percent sialylation at FcF241A and clinical score (Figure 8B), half-life (Figure 8C), AUClast (Figure 8D), and clearance (Figure 8E). Red trendlines were generated by simple linear regression, and the corresponding R2 and p-values ​​are also shown. Day 7 clinical score correlations were generated from data obtained in WT mice, and other correlations were generated from data in Tg32 mice. [Figure 8D] Figure 8 shows plots demonstrating the half-life and bioavailability of different F241A Fc glycoforms. Correlations between percent sialylation at FcF241A and clinical score (Figure 8B), half-life (Figure 8C), AUClast (Figure 8D), and clearance (Figure 8E). Red trendlines were generated by simple linear regression, and the corresponding R2 and p-values ​​are also shown. Day 7 clinical score correlations were generated from data obtained in WT mice, and other correlations were generated from data in Tg32 mice. [Figure 8E]Figure 8 shows plots demonstrating the half-life and bioavailability of different F241A Fc glycoforms. Correlations between percent sialylation at FcF241A and clinical score (Figure 8B), half-life (Figure 8C), AUClast (Figure 8D), and clearance (Figure 8E). Red trendlines were generated by simple linear regression, and the corresponding R2 and p-values ​​are also shown. Day 7 clinical score correlations were generated from data obtained in WT mice, and other correlations were generated from data in Tg32 mice. [Figure 9A] Figures 9A-9C show examples and plots demonstrating the protective effect of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. (Figure 9A) Sequence alignment of WT Fc (SEQ ID NO: 1) and FcAbdeg (SEQ ID NO: 5), with the thin box indicating the location of the Abdeg mutation and the thicker box indicating the location of the N297 glycosylation site. [Figure 9B] 9A-9C show illustrations and plots demonstrating the protective effect of the F241A / B4ST6 Fc polypeptide and the FcAbdeg polypeptide (SEQ ID NO: 5) in vivo (FIG. 9B). An illustration of a human IgG Fc structure with the positions of the Abdeg mutation (open hexagon) and N297 (hatched hexagon) marked, showing their relative distance from each other and different locations on the exterior versus interior of the Fc structure. [Figure 9C] Figures 9C and 9D show examples and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. WT Fc, F241A / B4ST6 Fc, and FcAbdeg were loaded onto an anti-human Fc biosensor, and the binding kinetics to mouse (Figure 9C) and human FcRn (Figure 9D) were examined by surface plasmon resonance (SPR). Dissociation constants (KD) are plotted as μM (solid bars), and association constants (KA) are plotted as Ms-1 (hatched bars). [Figure 9D]Figures 9C and 9D show examples and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. WT Fc, F241A / B4ST6 Fc, and FcAbdeg were loaded onto an anti-human Fc biosensor, and the binding kinetics to mouse (Figure 9C) and human FcRn (Figure 9D) were examined by surface plasmon resonance (SPR). Dissociation constants (KD) are plotted as μM (solid bars), and association constants (KA) are plotted as Ms-1 (hatched bars). [Figure 9E] Figures 9E and 9F show illustrations and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. Female WT C57BL / 6 (Figure 9E) and humanized homozygous FcRn(Tg32) mice (Figure 9F) were administered a single dose of 50 mg / kg or 100 mg / kg F241A / B4ST6 Fc or 10 mg / kg FcAbdeg, and serum mouse IgG was measured by ELISA up to 7 days post-dose. [Figure 9F] Figures 9E and 9F show illustrations and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. Female WT C57BL / 6 (Figure 9E) and humanized homozygous FcRn(Tg32) mice (Figure 9F) were administered a single dose of 50 mg / kg or 100 mg / kg F241A / B4ST6 Fc or 10 mg / kg FcAbdeg, and serum mouse IgG was measured by ELISA up to 7 days post-dose. [Figure 9G]Figure 9G shows an illustration and plots demonstrating the protective effects of F241A / B4ST6 Fc polypeptide and FcAbdeg polypeptide (SEQ ID NO: 5) in vivo. (Figure 9G) SIGN-R1- / - and hDC-SIGN+ mouse bone marrow-derived macrophages (BMDMs) had their Fcγ receptors blocked and were then incubated with PBS, F241A / B4ST6 Fc, or FcAbdeg, and binding was detected by FACS using an anti-human IgG Fc antibody. The plot shows the percentage of cells bound by the added Fc. [Figure 9H] Figures 9H-9J show examples and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. Female WT C57BL / 6 mice (n=5 per group) and female and male SIGN-R1- / - mice (n=5 per group) were administered arthritic K / BxN serum in a prophylactic manner in parallel with PBS, 1 g / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, or 10 mg / kg FcAbdeg, and joint swelling was clinically scored for 10 days. (Figure 9H) Clinical scores of joint inflammation over 10 days in female WT C57BL6 mice (n=5 mice per group). [Figure 9I] Figures 9H-9J show examples and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. Female WT C57BL / 6 mice (n=5 per group) and female and male SIGN-R1- / - mice (n=5 per group) were administered arthritic K / BxN serum in a prophylactic manner in parallel with PBS, 1 g / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, or 10 mg / kg FcAbdeg, and joint swelling was clinically scored for 10 days. (Figure 9I) Clinical scores of joint inflammation over 10 days in female SIGN-R1- / - mice (n=5 mice per group). [Figure 9J]Figures 9H-9J show examples and plots demonstrating the protective effects of F241A / B4ST6 Fc and FcAbdeg polypeptides (SEQ ID NO: 5) in vivo. Female WT C57BL / 6 mice (n = 5 per group) and female and male SIGN-R1- / - mice (n = 5 per group) were administered arthritic K / BxN serum in a prophylactic manner in parallel with PBS, 1 g / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, or 10 mg / kg FcAbdeg, and joint swelling was clinically scored for 10 days. (Figure 9J) Clinical scores for joint inflammation on day 7, representing the peak of disease, are plotted for each group. Bar graphs are plotted as means and standard deviations. Statistical significance was assessed using ordinary one-way analysis of variance and Tukey's multiple comparisons. [Figure 10A] Figure 10 shows illustrations and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10A) Female WT C57BL / 6 mice (n=5) were administered arthritogenic K / BxN serum on day 0, followed by PBS, 1 g / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, or 10 mg / kg FcAbdeg in a therapeutic manner on day 2. Joint swelling was clinically scored for 12 days after K / BxN. [Figure 10B] Figure 10 shows illustrations and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10B) Clinical scores of joint inflammation shown over 12 days in female WT C57BL6 mice (n=4 or 5 mice per group). [Figure 10C] Figure 10 shows illustrations and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10C) Bar graph showing the clinical scores at day 8 (peak of disease) for each of the groups. [Figure 10D]Figure 10 shows an illustration and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10D) Female WT C57BL / 6 mice (n = 4-5) were administered 50 mg / kg F241A / B4ST6 Fc alone or in combination with increasing doses of FcAbdeg (1-10 mg / kg). Ten days later, serum human IgG Fc was detected by ELISA. [Figure 10E] Figure 10E shows an illustration and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. Female WT C57BL / 6 mice (n = 3-5) were administered arthritic K / BxN serum in a prophylactic manner in parallel with PBS, 1 mg / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, 1 mg / kg FcAbdeg, or a combination of F241A / B4ST6 Fc and FcAbdeg, and joint swelling was clinically scored for 10 days. Clinical scores of joint inflammation are shown over 10 days in female WT C57BL6 mice (n = 3-5 mice per group). [Figure 10F] Figure 10 shows illustrations and plots showing the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis (Figure 10F). Clinical scores for each group on day 6, representing the peak of disease, are plotted. [Figure 10G]Figure 10G shows an illustration and plots demonstrating the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10G) Female WT C57BL / 6 mice (n=5) were administered arthritogenic K / BxN serum on day 0, followed by a therapeutic dose of PBS, 1 g / kg IVIG, 50 mg / kg F241A / B4ST6 Fc, 1 mg / kg FcAbdeg, or a combination of F241A / B4ST6 Fc and FcAbdeg on day 2. Joint swelling was clinically scored for 12 days after K / BxN. Clinical scores of joint inflammation are shown over 12 days in female WT C57BL6 mice (n=5 mice per group). [Figure 10H] Figure 10 shows an illustration and plots showing the combinatorial preventive and therapeutic effects of F241 / B4ST6 Fc and FcAbdeg in an in vivo mouse model of arthritis. (Figure 10H) Clinical scores on day 9 for each group, representing the peak of disease, are plotted. Bar graphs are plotted as means and standard deviations. Statistical significance was assessed using ordinary one-way analysis of variance and Tukey's multiple comparisons. [Figure 11A] Figure 11A depicts skin blistering scores and FcγRIIB expression in peripheral neutrophils. The downward triangles in Figure 11A indicate the days on which treatment was administered: day 0 (3 hours before administration of pathogenic autoantibodies) and day 3. The percentage of total skin area affected by blisters (y-axis ± SEM) over the number of days after disease induction (x-axis) is plotted. Open circles, squares, and upward triangles represent treatment with PBS, 100 mg / kg FcF241A, or 10 mg / kg efgartigimod, respectively. Four animals were used per group. [Figure 11B]Figure 11B shows skin blistering scores and FcγRIIB expression on peripheral neutrophils. Figure 11B shows FcγRIIB expression on the surface of peripheral neutrophils in mice treated with PBS, FcF241A, or efgartigimod (MFI±SEM; n=4 per group). Statistical significance was determined by two-way ANOVA, and FcF241A was the only one to significantly (p<0.001) increase FcγRIIB expression on day 5 of the study compared with PBS. [Figure 12A] Figures 12A, 12B, and 12C show the results of fluorescence-activated cell sorting (FACS) analysis of the kinetics of FcγRIIB upregulation on B cells and neutrophils. FACS plots assessing FcγRIIB expression (MFI ± SEM; n = 4 per group) on the surface of peripheral naive B cells, mature B cells, and neutrophils, respectively, in mice treated with PBS, NVG-2089, or efgartigimod are shown in Figures 12A, 12B, and 12C. Data are plotted against days after induction of epidermolysis bullosa acquisita (EBA) on the x-axis. Statistical significance was determined by two-way ANOVA with multiple comparisons. [Figure 12B] Figures 12A, 12B, and 12C show the results of fluorescence-activated cell sorting (FACS) analysis of the kinetics of FcγRIIB upregulation on B cells and neutrophils. FACS plots assessing FcγRIIB expression (MFI ± SEM; n = 4 per group) on the surface of peripheral naive B cells, mature B cells, and neutrophils, respectively, in mice treated with PBS, NVG-2089, or efgartigimod are shown in Figures 12A, 12B, and 12C. Data are plotted against days after induction of epidermolysis bullosa acquisita (EBA) on the x-axis. Statistical significance was determined by two-way ANOVA with multiple comparisons. [Figure 12C]Figures 12A, 12B, and 12C show the results of fluorescence-activated cell sorting (FACS) analysis of the kinetics of FcγRIIB upregulation on B cells and neutrophils. FACS plots assessing FcγRIIB expression (MFI ± SEM; n = 4 per group) on the surface of peripheral naive B cells, mature B cells, and neutrophils, respectively, in mice treated with PBS, NVG-2089, or efgartigimod are shown in Figures 12A, 12B, and 12C. Data are plotted against days after induction of epidermolysis bullosa acquisita (EBA) on the x-axis. Statistical significance was determined by two-way ANOVA with multiple comparisons. [Figure 13] Figure 1 shows images of hematoxylin and eosin (H&E) stained tissue sections from ear tissue biopsies. Skin samples from the area of ​​a mouse ear affected by blisters were biopsied and H&E stained for infiltrating immune cells. The top, middle, and bottom rows represent images from mice treated with vehicle control, FcF241A, and efgaltigimod, respectively. Images are annotated to reveal cartilage, blood, dead skin, and blisters. [Figure 14A] 14A-14B show the results of experiments assessing changes in immune cell populations present in skin biopsies. The mean percentage (+SEM) of neutrophils (FIG. 14A) and CD62L-high expressing monocytes (FIG. 14B) present in ear skin lesions is shown on the y-axis, plotted against days after epidermolysis bullosa acquisita (EBA) induction on the x-axis. Statistical significance was determined by two-way ANOVA with multiple comparisons. [Figure 14B] 14A-14B show the results of experiments assessing changes in immune cell populations present in skin biopsies. The mean percentage (+SEM) of neutrophils (FIG. 14A) and CD62L-high expressing monocytes (FIG. 14B) present in ear skin lesions is shown on the y-axis, plotted against days after epidermolysis bullosa acquisita (EBA) induction on the x-axis. Statistical significance was determined by two-way ANOVA with multiple comparisons. [Figure 15]Graph showing mouse body weights (mean + SEM normalized to study day 0) (shown on the y-axis) after experimental autoimmune encephalomyelitis (EAE) induction plotted against days after treatment initiation (shown on the x-axis) for each treatment group (n=7). Treatment groups are as indicated in the figure legend. Statistical significance compared to the vehicle control group is indicated by an asterisk and was determined using two-way analysis of variance. [Figure 16A] FIG. 16A is a graph showing the plot clinical score (mean+SEM) (shown on the y-axis) against the number of days since initiation of treatment (shown on the x-axis) for each treatment group (n=7). [Figure 16B] Figure 16B is a bar graph showing the mean AUC (+SEM) of clinical scores over the entire time course of the study. Treatment groups are defined in the figure legend. Statistical significance compared to the vehicle control group is indicated by an asterisk and is determined using two-way or one-way ANOVA, as indicated. DETAILED DESCRIPTION OF THE INVENTION

[0043] Inflammatory disorders, including autoimmune diseases, involve the abnormal activation and subsequent migration of leukocytes to affected areas of the body. These conditions encompass a wide range of illnesses that affect the lives of millions of people worldwide. Although various treatments are currently available, many have significant side effects or are only insufficiently effective in alleviating symptoms.

[0044] Immunoglobulin G (IgG) has long been recognized to mediate both pro- and anti-inflammatory activities through interactions mediated by its fragment crystallizable (Fc) region. While Fc-FcγR interactions are responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies, intravenous gamma globulin (IVIG) and its component Fc fragments are anti-inflammatory and are widely used to suppress inflammation in disease states. Glycosylation of IgG has been proposed to be important for regulating its cytotoxic and inflammatory potential. For example, previous studies have demonstrated that the anti-inflammatory activity of IVIG is a property of the Fc fragment and its attached N-glycan sialic acid (SA) moieties, indicating a combined requirement for a specific polypeptide backbone and glycosylation profile for its anti-inflammatory effect.

[0045] Sialylation of recombinant therapeutic glycoproteins, e.g., Fc polypeptides, is generally performed in mammalian or mammalian cell lines capable of reproducing mammalian-like glycosylation profiles. The addition of terminal SA residues at the N-glycans of the Fc heavy chain, particularly at asparagine 297 (Asn297), has been shown to affect various in vivo parameters of Fc polypeptides, including absorption, blood half-life, and clearance, as well as their immunogenic or immunosuppressive properties. Glycoengineering of Fc polypeptides with high levels of sialylation has faced significant obstacles, including limited and non-uniform sialylation of the polypeptide pool.

[0046] In some embodiments, disclosed herein are modified, hypersialylated Fc polypeptides having an amino acid substitution from phenylalanine (F) to an aliphatic amino acid residue (e.g., alanine, glycine, isoleucine, leucine, proline, valine, and methionine) at amino acid position 241 (F241A; numbered according to Kabat) of the Fc heavy chain. Further disclosed are methods of producing the modified Fc polypeptides using a recombinant expression system comprising one or more (e.g., one, two, three, or more) nucleic acid expression vectors and a mammalian host cell. Furthermore, the present disclosure provides methods for treating an inflammatory disease or disorder (e.g., an autoimmune disorder, such as arthritis or idiopathic thrombocytopenic purpura (ITP)) using the therapeutic agents or compositions disclosed herein. A subject with an inflammatory disease or disorder is treated according to the methods disclosed herein by administering the therapeutic agent or composition to the subject by any acceptable route.

