Methods of treating autoimmune disorders using modified f c polypeptides with enhanced sialylation

Abstract

Methods of treating autoimmune disorders affecting the skin and / or the kidney, e.g., epidermolysis bullosa acquisita (EBA) or glomerulonephritis in a human subject thereof, using a population of 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.

Description

[0001] Cross-reference to related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 564,940, filed March 13, 2024, the disclosure of which is hereby incorporated, in its entirety, by reference for all purposes.

[0002] sequence list This application contains a sequence list that has been electronically submitted in XML format. The sequence list XML is hereby incorporated in its entirety by reference. The XML file was created on February 19, 2025, named NVG-005WO_SL.xml, and has a size of 16,494 bytes.

[0003] background Inflammatory disorders, including autoimmune diseases, are disturbances involving the abnormal activation of white blood cells and their subsequent migration to affected areas of the body.

[0004] For example, acquired epidermolysis bullosa (EBA) is an autoimmune cutaneous disease that causes blistering of the skin and mucous membranes. EBA is caused by autoantibodies targeting type VII collagen (a component that anchors fibrils), leading to epidermal detachment and fragility. Current treatment options focus on managing patients' wounds and pain through nonspecific immunosuppression, which in many cases does not lead to remission.

[0005] As another example, glomerulonephritis (GN) is a group of immune-mediated diseases characterized by damage to the glomerular compartments of the nephrons in the kidney. GN is a leading cause of end-stage renal disease worldwide and is associated with significant morbidity and mortality. Untreated acute GN can lead to chronic kidney disease and irreversible renal failure. Immune-mediated GN, which accounts for the majority of forms, is characterized by a pathogenic immune response against renal autoantigens or systemic autoimmune manifestations within the kidney. Most treatment strategies still employ corticosteroids and cytotoxic agents, which aim to suppress the entire immune system.

[0006] There is still a need for improved therapeutic agents for certain inflammatory disorders affecting the skin or kidneys, such as acquired bullous epidermolysis or immune-mediated GN.

[0007] Overview This disclosure provides methods for treating autoimmune disorders affecting the skin and / or kidneys in subjects (e.g., human subjects) who have a corresponding need.

[0008] In one aspect, a method is provided for treating an autoimmune disorder affecting the skin and / or kidneys in a human subject with a corresponding need, the method comprising: administering to the subject a therapeutically effective amount of a population of modified Fc peptides or a pharmaceutical composition thereof, each modified Fc peptide having (i) an amino acid sequence at least 75% identical to the sequence of SEQ ID NO: 2 and (ii) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), the population comprising at least 50% of modified Fc peptides having a sialic acid (SA) moiety attached to an N-glycan via an α(2,6) bond.

[0009] In some implementations, the aliphatic amino acid residue at position 241 is alanine (Ala; F241A).

[0010] In some embodiments, the N-glycan is attached to asparagine (Asn) at amino acid residue 297 of the modified Fc polypeptide (Asn297; according to Kabat number; corresponding to amino acid residue 88 of SEQ ID NO: 2).

[0011] In some embodiments, at least 60%, at least 70%, at least 80%, or at least 90% of the modified Fc polypeptide comprises an SA moiety attached to the N-glycan of the Fc polypeptide via an α(2,6) bond.

[0012] In some implementations, the N-glycan of the modified Fc peptide is monosialylated or disialylated.

[0013] In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the modified Fc polypeptide comprises a monosialylated N-glycan, said monosialylated N-glycan comprising an SA moiety attached via an α(2,6) bond. In some embodiments, at least 30% of the modified Fc polypeptide comprises a monosialylated N-glycan, said monosialylated N-glycan comprising an SA moiety attached via an α(2,6) bond. In some embodiments, at least 40% of the modified Fc polypeptide comprises a monosialylated N-glycan, said monosialylated N-glycan comprising an SA moiety attached via an α(2,6) bond. In some embodiments, at least 50% of the modified Fc polypeptide comprises a monosialylated N-glycan, said monosialylated N-glycan comprising an SA moiety attached via an α(2,6) bond. In some embodiments, at least 60% of the modified Fc polypeptide comprises a monosialylated N-glycan containing an SA moiety attached via an α(2,6) bond. In some embodiments, at least 70% of the modified Fc polypeptide comprises a monosialylated N-glycan containing an SA moiety attached via an α(2,6) bond. In some embodiments, at least 80% of the modified Fc polypeptide comprises a monosialylated N-glycan containing an SA moiety attached via an α(2,6) bond. In some embodiments, at least 90% of the modified Fc polypeptide comprises a monosialylated N-glycan containing an SA moiety attached via an α(2,6) bond.

[0014] In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 30% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 40% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 50% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 60% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 70% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 80% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, at least 90% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds.

[0015] In some embodiments, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, at least about 60% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, at least about 70% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, at least about 80% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, at least about 90% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, about 100% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, the galactose moiety is attached to the α(1,3) arm and / or α(1,6) arm of the N-glycan. In some embodiments, the galactose moiety is a branched-chain galactose moiety.

[0016] In one aspect, a method is provided for treating an autoimmune disorder affecting the skin and / or kidneys in a human subject with a corresponding need, the method comprising: administering to the human subject a therapeutically effective amount of a population of modified Fc peptides or a pharmaceutical composition thereof, each modified Fc peptide having (i) an amino acid sequence at least 75% identical to the sequence of SEQ ID NO: 2 and (ii) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), the population comprising: about 40% of modified Fc peptides having a sialic acid (SA) moiety attached to an N-glycan of the modified Fc peptide via an α(2,3) bond.

[0017] In some implementations, the aliphatic amino acid residue at position 241 is alanine (Ala; F241A).

[0018] In some embodiments, the modified Fc peptide is an IgG1 Fc peptide. In some embodiments, the modified Fc peptide is an IgG3 Fc peptide.

[0019] In some implementations, autoimmune disorders affect the skin, such as acquired epidermal bullae (EBA).

[0020] In some implementations, autoimmune disorders affect the kidneys, such as immune-mediated glomerulonephritis.

[0021] In some implementations, the population or pharmaceutical composition has a half-life of at least 3.5 days after administration to human subjects.

[0022] In some implementations, the population or pharmaceutical composition has a half-life of at least 4 days after administration to human subjects.

[0023] In some embodiments, the population or pharmaceutical composition is cleared from the circulation of a human subject at a rate not exceeding 45 mL / day / kg, not exceeding 40 mL / day / kg, not exceeding 35 mL / day / kg, not exceeding 30 mL / day / kg, or not exceeding 25 mL / day / kg.

[0024] In some embodiments, the concentration (AUC) of the modified Fc peptide over time is at least 480 days × mg / mL between 1 day and 35 days after administration of the population or pharmaceutical composition to human subjects.

[0025] In some embodiments, 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 day and 35 days after administration of the population or pharmaceutical composition to human subjects.

[0026] In some embodiments, the provided method further includes administering an additional therapeutic agent to a human subject. The additional therapeutic agent may be administered to the human subject before, during, or after administration of the group or pharmaceutical composition.

[0027] 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.

[0028] In some embodiments, the second modified Fc polypeptide comprises amino acid substitutions of M252Y, S254T, T256E, H433K, and N434F.

[0029] In some embodiments, the second modified Fc polypeptide has an amino acid sequence as shown in SEQ ID NO: 5.

[0030] In some embodiments, the second modified Fc peptide is administered at a dose of 1 mg / kg to 20 mg / kg. In some embodiments, the second modified Fc peptide is administered at a dose of 10 mg / kg. In some embodiments, the second modified Fc peptide is administered once weekly for four weeks.

[0031] In some implementations, additional therapeutic agents are selected from the group consisting of: anti-inflammatory agents, immunosuppressants, analgesics, disease-modifying antirheumatic drugs (DMARDs), anti-irritants, platelet-enhancing drugs, thrombopoietin receptor (TPOR) agonists, physical therapy, and surgery.

[0032] In some implementations, the anti-inflammatory agent is selected from the group consisting of: nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, anti-inflammatory antibodies or their antigen-binding fragments, anti-inflammatory cytokines, kinase inhibitors, and intravenous immunoglobulins (IVIG).

[0033] In one aspect, a population of modified Fc peptides or a pharmaceutical composition thereof is provided, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), the population comprising: at least 60% of modified Fc peptides having a sialic acid (SA) moiety attached to an N-glycan of the modified Fc peptide via an α(2,6) bond, for use as a medicament for treating autoimmune disorders affecting the skin and / or kidneys in human subjects with corresponding needs.

[0034] In one aspect, a group of modified Fc peptides or a pharmaceutical composition thereof are provided, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), comprising: at least 60% of the modified Fc peptide having a sialic acid (SA) moiety attached to an N-glycan of the modified Fc peptide via an α(2,6) bond, for treating autoimmune disorders affecting the skin and / or kidneys in human subjects with corresponding needs.

[0035] In some implementations, autoimmune disorders affect the skin, such as acquired epidermal bullae (EBA).

[0036] In some implementations, autoimmune disorders affect the kidneys, such as immune-mediated glomerulonephritis. Brief description of the attached diagram Figure 1 A diagram and nomenclature of a representative biantennary N-glycan structure present on asparagine 297 (Asn297) of the heavy chain of the Fc polypeptide of this disclosure are shown. The exemplified glycoforms are covalently attached to the end of the constant region 2 (CH2) domain of the heavy chain of the Fc polypeptide at the base of each glycan structure. All subsequent descriptions of the N-glycans are provided as straight-chain or branched sequences of carbohydrates starting from Asn297 (e.g., Asn297 → carbohydrate 1 → carbohydrate 2, etc.). The carbohydrate closest to Asn297 is considered to be... Proximal Those carbohydrates farther from Asn297 are considered to be... remote And those carbohydrates furthest from Asn297 are considered to be... terminalThe G0F glycoform (i.e., the core glycan) comprises an N-acetylglucosamine (GlcNAc) and a mannose (Man) core, which is modified by fucose (Fuc) residues at the nearest GlcNAc moiety. The G0 glycoform is identical to the G0F glycoform except for the absence of the Fuc moiety in 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 in which five mannose moieties are attached to the core glycan. The G1F glycoform comprises a core glycan in which a single terminal galactose (Gal) is 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 in the core glycan. The G2F glycoform includes 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 portion of the core glycan. The G2F SA glycoform contains a core G2F glycan structure, with a single terminal sialic acid (SA) portion attached to one of the Gal portions (either the α(1,6) or α(1,3) arm of the branched N-glycan). The SA is linked to the Gal via either an α(2,3) or α(2,6) bond. The G2FSA2 glycoform contains a core G2F glycan structure, with a terminal SA portion attached to each Gal portion (both the α(1,6) and α(1,3) arms of the branched N-glycan). The SA is linked to the Gal via either an α(2,3) or α(2,6) bond.

[0038] Figures 2A-2B This is a bar graph showing the level of α(2,3) sialylation after transient transfection of Chinese hamster ovary (CHO) cells with wild-type (WT) Fc peptide or a mutant Fc peptide (IgG1 isotype), wherein the mutant Fc peptide has an alanine (A) substituted for phenylalanine (F) at amino acid position 241 (F241), corresponding to amino acid residue 32 of SEQ ID NO: 2. WT Fc mainly comprises glycans terminated at GlcNAc (G0 or G0F; 49%) or terminated at GlcNAc containing a single Gal (G1 or G1F; 39%), but rarely or no bis-Gal (G2F) glycoforms or SA-containing glycoforms (<1.0%). Figure 2A The F241A mutation results in a significant enhancement of Gal addition to the Fc domain, with 22% of the peptides containing mono-2,3 SA (G2FSA) and 19% containing di-2,3 SA (G2FSA2). Figure 2B ).

[0039] Figures 3A-3D This demonstrates the recombinant expression of Fc by stable transfection of Chinese hamster ovary (CHO) cells using a 1 L culture tank. F241AA bar chart of N-glycan analysis of the mutant. It shows the Fc expressed alone. F241A Bar chart of N-glycan analysis of mutants (pool 1); Figure 3A The Fc expression combined with β-galactosidase α-2,6-sialyltransferase 1 is shown. F241A Bar chart of N-glycan analysis of mutants (ST6GAL1; pool 2; Figure 3B The image shows Fc expressed in combination with ST6GAL1 and β-1,4-galactosyltransferase 1. F241A Bar chart of N-glycan analysis of mutants (B4GALT1; Pool 3; Figure 3C The image shows Fc co-expression with siRNA targeting the SA transporter (solute carrier family 35 member A1). F241A Bar chart of N-glycan analysis of mutants (SLC35A1 KD; Pool 4; Figure 3D Pool 1 contained 18% mono-α(2,3)-sialylated, 5% di-α(2,3)-sialylated, and 47% with one or two terminal Gals. Pool 2 contained 30% mono-α(2,6)-sialylated, 27% di-α(2,6)-sialylated, and 7% with one or two terminal Gals. Pool 3 contained 3% mono-α(2,6)-sialylated, 81% di-α(2,6)-sialylated, and 0% with terminal Gals. Pool 4 contained 0% sialylated, and 84% with one or two terminal Gals.

[0040] Figures 4A-4C This study presents independently validated blots providing the glycan structures of pools 1-4 (P1-P4) from 1 L of stably transfected CHO culture. Images of Coomassie staining for each of P1-P4 are also shown. Figure 4A The use of biotinylated elderberry () is shown. Sambucus nigra (SNA) lectin staining to detect the blots of each of P1-P4 in α(2,6) SA. Figure 4B P1-P4 use Korean locust ( Maackia amurensis (MAL I) lectin staining to detect terminal galactose ( Figure 4C ).