[0047] Immunoglobulin and Fc glycosylation IgG is a glycoprotein composed of two identical heavy chains and two light chains, each consisting of a variable and constant domain. IgG contains a single N-linked glycan at asparagine 297 (Asn297) in the CH2 domain of each of its two heavy chains. The covalently attached complex polysaccharide is composed of a core biantennary pentasaccharide containing N-acetylglucosamine (GlcNAc) and mannose (Man). Further modifications of the core carbohydrate structure are observed in serum antibodies, including fucose (Fuc), bisecting GlcNAc, galactose (Gal), and variable terminal sialic acid (SA) moieties. Thus, more than 40 different glycoforms have been detected covalently attached to this single glycosylation site. Glycosylation of IgG is known to be essential for binding to all FcγRs by maintaining the open conformation of the two heavy chains. This IgG glycosylation for FcγR binding is thought to explain the inability of deglycosylated IgG antibodies to mediate inflammatory responses, such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and release of inflammatory mediators in vivo. That individual IgG glycoforms may contribute to the modulation of inflammatory responses is suggested by the altered affinity of individual FcγRs and the resulting effects on cytotoxicity reported for IgG antibodies containing or lacking Fuc. An association between autoimmune conditions and specific glycosylation patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and autoimmune vasculitis, where decreased galactosylation and sialylation of IgG antibodies have been reported.

[0048] composition In some embodiments, the polypeptides have the sequence of a variant of the wild-type human IgG Fc polypeptide of SEQ ID NO: 1 and have a high level of sialylation, e.g., at least 60% alpha(2,6) sialylation (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Disclosed herein are compositions (e.g., therapeutic compositions) comprising modified Fc polypeptides having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) or about 40% α(2,3) sialylation (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%). Also disclosed are pharmaceutical compositions comprising the modified Fc polypeptides and a pharmaceutically acceptable carrier, excipient, or diluent.

[0049] Fc polypeptide variants In some embodiments, the compositions described herein comprise a variant (i.e., modified) Fc polypeptide (e.g., an IgG1 Fc polypeptide) that contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions relative to its wild-type / parent amino acid sequence of SEQ ID NO: 1 (the bold, underlined phenylalanine residue (N) corresponds to Asn297, to which the Fc N-glycan is attached; numbered according to the Kabat system). KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0050] In some embodiments, the one or more amino acid substitutions is a phenylalanine (Phe; F) at position 241 of the Fc polypeptide (corresponding to amino acid position 32 of SEQ ID NO: 1; numbered according to the Kabat system). In some embodiments, the Phe at position 241 of the modified Fc polypeptide is substituted with an aliphatic amino acid residue (e.g., alanine, glycine, valine, leucine, isoleucine, and proline). In some embodiments, the Phe at position 241 of the modified Fc polypeptide is substituted with an alanine (Ala or A; F241A substitution). In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has the amino acid sequence of SEQ ID NO:2, or a variant thereof having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2. In some embodiments, a modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2. In some embodiments, a modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2.In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 98% (e.g., at least 98%, 99% or more) sequence identity to SEQ ID NO: 2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution (shown below in bold and underlined). A ), which has the amino acid sequence of SEQ ID NO:2 as shown below. KVDKKVEPKSCDKTHTCPPCPAPELLGGPSV A LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0051] In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 6. In some embodiments, a modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 6. In some embodiments, a modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 6. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:6. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:6. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence with at least 98% (e.g., at least 98%, 99% or more) sequence identity to SEQ ID NO:6. In some embodiments, the modified Fc polypeptide comprises an F241A substitution (shown below in bold and underlined) A ), which has the amino acid sequence of SEQ ID NO:6 as shown below. KVDKRVEPKSCDKTHTCPPCPAPELLGGPSV A LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6)

[0052] In some embodiments, the amino acid composition of the modified Fc polypeptides described herein is altered without destroying the ability of the polypeptide to bind to its respective receptor and elicit its respective cellular response. For example, some embodiments contain one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are known. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, for example, a predicted nonessential amino acid residue in SEQ ID NO: 2 is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for their ability to bind to the respective Fc receptors and elicit the corresponding cellular response to identify mutants that retain the desired activity.

[0053] In some embodiments, the modified Fc polypeptide is an IgG1 isotype. In some embodiments, the modified Fc polypeptide is an IgG3 isotype.

[0054] Sialylation of modified Fc polypeptides Glycosylation of immunoglobulins has been shown to have profound effects on their effector function, structural stability, and secretion rate from antibody-producing cells. The carbohydrate groups responsible for these properties are generally attached to the constant (C) region of the antibody. For example, glycosylation of IgG at Asn297 in the CH2 domain is required for the full ability of IgG to activate the classical pathway of complement-dependent cytolysis.

[0055] Each antibody has a distinct array of N-linked glycan structures that variably affect protein assembly, secretion, and function. These N-linked glycans vary considerably depending on the degree of processing and, in some embodiments, contain high mannose and complex biantennary oligosaccharides with or without bisecting GlcNAc and core fucose (Fuc) residues. There is usually heterogeneous processing of the core oligosaccharide structure attached to a particular glycosylation site, resulting in even monoclonal antibodies existing as multiple glycoforms. Similarly, major differences in antibody glycosylation have been shown to occur between antibody-producing cell lines, with minor differences observed for a given cell line grown under different culture conditions.

[0056] The presence of sialic acid (SA) residues on the N-glycans of Fc polypeptides has recently been identified as a key factor in mediating the anti-inflammatory effects of intravenous immunoglobulin (IVIG), which have been demonstrated in certain autoimmune diseases. This anti-inflammatory effect is proposed to be mediated by binding of the sialylated Fc component of IVIG to the inhibitory FcγRIIB and DC-SIGN receptors. Removal of IVIg sialic acid results in loss of protection in animal models of multiple sclerosis, rheumatoid arthritis, Guillain-Barré syndrome, and idiopathic thrombocytopenic purpura (ITP), despite retaining normal circulating half-life and binding to FcRn. Conversely, excessive sialylation of IVIg increases the potency of its anti-inflammatory activity by 10-30-fold in several different animal models of autoimmune disease. The biological consequences of binding to and activating type II Fc receptors are IL-33 release, upregulation of inhibitory FcγRIIB, and T regulatory cell expansion. In mice, knockout of SIGNR1 (the mouse homolog of DC-SIGN) blocks the anti-inflammatory activity of IVIg. Blockade of the IL33 receptor, knockout of FcγRIIB, or depletion of T regulatory cells also blocks the anti-inflammatory properties of IVIg in various settings. However, only a small fraction of IgG in IVIG has SA-terminated glycans, thereby requiring high doses (1–2 g / kg) of IVIG to elicit a therapeutic anti-inflammatory effect. Previous efforts to generate highly sialylated pools of IgG Fc have focused on purifying the sialylated fraction of Fc polypeptides from IVIG. However, no methods are available for recombinantly producing pure highly sialylated Fc peptides with a defined level of sialylation.

[0057] The present disclosure provides methods for enhancing sialylation of an IgG Fc polypeptide by providing a modified Fc polypeptide (e.g., the F241A Fc mutant of SEQ ID NO: 2 or a variant thereof) in a recombinant expression system (e.g., one or more nucleic acid expression vectors introduced into a host cell, e.g., a mammalian host cell) alone or in combination with one or more (e.g., one, two, or more) recombinant glycosyltransferases (e.g., ST6GAL1 and B4GALT1) under conditions and for a time sufficient to result in a desired level of Fc sialylation. In some embodiments, the one or more recombinant glycosyltransferases is ST6GAL1. The activity of ST6GAL1 results in 6-sialylated oligosaccharides comprising 6-sialylated galactose. The term "ST6GAL1" refers to a sialyltransferase capable of attaching SA to the sixth atom of an acceptor polysaccharide. In some embodiments, the one or more recombinant glycosyltransferases is B4GALT1. The term "B4GALT1" refers to an enzyme belonging to the family of beta-1,4-galactosyltransferases that transfers galactose in a β(1,4) linkage to acceptor sugars such as GlcNAc, Glc, and Xyl. In some embodiments, the one or more recombinant glycosyltransferases are ST6GAL1 and B4GALT1.

[0058] The disclosed methods are used to achieve a particular level of Fc sialylation. In some embodiments, modified Fc polypeptides of the present disclosure are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% sialylated Fc polypeptides within the pool. In some embodiments, modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 40% sialylated Fc polypeptides within the pool. In some embodiments, modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 45% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 50% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 55% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 60% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 65% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 70% sialylated Fc polypeptides within the pool.In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 75% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 80% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 85% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 90% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having at least 95% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having about 100% sialylated Fc polypeptides within the pool. In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of Fc polypeptides having between 40% and 100% sialylated Fc polypeptides within the pool, e.g., between 45% and 50%, between 50% and 55%, between 55% and 60%, between 60% and 65%, between 65% and 70%, between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%.

[0059] In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of modified Fc polypeptides comprising at least 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) Fc polypeptides having SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 65% (e.g., at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptide comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages.In some embodiments, at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via α(2,6) linkages. In some embodiments, at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, at least 98% (e.g., at least 98%, 99% or more) of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, at least 99% of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, about 100% of the modified Fc polypeptides comprise SA moieties attached to N-glycans of the Fc polypeptide via an α(2,6) linkage. In some embodiments, the N-glycans of the modified Fc polypeptides are monosialylated or disialylated. In some embodiments, at least 30% of the modified Fc polypeptides comprise monosialylated N-glycans comprising SA moieties attached via an α(2,6) linkage.In some embodiments, at least 30% of the modified Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage, hi some embodiments, about 90% of the modified Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) linkage.

[0060] In some embodiments, at least about 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise a galactose moiety. In some embodiments, at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides comprise a galactose moiety. In some embodiments, about 100% of the modified Fc polypeptides comprise a galactose moiety. In some embodiments, the galactose moiety is attached to the α(1,3) arm and / or the α(1,6) arm of the N-glycan. In some embodiments, the galactose moiety is a branched galactose moiety.

[0061] In some embodiments, the modified Fc polypeptides are expressed and / or cultured under conditions and for a time sufficient to generate a pool of modified Fc polypeptides comprising about 40% (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%) of Fc polypeptides having SA moieties attached to N-glycans of the modified Fc polypeptides via α(2,3) linkages.

[0062] In some embodiments, the N-glycan is attached to an asparagine (Asn) at amino acid residue 297 of the polypeptide (Asn297; numbered according to Kabat and corresponding to amino acid residue 88 of SEQ ID NO:2).

[0063] Treatment method Selecting a target In some embodiments, disclosed herein are methods for treating or preventing an inflammatory disease or disorder in an individual in need thereof. The term "inflammatory disease or disorder" refers to a disease or disorder (e.g., an autoimmune disease or disorder) characterized by abnormal or unwanted inflammation. In some embodiments, the autoimmune disease or disorder is a T cell-mediated autoimmune disease or disorder (e.g., multiple sclerosis and type I diabetes). The term "T cell-mediated disease" refers to any inflammatory disorder characterized by abnormally low levels of functionally active regulatory T (Treg) cells or abnormally activated effector T cells. In some embodiments, the autoimmune disease or disorder is arthritis (e.g., rheumatoid arthritis and psoriatic arthritis). In some embodiments, the autoimmune disease or disorder is idiopathic thrombocytopenic purpura (ITP).

[0064] Glycosylation of IgG has been proposed to be important for regulating its cytotoxic and inflammatory potential. For example, the anti-inflammatory activity of IVIG has been shown to be a property of the terminal SA residues on the Fc fragment and its attached N-glycans, indicating a combined requirement for a specific polypeptide backbone and glycosylation profile for its anti-inflammatory effect. Surprisingly, the inventors have shown that the disclosed compositions containing high levels of α(2,6) (e.g., at least 60%) or α(2,3) sialylation (e.g., about 40%) have robust therapeutic effects in mouse models of arthritis and ITP, thereby highlighting the potential clinical efficacy of the disclosed compositions for the treatment of inflammatory diseases or disorders.

[0065] In some embodiments, the compositions and methods described herein are used to treat patients who exhibit symptoms of an inflammatory disease or disorder. The compositions and methods described herein can also be used to treat patients who are in remission from an inflammatory disease or disorder. Additionally, the compositions and methods described herein can also be administered as a preventative treatment to patients at risk of developing an inflammatory disease or disorder.

[0066] Administration route In some embodiments, the modified Fc polypeptides described herein or compositions containing them are administered to a subject with an inflammatory disease or disorder by various routes, for example, intravenously, intradermally, subcutaneously, percutaneously, transdermally, intramuscularly, transmucosally, or intraosseously. In some embodiments, the compositions described herein are administered to a subject systemically (e.g., intravenously). In some embodiments, the compositions described herein are administered to a subject locally (e.g., to the site of inflammation). The most suitable administration route in any given case will vary depending on the particular composition administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time and route of administration), the patient's age, physique, and sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. Multiple administration routes may be used to treat a single subject. Multiple routes of administration may be used at one time to treat a single subject, or a subject may be treated initially via one route of administration and then, for example, one week, two weeks, one month, six months, or one year later via another route of administration at a second appointment. The compositions of the present disclosure may be administered to a subject once or more than once (e.g., 2-10 times) per week, month, or year for treatment.

[0067] Dosage The amount of a modified Fc polypeptide disclosed herein or a composition containing same used in conjunction with the methods of treatment disclosed herein will typically be a therapeutically effective amount. By way of non-limiting example, an effective amount is an amount sufficient to reduce symptoms of an inflammatory disease or disorder, including, for example, edema, congestion, erythema, bruising, tenderness, rigidity, swelling, fever, chills, nasal congestion, headache, difficulty breathing, fluid retention, blood clots, loss of appetite, increased heart rate, granuloma formation, fibrinous, pus, non-viscous serous fluid, ulcers, increased production of autoreactive effector immune cells, inflammation, or pain.

[0068] The appropriate effective amount of the composition to be administered for a particular application of the disclosed methods can be determined, for example, by using the guidance provided herein. For example, the effectiveness of the compositions disclosed herein in treating symptoms of an inflammatory disease or disorder can, in some embodiments, be determined by observing one or more clinical symptoms and / or physiological indicators associated with the condition. The response of an individual with an inflammatory disease or disorder to treatment can be monitored by determining the severity of the symptoms or by determining the frequency of autoreactive T cells in a sample obtained from an individual with an inflammatory disease or disorder (e.g., an autoimmune disease or disorder). The severity of symptoms of an autoimmune disease or disorder may be correlated with the number of autoreactive T cells. Furthermore, an increase in the number of autoreactive T cells in a sample can be used as an indicator for applying a treatment intended to minimize the severity of symptoms and / or treat the autoimmune disease or disorder before symptoms appear. As another example, the effectiveness of the compositions disclosed herein in treating symptoms of an autoimmune disease or disorder can be determined by relying on clinical experience with existing T cell infusion therapies. Improvement of an autoimmune disease or disorder may also be indicated by a reduced need for concomitant therapy. Those skilled in the art will know the appropriate symptoms or indicators associated with a particular autoimmune disease or disorder and will know how to determine whether an individual is a candidate for treatment as disclosed herein. The individual's condition can be monitored throughout the course of therapy, and the effective amount of the compound or composition disclosed herein administered can be adjusted accordingly.

[0069] In some embodiments, a therapeutically effective amount of a composition disclosed herein reduces symptoms associated with an inflammatory disease or disorder by, for example, at least 10%, at least %, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, a therapeutically effective amount of a composition disclosed herein reduces symptoms associated with an inflammatory disease or disorder by, for example, up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to %, or up to 100%. In some embodiments, a therapeutically effective amount of a composition disclosed herein reduces symptoms associated with an inflammatory disease or disorder by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about %, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about %, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about %, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%. In some embodiments, a therapeutically effective amount of a composition disclosed herein is a dosage sufficient to reduce symptoms associated with an inflammatory disease or disorder, e.g., for at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months or more.