[0041] Figures 5A-5F This illustrates the effect of sialylation on Fc in mice. F241A A graph showing the effects of mutant exposure. These experiments were conducted using Fc cells generated by stable transfection of 10 mL CHO cell culture pools. F241AThe materials were prepared as follows: Pool 1 material (F241A alone) contained 15% mono-2,3-sialic acid and 20% di-2,3-sialic acid. Pool 2 material (F241A + ST6GAL1) contained 31% mono-2,6-sialic acid and 35% di-2,6-sialic acid. Pool 3 material (F241A + ST6GAL1 + B4GALT1) contained 3% mono-2,6-sialic acid and 90% di-2,6-sialic acid. Pool 4 material (F241A + SLC35A1 KD) contained 0% mono-sialic acid and 0% di-sialic acid. The y-axis on a logarithmic scale shows the FcRn levels in JAX-014565 mice (mouse FcRn homozygous knockout, human FcRn hemizygous, Tg32 strain) after intravenous (IV) bolus administration of pools 2 (F241A + ST6GAL1), 3 (F241A + ST6GAL1 + B4GALT1), and 4 (F241A + SLC35A1 KD). F241A Graph showing the change in serum concentration (μg / mL) of mutants over time. Figure 5A ).and Figure 5A The same data is shown on a linearly scaled y-axis. Figure 5B The maximum exposure, as measured by the area under the curve (AUC), was achieved in pool 3 material with 93% α(2,6) sialylation. The effects of intravenous (IV) administration of pool 1 (F241A only), pool 3 (F241A+ST6GAL1+B4GALT1), and IgG1 Fc domains (efgartigimod (EFG); Argenx) with ABDEG mutations (M252Y / S254T / T256E / H433K / N434F) to enhance FcRn binding in JAX-014565 mice are shown. F241A Graph showing the change in serum concentration (μg / mL) of mutants over time. Figure 5C (), shown on a logarithmic scale along the y-axis. (and) Figure 5C The same data is shown on a linearly scaled y-axis. Figure 5D ). 93% α(2,6) sialylated Fc F241A (Pool 3) showed the greatest exposure in these human FcRn mice. The ERG Fc domain had the lowest AUC, similar to... Figures 5A-5B The unsialylated F241A (pool 4) is shown. The levels of Fc in male and female CD1 mice after intravenous (IV) administration of pool 1 (F241A 2,3 sialylated) or pool 3 (F241A 2,6 sialylated) are shown. F241A A graph showing the average concentration (μg / mL) of the mutant over time. Figure 5E). In Fc receiving α(2,3) sialylation (hollow triangle and solid triangle) F241A Or α(2,6) sialylated (hollow and solid circles) Fc F241A There was no significant difference in exposure levels between male and female mice. However, the α(2,6)-sialyzed Fc pool had greater exposure than the α(2,3)-sialyzed Fc pool, although this difference may be attributed to the degree of sialyzation rather than the α(2,3) bond versus the α(2,6) bond. Repeated administration (100 mg / kg weekly for 4 weeks) of α(2,3)-sialyzed and α(2,6)-sialyzed Fc pools in CD1 mice is shown. F241A mutant ( Figure 5F A bar chart showing the effect of α(2,3) sialylation on Fc. F241A In the administration of α(2,6) sialylated Fc F241A Subsequently, in male and female CD1 mice, in C min Greater exposure was achieved at both (before administration, day 21) and 24 hours after the fourth administration (24 hours after administration, day 22).

[0042] Figures 6A-6F The Fc depicting sialylation is shown. F241A A graph showing the therapeutic effects of the peptide in a mouse model of arthritis. A graph showing 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 transiently transfected (41%) α(2,3) sialylated F241A material is also included. Figure 6A ). =On day 6, Fc with IVIG and α(2,3) sialylated F241A The difference in clinical scores between them was (p = 0.002); =On day 7, Fc with IVIG and α(2,3) sialylated F241A The difference in clinical scores between IVIG and α(2,3) sialylated Fc was p = 0.04; for all other days, the difference in clinical scores between IVIG and α(2,3) sialylated Fc was p = 0.04. F241A There were no significant differences between the peptides. Inflammation peaked on day 7 in PBS-treated mice. Therefore, clinical scores on days 7 and 8 of the study were also significantly higher. Figures 6B-6C The results are shown in box plots, which are represented as the mean and the standard error of the mean. Relative to PBS, 1 g / kg IVIG and 50 mg / kg Fc... F241ABoth significantly suppressed inflammation. These data suggest that F241A is approximately 20 times more potent than IVIG in the K / BxN serum transfer model of arthritis. A graph showing 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 culture is presented. Figure 6D ; an asterisk indicates a statistically significant difference compared to IVIG; p < 0.05). As determined by an unpaired t-test, from day 5 onwards, IVIG showed significantly greater anti-inflammatory activity than F241A α(2,3) sialylated Fc (p < 0.01). In contrast, only from day 8 onwards, IVIG was significantly different from F241A α(2,6) sialylated Fc (unpaired t-test, p < 0.05). Clinical scoring plots show that the peak of inflammation in PBS-treated animals occurred on day 6 ( Figure 6E ) and the 7th day ( Figure 6F ), α(2,6) sialylated Fc F241A Fc that leads to sialylation relative to α(2,3) F241A Significantly less inflammation Figures 6E-6F ).

[0043] Figure 7 Box-and-whisker plots show 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. 2,3-sialylated Fc F241A The material is as effective as IVIG at 10 times lower concentrations.

[0044] Figures 8A-8E It shows the different Fc F241A A graph showing the half-life and bioavailability of sugar forms. Figure 8A Female humanized FcRn mice (n = 6 per group) were administered 20 mg / kg Fc F241A One of the four products at a time: (1) a single Fc F241A (2) Fc F241A + ST6GAL1;(3) Fc F241A + B4GALT1 + ST6GAL1; or (4) Fc F241A+SLC35A1 siRNA. Serum concentrations of hIgG Fc were measured by ELISA on days 1, 3, and 7, and at weeks 2 (14 days), 3 (21 days), and 5 (35 days) after administration, until the serum concentration decreased to below the detection threshold of 1 µg / ml. From the obtained data, half-life, area under the curve (AUClast) from time of administration to the last measurable concentration, and clearance were calculated for each Fc (see also Table 4). Data were plotted as mean with standard deviation. Statistical analysis was performed using a standard one-way ANOVA with Tukey multiple comparisons. Fc values ​​are shown. F241A Percentage of sialylation on the surface and clinical score ( Figure 8B ),half life( Figure 8C ), AUClast ( Figure 8D ) and clearance rate ( Figure 8E The correlation between the two is shown. The trend line is generated by simple linear regression, and the corresponding R² and p-values ​​are also shown. Figures 8B-8E Correlation of clinical scores on day 7 () Figure 8B The data were generated from data obtained in WT mice, while other correlations were generated from data in Tg32 mice. Figures 8C-8E ).

[0045] Figure 9A A graph showing serum IgG Fc concentrations (shown on the y-axis) measured by ELISA versus the number of days following IgG Fc administration (shown on the x-axis) for each treatment group is presented. Treatment groups are shown in the legend. Statistical significance relative to the control group was determined by a standard one-way ANOVA. The data demonstrate that the asialylated glycoprotein receptor (ASGPR) represents the major clearance pathway for non-sialylated human IgG1 Fc. Figure 9B The Fc shown is determined by biological layer interference. F241A / siSLC or Fc F241A / B4ST6 Association constant (K) with asialic acid glycoprotein receptor (ASGPR) a M -1 ).

[0046] Figure 10A and Figure 10B To illustrate the use of Fc F241A A graph showing the clinical scores of K / BxN mice (arthritis model) treated with different glycoforms. Figure 10A The description shows the administration of PBS or 50 mg / kg of IVIg, Fc F241A 、Fc F241A / ST6 、Fc F241A / B4ST6 or Fc F241A / siSLC A bar chart of clinical scores of K / BxN treated mice (N = 5) on day 8 (the peak of the disease). Figure 10BClinical scores of mice treated with K / BxN (N = 5 per group) across 10 days were depicted. P-values ​​were determined using a standard one-way ANOVA with Tukey multiple comparisons.

[0047] Figures 11A-11L It shows Fc F241A / B4ST6 peptides and Fc Abdeg Illustrations and figures showing the protective effect of the polypeptide (SEQ ID NO: 5) in vivo. Figure 11A The diagram shows WT Fc (SEQ ID NO: 1) and Fc. Abdeg Sequence alignment of (SEQ ID NO: 5), where the thin box indicates the location of the Abdeg mutation and the thick box indicates the location of the N297 glycosylation site. Figure 11B A diagram of the human IgG Fc structure is shown, with the locations of the Abdeg mutation (empty hexagon) and N297 (striped hexagon) marked, showing their relative distances to each other and their different positions on the outside of the Fc structure relative to the inside. Figure 11C and Figure 11D )WT Fc、Fc F241A / B4ST6 and Fc Abdeg It was loaded onto an anti-human Fc biosensor and examined for its correlation with mouse FcRn via surface plasmon resonance (SPR). Figure 11C ) and human FcRn ( Figure 11D The binding dynamics of ). Dissociation constant (K) D The associativity constant (KA) is plotted as µM (above), and the association constant (KA) is plotted as Ms. -1 (See image below). Figure 11E and Figure 11F Female WT C57BL / 6 Figure 11E ) and humanized homozygous FcRn (Tg32) mice ( Figure 11F ) was administered a single dose of 50 mg / kg or 100 mg / kg of Fc F241A / B4ST6 Or 10 mg / kg of Fc Abdeg Normalized serum mouse IgG levels were measured by ELISA from day 0 to day 7 after drug administration (shown on the y-axis). Figure 11G SIGN-R1 - / - and hDC-SIGN + Fcγ receptors in mouse bone marrow-derived macrophages (BMDM) were blocked, and then reacted with PBS and Fcγ receptors. F241A / B4ST6 or Fc Abdeg The cells were incubated together, and binding was detected by FACS using an anti-human IgG Fc antibody. The bar graph shows the percentage of cells bound by the added Fc as a fold change in binding compared to PBS. Figure 11H and Figure 11I This demonstrates the detection of PBS and Fc by FACS. F241A / B4ST6 and Fc WT A graph showing the binding of the substance to the cell surface of mouse peritoneal macrophages (pMAC) (mean fluorescence intensity (MFI)). Figure 11H Showing from SIGN-R1 + MFI binds to the cell surface in mouse pMAC, and Figure 11I Showing from SIGN-R1 - MFI binds to the cell surface in mouse pMAC. Figures 11J-11L Female WT C57BL / 6 mice (n = 5 per group) and female and male SIGN-R1 mice - / - Mice (n=5 per group) were prophylactically administered arthritis-inducing K / BxN serum, as well as PBS, 1 g / kg IVIG, and 50 mg / kg Fc. F241A / B4ST6 Or 10 mg / kg Fc Abdeg The joint swelling was clinically scored for 10 days. Figure 11J A line graph showing the clinical scores of joint inflammation over 10 days in female WT C57BL6 mice (n = 5 mice per group) is presented. Figure 11K Female SIGN-R1 was shown. - / - Line graph of clinical scores for joint inflammation in mice (n = 5 mice per group) over 10 days. Figure 11L A bar chart showing the clinical scores of joint inflammation on day 7 for each group, representing the peak of the disease, is presented. The bars are plotted as mean and standard deviation. Statistical significance was assessed using a plain one-way ANOVA with Tukey multiple comparisons.

[0048] Figures 12A-12I This shows F241A / B4ST6 Fc and Fc Abdeg Illustrations and figures of the combined preventive and therapeutic effects in an in vivo mouse model of arthritis. Figure 12A An illustrative experimental diagram is shown to illustrate the preventative approach in the K / BxN model. Figure 12A (see above image) and treatment methods ( Figure 12A (See image below) Both check Fc F241A / B4ST6 and Fc Abdeg The effect. In short, female WT C57BL / 6 mice (n = 5) were administered arthritis-inducing K / BxN serum on day 0, followed by therapeutic administration of PBS, 1 g / kg IVIG, and 50 mg / kg Fc on day 2. F241A / B4ST6 Or 10 mg / kg Fc Abdeg Joint swelling was clinically scored for 12 days after K / BxN. Figure 12BClinical scores of joint inflammation shown over 12 days in female WT C57BL6 mice (n = 4 or 5 mice per group) are presented. Figure 12C A bar chart is shown, which illustrates the clinical score on day 8 (peak of inflammation) for each of the above groups. Figure 12D Serum Fc on day 10 is shown F241A Horizontal (y-axis) histogram. Female WT C57BL / 6 mice (n = 4–5) were administered alone or with escalating doses of Fc. Abdeg (1-10 mg / kg) combination of 50 mg / kg Fc F241A / B4ST6 Ten days later, serum human IgG Fc was detected by ELISA. Figure 12E This demonstrates the administration of PBS or 1 mg / kg Fc. Abdeg A graph showing the relative serum IgG titers (normalized against PBS) of mice (n = 3) on days 1, 3, and 7 after administration. The p-value was determined using a one-tailed Mann-Whitney test. Figure 12F Female WT C57BL / 6 mice (n = 3–5) were prophylactically administered arthritis-inducing K / BxN serum, along with PBS, 1 g / kg IVIG, and 50 mg / kg Fc. F241A / B4ST6 1 mg / kg Fc Abdeg or Fc F241A / B4ST6 and Fc Abdeg The combination of [various parameters] was used, and joint swelling was clinically scored over 10 days. Clinical scores of joint inflammation were displayed over 10 days in female WT C57BL6 mice (n=3-5 mice per group). Figure 12G Clinical scores on day 6 for each group, representing the peak of the disease, were plotted. Figure 12H On day 0, female WT C57BL / 6 mice (n = 5) were administered arthritis-inducing K / BxN serum, followed by therapeutic administration of PBS, 1 g / kg IVIG, and 50 mg / kg Fc on day 2. F241A / B4ST6 1 mg / kg Fc Abdeg or Fc F241A / B4ST6 With Fc Abdeg The combination of K / BxN was used. Clinical scoring of joint swelling was performed for 12 days after K / BxN. Clinical scores of joint inflammation across 12 days are shown in female WT C57BL6 mice (n = 5 mice per group). Figure 12I Clinical scores on day 9, representing the peak of the disease, were plotted for each group. Bar charts were generated as mean and standard deviation. Statistical significance was assessed using a plain one-way ANOVA with Tukey multiple comparisons.

[0049] Figure 13A and Figure 13B The skin blistering score and FcγRIIB expression in peripheral neutrophils were characterized. Figure 13A Inverted triangles represent the number of days of treatment (day 0 (3 hours before administration of the pathogenic autoantibody) and day 3). The percentage of total skin area affected by blisters is plotted within several days after disease induction (x-axis) (y-axis ± SEM). Hollow circles, squares, and equilateral triangles depict the effects of PBS, 100 mg / kg Fc, etc. F241A Alternatively, treat with 10 mg / kg edema. Use four animals per group. Figure 13B Depicting the use of PBS, Fc F241A FcgRIIB expression on the surface of peripheral neutrophils in mice treated with efgartijimod or MFI ± SEM (n = 4 per group). Statistical significance was determined by two-way ANOVA, where Fc F241A This uniquely led to a significant (p<0.001) increase in FcγRIIB expression relative to PBS on day 5 of the study.

[0050] Figures 14A-14C The results of fluorescence-activated cell sorting (FACS) analysis show the kinetics of FcγRIIB upregulation on B cells and neutrophils. Figure 14A , Figure 14B and Figure 14C The image shows FACS plots, evaluating the effects of PBS and Fc. F241A FcγRIIB expression on the surface of peripheral naive B cells, mature B cells, and neutrophils, respectively, in mice treated with eigenin (MFI ± SEM; n = 4 per group). Data were plotted on a graph with the number of days post-induction of acquired epidermolysis bullosa (EBA) as the x-axis. Statistical significance was determined by two-way ANOVA using multiple comparisons.

[0051] Figure 15 Images of tissue sections from ear tissue biopsies stained with hematoxylin and eosin (H&E) are shown. Skin samples from areas of the ear affected by blisters in mice were biopsied, and infiltrating immune cells were stained with H&E. The top, middle, and bottom rows depict tissue sections from ear biopsies stained with a medium control and Fc, respectively. F241A Images of mice treated with edema. Images are annotated to show cartilage, blood, dead skin, and blisters.

[0052] Figure 16A and Figure 16B The experimental results assessing changes in the immune cell population present in skin biopsies are shown. Neutrophils were present in ear skin lesions. Figure 16A ) and CD62L-high expressing monocytes ( Figure 16B The mean percentage (+SEM) is shown on the y-axis and plotted on the x-axis for the number of days following acquired epidermolysis bullosa (EBA) induction. Statistical significance was determined using a two-way ANOVA with multiple comparisons.

[0053] Figure 17A This is a graph showing the percentage change in body weight of mice following experimental autoimmune encephalomyelitis (EAE) induction (relative to the normalized mean + SEM on day 0 of the study) for each treatment group (n=7) over the number of days after treatment initiation (shown on the x-axis) (shown on the y-axis). The treatment groups are shown in the legend of the figure. Statistical significance relative to the vector control group is indicated by an asterisk and determined using two-way ANOVA and the Bonferroni post-hoc test.

[0054] Figure 17B This is a graph showing the percentage change in body weight (normalized relative to study day 0) of mice following induction of experimental autoimmune encephalomyelitis (EAE) for each treatment group (n = 7 per group, administered on days 5, 10, 15, and 20) against the number of days since treatment initiation (shown on the x-axis) (shown on the y-axis). Treatment groups are shown in the legend.

[0055] Figure 18A This is a graph showing the clinical scores (mean + SEM) plotted for each treatment group (n=7) based on the number of days after treatment initiation (shown on the x-axis) (shown on the y-axis). Figure 18B This is a bar chart showing the mean AUC of clinical scores (+SEM) throughout the study period. The treatment group is defined in the legend of the figure. Statistical significance relative to the treatment control group is indicated by an asterisk and determined using two-way or one-way ANOVA, as indicated.

[0056] Figure 18C A graph showing clinical scores (shown on the y-axis) for each treatment group (n = 7 per group, administered on days 5, 10, 15, and 20) versus the number of days since treatment initiation (shown on the x-axis; MOG peptide administered on day 0) is presented. Treatment groups are defined in the graph legend. Two-way ANOVA and Bonferroni post-hoc test were used to determine the p-values ​​of treatment groups relative to the mediator control group.

[0057] Figure 19 To demonstrate the effects of EAE-induced mice treated with saline (n=6) and 33 mg / kg Fc F241A A graph showing the clinical scores (shown on the y-axis) of the treatment groups (n=4) or 33 mg / kg WT Fc (n=4) versus the number of days after treatment initiation (shown on the x-axis).

[0058] Figure 20A To plot the results using phosphate-buffered saline (PBS), 1 g / kg IVIG, and 100 mg / kg Fc F241A In treated mice, Treg (CD4) + CD25 + FOXP3 + ) cells account for a certain percentage of total CD4 + A bar chart showing the frequency of cell percentages. Indicating Fc F241A The p-values ​​(P<0.05) between PBS controls were determined by a nonparametric T-test.

[0059] Figure 20B This diagram illustrates the experimental protocol for an immunization assay using the 2W antigen (a model antigen used to track specific clonal T cell responses). Treatments include saline, 2 g / kg IVIG, and 33 mg / kg Fc. F241A Or 10 mg / kg Fc F241A .

[0060] Figure 20C To describe the effects of saline, 2 g / kg IVIG, and 33 mg / kg Fc F241A Or 10 mg / kg Fc F241A A graph showing the absolute number of antigen-specific total T cells, Teff, and Treg in treated mice.

[0061] Figure 20D To describe the effects of saline, 2 g / kg IVIG, and 33 mg / kg Fc F241A Or 10 mg / kg Fc F241A A graph showing the ratio of Teff to Treg cells from the spleen of 2W antigen-specific cells in treated mice.

[0062] Figure 20E The results showed the use of saline, 2 g / kg IVIG, and 33 mg / kg Fc. F241A Or 10 mg / kg Fc F241A A graph showing the percentage of total spleen-derived 2W antigen-specific Treg cells expressing IL10 in treated mice.

[0063] Figure 21AAn overview of the experimental timeline for the nephrotoxic nephritis (NTN) mouse model. Mice were immunized with sheep IgG to develop a robust anti-sheep IgG response and subsequently challenged (day 0) with sheep anti-glomerular basement membrane (α-GBM) serum, which targets the kidneys with sheep:mouse IgG immune complexes, leading to kidney destruction. NTN-treated mice were administered PBS, IVIG, or 200 mg / kg, 100 mg / kg, or 50 mg / kg of Fc. F241A On day 7, the sheep's IgG+ blood urea nitrogen (BUN) level was measured. Figure 21B This is a graph showing α-sheep mouse IgG detected by ELISA in mouse serum on day 7 (shown on the y-axis) against different treatment interventions (shown on the x-axis). Figure 21C This is a graph showing the serum blood urea nitrogen (BUN) levels (mg / dL) in mice on day 7 (shown on the y-axis) for different treatment interventions (shown on the x-axis). p-values ​​were determined using standard one-way or two-way ANOVA and Tukey multiple comparisons.

[0064] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. Generally, terms used in conjunction with the techniques of immunology, oncology, cell and tissue culture, molecular biology, and protein chemistry described herein are those well-known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and do not limit any of the claimed subject matter. Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0065] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, references to “polypeptide” include more than one polypeptide.

[0066] Unless the context clearly indicates otherwise, as used herein, all numerical values ​​or ranges include all integers within or covering such ranges, as well as fractions of values ​​or integers within or covering such ranges. Thus, for example, references to a range of 90%–100% include 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, the range of 1-5,000 times includes 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, as well as 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, and so on.

[0067] As used in this article, “approximately” means a number that includes the figure and ranges from 10% below the figure to 10% above the figure. “Approximately” range means 10% below the lower limit of the range and 10% above the upper limit of the range.

[0068] As used herein, “administration” means providing or giving a therapeutic agent (e.g., a modified Fc peptide of this disclosure or a composition containing thereof) to a subject via any effective route. Exemplary routes of administration are described in the following sections.

[0069] As used herein, the term "effective amount" refers to the amount of the Fc peptide or composition of this disclosure sufficient, when administered to a subject, to induce the disclosed effect, such as achieving treatment, prognosis, or diagnosis of a disease (e.g., an autoimmune disorder) as described herein. When used alone or in combination, 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, alleviating, or improving the disease or disorder described herein) and on the subject being treated and the disease condition, the subject's weight and age, the severity of the disease condition, the method of administration, etc.

[0070] 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 the immunoglobulin heavy chain. In some embodiments, “Fc polypeptide” is the native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from position Pro230 to its C-terminus.

[0071] "Isolated" polypeptides or proteins or groups thereof refer to polypeptides or proteins or groups thereof that have been isolated from other proteins, lipids, and nucleic acids with which they are naturally associated. In some embodiments, the polypeptide / protein or group thereof constitutes at least 10% by dry weight of the purified article (i.e., any percentage between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), or mass spectrometry (MS). In some embodiments, the isolated polypeptides / proteins or groups thereof described herein are produced by recombinant DNA technology or by chemical methods. In some embodiments, the isolated polypeptide includes SEQ ID NO:2.

[0072] The “natural” or “parental” Fc region contains the same amino acid sequence as that of Fc regions found in nature. The “variant” or “modified” Fc region includes an amino acid sequence that differs from the amino acid sequence of the natural Fc region due to at least one amino acid modification, such as an amino acid substitution (e.g., F241A). In some embodiments, the modified Fc region has at least one amino acid substitution, for example, from about one to about ten amino acid substitutions, compared to the natural Fc region or the Fc region of the parent polypeptide. In some embodiments, the modified Fc region described herein will 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 with the natural Fc region and / or with the Fc region of the parent polypeptide.

[0073] The "sequence identity percentage (%)" relative 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 those in the reference polynucleotide or polypeptide sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum sequence identity percentage. Alignment for determining the nucleic acid or amino acid sequence identity percentage can be performed 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 sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared, are determined by any suitable means. For example, the sequence alignment computer program BLAST can be used to generate sequence identity percentage values. As an illustration, the sequence identity percentage of a given nucleic acid or amino acid sequence A with, and with, or against a given nucleic acid or amino acid sequence B (which may optionally be expressed as a given nucleic acid or amino acid sequence A having a certain sequence identity percentage with, and with, or against the given nucleic acid or amino acid sequence B) is calculated as follows: 100 multiplied by (fraction X / Y) Where X is the number of nucleotides or amino acids that are scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It should be understood 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, the percentage of sequence identity between A and B will not be equal to the percentage of sequence identity between B and A.

[0074] As used herein, the term “pharmaceutically acceptable” means those compounds, materials, compositions, and / or dosage forms suitable for contact with tissues of subjects, such as mammals (e.g., humans), without excessive toxicity, irritation, allergic reactions, and other problematic complications commensurate with a reasonable benefit / risk ratio.

[0075] The term "peptide" refers to a chain of amino acids. Peptides are not limited to products of a specific length. Peptides, oligopeptides, and proteins are included within the definition of a polypeptide, and these terms are used interchangeably herein unless otherwise specifically indicated. The term also covers chains of peptides that have post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In some embodiments, a polypeptide is a complete protein or a subfraction thereof.

[0076] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, particularly a human, who requires diagnosis, treatment, or therapy. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, domesticated and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some implementations, the mammal is a human. These terms do not require the supervision of a medical professional.

[0077] The term "therapeuticly effective amount" generally refers to the amount of a disclosed composition that effectively "treats" a disease or disorder in a subject or mammal. In some embodiments, the compositions described herein are administered to a subject in an amount that effectively produces 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 the amount that achieves at least part of the desired therapeutic or preventative effect in an organ or tissue. The amount of therapeutic agent required to bring about preventative and / or therapeutic treatment of a disease or disorder is not fixed in itself. In some embodiments, the amount of therapeutic agent administered varies depending on the type and extent of the disease and the size of the mammal suffering from the disease or disorder. When used in conjunction with a treatment method involving the administration of a therapeutic agent after the occurrence of symptoms of a disease or disorder in a subject, the term "therapeuticly effective" means that one or more signs or symptoms of the disease or disorder are improved or eliminated after treatment.

[0078] An effective response to this disclosure is achieved when a subject experiences partial or complete remission or reduction of the signs or symptoms of the disease, and in the case of treating a disease (e.g., an autoimmune disorder), specifically including, but not limited to, improvement of symptoms, cure, remission, prolongation of survival, or other objective responses. In some embodiments, depending on prognostic factors, including the number of relapses, disease stage, and other factors, the expected progression-free survival is measured in months to years. Prolonged survival includes, but is not limited to, a period of at least 1 month (mo.), 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. Optionally, in some embodiments, an effective response is when the subject's symptoms remain unchanged. Further indications for treatment are described in more detail below.

[0079] In some implementations, a therapeutic agent is administered as a preventative measure before the undesirable symptoms of a disease or disorder manifest, thereby preventing the disease or disorder from progressing or optionally delaying its development. Therefore, when used in conjunction with a preventative approach, the term "treatment-effective" means that, on average, a smaller number of subjects develop the undesirable disease or disorder or experience a progression in symptom severity after treatment.