[0070] Treatment effect In some embodiments, the compositions of the disclosure are effective in reducing immune cell (e.g., T cells, B cells, NK cells, ILC1s, ILC2s, ILC3s, monocytes, macrophages (M1 and M2), dendritic cells, or antigen-presenting cells) migration, reducing immune cell proliferation, reducing immune cell recruitment, increasing immune cell lymph node homing, reducing immune cell lymph node egress, reducing immune cell differentiation, reducing immune cell activation, reducing immune cell polarization, reducing immune cell cytokine production, reducing immune cell degranulation, reducing immune cell maturation, reducing immune cell AD, It is administered in an amount, and for a time, effective to result in a reduction in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of CC reduction, immune cell ADCP reduction, immune cell antigen presentation reduction, immune cell serotonin receptor expression reduction, treatment of an inflammatory disease or disorder, reduction of symptoms of an inflammatory disease or disorder, reduction in inflammation, reduction in autoantibody levels, increased organ function, and a reduction in the rate or number of relapses or acute exacerbations.

[0071] The reduction in symptom severity, in some embodiments, is any amount that achieves a therapeutic benefit in the patient. Treatment efficacy is measured over various time frames, including, for example, months to years, depending on prognostic factors, including the number of relapses, stage of the disease, and other factors.

[0072] Prolonged survival is another desirable treatment criterion, including, but not limited to, an increase in survival time of at least 1 month (month), about at least 2 months (months), about at least 3 months, about at least 4 months, about at least 6 months, about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 years or more. In some embodiments, overall survival is measured in months to years. In some embodiments, the subject's symptoms remain static or decrease.

[0073] Combination therapy The compositions disclosed herein, in some embodiments, are administered in combination with one or more (e.g., 1, 2, 3, 4, 5, or more) additional therapeutic agents or modalities for the treatment of a disease or disorder described herein (e.g., an inflammatory disease or disorder, e.g., an autoimmune disease or disorder).

[0074] In some embodiments, the one or more additional therapeutic agents are a second modified Fc polypeptide. In some embodiments, the second modified Fc polypeptide is a human IgG1 Fc polypeptide. In some embodiments, the second modified Fc polypeptide comprises one or more (e.g., 1, 2, 3, 4, or 5) mutations selected from the group consisting of M252Y, S254T, T256E, H433K, and N434F (numbered according to the EU index of Kabat, also known as "Abdeg" mutations - see Figures 9A and 9B; see also the bold, underlined residues in SEQ ID NO: 5 below). IgG1 Fcs containing the Abdeg mutation exhibit enhanced affinity for FcRn, thereby enabling the mutant Fc to outcompete native IgG for FcRN binding. As a result, the Fc Abdeg The polypeptide accelerates the depletion of circulating total IgG by saturating FcRn. In some embodiments, the second modified Fc polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO:5. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL KF HYTQKSLSLSPG (SEQ ID NO: 5) *Bold underlined amino acid residues correspond to Abdeg mutations (M252Y, S254T, T256E, H433K and N434F).

[0075] In some embodiments, an Fc polypeptide of the present disclosure is co-administered (e.g., sequentially or simultaneously) with a second modified Fc polypeptide of SEQ ID NO: 5 to a subject. In some embodiments, the second modified Fc polypeptide is administered to a subject in an amount of 1 to 20 mg / kg, e.g., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. In some embodiments, the second modified Fc polypeptide is administered to a subject weekly. In some embodiments, the second modified Fc polypeptide is administered to a subject for 1 to 8 weeks (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks).

[0076] In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, immunosuppressants, analgesics, anti-rheumatic agents such as disease-modifying anti-rheumatic drugs (DMARDs), counterirritants, platelet-increasing agents, thrombopoietin receptor (TPOR) agonists, physical therapy, surgery, or any combination thereof. In some embodiments, the anti-inflammatory agent is selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory antibodies or antigen-binding fragments thereof, anti-inflammatory cytokines, kinase inhibitors, IVIG, or any combination thereof. In some embodiments, the one or more additional therapeutic agents are prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or a biologic such as tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. For example, if the disease is RA, the second agent may be one or more of prednisone, prednisolone and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab.In some embodiments, the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylate (5-aminoalicylic acid, sulfasalazine, mesalamine, balsalazide, olsalazine), antibiotic, antihistamine, anti-TNFα (infliximab, adalimumab, certolizumab pegol, natalizumab), ustekinumab, azathioprine, belimumab, beta interferon, calcineurin inhibitor, certolizumab, corticosteroid (prednisone, methylprednisolone), cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumarate, glatiramer acetate These include acetaminophen, golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonists, long-acting beta-2 agonists, methotrexate, mitoxantrone, mycophenolate mofetil, natalizumab, NSAIDs, ocrelizumab, pimecrolimus, probiotics, retinoids, rituximab, salicylic acid, short-acting beta-2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab, and vedolizumab.

[0077] In some embodiments, the compositions of the present disclosure are administered alone or in combination with additional therapeutic agents, simultaneously or sequentially, depending on the condition to be treated.When two or more compositions are administered, the compositions are, for example, administered in combination (either sequentially or simultaneously).In some embodiments, one or more additional therapeutic agents are administered immediately before or after the composition, or 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 1 month or more before or after the administration of the composition.In some embodiments, the compositions are administered in a single dose or multiple doses.

[0078] Pharmaceuticals The present disclosure, in some embodiments, provides use of a composition described herein for the manufacture of a medicament for treating a condition, disease, or disorder described herein. In some embodiments, the medicament is formulated based on the physical characteristics of the subject requiring treatment and is formulated in single or multiple formulations based on the stage of the condition, disease, or disorder. The medicament, in some embodiments, is packaged in a suitable package with appropriate labeling for distribution to hospitals and clinics, the labeling indicating that the medicament is for treating a subject with a disease described herein. The medicament, in some embodiments, is packaged as a single or multiple unit. In some embodiments, the package includes instructions for the dosage and administration of the composition, as described below. The present disclosure is further directed to a medicament comprising a composition described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutically acceptable carrier, diluent, or excipient is selected from the group consisting of stabilizers, buffers, surfactants, bulking agents, solvents, tonicity or osmolality adjusters, antioxidants, adjuvants, and antimicrobial agents.

[0079] Manufacturing method In some embodiments, disclosed herein are methods for producing Fc polypeptides having high levels of sialylation, e.g., the modified Fc polypeptides disclosed herein. The modified Fc polypeptides of the present invention are, in some embodiments, produced under conditions that result in an increased amount of Fc sialylation at the Fc N-glycan (i.e., Asn297) compared to an unmodified (e.g., native / parent) Fc polypeptide.

[0080] Recombinant Expression Systems In some embodiments, disclosed herein are methods, systems, and vectors for achieving expression of recombinant polypeptides in host cells, particularly modified Fc polypeptides of the present disclosure.

[0081] In some embodiments, (1) (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2; and (ii) a modification having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2). Disclosed herein is a recombinant expression system comprising a nucleic acid expression vector (e.g., a plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising: (a) a first expression cassette comprising a first mammalian promoter operably linked to a polynucleotide encoding a selected Fc polypeptide; and (b) a second expression cassette comprising a second mammalian promoter operably linked to a polynucleotide encoding beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1). In some embodiments, the aliphatic amino acid at position 241 is Ala (F241A). The second expression cassette, in some embodiments, further comprises a polynucleotide encoding a beta-1,4-galactosyltransferase 1 (B4GALT1) enzyme. In some embodiments, the polynucleotide encoding the B4GALT1 enzyme is operably linked to a second promoter. In some embodiments, the second expression cassette further comprises an internal ribosome entry site (IRES) sequence located between the polynucleotide encoding the ST6GAL1 enzyme and the polynucleotide encoding the B4GALT1 enzyme. In some embodiments, the first promoter and the second promoter are each independently selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0082] In some embodiments, (1) (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2, and (ii) a modified F having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2). Disclosed herein is a recombinant expression system comprising a nucleic acid expression vector (e.g., a plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising: (a) a first expression cassette comprising a first mammalian promoter operably linked to a polynucleotide encoding a c polypeptide, a polynucleotide encoding ST6GAL1, and an IRES sequence located therebetween; and (b) a second expression cassette comprising a second mammalian promoter operably linked to a polynucleotide encoding a B4GAL1 enzyme. In some embodiments, the aliphatic amino acid at position 241 is Ala (F241A). In some embodiments, the first promoter and the second promoter are each independently selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0083] In some embodiments, (1) (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2, and (ii) a modified ribonucleotide having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2). Disclosed herein is a recombinant expression system comprising a nucleic acid expression vector (e.g., a plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising (a) a first expression cassette comprising a first mammalian promoter operably linked to a polynucleotide encoding an Fc polypeptide, a polynucleotide encoding B4GALT1, and an IRES sequence therebetween; and (b) a second expression cassette comprising a second mammalian promoter operably linked to a polynucleotide encoding the ST6GAL1 enzyme. In some embodiments, the aliphatic amino acid at position 241 is Ala (F241A). In some embodiments, the first promoter and the second promoter are each independently selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0084] In some embodiments, (1) a first polynucleotide encoding (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2, and (ii) a modified Fc polypeptide having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2). Disclosed herein is a recombinant expression system comprising a nucleic acid expression vector (e.g., a plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising an expression cassette comprising a mammalian promoter operably linked to (1) a first polynucleotide encoding the ST6GAL1 enzyme, (2) a second polynucleotide encoding the ST6GAL1 enzyme, (3) a first IRES sequence disposed between the first and second polynucleotides, (4) a third polynucleotide encoding the B4GALT1 enzyme, and (5) a second IRES sequence disposed between the second and third polynucleotides. In some embodiments, the aliphatic amino acid at position 241 is Ala (F241A). In some embodiments, the promoter is selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0085] In some embodiments, disclosed herein is a recombinant expression system comprising: (a) two or more (e.g., two, three, or more) nucleic acid expression vectors (e.g., plasmids, RNA vectors, viruses, viral vectors, or other suitable replicons) that together encode (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2 and (ii) a modified Fc polypeptide having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2), and (b) an ST6GAL1 enzyme and / or optionally (c) a B4GALT1 enzyme.

[0086] In some embodiments, the two or more (e.g., two, three, or more) nucleic acid expression vectors (e.g., plasmids, RNA vectors, viruses, viral vectors, or other suitable replicons) are two expression vectors. In some embodiments, the two expression vectors include: (a) a first expression vector comprising an expression cassette comprising a first promoter operably linked to a polynucleotide encoding (i) an amino acid sequence at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO: 2 and (ii) a modified Fc polypeptide having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO: 2); and (b) a second expression vector comprising an expression cassette comprising a second promoter operably linked to a polynucleotide encoding an ST6GAL1 enzyme, and optionally (c) a polynucleotide encoding a B4GALT1 enzyme. In some embodiments, the polynucleotide encoding the ST6GAL1 enzyme and the polynucleotide encoding the B4GALT1 enzyme are separated by an IRES sequence.

[0087] In some embodiments, the two expression vectors comprise: (a) a first expression vector comprising an expression cassette comprising a first promoter operably linked to (i) a polynucleotide encoding a modified Fc polypeptide having an amino acid sequence at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2 and (ii) an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2), and a polynucleotide encoding an ST6GAL1 enzyme; and (b) a second expression vector comprising an expression cassette comprising a second promoter operably linked to a polynucleotide encoding a B4GALT1 enzyme. In some embodiments, the polynucleotide encoding the modified Fc polypeptide and the polynucleotide encoding the ST6GAL1 enzyme are separated by an IRES sequence.

[0088] In some embodiments, the two expression vectors include (a) a first expression vector comprising an expression cassette comprising a first promoter operably linked to a polynucleotide encoding (i) an amino acid sequence at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO: 2 and (ii) a modified Fc polypeptide having an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO: 2), and a polynucleotide encoding a B4GALT1 enzyme; and (b) a second expression vector comprising an expression cassette comprising a second promoter operably linked to a polynucleotide encoding an ST6GAL1 enzyme. In some embodiments, the polynucleotide encoding the modified Fc polypeptide and the polynucleotide encoding the B4GALT1 enzyme are separated by an IRES sequence.

[0089] In some embodiments, the two or more (e.g., two, three, or more) nucleic acid expression vectors (e.g., plasmids, RNA vectors, viruses, viral vectors, or other suitable replicons) are three expression vectors. In some embodiments, the three expression vectors contain (a) (i) an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO:2 and (ii) a sequence at position 241 (numbered according to Kabat, corresponding to amino acid residue 32 of SEQ ID NO:2). (a) a first expression vector comprising an expression cassette comprising a first promoter operably linked to a polynucleotide encoding a modified Fc polypeptide having an aliphatic amino acid residue in the Fc region (a) of the Fc polypeptide; (b) a second expression vector comprising an expression cassette comprising a second promoter operably linked to a polynucleotide encoding an ST6GAL1 enzyme; and (c) a third expression vector comprising an expression cassette comprising a third promoter operably linked to a polynucleotide encoding a B4GALT1 enzyme.

[0090] In some embodiments, the polynucleotide encoding the ST6GAL1 enzyme encodes an ST6GAL1 enzyme having the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:3, as shown below. MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSS SKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREID DHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWEL WDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (SEQ ID NO: 3; UniProt ID number: P15907-1)

[0091] In some embodiments, the polynucleotide encoding the B4GALT1 enzyme encodes a B4GALT1 enzyme having the amino acid sequence of SEQ ID NO:4 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO:4, as shown below. MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLH PVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFL TINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS (SEQ ID NO: 4; UniProt ID number: P15291-1)

[0092] In some embodiments, a polynucleotide encoding any one of the polypeptides disclosed herein (e.g., a modified Fc polypeptide, ST6GAL1 enzyme, or B4GALT1 enzyme) is codon-optimized. Codon optimization refers to the process of modifying a nucleic acid sequence according to the principle that the frequency of occurrence of synonymous codons (e.g., codons encoding the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Sequences modified in this manner are referred to herein as "codon-optimized." This process can be performed on any of the sequences described herein to enhance expression or stability. The sequence surrounding the translation start site can be converted to a consensus Kozak sequence by any suitable method.

[0093] Expression vector A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into host cells. The expression vectors used in the compositions and methods described herein may contain one or more (e.g., one, two, three or more) polynucleotides encoding one or more (e.g., one, two, three or more) polypeptides of the present disclosure, and may further comprise one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more) nucleic acid elements used to regulate the expression of these substances and / or the integration of such polynucleotides into the genome of host cells.

[0094] In some embodiments, the vector is a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, bacteriophage, extrachromosomal element, minichromosome, or artificial chromosome. Alternatively, in some embodiments, the vector, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it is integrated. Certain vectors used for expression of one or more engineered polypeptides described herein, in some embodiments, include a plasmid containing regulatory sequences that direct gene transcription, such as a promoter and, optionally, an enhancer region. Other useful vectors for expression of one or more polypeptides of the present disclosure contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes contained in the expression vector. The expression vector suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. An example of a suitable selection marker is the glutamine synthetase (GS) gene. Additional examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, zeocin, nourseothricin, carbenicillin, tetracycline, streptomycin, and spectinomycin.

[0095] In some embodiments, the expression vector of the present disclosure further comprises a polynucleotide encoding a protein tag, such as a His-tag (e.g., 6x-His), a maltose binding protein tag, a SNAP tag, a FLAG tag, a halo tag, a fluorescent protein tag, etc.

[0096] viral vectors Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into host cells.Viral genomes are particularly useful vectors for gene delivery, because the polynucleotides contained in these genomes are usually integrated into the nuclear genome of host cells by systemic or specific transduction.These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., viral vectors of the Retroviridae family), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and double-stranded DNA viruses, including alphaviruses and adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma, avian C virus, mammalian C, B, and D viruses, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus.Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason-Pfizer simian virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0097] Regulatory elements The recognition and binding of a polynucleotide encoding one or more polypeptides disclosed herein by mammalian RNA polymerase is important for gene expression. Such elements may include sequence elements in the polynucleotide(s) that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription start site. Such sequence elements include, for example, mammalian promoters, specific transcription initiation factors, and sequences that are ultimately recognized and bound by RNA polymerase.

[0098] Polynucleotides suitable for use with the compositions and methods described herein also include those encoding the modified Fc polypeptides of the present disclosure downstream of a mammalian promoter. In some embodiments, the mammalian promoter is selected from the group consisting of an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

[0099] Alternatively, promoters derived from viral genomes can be used for stable expression of these substances in mammalian cells. Examples of functional viral promoters that can be used to drive mammalian expression of these substances include the adenovirus late promoter, vaccinia virus 7.5K promoter, simian virus 40 (SV40) promoter, cytomegalovirus promoter, herpes simplex virus (HSV) tk promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) promoters (e.g., murine CMV promoter).