[0080] As used herein, the terms “treatment,” “treating,” etc., in some contexts refer to the administration of an agent or the performance of a procedure for the purpose of achieving a certain effect. The effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of achieving partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” includes treating diseases or disorders in mammals, particularly humans (e.g., autoimmune disorders), and includes: (a) preventing the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases related to or caused by a primary disease); (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing its remission. The term treatment includes any sign of success in treating, improving, or preventing a disease or disorder, including any objective or subjective parameters such as reduction, relief, or attenuation of symptoms, or making the disease condition more tolerable for the patient, slowing the rate of deterioration or decline, or making the endpoint of deterioration less severe. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Accordingly, the term "treatment" includes the administration of an agent or composition of this disclosure to prevent or delay, alleviate, stop, or inhibit the development of symptoms or conditions associated with a disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a subject's disease, symptoms of the disease, or side effects of the disease. A subject's disease or disorder is "treated" if, after receiving a therapeutic dose of a therapeutic agent or composition of this disclosure, the patient exhibits an observable and / or measurable change in a parameter or symptom of the disease or disorder.

[0081] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors, such as plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Expression vectors suitable for the compositions and methods described herein comprise expression cassettes containing polynucleotide sequences and additional sequence elements, such as those for expressing proteins and optionally integrating these polynucleotide sequences into the genome of a host cell. Certain vectors, as described herein, that can be used to express one or more (e.g., 1, 2, 3, or more) recombinant polypeptides comprise plasmids containing regulatory sequences, such as promoter and enhancer elements that direct gene transcription. Other useful vectors for expressing the disclosed polypeptides comprise polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions (UTRs), internal ribosome entry sites (IRES), and polyadenylation signaling sites to guide efficient transcription of transgenes carried on the expression vector. Expression vectors suitable for the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. An example of a suitable selection marker is a glutamine synthase (GS) gene. Further examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, norcini, bleomycin, carbenicillin, tetracycline, streptomycin, and spectinomycin.

[0082] Detailed Explanation Inflammatory disorders, including autoimmune diseases, are disturbances involving the abnormal activation of immune cells and their subsequent migration to affected areas of the body. These conditions encompass a wide range of diseases affecting the lives of millions of people worldwide. Although many treatments are currently available, many have significant side effects or are not effective enough in relieving symptoms.

[0083] Immunoglobulin G (IgG) has long been thought to mediate both pro-inflammatory and anti-inflammatory activities through interactions mediated by its fragment crystallizable (Fc) region. While Fc:FcγR interactions are the cause of the pro-inflammatory properties of immune complexes and cytotoxic antibodies, intravenous immunoglobulin (IVIG) and its constituent Fc fragments are anti-inflammatory and widely used to suppress inflammation in disease states. Glycosylation of IgG has been proposed to be crucial for modulating its cytotoxic and inflammatory potential. For example, previous studies have shown that the anti-inflammatory activity of IVIG is a property of its Fc fragment and its linked N-glycan sialic acid (SA) moiety, highlighting the need for a specific combination of polypeptide backbone and glycosylation profiles for its anti-inflammatory effects.

[0084] Sialization of recombinant therapeutic glycoproteins such as Fc peptides is typically performed in mammalian cell lines capable of replicating mammalian or mammalian-like glycosylation profiles. The addition of terminal SA residues to the N-glycans of the Fc heavy chain, particularly to asparagine 297 (Asn297), has been shown to influence various in vivo parameters of Fc peptides, including absorption, blood half-life, and clearance, as well as their immunogenicity or immunosuppressive properties. Glycoengineering of Fc peptides with high levels of sialylation has faced significant challenges, including limited and non-uniform sialylation of the peptide pool.

[0085] In some embodiments, this document discloses modified and highly sialylated Fc polypeptides having an amino acid substitution at amino acid position 241 of the Fc heavy chain, ranging from phenylalanine (F) to aliphatic amino acid residues (e.g., alanine, glycine, isoleucine, leucine, proline, valine, and methionine) (F241; according to Kabat numbering). Methods for manufacturing modified Fc polypeptides using recombinant expression systems comprising one or more (e.g., 1, 2, 3, or more) nucleic acid expression vectors and mammalian host cells are also disclosed. Furthermore, this disclosure provides methods for treating autoimmune disorders (such as acquired epidermolysis bullosa or immune-mediated glomerulonephritis) using the therapeutic agents or compositions disclosed herein. Subjects suffering from autoimmune disorders are treated by administering the therapeutic agents or compositions to a subject via any acceptable route according to the methods disclosed herein.

[0086] Immunoglobulins and Fc glycosylation IgG is a glycoprotein composed of two identical heavy chains and two light chains, which include variable and constant domains. IgG contains a single N-linked glycan at the asparagine 297 (Asn297) site in the CH2 domain of each of its two heavy chains. The covalently linked complex carbohydrate consists of a core biantennary penta-saccharide comprising N-acetylglucosamine (GlcNAc) and mannose (Man). Further modifications to the core carbohydrate structure have been observed in serum antibodies containing fucose (Fuc), branched GlcNAc, galactose (Gal), and a variable terminal sialic acid (SA) moiety. Therefore, more than 40 different glycoforms have been detected covalently attached to this single glycosylation site. Glycosylation of IgG has been demonstrated to be essential for binding to all FcγRs by maintaining the open conformation of the two heavy chains. It is believed that this IgG glycosylation for FcγR binding, leading to deglycosylation, prevents IgG antibodies from mediating intracellularly triggered inflammatory responses such as antibody-dependent cytotoxicity (ADCC), phagocytosis, and the release of inflammatory mediators. Individual IgG glycoforms may contribute to the modulation of inflammatory responses, as suggested by altered affinity of individual FcγR against IgG antibodies containing or lacking Fuc and their indirect effects on cytotoxicity. A link between autoimmune status and specific glycosylation patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and autoimmune vasculitis, where reduced galactosylation and sialylation of IgG antibodies have been reported.

[0087] Composition In some embodiments, compositions (e.g., therapeutic compositions) are disclosed herein comprising a modified Fc polypeptide having a sequence of a variant of the wild-type human IgG Fc polypeptide of SEQ ID NO: 1 (or an allotype, e.g., an allotype comprising the amino acid sequence of SEQ ID NO: 8, 9, 11 and / or 13) and having a high level of sialylation, such as at least 50% α(2,6) sialylation (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 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) 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%).

[0088] Fc peptide variants In some embodiments, the compositions described herein comprise a variant (i.e., a modified) Fc polypeptide (e.g., an IgG1 Fc polypeptide) containing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions relative to the wild-type / parental amino acid sequence, such as the wild-type / parental amino acid sequence of SEQ ID NO: 1 (the bolded and underlined phenylalanine residues (N) correspond to Asn297, to which the Fc N-glycan is attached; according to the Kabat system numbering).

[0089] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1) In some embodiments, one or more amino acids are substituted at position 241 of the Fc polypeptide for phenylalanine (Phe; F) (corresponding to amino acid position 32 of SEQ ID NO:1; according to Kabat system numbering). In some embodiments, 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, Phe at position 241 of the modified Fc polypeptide is substituted with 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 with SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence having 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 with SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence in SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the sequence in SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) identical to the sequence in SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution and has an amino acid sequence having at least 98% (e.g., at least 98%, 99% or more) sequence identity with SEQ ID NO:2. In some embodiments, the modified Fc polypeptide comprises an F241A substitution (shown in bold and underlined below).A It has the amino acid sequence of SEQ ID NO:2, as shown below.

[0090] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSV A LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2) In some embodiments, the compositions described herein comprise variant (i.e., modified) Fc peptides (e.g., IgG1 Fc peptides) that are allotropic variants of the wild-type / parental amino acid sequence, such as allotropic variant K214R containing the amino acid sequence of SEQ ID NO: 8, and contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions (bold and underlined arginine residues (R) correspond to K214R; according to Kabat system numbering).

[0091] KVDK R VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:8) In some embodiments, one or more amino acids are substituted at position 241 of the Fc polypeptide for phenylalanine (Phe; F) (corresponding to amino acid position 32 of SEQ ID NO:8; according to Kabat system numbering). In some embodiments, 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, Phe at position 241 of the modified Fc polypeptide is substituted with alanine (Ala or A; F241A substitution).

[0092] In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions 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 with SEQ ID NO:6. In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions and has the amino acid sequence having 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 with SEQ ID NO:6. In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions 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 with SEQ ID NO: 6. In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 6. In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions and has an amino acid sequence having at least 98% (e.g., at least 98%, 99% or more) sequence identity with SEQ ID NO: 6. In some embodiments, the modified Fc polypeptide comprises K214R and F241A substitutions (shown hereinafter as bold and underlined R and A) and has the amino acid sequence of SEQ ID NO: 6, as shown below.

[0093] KVDK R VEPKSCDKTHTCPPCPAPELLGGPSV ALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:6) In some embodiments, the compositions described herein comprise variant (i.e., modified) Fc peptides (e.g., IgG1 Fc peptides) that are allotropic variants of the wild-type / parental amino acid sequence, such as allotropic variant D356E containing the amino acid sequence of SEQ ID NO: 9, and contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions (bold and underlined glutamic acid residues (E) correspond to D356E; according to Kabat system numbering).

[0094] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9) In some embodiments, one or more amino acids are substituted at position 241 of the Fc polypeptide for phenylalanine (Phe; F) (corresponding to amino acid position 32 of SEQ ID NO:9; according to Kabat system numbering). In some embodiments, 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, Phe at position 241 of the modified Fc polypeptide is substituted with alanine (Ala or A; F241A substitution).

[0095] In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions and has the amino acid sequence of SEQ ID NO: 10, 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 with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions and has an amino acid sequence having 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 with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions 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 with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions 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 with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions and has an amino acid sequence having at least 98% (e.g., at least 98%, 99% or more) sequence identity with SEQ ID NO: 10. In some embodiments, the modified Fc polypeptide comprises D356E and F241A substitutions (shown hereinafter as bolded and underlined E and A) and has the amino acid sequence of SEQ ID NO: 10, as shown below.

[0096] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSV ALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:10) In some embodiments, the compositions described herein comprise variant (i.e., modified) Fc peptides (e.g., IgG1 Fc peptides) that are allotropic variants of the wild-type / parental amino acid sequence, such as allotropic variant L358M containing the amino acid sequence of SEQ ID NO: 11, and contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions (bold and underlined methionine residues (M) correspond to L358M; according to Kabat system numbering).

[0097] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:11) In some embodiments, one or more amino acids are substituted at position 241 of the Fc polypeptide for phenylalanine (Phe; F) (corresponding to amino acid position 32 of SEQ ID NO: 11; according to Kabat system numbering). In some embodiments, 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, Phe at position 241 of the modified Fc polypeptide is substituted with alanine (Ala or A; F241A substitution).

[0098] In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions and has the amino acid sequence of SEQ ID NO: 12, 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 with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions and has an amino acid sequence having 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 with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions 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 with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions 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 with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions and has an amino acid sequence having at least 98% (e.g., at least 98%, 99% or more) sequence identity with SEQ ID NO: 12. In some embodiments, the modified Fc polypeptide comprises L358M and F241A substitutions (shown hereinafter as bold and underlined M and A) and has the amino acid sequence of SEQ ID NO: 12, as shown below.

[0099] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSV ALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:12) In some embodiments, the compositions described herein comprise variant (i.e., modified) Fc peptides (e.g., IgG1 Fc peptides) that are allotropic variants of the wild-type / parental amino acid sequence, such as allotropic variant A431G containing the amino acid sequence of SEQ ID NO: 13, and contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions (bold and underlined glycine residues (G) correspond to A431G; according to Kabat system numbering).

[0100] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE G LHNHYTQKSLSLSPGK (SEQ ID NO:13) In some embodiments, one or more amino acids are substituted at position 241 of the Fc polypeptide for phenylalanine (Phe; F) (corresponding to amino acid position 32 of SEQ ID NO: 13; according to Kabat system numbering). In some embodiments, 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, Phe at position 241 of the modified Fc polypeptide is substituted with alanine (Ala or A; F241A substitution).

[0101] In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions and has the amino acid sequence of SEQ ID NO: 14, 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 with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions and has an amino acid sequence having 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 with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions 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 with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions 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 with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions and has an amino acid sequence with at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions and has an amino acid sequence having at least 98% (e.g., at least 98%, 99% or more) sequence identity with SEQ ID NO: 14. In some embodiments, the modified Fc polypeptide comprises A431G and F241A substitutions (shown hereinafter as bold and underlined G and A) and has the amino acid sequence of SEQ ID NO: 14, as shown below.

[0102] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSV ALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE G LHNHYTQKSLSLSPGK (SEQ ID NO:14) IgG1 Fc containing the Abdeg mutation exhibits enhanced affinity for FcRn, thus allowing the mutant Fc to bind to FcRn superiorly to native IgG. Therefore, Fc Abdeg The peptide accelerates the depletion of circulating total IgG by saturating FcRn. In some embodiments, the modified Fc peptide contains phenylalanine (Phe; F) at position 241 of the Fc peptide (corresponding to amino acid position 20 of SEQ ID NO: 5; according to Kabat system numbering).

[0103] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL KF HYTQKSLSLSPG (SEQ ID NO:5) The bolded and underlined amino acid residues correspond to Abdeg mutations (M252Y, S254T, T256E, H433K, and N434F).

[0104] In some embodiments, the amino acid composition of the modified Fc peptides described herein is varied without impairing the peptide's ability to bind to the corresponding receptor and trigger the corresponding cellular response. For example, in some embodiments, it comprises one or more conserved amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known. These families include amino acids having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues, for example, in SEQ ID NO:2, are preferably replaced by 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 corresponding Fc receptor and trigger the corresponding cellular response to identify mutants that retain the desired activity.

[0105] In some embodiments, the modified Fc peptide is an IgG1 isotype. In some embodiments, the modified Fc peptide is an IgG3 isotype.

[0106] Sialization of modified Fc peptides Glycosylation of immunoglobulins has been shown to significantly influence their effector function, structural stability, and secretion rate from antibody-producing cells. The glycosylation 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 essential for IgG's full ability to activate the classical complement-dependent cytokine lysis pathway.

[0107] Each antibody possesses a range of distinct N-linked glycan structures that variably influence protein assembly, secretion, and function. In some embodiments, these N-linked glycans vary considerably depending on the degree of processing and include high-mannose and complex biantennary oligosaccharides with or without bivalent N-acetylglucosamine (GlcNAc) and core fucose (Fuc) residues. Typically, heterogeneous processing of the core oligosaccharide structure attached to specific glycosylation sites exists, resulting in even monoclonal antibodies existing in multiple glycoforms. Similarly, it has been shown that major differences in antibody glycosylation occur between antibody-producing cell lines, and even minute differences are observed for a given cell line grown under different culture conditions.

[0108] The presence of sialic acid (SA) residues on the N-glycan of the Fc polypeptide has recently been identified as a key factor in mediating the anti-inflammatory effects of intravenous immunoglobulin (IVIG), as demonstrated in several autoimmune diseases. This anti-inflammatory effect has been proposed to be mediated by the binding of the sialylated Fc component of IVIG to the inhibitory FcγRIIB receptor and the DC-SIGN receptor. Removal of IVIg sialic acid leads to loss of protective effects in animal models of multiple sclerosis, rheumatoid arthritis, Guillain-Barré syndrome, and idiopathic thrombocytopenic purpura (ITP), despite preservation of normal circulating half-life and binding to FcRn. Conversely, in several different animal models of autoimmune diseases, hypersialylation of IVIg increases the potency of anti-inflammatory activity by 10–30-fold. The biological results of binding to and activating the type II Fc receptor are IL-33 release, upregulation of the inhibitory FcγRIIB, and expansion of regulatory T cells. In mice, knockout of SIGN-R1 (a mouse homologue of DC-SIGN) blocked the anti-inflammatory activity of IVIg. Blocking the IL-33 receptor, knockout of FcγRIIB, or depletion of T regulatory cells also blocked the anti-inflammatory properties of IVIg under various conditions. However, only a small fraction of IgG in IVIG possesses a glycan that terminates at SA, thus requiring high-dose (1-2 g / kg) administration of IVIG to elicit a therapeutic anti-inflammatory effect. Previous efforts to produce highly sialylated IgG Fc pools have focused on purifying the sialylated fractions of Fc peptides from IVIG. However, no available method exists for the recombinant production of pure, highly sialylated Fc peptides with predetermined levels of sialylation.

[0109] This disclosure provides a modified Fc polypeptide (e.g., Fc of SEQ ID NO: 2) provided, alone or in combination with one or more (e.g., 1, 2 or more) recombinant glycosyltransferases (e.g., ST6GAL1 and B4GALT1) in a recombinant expression system (e.g., one or more nucleic acid expression vectors introduced into a host cell such as a mammalian host cell) under conditions and time sufficient to produce the desired level of Fc sialylation. F241A Methods for enhancing the sialylation of IgG Fc peptides (using mutants or variants thereof). In some embodiments, one or more recombinant glycosyltransferases are ST6GAL1. The activity of ST6GAL1 produces 6-sialylated oligosaccharides, including 6-sialylated galactose. The term "ST6GAL1" refers to a sialyltransferase capable of attaching SA to the sixth atom of the recipient polysaccharide. In some embodiments, one or more recombinant glycosyltransferases are B4GALT1. The term "B4GALT1" refers to an enzyme belonging to the family of β-1,4-galactosyltransferases that transfers galactose to the recipient sugar via a β(1,4) bond, such as GlcNAc, Glc, and Xyl. In some embodiments, one or more recombinant glycosyltransferases are ST6GAL1 and B4GALT1.

[0110] 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 with the amino acid sequence of SEQ ID NO:3, as shown below.

[0111] MIHTNLKKKFSCCVLVFLLFAVICWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSS SKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREID DHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWEL WDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (SEQ ID NO:3; UniProt ID No.: P15907-1) 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 with the amino acid sequence of SEQ ID NO:4, as shown below.

[0112] MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS (SEQ ID NO:4; UniProt ID No.: P15291-1) The disclosed methods are used to achieve specific levels of Fc sialylation. In some embodiments, the modified Fc peptides of this disclosure are expressed and / or cultured under conditions and for a time sufficient to produce Fc peptides, said Fc peptide pool having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% sialylated Fc peptides. In some embodiments, the modified Fc peptides are expressed and / or cultured under conditions and for a time sufficient to produce Fc peptides, said Fc peptide pool having at least 40% sialylated Fc peptides. In some embodiments, the modified Fc peptides are expressed and / or cultured under conditions and for a time sufficient to produce Fc peptides, said Fc peptide pool having at least 45% sialylated Fc peptides. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 50% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 55% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 60% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 65% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 70% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 75% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 80% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a sufficient time to produce an Fc peptide pool containing at least 85% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce an Fc peptide pool, the pool containing at least 90% sialylated Fc peptide.In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce an Fc peptide pool containing at least 95% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce an Fc peptide pool containing at least 100% sialylated Fc peptide. In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce a pool of Fc peptides, the pool containing sialylated Fc peptides at a concentration between 40% and 100%, such as 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%.

[0113] In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce a pool of modified Fc peptides, said pool 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 Fc peptides having an SA moiety of an N-glycan attached to the Fc peptide via an α(2,6) bond. 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 comprises the SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. 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 polypeptide comprises the SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. 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 polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. 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 polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond.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 polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. 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 polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. In some embodiments, at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or more) of the modified Fc polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. In some embodiments, at least 98% (e.g., at least 98%, 99%, or more) of the modified Fc polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. In some embodiments, at least 99% of the modified Fc polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. In some embodiments, approximately 100% of the modified Fc polypeptide comprises an SA moiety of the N-glycan attached to the Fc polypeptide via an α(2,6) bond. In some embodiments, the N-glycan of the modified Fc polypeptide is monosialylated or disialylated. In some embodiments, at least 30% of the modified Fc polypeptide comprises a monosialylated N-glycan containing an SA moiety attached via an α(2,6) bond. In some embodiments, at least 30% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds. In some embodiments, approximately 90% of the modified Fc polypeptide comprises a disialialylated N-glycan, said disialialylated N-glycan comprising two SA moieties attached via α(2,6) bonds.

[0114] 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 polypeptide contains 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 polypeptide comprises 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 polypeptide comprises 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 polypeptide comprises a galactose moiety. In some embodiments, about 100% of the modified Fc polypeptide comprises a galactose moiety. In some embodiments, the galactose moiety is attached to the α(1,3) arm and / or α(1,6) arm of the N-glycan. In some embodiments, the galactose moiety is a branched-chain galactose moiety.

[0115] In some embodiments, the modified Fc peptide is expressed and / or cultured under conditions and for a time sufficient to produce a pool of modified Fc peptides, said pool 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 peptides having the SA moiety of an N-glycan attached to the modified Fc peptide via an α(2,3) bond.

[0116] In some embodiments, the N-glycan is attached to asparagine (Asn) at amino acid residue 297 of the polypeptide (Asn297; according to the Kabat number; corresponding to amino acid residue 88 of SEQ ID NO:2).

[0117] Treatment Subjects In some embodiments, this document discloses methods for treating or preventing autoimmune disorders affecting the skin and / or kidneys in individuals with appropriate need. In some embodiments, the individuals with appropriate need are mammalian subjects (e.g., human subjects). The term "inflammatory disease or disorder" refers to a disease or disorder characterized by abnormal or unwanted inflammation (e.g., an autoimmune disease or disorder). In some embodiments, the autoimmune disease or disorder is a T-cell-mediated autoimmune disease or disorder. 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 triggered by autoantibodies. The term "autoantibody" or "autoantibodies" refers to immunoglobulins produced by the immune system that target one or more of an individual's own proteins.

[0118] In some implementations, autoimmune disorders affect the skin. For example, an autoimmune disorder can be acquired epidermolysis bullosa (EBA). Symptoms and signs of EBA include bullous lesions (e.g., typically in areas of minor trauma, such as the extensor surfaces of the elbows and the dorsal surfaces of the hands and feet), dystrophic nails, cicatricial alopecia, mucosal involvement, or ocular lesions. Symptoms and signs of EBA healing can also be used to identify patients with EBA, such as scarring, milia (superficial epidermal inclusion cysts), and hyperpigmentation. Methods for diagnosing EBA are known in the art and include, for example, analysis of skin biopsy samples and direct immunofluorescence to assess the deposition of IgG and complement in the basement membrane area.

[0119] In some implementations, autoimmune disorders affect the kidneys. For example, an autoimmune disorder can be immune-mediated nephritis, such as immune-mediated glomerulonephritis (GN), including proliferative glomerulonephritis (characterized by mesangial cell proliferation and influx of inflammatory cells) or membranous glomerulonephritis (characterized by matrix accumulation and thickening of the glomerular basement membrane (GBM) and capillary walls). GN can be acute or chronic. Signs and symptoms of GN include hypertension, proteinuria, hematuria, and nephrotic syndrome (presenting with massive proteinuria causing leg edema). Renal biopsy (e.g., for immunoglobulin, complement factor staining, or standard tissue staining) and / or renal ultrasound can be used to confirm GN. Additionally or optionally, increased levels of blood metabolites (e.g., creatinine and urea), blood tests (e.g., detecting leukopenia, acute-phase proteins, immunoglobulin levels, hypocomplementemia, autoantibodies, alloantibodies, anti-vaccine titers, HLA type, free light chains, monoclonal gammaglobulinosis, or pathogen-related changes), urinalysis, microbiological tests, and / or clinical history (e.g., history of autoimmune diseases, transplantation, and / or autoinflammatory disorders) may also be used to identify an individual as having GN.

[0120] It has been proposed that glycosylation of IgG is crucial for modulating its cytotoxic and inflammatory potential. For example, the anti-inflammatory activity of IVIG has been demonstrated by the properties of the Fc fragment on its linked N-glycan and the terminal SA residue, indicating a need for a combination of specific polypeptide backbones and glycosylation profiles for its anti-inflammatory effects. 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%) exhibit robust therapeutic activity in mouse models of EBA and immune-mediated glomerulonephritis, thus highlighting the potential clinical efficacy of the disclosed compositions for the treatment of autoimmune disorders.

[0121] The compositions and methods described herein may be used to treat patients presenting with symptoms of an autoimmune disorder, diagnosed with an autoimmune disorder, and / or in remission of an autoimmune disorder. Optionally or additionally, the compositions and methods described herein may be administered as prophylactic treatment to patients at risk of developing an autoimmune disorder.

[0122] Application route In some embodiments, the modified Fc peptides or compositions comprising them described herein are administered to subjects with autoimmune disorders via multiple routes, such as intravenous, intradermal, subcutaneous, percutaneous, transdermal, intramuscular, transmucosal, or intraosseous administration. In some embodiments, the compositions described herein are administered to the subject systemically (e.g., intravenously). In some embodiments, the compositions described herein are administered to the subject locally (e.g., at the site of inflammation). In any given case, the most suitable route of administration will depend on the specific composition administered, the patient, the method of formulation, the method of administration (e.g., timing and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. Multiple routes of administration can be used to treat a single subject. A single subject can be treated once using multiple routes of administration, or the subject can be treated first via one route of administration and then treated at a second appointment, such as 1 week later, 2 weeks later, 1 month later, 6 months later, or 1 year later via another route of administration. The compositions disclosed herein can be administered to a subject once or twice or more (e.g., 2-10 times) weekly, monthly or annually for treatment.

[0123] dose The amount of the modified Fc peptide disclosed herein, or a composition comprising it, used in conjunction with the treatment methods disclosed herein will generally be a therapeutically effective amount. As a non-limiting example, an effective amount is sufficient to alleviate the symptoms of an autoimmune disorder, including, for example, edema, congestion, erythema, bruising, tenderness, stiffness, swelling, fever, chills, nasal congestion, headache, dyspnea, fluid retention, blood clots, loss of appetite, increased heart rate, granuloma formation, fibrinous symptoms, pus, non-viscous serous fluid, ulceration, increased production of autoreactive effector immune cells, inflammation, or pain.

[0124] For a specific application of the disclosed methods, the appropriate effective amount of the composition to be administered can be determined, for example, using the guidelines provided herein. For instance, in some embodiments, the effectiveness of the disclosed compositions in treating symptoms of an autoimmune disorder is determined by observing one or more clinical symptoms and / or physiological indicators associated with the condition. The response of an individual with an autoimmune disorder to treatment can be monitored by determining the severity of their symptoms or by determining the frequency of self-reactive T cells in a sample from an individual with an autoimmune disorder (e.g., an autoimmune disease or disorder). The severity of symptoms of an autoimmune disease or disorder can be correlated with the number of self-reactive T cells. Furthermore, an increase in the number of self-reactive T cells in a sample can be used as an indication for applying a treatment intended to minimize the severity of symptoms and / or treat the autoimmune disease or disorder before the onset of symptoms. As another example, the effectiveness of the disclosed compositions in treating symptoms of the disclosed autoimmune disease or disorder can be determined by relying on clinical experience with existing T-cell infusion therapies. Improvement in an autoimmune disease or disorder can also be indicated by a reduced need for concurrent therapy. Those skilled in the art will recognize appropriate symptoms or indicators associated with a specific autoimmune disease or disorder and will know how to determine whether an individual is a candidate for a treatment as disclosed herein. An individual's condition can be monitored throughout the treatment process, and the effective amount of the compound or composition disclosed herein administered can be adjusted accordingly.