[0100] Once a polynucleotide encoding one or more recombinant polypeptides of the present disclosure has been extrachromosomally internalized by a host cell and / or integrated into the host cell's nuclear DNA, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the host cell to an exogenous chemical reagent, such as a substance that modulates the binding of transcription factors and / or RNA polymerase to a promoter, thus regulating gene expression. The chemical reagent can act to promote the binding of RNA polymerase and / or transcription factors to a promoter, for example, by removing promoter-bound repressor proteins. Alternatively, the chemical reagent can act to increase the affinity of the promoter for RNA polymerase and / or transcription factors, thereby increasing the transcription rate of genes located downstream of the promoter in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to host cells to promote gene expression according to established protocols.

[0101] Other gene regulatory elements that can be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences.Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides containing genes of interest, resulting in DNA adopting a three-dimensional orientation that is favorable for the binding of transcription factors and RNA polymerase at the transcription start site.Therefore, polynucleotides for use in the compositions and methods described herein include those encoding the modified Fc polypeptides of the present disclosure and also mammalian enhancer sequences.Many enhancer sequences derived from mammalian genes are currently known, including enhancers derived from genes encoding mammalian globin, elastase, albumin, α-fetal protein, and insulin.Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the CMV early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers can be spliced ​​into a vector containing a polynucleotide encoding a water-producing NADH oxidase, for example, at a position 5' or 3' to the gene. In a preferred orientation, the enhancer is placed 5' to the promoter, which is, in turn, positioned 5' to the polynucleotide encoding a modified Fc polypeptide of the present disclosure.

[0102] Other exemplary regulatory elements suitable for use with the expression vectors described herein include untranslated region (UTR, e.g., 5'UTR and / or 3'UTR) insulator elements, terminator elements, polyadenylation signals, response elements and other functional elements or signals.

[0103] host cell The modified Fc polypeptides of the present invention are, in some embodiments, produced in a host expression system, e.g., a host cell (e.g., a mammalian cell), capable of expressing the modified Fc polypeptides of the present disclosure and / or one or more glycosyltransferases (e.g., ST6GAL1 and / or B4GALT1). Typically, such host expression systems may include bacterial, fungal, plant, mammalian, or insect expression systems. In some embodiments, the host cell is a mammalian host cell, such as a Chinese hamster ovary (CHO) cell line (e.g., CHO-K1; ATCC CCL-61), a green monkey cell line (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)), a mouse cell (e.g., NS / 0), a baby hamster kidney (BHK) cell line (e.g., ATCC CRL-1632 or ATCC CCL-10), or a human cell (e.g., HEK293 (ATCC CRL-1573) or 293T (ATCC CRL-11268)), or any other suitable cell line available from a public repository, such as the American Type Culture Collection, Rockville, MD. Additionally, insect cell lines, e.g., Lepidoptera cell lines, e.g., Sf9, plant cell lines, fungal cell lines, e.g., yeasts such as Saccharomyces cerevisiae and Pichia pastoris, Hansenula species, or Bacillus species, e.g., B. subtilis, or bacterial expression systems based on E. coli, can be used. Modifications to the host cell may be required to ensure N-linked glycosylation and glycan maturation, resulting in complex biantennary sugars, as typically found in the Fc domain of human IgG. In some embodiments, the mammalian host cells described herein are transformed (e.g., transfected or transduced) with any one of the recombinant expression systems described herein.

[0104] Despite the availability of several other mammalian cell lines, the majority of recombinant therapeutic proteins produced today are manufactured in CHO cells. Their strengths include, for example, robust growth as adherent cells or in suspension, adaptability to serum-free and chemically defined media, high productivity, and an established history of regulatory approval for therapeutic recombinant protein production. They are also highly amenable to genetic modification, and methods used for cell transfection, recombinant protein expression, and clonal selection are well characterized. CHO cells also offer human-compatible post-translational modifications. As used herein, "CHO ​​cells" includes, but is not limited to, CHO-K1, CHO-DG44, CHO-M, CHO-S, CHO GS knockout, and variants and derivatives thereof.

[0105] Thus, in some embodiments, the host cells described herein are modified with one or more (e.g., one, two, three, or more) recombinant expression systems of the present disclosure to achieve a desired level of recombinant protein expression, including expression of a modified Fc polypeptide (e.g., F241A Fc) and / or one or more (e.g., one, two, or more) glycosyltransferases (e.g., ST6GAL1 and / or B4GALT1). In some embodiments, the host cells described herein are modified to express both the ST6GAL1 and B4GALT1 enzymes in a desired ratio. For example, host cells (e.g., mammalian host cells) are, in some embodiments, modified to achieve a ratio of ST6GAL1 and B4GALT1 enzyme expression of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20 (mol:mol). In some embodiments, the ratio of the amount of ST6GAL1 and B4GALT1 protein expression in a host cell is determined by the strength of the promoter sequence(s) to which each of these proteins is operably linked. In some embodiments, the ratio of the amount of ST6GAL1 and B4GALT1 protein expression in a host cell is determined by one or more regulatory sequences (e.g., enhancers, polyadenylation signals, terminators, insulators, UTR sequences, etc.) present in the vector(s) containing the enzyme-encoding polynucleotide(s). In some embodiments, the ratio of the amount of ST6GAL1 and B4GALT1 protein expression in a host cell is determined by the host cell type. In some embodiments, the ratio of the amount of ST6GAL1 and B4GALT1 protein expression in a host cell is determined by one or more factors specific to the host cell.

[0106] Additionally, the methods described herein can include maintaining host cell viability at a level sufficient to allow high levels of sialylation of the modified Fc polypeptides described herein. Without wishing to be bound by any theory, lysis of host cells in culture can result in the release of sialidase enzymes into the culture medium, which can reduce the overall level of Fc sialylation through sialidase-mediated cleavage of SA moieties from Fc glycans. Thus, the present disclosure provides methods for maintaining host cell viability at or above a predetermined value, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% viability after 10 to 20 days (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days) in culture. Host cell viability can be maintained and assessed using any suitable method.

[0107] Culture conditions In certain embodiments, disclosed herein are methods for culturing host cells of the present disclosure in a medium under conditions that allow expression of one or more polypeptides encoded by the polynucleotides disclosed herein, and purifying the one or more polypeptides from the cultured cells or cell medium. To increase the level of sialylation of the modified Fc polypeptides described herein, the culture conditions under which the host cells of the present disclosure are maintained can be optimized. For example, in some embodiments, to increase the SA content, the production rate is reduced and osmolality is generally maintained within lower limits appropriate for the particular host cells being cultured. In some embodiments, an osmolality in the range of about 250 mOsm to about 450 mOsm (e.g., between 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, and 440-450 mOsm) is appropriate for increasing SA content. Previous studies have reported that the SA content of sugar side chains attached to antibodies differs significantly when the antibodies are produced as ascites or in serum-free or serum-containing culture medium. Furthermore, others have shown that using different bioreactors for cell growth and medium oxygenation affected the amount of galactose and SA in the glycans attached to the antibody.

[0108] Furthermore, in some embodiments, the culture conditions are further modified to increase the sialylation rate of the polypeptides of the present disclosure. One method for enhancing Fc sialylation is by adding substances that enhance sialylation (e.g., by inhibiting sialidase activity), such as uridine, manganese, copper, dexamethasone, hydrocortisone, N-acetylmannoseamine, tetraacetylated ManNAc, N-azidoacetyl-D-mannosamine, 1,3,4-O-Bu3ManNAc, α(2,3)-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), siastatin B, fetuin, and glycerol.

[0109] In some embodiments, Fc sialylation is enhanced by modifying the culture conditions to a pH that favors sialylation, for example, a pH of about 7.2 (eg, 7.0, 7.1, 7.2, 7.3, or 7.4).

[0110] Methods for delivering recombinant nucleic acids into host cells The vectors disclosed herein can be introduced into cells by various methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. The gene encoding the polypeptide of the present disclosure can also be introduced into host cells by targeting the vector containing the gene encoding such substance to cell membrane phospholipids.

[0111] For example, electroporation can be used to permeabilize host cells by applying an electrostatic potential to the cells of interest. Host cells thus subjected to an external electric field are then susceptible to the uptake of exogenous nucleic acids. A similar technique, nucleofection, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells.

[0112] An additional technique useful for transfection of target cells is squeeze-poration, which induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to the applied stress. This technique has the advantage that no vector is required for delivery of nucleic acids into cells, such as target cells.

[0113] Lipofection represents another technique useful for transfecting target cells. This method involves loading nucleic acids into liposomes, which often exhibit cationic functional groups, such as quaternary or protonated amines, facing the exterior of the liposome. This leads to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids is to contact cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with the cell membrane include activated dendrimers, polyethyleneimine, and diethylaminoethyl (DEAE)-dextran. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of nucleic acids.

[0114] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also known as optical transfection, which involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases superior to, electroporation.

[0115] Impalefection is another technique that can be used to deliver genetic material into target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene intended for intracellular delivery is attached to the nanostructure surface. A tip with an array of these needles is then pressed against a cell or tissue. Cells impaled by the nanostructures can express the delivered gene(s).

[0116] Magnetofection can also be used to deliver nucleic acids to target cells. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, are fully biodegradable, and are coated with specific cationic molecules that vary depending on the application. The association with the nucleic acid vector is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by a magnet.

[0117] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves using sound waves (usually ultrasonic frequencies) to modify the permeability of the cell plasma membrane, thereby permeabilizing the cells and allowing polynucleotides to penetrate the cell membrane.

[0118] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles derived from the co-overexpression of the glycoprotein VSV-G, for example, together with a genome-modifying protein, for example, a nuclease, can be used to efficiently deliver proteins into cells, which then catalyze the site-specific cleavage of endogenous polynucleotide sequences, preparing the genome of the cell for covalent integration of a polynucleotide of interest, for example, a gene or regulatory sequence.

[0119] Protein purification The modified Fc polypeptides of the disclosure can be recovered and purified from recombinant cell culture by any suitable method, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography (HPLC) can also be used for purification.

[0120] The Fc polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Depending on the host used in a recombinant production procedure, the Fc polypeptides of the present disclosure may be glycosylated (e.g., sialylated).

[0121] Analytical methods for assessing Fc sialylation Fc sialylation can be assessed by any suitable assay. For example, sialylation of an Fc polypeptide is assessed by HPLC. In some embodiments, sialylation of a modified Fc polypeptide of the present disclosure is assessed by hydrophilic interaction liquid chromatography (HILIC). In some embodiments, sialylation of a modified Fc polypeptide of the present disclosure is assessed by MS. In some embodiments, sialylation of a modified Fc polypeptide of the present disclosure is assessed by size exclusion chromatography (SEC). In some embodiments, sialylation of a modified Fc polypeptide of the present disclosure is assessed by HPLC, HILIC, MS, SEC, HPLC-UV, or any combination thereof.

[0122] Additional Fc modifications The disclosed Fc polypeptides, in some embodiments, are further modified using techniques known in the art for various purposes (i.e., in addition to an aliphatic amino acid substitution at amino acid residue 241 of the Fc heavy chain, e.g., an F241A substitution). In some embodiments, the Fc polypeptides of the present disclosure are further modified to increase the half-life of the antigen-binding fragment in circulation when administered to a subject (e.g., a human). In some embodiments, the Fc region disclosed herein is modified to reduce or silence an effector function of the Fc polypeptide (e.g., antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), opsonization, phagocytosis, transcytosis, neutralization of infectivity, inflammation, mucosal immunity, and neonatal immunity). In some embodiments, the modified Fc polypeptides of the present disclosure are further modified to modulate binding of the Fc polypeptide to one or more Fc receptors.

[0123] The covalent modification of Fc polypeptide is also included herein.In some embodiments, where appropriate, covalent modification is carried out by chemical synthesis or by enzymatic or chemical cleavage of polypeptide.In some embodiments, other types of covalent modification are introduced by reacting targeted amino acid residue with organic derivatizing agent, which can react with selected side chain or N- or C-terminal residue.

[0124] Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0125] In some embodiments, histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful in some embodiments, and the reaction is performed in 0.1 M sodium cacodylate at pH 6.0 in some embodiments.

[0126] In some embodiments, lysinyl and amino-terminal residues are reacted with succinic or carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residue. Other suitable reagents for derivatizing alpha-amino-containing residues include transaminase-catalyzed reactions with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and glyoxylate.

[0127] In some embodiments, arginyl residues are modified by reaction with one or several conventional reagents, such as phenylglyoxal, α(2,3)-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents, in some embodiments, react with lysine groups as well as the arginine epsilon-amino group.

[0128] In some embodiments, specific modifications of tyrosyl residues are made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used in some embodiments to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be used to prepare labeled proteins for use in radioimmunoassays. 125 I or 131It is iodinated using I.

[0129] Carboxyl side groups (aspartyl or glutamyl) are specifically modified by reaction with carbodiimides (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Further, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0130] In some embodiments, glutaminyl and asparaginyl residues are deamidated to the corresponding glutamyl and aspartyl residues, respectively, which are deamidated under neutral or basic conditions.

[0131] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine and histidine side chains, acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0132] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the therapeutic agents of the present disclosure. These procedures do not require production of the Fc polypeptide in a host cell with glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, in some embodiments, the sugar(s) are attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, e.g., of cysteine, (d) free hydroxyl groups, e.g., of serine, threonine, or hydroxyproline, (e) aromatic residues, e.g., of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.

[0133] Another type of covalent modification involves linking the polypeptide to one of a variety of non-proteinaceous polymers, for example, polysaccharide polymers such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or dextran. In some embodiments, the Fc polypeptides of the present disclosure are modified by the addition of polyethylene glycol (PEG). In some embodiments, PEG modification (PEGylation) leads to one or more of improved circulation time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0134] Fatty acids and fatty acid esters are also suitable moieties for covalently modifying the Fc polypeptides of the present disclosure, and may be saturated or contain one or more units of unsaturation. Fatty acids suitable for modifying the Fc polypeptides disclosed herein include, for example, n-dodecanoate (C 12 , laurate), n-tetradecanoate (C 14 , myristate), n-octadecanoate (C 18 , stearate), n-eicosanoate (C 20 , arachidate), n-docosanoate (C 22 , behenate), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cis-Δ9-octadecanoate (C 18 , oleate), all-cis-Δ5,8,11,14-eicosatetraenoate (C 20 Suitable fatty acid esters include mono-esters of dicarboxylic acids containing a straight-chain or branched lower alkyl group. The lower alkyl group can contain from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

[0135] In some embodiments, the Fc polypeptides provided herein are conjugated or linked to a therapeutic moiety, an imaging or detectable moiety, or an affinity tag. Conjugating or linking the polypeptide can be performed using any suitable method. The association (binding) between the compound and the label includes any suitable means, including, but not limited to, covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the Fc polypeptide is conjugated to or recombinantly engineered with an affinity tag (e.g., a purification tag). Affinity tags, such as polyhistidine tags (e.g., His6), are suitable for use with the compositions and methods described herein.

[0136] Pharmaceutical Composition In some embodiments, there is provided (a) a population of modified Fc polypeptides, wherein each modified Fc polypeptide (i) has an amino acid sequence that is at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence of SEQ ID NO: 2, and (ii) has an aliphatic amino acid residue (e.g., Ala) at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO: 2), and is linked to the Fc polypeptide via an α(2,6) linkage. Disclosed herein are pharmaceutical compositions comprising: (a) a population of modified Fc polypeptides, the population comprising at least 60% (e.g., at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptides having sialic acid (SA) moieties attached to N-glycans; and (b) a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are useful for in vitro or in vivo analysis, or, in the case of pharmaceutical compositions, for administration to a subject in vivo or ex vivo to treat a subject having a disease or disorder (e.g., an inflammatory disease or disorder, e.g., an autoimmune disease or disorder) using the disclosed polypeptides.

[0137] In some embodiments, the carrier, diluent, or excipient is a stabilizer, buffer, surfactant, bulking agent, solvent, tonicity or osmolality adjuster, antioxidant, adjuvant, antimicrobial, or other suitable material. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration.