[0125] In some embodiments, a therapeutically effective amount of the composition disclosed herein reduces symptoms associated with autoimmune disorders by, for example, at least 10%, at least 20%, 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 the composition disclosed herein reduces symptoms associated with autoimmune disorders by, for example, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, or at most 100%. In some embodiments, a therapeutically effective amount of the composition disclosed herein reduces symptoms associated with autoimmune disorders by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, 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 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%. In some embodiments, the therapeutically effective dose of the compositions disclosed herein is a dose sufficient to alleviate symptoms associated with autoimmune disorders for a duration of, for example, at least one week, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, or longer.

[0126] Treatment effect In some embodiments, the methods of this disclosure result in a reduction of one or more of the following (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more): reduced migration of immune cells (e.g., T cells, B cells, NK cells, ILC1, ILC2, ILC3, monocytes, macrophages (M1 and M2), dendritic cells, or antigen-presenting cells), reduced immune cell proliferation, reduced immune cell recruitment, and increased immunity. The methods disclosed herein can reduce immune cell lymph node homing, decrease immune cell lymph node discharge, reduce immune cell differentiation, reduce immune cell activation, reduce immune cell polarization, reduce immune cell cytokine production, reduce immune cell degranulation, reduce immune cell maturation, reduce immune cell antibody-dependent cell (ADCC), reduce immune cell antibody-dependent cell (ADCP), reduce immune cell antigen presentation, reduce immune cell serotonin receptor expression, treat autoimmune disorders, alleviate symptoms of autoimmune disorders, reduce inflammation, lower autoantibody levels, increase organ function, and reduce the rate or frequency of relapses or flare-ups. In some embodiments, the methods of this disclosure result in a reduction in the remission of one or more symptoms of autoimmune diseases and / or a reduction in clinical scores associated with autoimmune diseases.

[0127] For example, in the context of autoimmune disorders affecting the skin (e.g., acquired epidermolysis bullosa), the methods disclosed herein can lead to a reduction in symptoms such as skin blistering. Similarly, in the context of kidney disease (e.g., immune-mediated GN), the methods disclosed herein can lead to improvements in kidney function.

[0128] In some implementations, the reduction in the severity of the aforementioned symptoms can be of any amount, provided that a therapeutic benefit is achieved in the patient. Treatment efficacy is measured over different timeframes, including, for example, months to years, depending on prognostic factors such as the number of relapses, the stage of the disease, and other factors.

[0129] Prolonged survival is another desired treatment benchmark, which includes, but is not limited to, an increase in survival time of at least 1 month (mo), approximately at least 2 months (mo), approximately at least 3 months, approximately at least 4 months, approximately at least 6 months, approximately at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 20 years, 30 years, 40 years, 50 years, or longer. In some implementations, total survival is measured in months to years. In some implementations, the subject's symptoms remain unchanged or improve.

[0130] Combination therapy In some embodiments, the compositions disclosed herein are administered in combination with one or more (e.g., 1, 2, 3, 4, 5 or more) additional therapeutic agents or forms for the treatment of the diseases or disorders described herein (e.g., autoimmune disorders, such as autoimmune diseases or disorders).

[0131] In some embodiments, one or more additional therapeutic agents are a second modified Fc peptide. In some embodiments, the second modified Fc peptide is a human IgG1 Fc peptide. In some embodiments, the second modified Fc peptide contains one or more (e.g., 1, 2, 3, 4, or 5) mutations (according to Kabat's EU index number; also known as "Abdeg" mutations - see [link to Kabat]). Figure 11A and Figure 11B (See also the bolded and underlined residues of SEQ ID NO: 5 below). IgG1 Fc containing the Abdeg mutation exhibits enhanced affinity for FcRn, thereby allowing the mutant Fc to bind to FcRn superiorly to native IgG. Therefore, Fc Abdeg The peptide accelerates the depletion of circulating total IgG by saturating FcRn. In some embodiments, the second modified Fc peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:5.

[0132] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL KF HYTQKSLSLSPG (SEQ ID NO:5) The bolded and underlined amino acid residues correspond to Abdeg mutations (M252Y, S254T, T256E, H433K, and N434F).

[0133] In some embodiments, the modified Fc peptides of this disclosure contain Abdeg mutations (M252Y, S254T, T256E, H433K, and N434F) and F241A mutations.

[0134] In some embodiments, the Fc peptide of this disclosure is administered to the subject co-administered (e.g., sequentially or simultaneously) with the second modified Fc peptide of SEQ ID NO:5. In some embodiments, the second modified Fc peptide is administered to the subject at an amount of 1-20 mg / kg, such as 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 peptide is administered to the subject weekly. In some embodiments, the second modified Fc peptide is administered to the subject for 1-8 weeks (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks).

[0135] In some embodiments, one or more additional therapeutic agents are selected from the group consisting of: anti-inflammatory agents, immunosuppressants, analgesics, antirheumatic drugs such as disease-modifying antirheumatic drugs (DMARDs), anti-stimulants, platelet-enhancing drugs, 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 their antigen-binding fragments, anti-inflammatory cytokines, kinase inhibitors, IVIG, FcRN inhibitors, complement inhibitors, or any combination thereof. In some implementations, one or more additional therapeutic agents are prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or biologics such as tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab, egamod, batoclimab, nipocalimab, riliprubart, empasiprubart, or tocilizumab. For example, if the disease is RA, the second dose can be one or more of the following: prednisone, prednisolone and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, tofacitinib, adalimumab, abatacept, anaerobicin, kineret, cetozumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab.In some implementations, additional agents include 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alenzusmab, aminosalicylates / esters (5-aminosalicylic acid, sulfasalazine, mesalazine, balsalazide, olsalazine), antibiotics, antihistamines, anti-TNFα drugs (infliximab, adalimumab, certolizumab pegol, natezumab), ustekinumab, azathioprine, belimumab, belimumab, β-interferon, and calcineurin inhibitors. Inhibitors, cetrus tuzumab, corticosteroids (prednisone, methylprednisolone), cromoglycine, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumarate, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonists, long-acting β2 agonists, methotrexate, mitoxantrone, mycophenolate mofetil, natetarizumab, NSAIDs, olizumab, pimecrolimus, probiotics, retinoids, rituximab, salicylic acid, short-acting β2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab, and vedolizumab.

[0136] In some embodiments, depending on the condition to be treated, the compositions of this disclosure are administered alone or in combination with other therapeutic agents, either simultaneously or sequentially. When two or more compositions are administered, the compositions are, for example, administered in combination (sequentially or simultaneously). In some embodiments, one or more additional therapeutic agents are administered immediately before or after the composition, or for a period of 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 longer before or after the application of the composition. In some embodiments, the compositions are administered as a single or multiple doses.

[0137] drug In some embodiments, this disclosure provides for the use of the compositions described herein in the preparation of a medicament for treating the condition, disease, or disorder described herein. In some embodiments, the medicament is formulated based on the physical characteristics of a subject requiring treatment and is formulated as a single or more formulations based on the stage of the condition, disease, or disorder. In some embodiments, the medicament is packaged in a suitable package with appropriate labeling for dispensing to hospitals and clinics, wherein the labeling indicates treatment for a subject suffering from the disease described herein. In some embodiments, the medicament is packaged as a single unit or more units. In some embodiments, instructions for dosage and administration of the composition are included together with the packaging described below. This disclosure also relates to medicaments comprising the compositions described herein and pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutically acceptable carriers, diluents, or excipients are selected from the group consisting of: stabilizers, buffers, surfactants, fillers, solvents, tonic or osmotic pressure regulators, antioxidants, adjuvants, and antimicrobial agents.

[0138] Manufacturing method In some embodiments, methods for producing Fc peptides with high levels of sialylation, such as the modified Fc peptides disclosed herein, are disclosed. In some embodiments, the modified Fc peptides of the present invention are produced under conditions that result in an increased amount of Fc sialylation at Fc N-glycans (i.e., Asn297) compared to unmodified (e.g., natural / parental) Fc peptides.

[0139] Recombinant expression system In some embodiments, this document discloses methods, systems, and vectors for expressing recombinant peptides, particularly the modified Fc peptides of this disclosure, in host cells.

[0140] In some embodiments, this document discloses a recombinant expression system comprising a nucleic acid expression vector (e.g., plasmid, RNA vector, virus, viral vector, or other suitable replicon), the nucleic acid expression vector comprising: (a) a first expression cassette containing a first mammalian promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (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) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to SEQ ID NO: 2). (a) amino acid residue 32); and (b) a second expression cassette comprising a second mammalian promoter operatively linked to a polynucleotide encoding β-galactosyltransferase 1 (ST6GAL1) enzyme. In some embodiments, the aliphatic amino acid residue at position 241 is Ala (F241A). In some embodiments, the second expression cassette further comprises a polynucleotide encoding β-1,4-galactosyltransferase 1 (B4GALT1) enzyme. In some embodiments, the polynucleotide encoding B4GALT1 enzyme is operatively linked to the second promoter. In some embodiments, the second expression cassette further comprises an internal ribosome entry site (IRES) sequence located between the polynucleotide encoding ST6GAL1 enzyme and the polynucleotide encoding B4GALT1 enzyme. In some implementations, the first and second promoters are each independently selected from the group consisting of: mouse cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) promoter, eukaryotic elongation factor 2 (EEF2) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, actin promoter, and ubiquitin promoter.

[0141] In some embodiments, this document discloses a recombinant expression system comprising a nucleic acid expression vector (e.g., plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising: (a) a first expression cassette containing a first mammalian promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (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) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to SEQ ID NO: 2). (a) amino acid residue 32), a polynucleotide encoding ST6GAL1 and the IRES sequence position therebetween; and (b) a second expression cassette containing a second mammalian promoter operatively linked to a polynucleotide encoding the B4GAL1 enzyme. In some embodiments, the aliphatic amino acid residue at position 241 is Ala (F241A). In some embodiments, the first and second promoters are each independently selected from the group consisting of: murine cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) promoter, eukaryotic elongation factor 2 (EEF2) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, actin promoter, and ubiquitin promoter.

[0142] In some embodiments, this document discloses a recombinant expression system comprising a nucleic acid expression vector (e.g., plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising: (a) a first expression cassette containing a first mammalian promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (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) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to SEQ ID NO: 2). (a) amino acid residue 32), a polynucleotide encoding B4GALT1 and the IRES sequence position therebetween; and (b) a second expression cassette containing a second mammalian promoter operatively linked to a polynucleotide encoding the ST6GAL1 enzyme. In some embodiments, the aliphatic amino acid residue at position 241 is Ala (F241A). In some embodiments, the first and second promoters are each independently selected from the group consisting of: murine cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) promoter, eukaryotic elongation factor 2 (EEF2) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, actin promoter, and ubiquitin promoter.

[0143] In some embodiments, this document discloses a recombinant expression system comprising a nucleic acid expression vector (e.g., plasmid, RNA vector, virus, viral vector, or other suitable replicon) comprising an expression cassette containing a mammalian promoter operatively linked to: (1) a first polynucleotide encoding a modified Fc polypeptide having: (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) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to SEQ ID NO: 2). (2) amino acid residue 32); (2) a second polynucleotide encoding the ST6GAL1 enzyme; (3) a first IRES sequence located between the first and second polynucleotides; (4) a third polynucleotide encoding the B4GALT1 enzyme; and (5) a second IRES sequence located between the second and third polynucleotides. In some embodiments, the aliphatic amino acid residue at position 241 is Ala (F241A). In some embodiments, the promoter is selected from the group consisting of: mouse cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) promoter, eukaryotic elongation factor 2 (EEF2) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, actin promoter, and ubiquitin promoter.

[0144] In some embodiments, this document discloses a recombinant expression system comprising 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: (a) a 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 at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2); and (b) an ST6GAL1 enzyme; and / or optionally (c) a B4GALT1 enzyme.

[0145] In some implementations, two or more (e.g., 2, 3 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 comprise: (a) a first expression vector comprising an expression cassette containing a first promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (i) an amino acid sequence identical to 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 sequence of SEQ ID NO: 2 and (ii) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2); and (b) a second expression vector comprising an expression cassette containing a second promoter operatively linked to a polynucleotide encoding an ST6GAL1 enzyme; and optionally (c) a polynucleotide encoding a B4GALT1 enzyme. In some implementations, the polynucleotide encoding the ST6GAL1 enzyme and the polynucleotide encoding the B4GALT1 enzyme are separated by an IRES sequence.

[0146] In some embodiments, the two expression vectors comprise: (a) a first expression vector comprising an expression cassette containing a first promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (i) an amino acid sequence identical to 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 sequence of SEQ ID NO: 2 and (ii) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2); and a polynucleotide encoding the ST6GAL1 enzyme; and (b) a second expression vector comprising an expression cassette containing a second promoter operatively linked to a polynucleotide encoding the B4GALT1 enzyme. In some implementations, the polynucleotide encoding the modified Fc polypeptide and the polynucleotide encoding the ST6GAL1 enzyme are separated by an IRES sequence.

[0147] In some embodiments, the two expression vectors comprise: (a) a first expression vector comprising an expression cassette containing a first promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (i) an amino acid sequence identical to 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 sequence of SEQ ID NO: 2 and (ii) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2) and a polynucleotide encoding the B4GALT1 enzyme; and (b) a second expression vector comprising an expression cassette containing a second promoter operatively linked to a polynucleotide encoding the ST6GAL1 enzyme. In some implementations, the polynucleotide encoding the modified Fc polypeptide and the polynucleotide encoding the B4GALT1 enzyme are separated by an IRES sequence.

[0148] In some embodiments, two or more (e.g., 2, 3 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 comprise: (a) a first expression vector comprising an expression cassette containing a first promoter operatively linked to a polynucleotide encoding a modified Fc polypeptide having: (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) an aliphatic amino acid residue at position 241 (according to the Kabat number; corresponding to SEQ ID NO: 2). (a) amino acid residue 32 of 2); (b) a second expression vector comprising an expression cassette containing a second promoter operatively linked to a polynucleotide encoding the ST6GAL1 enzyme; and (c) a third expression vector comprising an expression cassette containing a third promoter operatively linked to a polynucleotide encoding the B4GALT1 enzyme.

[0149] In some embodiments, the polynucleotide encoding any of the polypeptides disclosed herein (e.g., modified Fc polypeptides, ST6GAL1 enzymes, or B4GALT1 enzymes) is codon-optimized. Codon optimization is the process of modifying a nucleic acid sequence based on the principle that the frequency of synonymous codons (e.g., codons encoding the same amino acid) in the encoding DNA varies across species. Such codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. Sequences modified in this manner are referred to herein as "codon-optimized". This process can be performed on any sequence described in this specification to enhance expression or stability. The sequence around the translation start site can be converted into a common Kozak sequence by any suitable method.

[0150] expression carrier Various vectors have been developed for delivering polynucleotides encoding exogenous proteins into host cells. Expression vectors used in the compositions and methods described herein may comprise one or more (e.g., 1, 2, 3, or more) polynucleotides encoding one or more (e.g., 1, 2, 3, or more) polypeptides of the present disclosure, and may also comprise, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleic acid elements for regulating the expression of these agents and / or the integration of such polynucleotides into the genome of the host cell.

[0151] In some embodiments, the vector is a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as plasmids, bacteriophages, extrachromosomal elements, microchromosomes, or artificial chromosomes. Alternatively, in some embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the host cell genome and replicates along with the chromosome into which it has already been integrated. In some embodiments, certain vectors for expressing one or more engineered polypeptides described herein include plasmids containing regulatory sequences, such as promoters and optionally enhancer regions, which direct gene transcription. Other useful vectors for expressing one or more polypeptides of this disclosure contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability of mRNA transcribed from the genes or nuclear export. 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 the gene carried on the expression vector. Expression vectors suitable for the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. An example of a suitable selection marker is the glutamine synthase (GS) gene. Another example of a suitable biomarker is a gene encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, norsylmycin, bleomycin, carbenicillin, tetracycline, streptomycin, and spectinomycin.

[0152] In some embodiments, the expression vector of this disclosure further comprises a polynucleotide encoding a protein tag, such as a His tag (e.g., 6x-His; SEQ ID NO: 7), a maltose-binding protein tag, a SNAP tag, a FLAG tag, a halotag, a fluorescent protein tag, etc.

[0153] Viral vector Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes into host cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the host cell's nuclear genome via generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., retroviral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as piconemaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and poxviruses (e.g., vaccinia virus, modified Ankara vaccinia virus (MVA), fowlpox virus, and canarypox virus). Other viruses include, for example, norovirus, capsid virus, flavivirus, reovirus, papillomavirus, hepatotropic DNA virus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis sarcoma, avian type C virus, mammalian type C virus, mammalian type B virus, mammalian type D virus, tumor retroviruses, HTLV-BLV group, lentiviruses, alpha retroviruses, gamma retroviruses, and foam viruses. Other examples include murine leukosis virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukosis virus, feline leukosis virus, feline sarcoma virus, avian leukosis virus, human T-cell leukosis virus, baboon endogenous virus, gibbon ape leukosis virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0154] Adjustment element The recognition and binding of polynucleotides encoding one or more polypeptides disclosed herein by mammalian RNA polymerases is important for gene expression. Therefore, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerases and facilitate the assembly of transcription complexes at transcription initiation sites can be included within polynucleotides. Such sequence elements include, for example, mammalian promoters whose sequences are recognized and bound by specific transcription initiation factors and ultimately by RNA polymerases.

[0155] The polynucleotides suitable for use in the compositions and methods described herein also include those encoding 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: elongation factor 1α (EF1α) promoter, eukaryotic elongation factor 2 (EEF2) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, actin promoter, and ubiquitin promoter.

[0156] Alternatively, promoters derived from viral genomes can also be used for the stable expression of these agents in mammalian cells. Examples of functional viral promoters that can be used to promote the mammalian expression of these agents are the adenovirus late promoter, the vaccinia virus 7.5K promoter, the simian virus 40 (SV40) promoter, the cytomegalovirus promoter, the herpes simplex virus (HSV) tk promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Moloney virus promoter, the Epstein-Barr virus (EBV) promoter, the Rous sarcoma virus (RSV) promoter, and cytomegalovirus (CMV) promoters (e.g., the mouse CMV promoter).

[0157] After a polynucleotide encoding one or more recombinant polypeptides of this disclosure has been internalized extrachromosomally and / or incorporated into the nuclear DNA of a host cell, transcription of that polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the host cell to an external chemical agent, such as an agent that regulates the binding of transcription factors and / or RNA polymerases to promoters and thus regulates gene expression. The chemical agent can be used to promote the binding of RNA polymerases and / or transcription factors to promoters, for example, by removing repressor proteins that have already bound the promoter. Alternatively, the chemical agent can be used to enhance the promoter affinity for RNA polymerases and / or transcription factors, such that the transcription rate of genes downstream of the promoter increases in the presence of the chemical agent. Examples of chemical agents that enhance polynucleotide transcription through the above mechanisms are tetracycline and doxycycline. These agents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to host cells according to established protocols to promote gene expression.

[0158] Other gene regulatory elements that can be included in the polynucleotides used in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in the polynucleotides containing the gene of interest, causing the DNA to adopt a three-dimensional orientation that favors the binding of transcription factors and RNA polymerases at the transcription start site. Therefore, the polynucleotides used in the compositions and methods described herein include those encoding modified Fc polypeptides of the present disclosure and other mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, and examples are enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers used in the compositions and methods described herein also include enhancers derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples are the SV40 enhancer (bp 100-270) on the late origin of replication side, the CMV early promoter enhancer, the polyoma enhancer on the late origin of replication side, and the adenovirus enhancer. Enhancers can be spliced ​​into a vector containing a polynucleotide encoding NADH oxidase for water formation, for example, at position 5' or 3' of the gene. In a preferred orientation, the enhancer is positioned on the 5' side of the promoter, which is located at the 5' of the polynucleotide relative to the modified Fc polypeptide encoding the present disclosure.

[0159] Other exemplary regulatory elements applicable to the expression vectors described herein include untranslated region (UTR, such as 5'UTR and / or 3'UTR) insulator elements, terminator elements, polyadenylation signals, response elements, and other functional elements or signals.

[0160] host cells In some embodiments, the modified Fc peptides of the present invention are produced in a host expression system, such as a host cell (e.g., a mammalian cell), capable of expressing the modified Fc peptides 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 implementations, the host cell is a mammalian host cell, such as the Chinese hamster ovary (CHO) cell line (e.g., CHO-K1; ATCC CCL-61), the green monkey cell line (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g., NS / 0), young hamster kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10), or human cells (e.g., HEK293 (ATCC CRL-1573) or 293T (ATCC CRL-11268)), or any other suitable cell line available from a public collection such as the American Type Culture Collection (Rockville, Md.). Additionally, insect cell lines, such as lepidopteran cell lines, e.g., Sf9, plant cell lines, and fungal cell lines, such as yeast, e.g., Saccharomyces cerevisiae (Saccharomyces cerevisiae), can be used. Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris ), species of the genus *Hansenula* ( Hansenula spp.), or based on Bacillus ( Bacillus Bacterial expression systems, such as Bacillus subtilis ( B. subtilis ) or E. coli ( E. coli The host cell may need to be modified to ensure that N-linked glycosylation and glycan maturation occur to produce complex biantennary sugars, such as those typically present on the Fc domain of human IgG. In some embodiments, the mammalian host cells described herein are transformed (e.g., transfected or transduced) using any of the recombinant expression systems described herein.

[0161] Despite the availability of several other mammalian cell lines, most recombinant therapeutic proteins produced today are prepared in CHO cells. Their advantages include, for example, robust growth as adherent cells or in suspensions, adaptability to serum-free and chemically defined media, high productivity, and an established history of regulatory approvals for the production of therapeutic recombinant proteins. They are also well-suited for genetic modification, and the methods used for cell transfection, recombinant protein expression, and clonal selection are well-characterized. CHO cells also provide human-compatible post-translational modifications. As used herein, “CHO cells” include, but are not limited to, CHO-K1, CHO-DG44, CHO-M, CHO-S, CHO GS knockouts, and their variants and derivatives.

[0162] Therefore, in some embodiments, the host cells described herein are modified with one or more (e.g., 1, 2, 3 or more) recombinant expression systems of this disclosure to achieve the desired level of recombinant protein expression, including modified Fc peptides (e.g., Fc F241A The expression of one or more (e.g., 1, 2 or more) glycosyltransferases (e.g., ST6GAL1 and / or B4GALT1). In some embodiments, the host cells described herein are modified such that both ST6GAL1 and B4GALT1 enzymes are expressed in a desired ratio. For example, in some embodiments, the host cell (e.g., a mammalian host cell) is modified to achieve a ST6GAL1 and B4GALT1 enzyme expression 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 ratio of ST6GAL1 to B4GALT1 protein expression in host cells is determined by the strength of each operatively linked promoter sequence of these proteins. In some embodiments, the ratio of ST6GAL1 to B4GALT1 protein expression in host cells is determined by one or more regulatory sequences present in a vector containing polynucleotides encoding enzymes (e.g., enhancers, polyadenylation signals, terminators, insulators, UTR sequences, etc.). In some embodiments, the ratio of ST6GAL1 to B4GALT1 protein expression in host cells is determined by the host cell type. In some embodiments, the ratio of ST6GAL1 to B4GALT1 protein expression in host cells is determined by one or more factors inherent to the host cell.

[0163] Furthermore, the methods described herein may include maintaining host cell viability at levels sufficient to allow for high levels of sialylation of the modified Fc polypeptides described herein. Without wishing to be bound by any theory, lysis of host cells in the culture may result in the release of sialylase into the culture medium, which may reduce the overall level of Fc sialylation through sialylase-mediated cleavage of the SA moiety from the Fc glycan. Therefore, this disclosure provides methods for maintaining host cell viability at or above predetermined values, such as at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, of viability after 10 to 20 days of culture (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). Any suitable method may be used to maintain and assess host cell viability.

[0164] Cultivation conditions In some embodiments, this document discloses methods for culturing host cells of the present disclosure in a culture medium under conditions that allow expression of one or more polypeptides encoded by the polynucleotides disclosed herein, and for purifying said one or more polypeptides from the cultured cells or the culture medium of the cells. To increase the level of sialylation of the modified Fc polypeptides described herein, the culture conditions for maintaining the host cells of the present disclosure can be optimized. For example, in some embodiments, to increase SA content, productivity is reduced, and the osmotic concentration is generally maintained within a lower margin suitable for the specific host cells being cultured. In some implementations, the range is from about 250 mOsm to about 450 mOsm (e.g., between 250-260 mOsm, between 260-270 mOsm, between 270-280 mOsm, between 280-290 mOsm, between 290-300 mOsm, between 300-310 mOsm, between 310-320 mOsm, between 320-330 mOsm, between 330-340 mOsm, between 340-350 mOsm, between 350-360 mOsm, between 360-370 mOsm, between 370-380 mOsm, between 380-390 mOsm, between 390-400 mOsm, between 400-410 mOsm). Osmolar concentrations (between 410-420 mOsm, 420-430 mOsm, 430-440 mOsm, and 440-450 mOsm) are suitable for increased SA content. Previous studies have reported that the SA content in antibody-linked sugar side chains differs significantly depending on whether the antibody is produced as ascites or in serum-free or serum-containing media. Furthermore, others have shown that using different bioreactors for cell growth and oxygenation of the culture medium affects the amount of galactose and SA in antibody-linked glycans.

[0165] Furthermore, in some embodiments, the culture conditions are further modified to increase the rate of sialylation of the polypeptides of this disclosure. One method for enhancing Fc sialylation is by adding an agent that enhances sialylation (e.g., by inhibiting sialylase activity) to the culture medium, such as, for example, uridine, manganese, copper, dexamethasone, hydrocortisone, N-acetylmannosamine, tetraacetylmannolamine, N-azidoacetyl-D-mannosamine, 1,3,4-O-Bu3Mannolamine, α(2,3)-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), sialyl sialidin B, fetoglobulin, and glycerol.

[0166] In some implementations, Fc sialylation is enhanced by changing the culture conditions to a pH that favors sialylation, such as about 7.2 (e.g., 7.0, 7.1, 7.2, 7.3, or 7.4).

[0167] Methods for delivering recombinant nucleic acids to host cells The vectors disclosed herein can be introduced into cells via a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and encapsulation in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipid transfection, and direct uptake. Genes encoding peptides of this disclosure can also be introduced into host cells by targeting cell membrane phospholipids with a vector containing a gene encoding such an agent.

[0168] For example, electroporation can be used to permeate host cells by applying an electrostatic potential to the cells of interest. Host cells subjected to an external electric field in this way then tend to take up exogenous nucleic acids. A similar technique, nuclear transfection, uses an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells.