[0138] Pharmaceutical formulations comprising Fc polypeptides identified by the methods described herein are prepared for storage, in some embodiments, in the form of a lyophilized formulation or an aqueous solution, by mixing the protein having the desired degree of purity with an optional physiologically acceptable carrier, diluent, or excipient. Acceptable carriers, diluents, or excipients are those that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, phosphate, citrate, histidine, TRIS, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, e.g., methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., proline, glycine, glutamine, asparagine, histidine, arginine, or lysine; glucose, monosaccharides, disaccharides and other carbohydrates, including sucrose, mannose or dextrins; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, e.g., TWEEN®, PLURONICS®, polyethylene glycol (PEG), polysorbates (e.g., For example, polysorbates 20, 40, 60, and 80) and poloxamers (e.g., P101, P105, P108, P122, P123, P124, P181, P182, P183, P184, P185, P188, P212, P215, P217, P231, P234, P235, P237, P238, P282, P284, P288, P331, P333, P334, P335, P338, P401, P402, P403, and P407). In some embodiments, the pharmaceutical composition is stable as a liquid solution at room temperature.

[0139] An acceptable carrier is physiologically acceptable to the subject to which it is administered and retains the therapeutic properties of the compound in / with which it is administered. Acceptable carriers and their formulations are generally described in Remington's Pharmaceutical Sciences, supra. One exemplary carrier is physiological saline. The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating agent, that is involved in the retention or transport of the target compound from the administration site in one organ or body part to another organ or body part, or in an in vitro assay system. Each carrier is acceptable in the sense of being compatible with the other ingredients in the formulation and not toxic to the subject to which it is administered. An acceptable carrier should not alter the specific activity of the target compound.

[0140] In some embodiments, the pharmaceutical compositions disclosed herein further comprise an acceptable additive to improve the stability of the compound in the composition and / or to control the release rate of the composition. The acceptable additive does not alter the specific activity of the compound of interest. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, the acceptable additive is combined with an acceptable carrier and / or excipient, such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants, such as polysorbate 20 or polysorbate 80, to increase the stability of the polypeptide and reduce gelation of the solution. In some embodiments, the surfactant is added to the composition in an amount of 0.01% to 5% of the solution. The addition of such an acceptable additive increases the stability and half-life of the composition during storage.

[0141] In some embodiments, the pharmaceutical compositions disclosed herein contain an isotonic buffer, such as a phosphate, acetate, histidine, or TRIS buffer, in combination with a tonicifying and stabilizing agent, such as a polyol, sorbitol, sucrose, or sodium chloride. In some embodiments, the tonicity agent is present in the composition in an amount of about 5%.

[0142] In some embodiments, the pharmaceutical compositions disclosed herein comprise a surfactant at 0.01-0.02% w / v to prevent aggregation, for stabilization, etc.

[0143] In some embodiments, the pH of the pharmaceutical compositions disclosed herein ranges from 4.5 to 6.5 or 4.5 to 5.5.

[0144] In some embodiments, the pharmaceutical compositions disclosed herein also contain more than one active compound as needed for the indication being treated, e.g., those with complementary activities that do not adversely affect each other. Such molecules are suitably present in any combination in amounts that are effective for the intended purpose.

[0145] In some embodiments, the active ingredient is encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.

[0146] Suspension and crystalline forms of the polypeptide are also contemplated herein, and any suitable method may be used to create suspension and crystalline forms.

[0147] In some embodiments, the pharmaceutical compositions disclosed herein are sterile.In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by any suitable conventional sterilization technique.For example, sterilization can be easily achieved by filtration through a sterile filtration membrane.In some embodiments, the solution obtained is packaged for use under aseptic conditions, or filtered, and lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0148] Lyophilization is used in some embodiments to stabilize polypeptides for long-term storage, such as when the polypeptide is relatively unstable in a liquid composition.

[0149] In some embodiments, excipients, such as polyols (including mannitol, sorbitol, and glycerol), sugars (including glucose and sucrose), and amino acids (including alanine, glycine, and glutamic acid), act as stabilizers for lyophilized products. Polyols and sugars are also used in some embodiments to protect polypeptides from damage induced by freezing and drying, and to enhance stability during storage in a dry state. Sugars are, in some embodiments, effective during both the lyophilization process and storage. Other classes of molecules, including monosaccharides and disaccharides and polymers such as PVP, have also been reported as stabilizers for lyophilized products.

[0150] For injection, in some embodiments, the pharmaceutical composition disclosed herein is a powder suitable for reconstitution with appropriate solution as described above.Examples of these include, but are not limited to, freeze-dried, rotary-dried or spray-dried powder, amorphous powder, granules, precipitate.For injection, the composition optionally contains stabilizer, pH modifier, surfactant, bioavailability modifier and combinations thereof.

[0151] In some embodiments, sustained-release preparations are prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, and these matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and ethyl-L-glutamate, injectable microspheres composed of non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as lactic acid-glycolic acid copolymers and leuprolide acetate, and poly-D-(-)-3-hydroxybutyric acid.Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for 100 days, while certain hydrogels release proteins for shorter periods.Encapsulated polypeptides remain in the body for a long time, but they may denature or aggregate due to exposure to humidity at 37°C, resulting in the loss of biological activity and possible changes in immunogenicity. Rational strategies devised for stabilization vary in some cases depending on the mechanism involved: for example, if the aggregation mechanism is found to be intermolecular S-bond formation via thio-disulfide exchange, stabilization is in some cases achieved by modifying sulfhydryl residues, lyophilization from acidic solution, controlling water content using appropriate additives, and developing specific polymer matrix compositions.

[0152] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, fast-releasing, long-acting, or sustained-releasing as described herein, hi some embodiments, the pharmaceutical compositions disclosed herein are formulated for controlled release or for sustained release.

[0153] Pharmaceutical compositions are administered by injection, including, but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, intracerebrospinal, intraosseous, or intramuscular injection. Excipients and carriers for use in formulating compositions for various injections are contemplated herein. The following description is merely exemplary and does not limit the scope of the compositions. Injectable compositions include, but are not limited to, aqueous solutions (if water soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, or phosphate-buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Fluidity is maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, are included in the composition. The resulting solution is packaged for use as is or lyophilized, and in some embodiments, the lyophilized preparation is subsequently combined with a sterile solution prior to administration. For intravenous injection or injection at the site of pain, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. For example, any suitable solution using an isotonic medium, such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. In some embodiments, sterile injectable solutions are prepared by incorporating the required amount of the active ingredient in an appropriate solvent with one or a combination of the above-listed ingredients, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0154] In some embodiments, the composition is administered intravenously, such as by injection of a unit dose. For injection, in some embodiments, the active ingredient is in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability. In some embodiments, for example, an isotonic vehicle is used to prepare a suitable solution, such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. Furthermore, in some embodiments, the composition is administered by aerosolization.

[0155] For parenteral administration, polypeptides are formulated in a unit-dose injectable form (e.g., solution, suspension, or emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate are also used. In some embodiments, liposomes are used as carriers. The vehicle contains small amounts of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. Polypeptides are usually formulated in such vehicles at a concentration of about 1 mg / mL to 10 mg / mL.

[0156] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, for example, to increase shelf life during storage. When a composition is contemplated for use in any of the medicaments or methods provided herein, in some embodiments, the composition is contemplated to be substantially free of pyrogens, so that the composition does not cause inflammatory or dangerous allergic reactions when administered to a human subject.

[0157] In some embodiments, acceptable carriers contain compounds that stabilize, increase, or delay absorption or clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low-molecular-weight proteins; compositions that reduce peptide clearance or hydrolysis; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. In some embodiments, surfactants are also used to stabilize, increase, or decrease the absorption of pharmaceutical compositions containing liposomal carriers. In some embodiments, the compound is complexed with the composition to make it resistant to acidic and enzymatic hydrolysis, or the compound is complexed with an appropriately resistant carrier, such as a liposome, to protect it from digestion. Protecting the compound from digestion can be achieved using any suitable method.

[0158] Packages, kits and pre-filled containers Also provided herein are kits containing one or more of the therapeutic agents described above, which in some embodiments comprise a modified, hypersialylated Fc polypeptide of the present disclosure or a composition containing same in suitable container means.

[0159] In some embodiments, a container means is provided that contains the composition described herein.In some embodiments, the container means is any suitable container that can contain liquid or lyophilized compositions, including but not limited to vials, syringes, bottles, and intravenous (IV) bags or ampoules.The syringe can hold any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc or more.

[0160] Provided herein are kits comprising the composition(s) described herein. In some embodiments, provided herein are kits containing the compositions described herein and, optionally, an additional therapeutic agent for treating a subject with an inflammatory disease or disorder.

[0161] In some embodiments, provided herein are kits for treating an inflammatory disease or disorder containing a composition described herein and a label attached to or packaged with the container, optionally describing use of the composition in combination with an additional therapeutic agent.

[0162] In some embodiments, the container means of the kits will generally include at least one vial, test tube, flask, bottle, ampoule, syringe, intravenous (IV) bag, and / or other container means, into which at least one Fc polypeptide of the present disclosure, or a composition containing same, may be placed and / or appropriately aliquoted.

[0163] In some embodiments, the kit includes a means for containing at least one Fc polypeptide and / or composition containing same in close confinement for commercial sale. In some embodiments, such containers include injection-molded and / or blow-molded plastic containers into which the desired vials are retained. In some embodiments, the kit also includes printed materials for using the materials in the kit.

[0164] In some embodiments, the package and kit further comprises a buffer, a preservative and / or a stabilizer in the pharmaceutical formulation. In some embodiments, each component of the kit is enclosed in an individual container, and all of the various containers are in a single package. In some embodiments, the kit of the present disclosure is designed for cryogenic or room temperature storage.

[0165] Additionally, in some embodiments, the preparation contains a stabilizer to increase the shelf life of the kit, such as bovine serum albumin (BSA). If the composition is lyophilized, the kit in some embodiments contains an additional preparation of a solution for reconstituting the lyophilized preparation. Acceptable reconstitution solutions include, for example, pharmaceutically acceptable phosphate-buffered saline (PBS).

[0166] In some embodiments, the packages and kits further include one or more components for an assay, such as an ELISA assay, an HPLC assay, or a mass spectrometry assay. Samples to be tested in this application include, for example, blood, plasma, serum, tissue sections and secretions, urine, lymph and its products. In some embodiments, the packages and kits further include one or more components for sample collection (e.g., a syringe, a cup, a swab, etc.).

[0167] In some embodiments, the packages and kits further include labeling specifying information required by the U.S. FDA or similar regulatory agency, such as product description, dosage and mode of administration, and / or indications for treatment. In some embodiments, the packages provided herein include any of the compositions as described herein.

[0168] The term "packaging material" refers to a physical structure that houses the components of the kit. In some embodiments, the packaging material maintains the sterility of the components and is made from materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, etc.). In some embodiments, the label or insert includes appropriate written instructions (e.g., instructing the user of the kit to practice one or more methods disclosed herein). The kit, in some embodiments, further includes labels or instructions for using the kit components in any of the methods of the present disclosure. In some embodiments, the kit includes the compound in a pack or dispenser together with instructions for administering the compound in the methods described herein.

[0169] In a further embodiment, the kit further comprises container means for one or more additional therapeutic agents for an inflammatory disease or disorder.

[0170] In some embodiments, the instructions include instructions for practicing any of the methods described herein, including treatment methods, hi some embodiments, the instructions further include indications for a satisfactory clinical endpoint or any adverse symptoms that occur, or additional information required by a regulatory agency, such as the Food and Drug Administration, for use in human subjects.

[0171] The instructions, in some embodiments, are on "printed material," such as paper or cardboard within or attached to the kit, or on labels attached to the kit or packaging materials, or to vials or tubes containing the kit components. The instructions, in some embodiments, are further contained on computer-readable media, such as CD-ROMs, DVDs, flash memory devices, solid-state memories, magnetic disks and disk devices, magnetic tape, cloud computing systems and services, and the like. In some cases, the programs and instructions are encoded on the media permanently, substantially permanently, semi-permanently, or non-transitory. [Example]

[0172] The following examples are put forth to provide those of ordinary skill in the art with a description of how to use, make, and evaluate the compositions and methods described herein, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. [Example]

[0173] The F241A Fc mutant exhibits increased levels of α(2,3) sialylation compared to the wild-type Fc domain Human IgG1 wild-type (WT) Fc domains or Fc domains with the F241A mutation were transiently expressed in Chinese hamster ovary (CHO) cells. Asparagine 297 (Asn297), a site of biantennary glycans, is post-translationally modified to manipulate the level of sialic acid linkage under normal physiological conditions. These recombinant Fc domains were purified by size-exclusion chromatography (SEC) and suspended in phosphate-buffered saline (PBS). N-glycan profiling was performed by high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) using known standards to define the structure of the biantennary sugar molecule attached to Asn297. The N-glycan structures identified using this analysis are illustrated in Figure 1.

[0174] The percentage of Fc polypeptides with the glycan structure defined at Asn297 is presented in Figures 2A-2B. WT Fc polypeptides contain primarily N-glycans terminating in N-acetylglucosamine (GlcNAc; 49%) or a single galactose-containing GlcNAc moiety (G1F; 39%), with little to no SA-containing glycoforms (<1.0%). In comparison, introduction of the F241A mutation into Fc polypeptides resulted in a substantial enhancement of galactose addition, with 22% of polypeptides containing mono-2,3SA (G2FSA) and 19% of molecules containing di-2,3SA (G2FSA2). [Example]

[0175] High levels of .ALPHA.(2,6) sialylation of the F241A Fc mutant in a recombinant expression system Human IgG1 Fc domains with a phenylalanine (F) to alanine (A) substitution at amino acid position 241 (F241A) were stably expressed in CHO cells in both 10 mL and 1 L cultures. To manipulate the N-glycan structure present at Asn297, Fc-F241A was expressed alone (pool 1), together with the beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) enzyme (pool 2), together with ST6GAL1 and beta-1,4-galactosyltransferase 1 (B4GALT1) enzymes (pool 3), or together with siRNA targeting the sialic acid transporter, solute carrier family 35 member A1 (SLC35A1 KD; pool 4). These recombinant Fc-F241A glycovariants were purified by size-exclusion chromatography (SEC) and suspended in phosphate-buffered saline (PBS). N-glycan profiling was performed by high performance liquid chromatography (HPLC) followed by mass spectrometry (MS) using known standards to define the structure of the biantennary N-glycan attached to Asn297.

[0176] Data are expressed as the percentage of Fc molecules containing the defined glycan structures obtained from 1 L of CHO culture. On the x-axis, glycan structures are defined as G0 (terminating with GlcNAc), G0F (terminating with GlcNAc with fucose), G1 (terminating with one galactose), G1F (terminating with one galactose and fucose), G2 (terminating with two galactoses), G2F (terminating with two galactoses and fucose), G2FSA (terminating with one sialic acid and fucose), and G2FSA2 (terminating with two sialic acids and fucose). Sialic acid is attached via an α(2,3) linkage in pool 1 and via an α(2,6) linkage in pools 2 and 3. When recombinant human IgG1 Fc domains bearing F241A were expressed alone in CHO cells (pool 1), 18% of the Fc polypeptides in the pool were mono-α(2,3)-sialylated, 5% were di-α(2,3)-sialylated, and 47% had terminal galactose (Figure 3A). When coexpressed with ST6GAL1 (pool 2), 30% of the Fc polypeptides were mono-α(2,6)-sialylated, 27% were di-α(2,6)-sialylated, and 7% had terminal galactose (Figure 3B). When coexpressed with ST6GAL1 and B4GALT1 (pool 3), 3% of the Fc polypeptides were mono-α(2,6)-sialylated, 81% were di-α(2,6)-sialylated, and 0% had terminal galactose (Figure 3C). When coexpressed with siRNA targeting SLC35A1 (pool 4), the Fc domain was completely devoid of sialic acid, and 84% of the Fc N-glycans terminated in galactose (Fig. 3D).

[0177] To independently verify the glycan structures associated with pools 1–4 generated from 1 L of stable CHO cultures, recombinant Fc-F241A glycovariants were purified by size-exclusion chromatography and suspended in PBS. Five micrograms of each of Fc-F241A glycovariant pools P1, P2, P3, and P4 were separated by SDS-PAGE under reducing conditions, Coomassie stained (Figure 4A), transferred to nitrocellulose membranes, and incubated with biotinylated Sambucus nigra (SNA) lectin to detect α(2,6) sialic acid (Figure 4B), or transferred to nitrocellulose membranes and incubated with biotinylated MAL I lectin to detect terminal galactose (Figure 4C). The lectin was detected using an ALP-conjugated goat anti-biotin antibody. These findings are consistent with the HPLC / MS analysis, which confirmed that pools 2 and 3 uniquely contain 2,6 sialic acid, whereas pools 1 and 4 do not, but rather display more terminal galactose. [Example]

[0178] Fc sialylation improves exposure to modified Fc polypeptides in mice To determine the pharmacokinetics of the sialylated F241A Fc mutant polypeptide in vivo, 7-8 week-old female JAX-014565 mice (mouse FcRN homozygous knockout, human FcRN hemizygous, Tg32 strain) were divided into three groups of six mice each. Mice received a single intravenous (IV) bolus dose via the tail vein of 20 mg / kg of recombinant human IgG1 Fc domain (10 mL stable CHO cultures) with F241A from pool 2 (66% α(2,6) sialylated), pool 3 (93% α(2,6) sialylated), and pool 4 (0% sialylated). Blood was collected at each of seven time points (three mice per time point): pre-dose, 0.5 h, 1, 3, 7, 21, and 35 days. Serum was prepared according to standard procedures and transferred to Eppendorf tubes for storage. The concentration of F241A Fc polypeptide in each serum sample was determined by ELISA. Briefly, a 96-well plate was coated with 1 μg of anti-human Fc-specific capture antibody overnight at 4°C. The plate was washed three times with blocking buffer and then incubated with blocking buffer (80 μL per well) at room temperature with shaking (500 rpm) for 2 hours. The plate was washed again, and standards were diluted (starting at 300 ng / mL, with serial 1:3 dilutions for seven additional concentrations) and applied to the plate to generate a calibration curve. Samples were diluted as needed with dilution buffer (minimum 20× dilution), applied to the same plate, and then incubated with 1:50,000 diluted detection antibody at room temperature for 1 hour (500 rpm shaking). The washed plate was then developed by adding 25 μL / well of TMB substrate and incubating for 5–10 minutes. Quenching was achieved by adding 25 μL / well of 4N sulfuric acid, and the absorbance was read at 450 nm using a plate reader. Graphical analysis was performed using Prism software to determine the area under the curve (AUC).

[0179] In Figures 5A-5B, serum concentrations (μg / mL) are plotted over time (days) for each of Fc-F241A Pool 2 (open squares), Pool 3 (filled circles), and Pool 4 (filled triangles). Data were presented using concentrations on a logarithmic scale (Figure 5A) and a linear scale (Figure 5B). These pools were directly compared as a means of assessing the effect of the degree of α(2,6) sialylation on pharmacokinetics in FcRN mice. The AUCs for each of these Fc-F241A glycovariants are summarized in Table 1 below and demonstrate that the degree of sialylation is directly proportional to exposure in these mice. Unsialylated Fc Pool 4 had the lowest AUC, followed by Pool 2, which was 66% α(2,6) sialylated. Maximum exposure was achieved with Pool 3, which was 93% α(2,6) sialylated.

[0180] [Table 1]

[0181] To compare the pharmacokinetic differences between the α(2,3)- and α(2,6)-sialylated F241A Fc mutants, 7-8 week-old female JAX-014565 mice were divided into three groups of six mice each. Mice were given a single IV bolus injection via the tail vein of recombinant human IgG1 Fc domain (20 mg / kg) with F241A from stable 10 mL CHO cultures of Pool 1 (35% α(2,3)-sialylated) or Pool 3 (93% α(2,6)-sialylated) material, and a human IgG1 Fc domain (efgaltigimod (EFG)) with Abdeg mutations (M252Y / S254T / T256E / H433K / N434F) to enhance FcRn binding, transiently expressed by CHO cells (0% sialylated). Blood was collected (three mice per time point) at six time points: pre-administration, 0.5 hours, 1, 3, 7, and 14 days. Serum was prepared according to standard procedures and transferred to tubes for storage. The concentration of F241A Fc in each serum sample was determined by ELISA as described above.

[0182] The concentration-time profiles of each of the Fc-F241A pools 1, 3, and EFG Fc are shown (Figures 5C-5D). Data are graphed using concentrations on a logarithmic scale (Figure 5C) and a linear scale (Figure 5D). The observed difference in AUC between α(2,3)- and α(2,6)-sialylated Fc-F241A is likely due to differences in the degree of sialylation rather than differences in α(2,3) versus α(2,6) sialylation, given the similar effect observed when titrating the level of α(2,6) sialylation. The 93% α(2,6)-sialylated Fc-F241A demonstrated the greatest exposure in these mice. The EFG Fc domain had the lowest AUC, similar to non-sialylated F241A (pool 4), as shown elsewhere in Figures 5A-5B. The AUC values ​​for each Fc pool are provided in Table 2 below.

[0183] [Table 2]

[0184] Similar experiments were then performed in male and female CD1 mice using stable CHO 1L-cultured Fc-F241A material as follows. Five- to seven-week-old male and female CD1 mice were divided into two groups (six males and six females each) and dosed with 20 mg / kg of Pool 1 Fc-F241A (23% α(2,3)-sialylated) or Pool 3 Fc-F241A (84% α(2,6)-sialylated) via a single IV bolus injection. Blood was collected by retro-orbital bleeding at six time points: pre-dose, 1 h, 4 h, and 1, 3, 7, and 14 days post-dose (three mice per time point). Serum was prepared according to standard procedures and transferred to tubes for storage. The concentration of F241A in each serum sample was determined by ELISA as described above.

[0185] As shown in the concentration-time profiles (Figure 5E) and in Table 3 below, there was no apparent difference in exposure between male and female mice administered either α(2,3)-sialylated (open and closed triangles) or α(2,6)-sialylated (open and closed circles) Fc-F241A. However, as previously observed, the α(2,6)-sialylated material had a greater exposure relative to the α(2,3)-sialylated material, although this difference is likely due to the degree of sialylation rather than the α(2,3) versus α(2,6) linkage.

[0186] [Table 3]

[0187] Finally, the effect of repeated dosing of α(2,6)-sialylated Fc-F241A on exposure to Fc-241A was tested in CD1 mice. Five- to seven-week-old male and female CD1 mice were divided into three groups (six males and six females each) and dosed (IV bolus injection every four weeks) with 100 mg / kg of Pool 1 Fc-F241A (23% α(2,3)-sialylated) or 100 mg / kg of Pool 3 Fc-F241A (84% α(2,6)-sialylated). Blood was collected on day 21 immediately before the fourth dose and on day 22, 24 hours after the fourth dose. Serum was prepared according to standard procedures and transferred to tubes for storage. The concentration of F241A in each serum sample was determined by ELISA as described above.

[0188] C in male and female CD1 mice after dosing with α(2,3)-sialylated Fc-F241A versus α(2,6)-sialylated Fc-F241A. minGreater exposures were achieved both at the first and 24 hours after the fourth dose (Figure 5F). Notably, 100 mg / kg of α(2,6)sialylated material achieved a minimum exposure of greater than 200 μg / mL, which is twice the concentration required for engagement of the DC-SIGN mechanism in cell-based assays. [Example]

[0189] α(2,6) Sialylation of F241A Fc improves the efficacy of anti-inflammatory activity in a murine model of arthritis To determine the therapeutic efficacy of the sialylated F241A Fc mutant polypeptide in inflammatory disease, a mouse model of arthritis was used. KRN transgenic heterozygous mice on a C57BL / 6 background were crossed with NOD / SHiLtJ mice to generate K / BxN mice. Serum samples from animals with inflamed joints were pooled from mice (6–9 weeks of age) that spontaneously developed inflammation. The pooled serum was frozen in aliquots and used for all experiments presented herein. Joint inflammation was induced in C57BL / 6 mice by intravenous injection of 4–200 μL of pooled K / BxN serum. Arthritis symptoms were scored daily as 0 (unaffected), 1 (swelling of one joint), 2 (swelling of more than one joint), and 3 (severe swelling of the entire paw). Each paw was scored, and the sum of all four paws was used to generate a clinical score. The scores generated by two independent technicians were averaged to generate a final clinical score. Fc-F241A produced by transient transfection in CHO cells was used in these experiments. For treatment conditions, mice received a single dose of 1 g / kg IVIG or 50 mg / kg (41%) α(2,3)sialylated F241A (transiently expressed in CHO cells) via tail vein bolus injection 1 h before administration of KBxN serum.

[0190] Mice (four per group) were randomized for treatment with a single dose of 1 g / kg IVIG or 50 mg / kg F241A. Clinical scores for the IVIG- and F241A Fc-treated groups were not significantly different from each other except on days 6 and 7, as determined by unpaired t-tests (Figure 6A). Peak inflammation was achieved by day 7 in PBS-treated mice. Therefore, clinical scores for study days 7 and 8 are shown separately as box plots expressed as the mean and standard error of the mean in Figures 6B-6C. Both 1 g / kg IVIG and 50 mg / kg F241A significantly suppressed inflammation relative to PBS. These data suggest that F241A is approximately 20-fold more potent than IVIG in the K / BxN serum transfer model of arthritis.

[0191] A similar experiment using Fc-F241A stable CHO 1 L culture material was then repeated using 50 mg / kg (84%) α(2,6)-sialylated F241A Fc. The two different F241A glycoforms evaluated herein differed in two respects. First, one recombinant human IgG1 Fc domain contained α(2,3)-linked SA (Pool 1), while the other contained α(2,6)-linked SA (Pool 3). Second, the percentage of Fc molecules containing SA, as determined by HPLC, was approximately 23% of the α(2,3)-linked material, whereas approximately 84% of the α(2,6)-linked material contained SA. Mice (5–6 per group) were randomized for treatment with a single dose of 1 g / kg IVIG, 50 mg / kg α(2,3)sialylated F241A Fc, or 50 mg / kg α(2,6)sialylated F241A Fc. IVIG demonstrated significantly greater anti-inflammatory activity than F241A α(2,3)sialylated Fc from day 5 onward, as determined by unpaired t-test (p<0.01; Figure 6D). In comparison, IVIG was only significantly different from F241A α(2,6)sialylated Fc from day 8 onward (unpaired t-test; p<0.05). On days 6 and 7, which are the peaks of inflammation for PBS-treated animals, α(2,6) sialylated F241A Fc resulted in significantly less inflammation than α(2,3) sialylated F241A Fc (Figures 6E-6F). [Example]

[0192] Sialylated F241A Fc polypeptide is more potent than IVIG in a mouse model of idiopathic thrombocytopenic purpura To determine the therapeutic efficacy of the sialylated F241A Fc mutant polypeptide in another inflammatory disease, a mouse model of idiopathic thrombocytopenic purpura (ITP) was used. Mouse 6A6-IgG2a anti-mouse platelet antibody was produced in 293T cells by transient transfection in serum-free medium and then purified using protein G beads. Female C56BL / 6 mice aged 6-8 weeks (n = 5 per group) received a single tail vein injection of 4 μg of 6A6 antibody at time zero. Mice were randomized and received PBS, 1 g / kg IVIG, or 100 mg / kg F241A Fc with α(2,3) sialylation (F241A α(2,3) sia), transiently expressed in CHO cells, 41% sialylated, via tail vein injection 2 hours before 6A6 administration. Platelet counts were monitored prior to 6A6 administration on study day 0, and again at 24, 48, and 72 hours after 6A6 administration. Box and whisker plots were generated showing first and third quartiles, median, and minimum and maximum values.

[0193] In this model of ITP, baseline platelet counts were similar across all three treatment groups on day 0 (Figure 7). After administration of 6A6 antiplatelet antibody, platelets were >90% depleted by 24 hours in PBS control mice, returned to approximately 40% of pre-6A6 levels by 48 hours, and returned to normal by 72 hours. Treatment with 1 mg / kg IVIG or 100 mg / kg α(2,3) sialylated F241A Fc provided statistically significant protection from platelet loss 24 and 48 hours after 6A6 administration, as determined by an unpaired t-test (p<0.01). These findings demonstrate that α(2,3) sialylated F241A Fc is approximately 10-fold more potent than IVIG in this model of ITP. [Example]

[0194] Terminal sialylation of F241A Fc enhances half-life and bioavailability in vivo Full-length IgG is known to have an extended serum half-life of up to 21 days due to its interaction with FcRn, which recycles IgG into the circulation after cellular uptake. In contrast, Fc fragments are known to have a shorter serum half-life. Therefore, to test whether sialylation of F241A Fc affected its serum half-life, a single intravenous 20 mg / kg dose of each of the various F241A Fc glycoforms or wild-type (WT) Fc was administered to humanized FcRn Tg32 (hFcRn) mice, and the amount of human IgG1 Fc in the circulation was quantified over time. Mice were randomly assigned to receive a single intravenous injection of one glycoform of F241A Fc at 20 mg / kg, with six mice per group. Blood was collected for serum isolation by retro-orbital bleeding at 0 min, 30 min, 1 day, 5 days, 7 days, 14 days, 21 days, and 35 days after administration. Mice were euthanized and blood was collected via cardiac puncture. Depending on the exposure achieved, subsequent time points were not plotted if values ​​fell below the limit of quantification (1 µg / mL). Not all mice were bled at any time point, but each mouse contributed 3–4 times over the course of the study. Serum samples were stored at -80 °C prior to quantification of the concentration of F241A Fc in mouse serum using ELISA.

[0195] After administration, both sialylated WT Fc and F241A Fc (F241A / siSLC Fc) produced in cells expressing small interfering RNA (siRNA) against the Golgi sialic acid transporter, SLC35A1, were rapidly cleared from the circulation and were undetectable after 14 and 10 days, corresponding to half-lives of 2.71 and 1.43 days, respectively (Figure 8A and Table 4). These preparations had areas under the plasma concentration-time curve to the last measurable plasma concentration (AUC) of 322 and 118 d*mg / mL and clearance rates of 61.3 and 168.3 mL / day / kg, respectively (Figure 8A and Table 4). F241A Fc had a half-life of 2.96 days, an AUC of 297 d*mg / mL, and a clearance of 42.1 mL / day / kg (Figure 8A and Table 4). F241A Fc (F241A / ST6 Fc) produced in cells expressing the α-2,6-sialyltransferase ST6GAL1 had a serum half-life of 3.72 days, an AUC of 483 days*mg / mL, and a clearance rate of 40.8 mL / day / kg. F241A Fc (F241A / B4ST6 Fc) produced in cells expressing the β1,4-galactosyltransferase B4GALT1 in parallel with ST6GAL1 showed the longest serum retention, with a half-life of 5.2 days, a decline to concentrations below 1 μg / mL only after 35 days post-administration, and an AUC of 782 days*mg / mL and a clearance rate of 24.8 mL / day / kg (Figure 8A and Table 4).

[0196] [Table 4]

[0197] The percent sialylation of the N297 glycan on F241A Fc correlated with anti-inflammatory activity (R 2 = 0.0042; Figure 8B), half-life (R 2 = 0.7952; Figure 8C), AUClast (R 2 = 0.83; Figure 8D) and the clearance rate (R 2= 0.5816; Figure 8E). These results were surprising because IgG half-life is thought to be primarily regulated by FcRn. Furthermore, Fc glycans are buried in a pocket at the top of the CH2 domain (Figure 9B) and are thought to be largely inaccessible. Based on these results, the F241A / B4ST6 Fc preparation, which has approximately 90% α-2,6-sialylation at the N297 glycan, was selected for use in further analysis and experiments. [Example]

[0198] Preferential receptor ligation binds F241A Fc and Fc Abdeg are distinguished Fc Abdeg Efgartigimod (efgartigimod) is a recently FDA-approved therapeutic agent that accelerates the depletion of circulating total IgG by saturating FcRn and is currently approved for treating myasthenia gravis. Abdeg contains "Abdeg" mutations (M252Y, S254T, T256E, H433K, and N434F) in the region of the Fc that binds to FcRn, allowing it to outcompete native IgG (Figures 9A and 9B). The following experiments were performed to demonstrate that Fc Abdeg and F241A / B4ST6 Fc functioned through similar receptors and cellular pathways.

[0199] First, WT Fc, F241A / B4ST6 Fc, and Fc Abdeg The interaction between FcRn and F241A / B4ST6 Fc was examined using surface plasmon resonance (SPR) with mouse and human FcRn. WT Fc and F241A / B4ST6 Fc bound similarly to mouse (Figure 9C) and human (Figure 9D) FcRn. In contrast, Fc AbdegThe dissociation constants (KD) of F241A / B4ST6 Fc are 267-fold and 39-fold lower than those of F241A / B4ST6 Fc for mouse FcRn (Figure 9C) and human FcRn (Figure 9D), respectively. We then examined the effects of both mutant Fc on circulating mouse IgG via FcRn. C57BL / 6 and hFcRn(Tg32) mice were injected with F241A / B4ST6 Fc or Fc Abdeg A single intravenous injection of F241A / B4ST6 Fc was given, and serum mIgG was determined by ELISA after dosing. In wild-type C57BL / 6 mice with murine FcRn, F241A / B4ST6 Fc treatment did not reduce serum IgG titers (Figure 9E). In contrast, Fc Abdeg induced a significant, immediate, and sustained reduction in IgG titers (Fig. 9E). F241A / B4ST6 Fc did not affect IgG serum titers in hFcRn(Tg32) mice (Fig. 9F). However, Fc Abdeg induced a transient decrease in IgG titers on day 1. These results suggest that Fc receptors on mFcRn Abdeg Consistent with its high affinity for IgG1 and relatively low affinity for hFcRn, multiple doses are required in humans to produce long-term reductions in serum IgG.

[0200] Both IVIG and sialylated IgG Fc require the mouse C-type lectin-specific ICAM-3-grabbing nonintegrin-related-1 (SIGN-R1) to mediate anti-inflammatory activity in vivo. This requirement can be circumvented by introduction of the SIGN-R1 human orthologue, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Thus, F241A / B4ST6 Fc and Fc Abdeg The ability of both SIGN-R1 and SIGN-R2 to bind to DC-SIGN was examined by cell-binding assay. - / - and hDC-SIGN + / SIGN-R1 - / - The cells were cultured in PBS, F241A / B4ST6 Fc, or Fc AbdegThe presence of the receptor was confirmed by flow cytometry by staining hCD209 using Alexa Fluor 647 anti-hCD209, clone 9E9A8 antibody. Cells were cultured overnight at 37°C and 5% CO2 in high-glucose DMEM medium supplemented with 1x antibiotic-antimycotic and 1x FBS. The next day, the supernatant was discarded, and cells were detached using enzyme-free dissociation buffer. The suspended cells were collected and centrifuged at 400 rpm for 10 minutes. The pellet was resuspended and washed with binding buffer (1x TBS, 1 nM CaCl2, 2.5% FBS, and 0.05% sodium azide). Fc receptors were blocked by adding anti-mouse CD16 / 32 antibody clone 93. After blocking, cells were washed with binding buffer and then 100 μg / mL of Fc Abdeg and F241A / B4ST6 Fc were added, which was then incubated on ice for 1 hour. After that, the cells were washed again with binding buffer. Abdeg Since both F241A / B4ST6 Fc and F241A / B4ST6 Fc are IgG Fc-based constructs, the cells were then stained with either an APC- or PE-conjugated antibody against hIgGFc, clone M1310G05. Staining was performed on ice in the dark for 30 minutes. After staining, the cells were washed with binding buffer, fixed in 2% formaldehyde, and then analyzed. BMDMs were analyzed by flow cytometry to detect surface-bound IgG. F241A / B4ST6 Fc or Fc Abdeg SIGN-R1 incubated with - / - No bound IgG was detected on BMDMs (Figure 9G). However, hDC-SIGN treated with F241A / B4ST6 Fc + / SIGN-R1 - / - In BMDM, IgG was detected, but Fc Abdeg It was not detected when using (Figure 9G).

[0201] F241A / B4ST6 Fc and Fc AbdegFor in vivo evaluation of mechanistic differences in the activity of - / - Mice were treated with PBS, high-dose IVIG, F241A / B4ST6 Fc, or Fc Abdeg Subsequently, K / BxN serum was administered, and paw swelling was monitored over 10 days (Figures 9H-9J). Each paw was given a clinical score of 0 to 3, with a score of 0 representing no inflammation in the paw and a score of 3 representing severe inflammation in all joints of the paw. The average score of all four paws for each mouse in the treatment group is shown. In WT mice, significant paw swelling was observed only in PBS-treated mice (Figures 9H and 9J). SIGN-R1 - / - In mice, swelling was observed in PBS-, IVIG-, and F241A / B4ST6 Fc-treated mice (Fig. 9I and Fig. 9J). Abdeg SIGN-R1 was administered - / - Mice had significantly less inflammation as measured by clinical scores, including at the peak of the disease (day 7; Figure 9J). Collectively, these results support the conclusion that F241A / B4ST6 Fc and Fc Abdeg We demonstrate that IL-16 suppresses autoantibody-induced inflammation through distinct molecular pathways. [Example]

[0202] F241A / B4ST6 Fc and Fc Abdeg Combinatorial anti-inflammatory activity of F241A / B4ST6 Fc and Fc Abdeg To investigate whether F241A / B4ST6 Fc and Fc can effectively attenuate autoantibody-induced inflammation when co-administered in vivo, Abdeg The effects of mutant Fc were evaluated in the K / BxN model in both preventative (Fig. 10A, top panel) and therapeutic (Fig. 10A, top panel) manners. Both mutant Fc significantly protected against inflammation in a manner comparable to high-dose IVIG, regardless of the timing of administration (Fig. 9J, Fig. 10B, and Fig. 10C).

[0203] Fc Abdegsuppresses IgG-mediated inflammation by saturating FcRn, thereby reducing total IgG titers and its markedly enhanced affinity for mFcRn, thus Abdeg was tested for its ability to lead to more rapid clearance of F241A / B4ST6 Fc from the circulation. Abdeg To determine dose combinations where clearance effects of F241A / B4ST6 Fc do not interfere with activity or half-life of F241A / B4ST6 Fc, C57BL / 6 mice were administered decreasing doses of Fc. Abdeg The mice were administered 50 mg / kg of F241A / B4ST6 Fc in combination with IgG, and human Fc titers were measured 10 days after administration (Figure 10D). Abdeg caused a dose-dependent decrease in circulating F241A / B4ST6 Fc as measured by ELISA. However, at 1 mg / kg, i.e., 1 / 10 of the clinical dose, Fc Abdeg induced the least clearance of circulating F241A / B4ST6 Fc.

[0204] Fc Abdeg Since mouse FcRn exhibits at least 10-fold enhanced affinity compared to human FcRn, we reasoned that a 10-fold lower dose would be more representative of the human system and would not deplete F241A / B4ST6 Fc titers. Therefore, C57BL / 6 mice were administered PBS, high-dose IVIG, 50 mg / kg F241A / B4ST6 Fc, 1 mg / kg Fc Abdeg or 50 mg / kg F241A / B4ST6 Fc and 1 mg / kg Fc Abdeg In the prevention model, IVIG, 50 mg / kg F241A / B4ST6 Fc, and 1 mg / kg Fc were administered together, followed by K / BxN serum. Abdeg All effectively reduced inflammation compared to PBS-treated mice (Figures 10E and 10F). Surprisingly, co-administered F241A / B4ST6 Fc and Fc Abdeg Mice given the Fc-Fc12 antibody showed a greater reduction in inflammation than either Fc alone.

[0205] The combined effect of the above Fc mutants as anti-inflammatory biologics was further tested in a therapeutic K / BxN model. As described above, mice were treated with arthritic K / BxN serum on day 0, followed by PBS, high-dose IVIG, 50 mg / kg F241A / B4ST6 Fc, 1 mg / kg Fc, or 1 mg / kg Fc. Abdeg or 50 mg / kg F241A / B4ST6 Fc and 1 mg / kg Fc Abdeg on day 2 and paw swelling was monitored over the next few days (Figures 10G and 10H). Co-administration reduced inflammation by day 4, 6 days after treatment. Unexpectedly, this was significantly different from treatment with either Fc alone. These data support the conclusion that F241A / B4ST6 Fc and Fc Abdeg 10 shows that combined administration of Fc results in an enhanced anti-inflammatory response in vivo compared to the effect of either Fc alone. [Example]

[0206] Fc in antibody transfer models of pemphigoid disease F241A Effectiveness Autoantibody-mediated pemphigoid disease was modeled in mice by repeated administration of rabbit anti-mouse type VII collagen polyclonal antibody (a pathogenic autoantibody found in epidermolysis bullosa acquisita). Treatment was administered on study day 0 (3 hours before the first dose of pathogenic autoantibody) and again on day 3.

[0207] Using this model of bullous pemphigoid disease, we demonstrated that 100 mg / kg FcRN inhibitor efgartigimod (10 mg / kg) prevented autoantibody-mediated blister formation. F241A The potential for Fc to protect against autoantibody-mediated skin blistering is presented over time in Figure 11A. As shown, when treated with the PBS control (open circles), the percentage of total skin area affected by blisters increases from approximately 1% on day 4 to 3-4% on days 9-10. F241AWhen treated with efgartigimod, a maximum blister formation of 1-2% was observed on days 9-10, representing a significant reduction in blister formation compared to the PBS control. Efgartigimod resulted in more moderate disease control, with a maximum blister formation of 2-3% observed on days 9-10.

[0208] In this model, immune complexes with collagen type VII antigens form in the basement membrane region, resulting in cross-linking of FcgRIV receptors on the surface of infiltrating neutrophils, which leads to a significant inflammatory response and subsequent blister formation. Importantly, the inhibitory FcgRIIB receptor is required for protection (Kasperkiewicz, Nimmerjahn et al. 2012). Fc F241A The therapeutic mechanism is thought to involve upregulation of FcgRIIB on B cells and myeloid cells, including neutrophils, which are key mediators of disease in this model. Therefore, we evaluated treatment-mediated changes in FcgRIIB cell surface expression on circulating B cells and myeloid cells. As shown in Figure 11B, Fc F241A independently resulted in a statistically significant increase in circulating peripheral blood neutrophil surface expression of FcgRIIB relative to the PBS control. Efgartigimod did not induce FcgRIIB expression.

[0209] Fc by all reactive leukocyte populations in the blood F241A The kinetics of FcgRIIB cell surface expression induced by FcgRIIB are shown in Figures 12A-12C. These included naive B cells (Figure 12A), activated mature B cells (Figure 12B), and neutrophils (Figure 12C). The data are from FcgRIIB cell surface expression induced by FcgRIIB. F241A However, these results suggest that it has the potential to maintain elevated FcgRIIB cell surface expression for 7 to 9 days after the last dose (study day 3).

[0210] At the end of the study (day 13), biopsies from the affected ear skin were collected from the mice, H&E staining was performed, and the images are shown in Figure 13. PBS control (top row), Fc F241ATwo representative images from the Fc (middle row) and efgartigimod (bottom row) treated groups are shown. The images are annotated to highlight cartilage, areas affected by blisters, pooled blood, and dead skin. F241A The effect of treatment with Fc leads to almost complete protection from skin damage caused by pathogenic autoantibodies. F241A Both efgartigimod and efgartigimod led to an early reduction in neutrophil infiltration into skin tissue, resulting in an increase in Fc F241A resulted in a significant reduction relative to the PBS control on days 3-5 of the study (Figure 14A). F241A Both efgartigimod and efgartigimod resulted in a significant reduction in infiltrating CD62 ligand-positive monocytes in skin tissues on days 3 to 13 (Fig. 14B).

[0211] Fc F241A is effective in a mouse model of pemphigoid disease and is associated with increased FcgRIIB surface expression on circulating neutrophils and reduced skin infiltration of neutrophils, the immune cells that drive disease in this model. These data support the FcgRIIB-mediated downregulation of the immune checkpoint receptor FcgRIIB. F241A These findings suggest that IL-1-mediated upregulation may be therapeutically significant in patients with pemphigoid diseases, including bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). [Example]

[0212] Fc F241A is protective in the MOG35-55-induced EAE model of T cell-mediated neuroinflammation in mice Experimental autoimmune encephalomyelitis (EAE) was induced in mice, followed by randomization into treatment groups (n=7 per group). Body weights were recorded daily starting on study day 0 through study day 21, and data were normalized to their baseline body weights on study day 0. As shown in Figure 15, all treatment groups demonstrated an initial decline in body weight (approximately 10%) in the first few days after EAE induction, followed by recovery. As the disease developed, the mean body weight of vehicle-treated mice declined to a greater extent than any of the intervention groups. However, this was largely not statistically significant. Overall, the results suggest that Fc F241A This suggests that cerebrospinal fluid (C1) and cerebrospinal fluid (C2) show a non-significant trend in protection from weight loss.

[0213] Clinical inflammation scores are presented in Figures 16A and 16B. As shown in Figure 16A, the onset of measurable disease occurred on study day 13 for the vehicle control group and rapidly increased to a plateau on study day 19. FTY-720, high-dose IVIg, and Fc F241A Significant protection was observed for treated mice over the entire course of the study. The area under the curve (AUC) for the entire time course of inflammation scores for each group is shown separately in Figure 16B. All three therapeutic interventions demonstrated significant protection (p<0.001) compared to the vehicle control. There were no significant differences in protection among these three therapeutic intervention groups at any time point.

[0214] These data suggest that Fc suppresses T cell-mediated neuroinflammation in the EAE model. F241A The protective activity of IVIg and Fc is confirmed, demonstrating a mechanism involving the management of effector T cell-mediated inflammation. Without wishing to be bound by any particular theory, F241AThe protective effect of IVIg may be a function of its ability to expand Tregs, as previously reported for sialylated IVIg and Fc-F241A mechanisms (Fiebiger BM et al., "Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs." Proc Natl Acad Sci U S A. 2015 May 5;112(18):E2385-94.).

[0215] Other embodiments Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the present disclosure has been described in connection with specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the present disclosure. Other embodiments are within the scope of the claims.

Claims

1. 1. A population of modified Fc polypeptides, each modified Fc polypeptide having (i) an amino acid sequence that is at least 75% identical to the sequence of SEQ ID NO:2 and (ii) an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2), wherein at least 60% of the modified Fc polypeptides have a sialic acid (SA) moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

2. 2. The population of modified Fc polypeptides of claim 1, wherein the aliphatic amino acid at position 241 is alanine (Ala, F241A).

3. 3. The population of modified Fc polypeptides of claim 1 or 2, wherein the N-glycan is attached to an asparagine (Asn) at amino acid residue 297 of the polypeptide (Asn297, numbered according to Kabat and corresponding to amino acid residue 88 of SEQ ID NO:2).

4. 4. The population of modified Fc polypeptides of any one of claims 1 to 3, wherein at least 70% of the Fc polypeptides comprise an SA moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

5. 5. The population of modified Fc polypeptides of any one of claims 1 to 4, wherein at least 80% of the Fc polypeptides comprise an SA moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

6. 6. The population of modified Fc polypeptides of any one of claims 1 to 5, wherein at least 90% of the Fc polypeptides comprise an SA moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

7. 7. The population of modified Fc polypeptides according to any one of claims 1 to 6, wherein the N-glycans of said Fc polypeptides are monosialylated or disialylated.

8. 8. The population of modified Fc polypeptides of claim 7, wherein at least 30% of the Fc polypeptides comprise monosialylated N-glycans comprising an SA moiety attached via an α(2,6) linkage.

9. 8. The population of modified Fc polypeptides of claim 7, wherein at least 30% of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) bond.

10. 10. The population of modified Fc polypeptides of claim 9, wherein approximately 90% of the Fc polypeptides comprise disialylated N-glycans comprising two SA moieties attached via an α(2,6) bond.

11. 11. The population of modified Fc polypeptides of claim 1, wherein at least about 60% of the Fc polypeptides comprise a galactose moiety.

12. 12. The population of modified Fc polypeptides of claim 1, wherein at least about 70% of the Fc polypeptides comprise a galactose moiety.

13. 13. The population of modified Fc polypeptides of claim 1, wherein at least about 80% of the Fc polypeptides comprise a galactose moiety.

14. 14. A population of modified Fc polypeptides according to any one of claims 1 to 13, wherein at least about 90% of the Fc polypeptides comprise a galactose moiety.

15. 15. The population of modified Fc polypeptides of claim 1, wherein approximately 100% of the Fc polypeptides comprise a galactose moiety.

16. 16. The population of modified Fc polypeptides of any one of claims 11 to 15, wherein the galactose moiety is attached to the α(1,3) arm and / or the α(1,6) arm of the N-glycan.

17. 17. The population of modified Fc polypeptides according to any one of claims 11 to 16, wherein the galactose moieties are branched galactose moieties.

18. 1. A population of modified Fc polypeptides, each modified Fc polypeptide having (i) an amino acid sequence that is at least 75% identical to the sequence of SEQ ID NO:2 and (ii) an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2), wherein approximately 40% of the modified Fc polypeptides have an SA moiety that is attached to an N-glycan of the Fc polypeptide via an α(2,3) bond.

19. 17. The population of modified Fc polypeptides of claim 16, wherein the aliphatic amino acid at position 241 is Ala (F241A).

20. 20. The population of modified Fc polypeptides of any one of claims 1 to 19, wherein the Fc polypeptide is IgG1.

21. 20. The population of modified Fc polypeptides of any one of claims 1 to 19, wherein the Fc polypeptide is IgG3.

22. 22. A pharmaceutical composition comprising: (a) the population of any one of claims 1 to 21; and (b) a pharmaceutically acceptable carrier, diluent, or excipient.

23. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutically acceptable carrier, diluent or excipient is selected from the group consisting of stabilizers, buffers, surfactants, bulking agents, solvents, tonicity or osmolality adjusters, antioxidants, adjuvants and antimicrobial agents.

24. 24. A method of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22 or 23.

25. 25. The method of claim 24, wherein the inflammatory disease or condition is an autoimmune disease or condition.

26. 26. The method of claim 24 or 25, wherein the inflammatory disease or condition is arthritis.

27. 26. The method of claim 24 or 25, wherein the inflammatory disease or condition is immune thrombocytopenia (ITP).

28. 28. The method of any one of claims 24 to 27, wherein the population or pharmaceutical composition has a half-life of at least 3.5 days following administration of the population or pharmaceutical composition to the subject.

29. 29. The method of any one of claims 24 to 28, wherein the population or pharmaceutical composition has a half-life of at least 4 days following administration of the population or pharmaceutical composition to the subject.

30. 30. The method of any one of claims 24 to 29, wherein the population or the pharmaceutical composition is cleared from the circulation of the subject at a rate of 45 mL / day / kg or less.

31. 31. The method of any one of claims 24 to 30, wherein the population or the pharmaceutical composition is cleared from the circulation of the subject at a rate of 40 mL / day / kg or less.

32. 32. The method of any one of claims 24 to 31, wherein the population or the pharmaceutical composition is cleared from the circulation of the subject at a rate of 35 mL / day / kg or less.

33. 33. The method of any one of claims 24 to 32, wherein the population or the pharmaceutical composition is cleared from the circulation of the subject at a rate of 30 mL / day / kg or less.

34. 34. The method of any one of claims 24 to 33, wherein the population or the pharmaceutical composition is cleared from the circulation of the subject at a rate of 25 mL / day / kg or less.

35. 35. The method of any one of claims 24 to 34, wherein the concentration of Fc polypeptide over time (AUC) is at least 480 days x mg / mL between 1 and 35 days after administration of the population or the pharmaceutical composition to the subject.

36. 36. The method of claim 35, wherein the AUC is at least 500 days x mg / mL, at least 550 days x mg / mL, at least 600 days x mg / mL, at least 650 days x mg / mL, at least 700 days x mg / mL, at least 750 days x mg / mL, at least 800 days x mg / mL, or at least 850 days x mg / mL between 1 and 35 days after administration of the population or the pharmaceutical composition to the subject.

37. 37. The method of any one of claims 24 to 36, further comprising administering to the subject an additional therapeutic agent.

38. 38. The method of claim 37, wherein the additional therapeutic agent is administered to the subject prior to, simultaneously with, or after administration of the population or the pharmaceutical composition.

39. 39. The method of claim 37 or 38, wherein the additional therapeutic agent is a second modified Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, H433K and N434F.

40. 40. The method of claim 39, wherein the second modified Fc polypeptide comprises the amino acid substitutions M252Y, S254T, T256E, H433K and N434F.

41. 41. The method of claim 39 or 40, wherein the second modified Fc polypeptide has the amino acid sequence shown as SEQ ID NO:

5.

42. 42. The method of any one of claims 39 to 41, wherein the second modified Fc polypeptide is administered at a dose of 1 mg / kg to 20 mg / kg.

43. 43. The method of any one of claims 39 to 42, wherein the second modified Fc polypeptide is administered at a dose of 10 mg / kg.

44. 44. The method of any one of claims 39 to 43, wherein the second modified Fc polypeptide is administered once a week for four weeks.

45. 45. The method of any one of claims 37 to 44, wherein the additional therapeutic agent is selected from the group consisting of anti-inflammatory drugs, immunosuppressants, analgesics, disease-modifying antirheumatic drugs (DMARDs), counterirritants, platelet-increasing agents, thrombopoietin receptor (TPOR) agonists, physical therapy, and surgery.

46. 46. ​​The method of claim 45, wherein the anti-inflammatory drug is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, an anti-inflammatory antibody or antigen-binding fragment thereof, an anti-inflammatory cytokine, a kinase inhibitor, and intravenous immunoglobulin (IVIG).

47. 1. A nucleic acid expression vector comprising: (a) a first expression cassette comprising a first mammalian promoter operably linked to a polynucleotide encoding a modified Fc polypeptide having (i) an amino acid sequence that is at least 75% identical to the sequence of SEQ ID NO:2 and (ii) an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2); (b) a second expression cassette comprising a second mammalian promoter operably linked to a polynucleotide encoding a beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) enzyme; and A nucleic acid expression vector comprising:

48. 48. The nucleic acid expression vector of claim 47, wherein the aliphatic amino acid at position 241 is Ala (F241A).

49. 49. The nucleic acid expression vector of claim 48, wherein the second expression cassette further comprises a polynucleotide encoding a beta-1,4-galactosyltransferase 1 (B4GALT1) enzyme.

50. 50. The nucleic acid expression vector of claim 49, wherein the polynucleotide encoding the B4GALT1 enzyme is operably linked to a second promoter.

51. 51. A nucleic acid expression vector described in any one of claims 47 to 50, wherein the second expression cassette further comprises an internal ribosome entry site (IRES) sequence located between the polynucleotide encoding the ST6GAL1 enzyme and the polynucleotide encoding the B4GALT1 enzyme.

52. 52. The nucleic acid expression vector of any one of claims 47 to 51, wherein the first promoter and the second promoter are each independently selected from the group consisting of a murine cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a eukaryotic elongation factor 2 (EEF2) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a phosphoglycerate kinase (PGK) promoter, an actin promoter, and a ubiquitin promoter.

53. 53. A mammalian host cell comprising an expression vector according to any one of claims 47 to 52.

54. 54. The mammalian host cell of claim 53, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

55. 54. The mammalian host cell of claim 53, wherein the mammalian host cell is a human embryonic kidney 293 (HEK293) cell.

56. 1. A method for producing a population of modified Fc polypeptides, wherein each modified Fc polypeptide has (i) an amino acid sequence having at least 75% identity to SEQ ID NO:2 and (ii) an aliphatic amino acid residue at position 241 (numbered according to Kabat and corresponding to amino acid residue 32 of SEQ ID NO:2); 43. A method comprising: (a) culturing a mammalian host cell of any one of claims 40 to 42 under conditions and for a period of time that induces expression of the first expression cassette and the second expression cassette, thus producing a population of modified Fc polypeptides; and (b) purifying the population of modified Fc polypeptides.

57. 57. The method of claim 56, wherein the aliphatic amino acid at position 241 is Ala (F241A).

58. 58. The method of claim 56 or 57, wherein at least 60% of the modified Fc polypeptides have an SA moiety attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

59. 59. The method of any one of claims 56 to 58, wherein at least 70% of the Fc polypeptides comprise an SA moiety that is attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

60. 60. The method of any one of claims 56 to 59, wherein at least 80% of the Fc polypeptides comprise an SA moiety that is attached to an N-glycan of the Fc polypeptide via an α(2,6) linkage.

61. 61. The method of any one of claims 56 to 60, wherein at least 90% of the polypeptides comprise SA moieties attached to N-glycans via α(2,6) linkages.

62. 62. The method of any one of claims 56 to 61, wherein the first promoter and the second promoter are each independently selected from the group consisting of a mouse CMV promoter, an EF1α promoter, an EEF2 promoter, a GAPDH promoter, a PGK promoter, an actin promoter, and a ubiquitin promoter.

63. 63. The method of any one of claims 56 to 62, wherein the first expression cassette and / or the second expression cassette each independently comprise one or more regulatory sequences selected from the group consisting of a 5' untranslated region (UTR), a 3' UTR, an enhancer, an insulator, an intron, an RNA transport element, a polyadenylation signal, an internal ribosome entry site (IRES), and a transcription terminator.

64. 64. The method of any one of claims 56 to 63, wherein the N-glycans of the Fc polypeptide are monosialylated or disialylated.

65. 65. The method of claim 64, wherein about 30% of the Fc polypeptide comprises monosialylated N-glycans comprising an SA moiety attached via an α(2,6) linkage.

66. 65. The method of claim 64, wherein about 30% of the Fc polypeptide comprises disialylated N-glycans comprising two SA moieties attached via an α(2,6) bond.

67. 67. The method of claim 66, wherein about 90% of the Fc polypeptide comprises disialylated N-glycans comprising two SA moieties attached via an α(2,6) bond.

68. 68. The method of any one of claims 56 to 67, wherein at least about 60% of the Fc polypeptides comprise a galactose moiety.

69. 69. The method of any one of claims 56 to 68, wherein at least about 70% of the Fc polypeptides comprise a galactose moiety.

70. 70. The method of any one of claims 56 to 69, wherein at least about 80% of the Fc polypeptides comprise a galactose moiety.

71. 71. The method of any one of claims 56 to 70, wherein at least about 90% of the Fc polypeptides comprise a galactose moiety.

72. 72. The method of any one of claims 56 to 71, wherein about 100% of the Fc polypeptides comprise a galactose moiety.

73. 73. The method of any one of claims 56 to 72, wherein the galactose moiety is attached to the α(1,3) arm and / or the α(1,6) arm of the N-glycan.

74. 74. The method of any one of claims 56 to 73, wherein the galactose moiety is a branched galactose moiety.

75. 75. The method of any one of claims 56 to 74, wherein the mammalian host cell comprises ST6GAL1 and B4GALT1 in a ratio of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20 (mol:mol).

76. 76. The method of any one of claims 56 to 75, wherein the mammalian host cell is in a population of mammalian host cells wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the mammalian host cells remain viable for 10 to 20 days after initiation of step (a).

77. 77. The method of any one of claims 56 to 76, further comprising contacting the mammalian host cell with an additive that enhances sialylation of an Fc polypeptide.

78. The additives include uridine, manganese, copper, dexamethasone, hydrocortisone, N-acetylmannosamine, tetraacetylated ManNAc, N-azidoacetyl D-mannosamine, 1,3,4-O-Bu ３ 78. The method of claim 77, wherein the hydroxyl group is selected from the group consisting of ManNAc, α(2,3)-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), siastatin B, fetuin, and glycerol.

79. 79. The method of any one of claims 56 to 78, wherein the glycosylation of N-glycans is determined using HPLC, MS, or a combination thereof.

80. 80. The method of claim 79, wherein the HPLC is hydrophilic interaction liquid chromatography (HILIC).

81. 24. A population according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22 or 23 for use as a medicament.

82. 24. A population according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 22 or 23 for use in the treatment of an inflammatory disease or condition in a subject in need thereof.

83. 83. The population or pharmaceutical composition of claim 82, wherein the inflammatory disease or condition is an autoimmune disease or condition.

84. 84. The population or pharmaceutical composition of claim 82 or 83, wherein the inflammatory disease or condition is arthritis.

85. 84. The population or pharmaceutical composition of claim 82 or 83, wherein the inflammatory disease or condition is immune thrombocytopenia (ITP).

86. 86. The population or pharmaceutical composition of any one of claims 81 to 85, having a half-life of at least 3.5 days following administration of the population or pharmaceutical composition to a subject.

87. 87. The population or pharmaceutical composition of claim 86, which has a half-life of at least 4 days after administration of the population or pharmaceutical composition to a subject.

88. 88. The population or pharmaceutical composition of any one of claims 81 to 87, which, following administration to a subject, is cleared from the subject's circulation at a rate of 45 mL / day / kg or less.

89. 89. The population or pharmaceutical composition of claim 88, which is cleared from the circulation of the subject at a rate of 40 mL / day / kg or less.

90. 90. The population or pharmaceutical composition of claim 89, which is cleared from the circulation of the subject at a rate of 35 mL / day / kg or less.

91. 91. The population or pharmaceutical composition of claim 90, which is cleared from the circulation of the subject at a rate of 30 mL / day / kg or less.

92. 92. The population or pharmaceutical composition of claim 91, which is cleared from the circulation of the subject at a rate of 25 mL / day / kg or less.

93. 93. The population or pharmaceutical composition of any one of claims 81 to 92, wherein the concentration of the Fc polypeptide over time (AUC) is at least 480 days x mg / mL between 1 and 35 days after administration of the population or pharmaceutical composition to a subject.

94. 94. The population or pharmaceutical composition of claim 93, wherein the AUC is at least 500 days×mg / mL, at least 550 days×mg / mL, at least 600 days×mg / mL, at least 650 days×mg / mL, at least 700 days×mg / mL, at least 750 days×mg / mL, at least 800 days×mg / mL or at least 850 days×mg / mL between 1 and 35 days after administration of the population or pharmaceutical composition to a subject.

95. 95. The population or pharmaceutical composition of any one of claims 81 to 94, wherein an additional therapeutic agent is administered to the subject.

96. 96. The population or pharmaceutical composition of claim 95, wherein the additional therapeutic agent is administered to the subject prior to, simultaneously with, or after administration of the population or pharmaceutical composition.

97. 97. The population or pharmaceutical composition of claim 95 or 96, wherein the additional therapeutic agent is a second modified Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, H433K and N434F.

98. 98. The population or pharmaceutical composition of claim 97, wherein the second modified Fc polypeptide comprises the amino acid substitutions M252Y, S254T, T256E, H433K and N434F.

99. 99. The population or pharmaceutical composition of claim 98, wherein the second modified Fc polypeptide has the amino acid sequence shown as SEQ ID NO:

5.

100. 100. The population or pharmaceutical composition of any one of claims 97 to 99, wherein the second modified Fc polypeptide is administered to the subject at a dose of 1 mg / kg to 20 mg / kg.

101. The population or pharmaceutical composition of claim 100, wherein the second modified Fc polypeptide is administered to the subject at a dose of 10 mg / kg.

102. 102. The population or pharmaceutical composition of any one of claims 97 to 101, wherein the second modified Fc polypeptide is administered to a subject once a week for four weeks.

103. 103. The population or pharmaceutical composition of any one of claims 95 to 102, wherein the additional therapeutic agent is selected from the group consisting of anti-inflammatory drugs, immunosuppressants, analgesics, disease-modifying antirheumatic drugs (DMARDs), counterirritants, platelet-increasing agents, thrombopoietin receptor (TPOR) agonists, physical therapy, and surgery.

104. 104. The population or pharmaceutical composition of claim 103, wherein the anti-inflammatory agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, an anti-inflammatory antibody or antigen-binding fragment thereof, an anti-inflammatory cytokine, a kinase inhibitor, and intravenous immunoglobulin (IVIG).

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