[0169] Another technique that can be used to transfect target cells is the squeeze-perforation method. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. The advantage of this technique is that it does not require a vector to deliver nucleic acids into cells such as target cells.

[0170] Lipid transfection represents another technique that can be used to transfect target cells. This method involves loading nucleic acids into liposomes, which typically present cationic functional groups, such as quaternary ammonium or protonated amines, to their exterior. This leads to the uptake of exogenous nucleic acids, for example, through direct fusion of the liposome with the cell membrane or through endocytosis of the complex. A similar technique that utilizes ionic interactions with the cell membrane to trigger the uptake of exogenous nucleic acids is to contact cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for cell membrane interactions are activated dendritic polymers, polyethyleneimine, and diethylaminoethyl (DEAE)-glucan. Magnetic beads are another tool that can be used to transfect target cells in a gentle yet efficient manner because this method utilizes an applied magnetic field to guide nucleic acid uptake.

[0171] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection, also known as optical transfection. This technique involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeate the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to electroporation and has been found to be superior to electroporation in some cases.

[0172] Impalefection is another technique that can be used to deliver genetic material to 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 surface of the nanostructure. A chip with an array of these needles is then pressed onto a cell or tissue. Cells pierced by the nanostructure can express the delivered gene.

[0173] Magnetic transfection can also be used to deliver nucleic acids to target cells. The principle of magnetic transfection is to associate nucleic acids with cationic magnetic nanoparticles. These nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules that vary depending on the application. Their association with the nucleic acid carrier is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then focused onto the target cells by the influence of an external magnetic field generated by a magnet.

[0174] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves using sound (usually ultrasound frequencies) to alter the permeability of the cell membrane to make the cell permeable and allow polynucleotides to penetrate the cell membrane.

[0175] Microvesicles represent another potential medium for modifying the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of the glycoprotein VSV-G with, for example, genome-modifying proteins such as nucleases can be used to efficiently deliver proteins into cells that subsequently catalyze site-specific cleavage of endogenous polynucleotide sequences, thereby preparing the cell's genome for covalent incorporation of polynucleotides of interest, such as gene or regulatory sequences.

[0176] Protein purification The modified Fc peptides of this disclosure can be recovered and purified from recombinant cell cultures by any suitable method, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (HPLC) can also be used for purification.

[0177] The Fc peptides of this invention include naturally purified products, products of chemical synthesis processes, and products generated from eukaryotic hosts via recombinant technology, such as yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production process, the Fc peptides of this disclosure are glycosylated (e.g., sialylated).

[0178] Analytical methods for assessing Fc sialic acidification Fc sialylation can be assessed by any suitable assay. For example, HPLC can be used to assess the sialylation of Fc peptides. In some embodiments, hydrophilic interaction liquid chromatography (HILIC) is used to assess the sialylation of the modified Fc peptides of this disclosure. In some embodiments, MS is used to assess the sialylation of the modified Fc peptides of this disclosure. In some embodiments, size exclusion chromatography (SEC) is used to assess the sialylation of the modified Fc peptides of this disclosure. In some embodiments, HPLC, HILIC, MS, SEC, HPLC-UV, or any combination thereof are used to assess the sialylation of the modified Fc peptides of this disclosure.

[0179] Other Fc modifications In some embodiments, the disclosed Fc peptide is further modified using techniques known in the art for a variety of purposes (i.e., in addition to aliphatic amino acid substitutions, such as F241A substitution, at amino acid residue 241 of the Fc heavy chain). In some embodiments, the Fc peptide of this disclosure is further modified to increase the circulating half-life of the antigen-binding fragment when administered to a subject (e.g., a human). In some embodiments, the Fc region disclosed herein is modified to reduce or silence the effector functions of the Fc peptide (e.g., antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), opsonization, phagocytosis, transcellularity, infection neutralization, inflammation, mucosal immunity, and neonatal immunity). In some embodiments, the modified Fc peptide of this disclosure is further modified to modulate the binding of the Fc peptide to one or more Fc receptors.

[0180] This document also includes covalent modifications of Fc peptides. In some embodiments, covalent modifications are performed, if applicable, by chemical synthesis or by enzymatic or chemical cleavage of the peptide. In some embodiments, other types of covalent modifications are introduced by reacting the targeted amino acid residues with an organic derivatizer capable of reacting with selected side chain or N-terminal or C-terminal residues.

[0181] Cysteine ​​residues most commonly react with α-haloacetic esters (and corresponding amines) such as chloroacetic acid or chloroacetamide to yield carboxymethyl or carboxamide methyl derivatives. Cysteine ​​residues are also derivatized by reactions with: bromotrifluoroacetone, α-bromo-β-(5-imidozoyl))propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 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.

[0182] In some embodiments, the histidine residue is derivatized by reacting with diethyl pyrocarbonate at pH 5.5–7.0, as this agent is relatively specific for the histidine side chain. In some embodiments, p-bromobenzoyl bromide is also available; in some embodiments, the reaction is carried out at pH 6.0 in 0.1 M sodium dimethylarsinate.

[0183] In some embodiments, the lysyl residue and the amino-terminal residue react with succinic anhydride or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing α-amino-containing residues include imine esters such as methylpyridinium imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0184] In some embodiments, arginine residues are modified by reacting with one or more conventional reagents, such as phenylglyoxal, α(2,3)-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, the derivatization of arginine residues requires the reaction to be carried out under basic conditions. Furthermore, in some embodiments, these reagents react with the lysine group and the ε-amino group of arginine.

[0185] In some embodiments, due to a particular interest in introducing spectral labeling into tyrosine residues, the tyrosine residues are specifically modified by reaction with aromatic diazonium compounds or tetranitromethane. In some embodiments, most commonly, N-acetimidazole and tetranitromethane are used to form O-acetyltyrosine substances and 3-nitro derivatives, respectively. 125 I or 131 Iodized tyrosine residues were used to prepare a marker protein for radioimmunoassay.

[0186] The carboxyl side group (aspartic or glutamine) is specifically modified by reacting with a carbodiimide (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-azacation-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartic and glutamine residues are converted to asparaginic and glutamine-acyl residues by reacting with ammonium ions.

[0187] In some embodiments, the glutamine acyl residue and the asparagine acyl residue are deamidated to the corresponding glutamine acyl residue and asparagine acyl residue, respectively. These residues are deamidated under neutral or basic conditions.

[0188] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of the lysine, arginine, and histidine side chains, acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

[0189] Another type of covalent modification involves chemically or enzymatically coupling a glycoside to a therapeutic agent of this disclosure. These procedures do not require the generation of Fc polypeptides in host cells capable of performing N-linked or O-linked glycosylation. In some embodiments, depending on the coupling mode used, the sugar is attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group such as cysteine, (d) a free hydroxyl group such as serine, threonine, or hydroxyproline, (e) an aromatic residue such as phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine.

[0190] Another type of covalent modification involves linking the peptide to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene polyols, polyoxyethylene sorbitol, polyoxyethylene glucose, polyoxyethylene glycerol, polyoxyethylene olefins, or polysaccharide polymers such as dextran. In some embodiments, the Fc peptides of this disclosure are modified by adding polyethylene glycol (PEG). In some embodiments, PEG modification (PEGylation) results in one or more of the following: improved cycle time, improved solubility, improved proteolytic tolerance, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0191] Fatty acids and fatty acid esters are also suitable components for covalently modifying the Fc peptides of this disclosure, and can be saturated or contain one or more unsaturated units. Suitable fatty acids for modifying the Fc peptides disclosed herein include, for example, n-dodecanoic acid (C... 12 Lauric acid), n-Tetradecanoic acid (C14 Myristic acid), n-octadecanoic acid (C 18 Stearic acid), n-eicosanoic acid (C 20 Arachidonic acid), n-docosalicylic acid (C 22 behenic acid), n-triacontanoic acid (C 30 ), n-Tetradecanoic acid (C 40 ), cis-Δ9-octadecanoic acid (C 18 oleic acid), all-cis-Δ5,8,11,14-eicosanoatetraenoic acid (C 20 Arachidonic acid, octanoic acid, tetradecanoic acid, octadecanoic acid, docosanoic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing straight-chain or branched lower alkyl groups. The lower alkyl groups may contain from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

[0192] In some embodiments, the Fc peptides provided herein are conjugated or linked to therapeutic, imaging, or detectable moieties or affinity tags. Conjugation or linking of the peptide can be performed using any suitable method. Association (binding) between the compound and the tag includes any suitable means, including but not limited to covalent and non-covalent interactions, chemical conjugation, and recombinant techniques. In some embodiments, the Fc peptides are conjugated or recombinantly engineered to affinity tags (e.g., purification tags). Affinity tags, such as, for example, multihistidine tags (e.g., 6x-His; SEQ ID NO: 7), are suitable for the compositions and methods described herein.

[0193] Pharmaceutical Composition In some embodiments, this document discloses a method for treating a disease or disorder in an individual with a corresponding need, the method comprising: (a) a modified population of Fc peptides, each modified Fc peptide having (i) an amino acid sequence identical to 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 sequence of SEQ ID NO: 2, and (ii) an aliphatic amino acid residue (e.g., Ala) at position 241 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), comprising: at least 50% (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 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 a modified Fc polypeptide having a sialic acid (SA) moiety of an N-glycan attached to the modified Fc polypeptide via an α(2,6) bond; and (b) a pharmaceutically acceptable carrier, diluent or excipient. Such compositions may be used for in vitro or in vivo analysis, or, in the case of pharmaceutical compositions, for in vivo or ex vivo administration to subjects to treat subjects with a disease or disorder (e.g., an autoimmune disorder, such as an autoimmune disease or disorder) with the disclosed peptide.

[0194] In some implementations, the carrier, diluent, or excipient is a stabilizer, buffer, surfactant, filler, solvent, tension or osmotic pressure regulator, antioxidant, adjuvant, antimicrobial agent, or other suitable material. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other substance will depend on the route of application.

[0195] In some embodiments, pharmaceutical formulations comprising Fc peptides identified by the methods described herein are prepared for storage by mixing a protein of desired purity with an optional physiologically acceptable carrier, diluent, or excipient, in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, diluents, or excipients are those that are non-toxic to the recipient at the doses and concentrations used, and include buffers such as acetates, phosphates, citrates, histidine, TRIS, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as proline, glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelates Compounds such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, polyethylene glycol (PEG), polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80) and poloxamer (e.g., P1). 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 4°C. In some embodiments, the pharmaceutical composition is stable as a liquid solution at room temperature.

[0196] An acceptable delivery vehicle is physiologically acceptable to the subject to which it is administered and retains the therapeutic properties of the compound administered with or therein. Acceptable delivery vehicles and their formulations are generally described, for example, in the following: Remington's Pharmaceutical SciencesIbid. An exemplary delivery vehicle is physiological saline. As used herein, the phrase “pharmaceutically acceptable delivery vehicle” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which participates in the delivery or transport of the subject compound from a site of application in one organ or part of the body to another organ or part of the body, or in an in vitro assay system. Each delivery vehicle is “acceptable” in the sense that it is compatible with other components of the formulation and does not cause harm to the subject to which it is administered. An acceptable delivery vehicle should also not alter the specific activity of the subject compound.

[0197] In some embodiments, the pharmaceutical compositions disclosed herein further comprise acceptable additives to improve the stability of the compounds in the composition and / or control the release rate of the composition. Acceptable additives do not alter the specific activity of the subject compound. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbitol, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, the acceptable additives are combined with acceptable carriers and / or excipients such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase peptide stability and reduce gelation of the solution. In some embodiments, the surfactant is added to the composition in an amount from 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0198] In some embodiments, the pharmaceutical compositions disclosed herein comprise isotonic buffers such as phosphates, acetates, histidines, or TRIS buffers, combined with tensile agents such as polyols, sorbitol, sucrose, or sodium chloride, which increase tensile strength and provide stabilization. In one embodiment, the tensile agent is present in the composition at approximately 5%.

[0199] In some embodiments, the pharmaceutical compositions disclosed herein contain 0.01% w / v to 0.02% w / v of a surfactant to prevent aggregation and for stabilization.

[0200] In some embodiments, the pH of the pharmaceutical compositions disclosed herein is in the range of 4.5-6.5 or 4.5-5.5.

[0201] In some embodiments, the pharmaceutical compositions disclosed herein also contain more than one active polypeptide necessary for the therapeutic indication, such as those with complementary activities that do not adversely affect each other. Such polypeptides are suitably combined in amounts effective for the intended purpose.

[0202] In some embodiments, the active ingredient is encapsulated in microcapsules, for example, by coagulation technology or by interfacial polymerization, such as hydroxymethyl cellulose microcapsules or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively; or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse droplet emulsions.

[0203] This paper also envisions suspension and crystalline forms of peptides; any suitable method can be used to prepare suspensions and crystalline forms.

[0204] In some embodiments, the pharmaceutical compositions disclosed herein are sterile. In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by conventional or any suitable sterilization technique. For example, sterilization can be easily accomplished by filtration through a sterile filter membrane. In some embodiments, the resulting solution is packaged for use or filtered and lyophilized under sterile conditions, and the lyophilized product is combined with the sterile solution prior to administration.

[0205] In some implementations, freeze-drying is used to stabilize the peptide for long-term storage, such as when the peptide is relatively unstable in a liquid composition.

[0206] In some embodiments, excipients such as, for example, polyols (including mannitol, sorbitol, and glycerol); sugars (including glucose and sucrose); and amino acids (including alanine, glycine, and glutamic acid) act as stabilizers for freeze-dried products. In some embodiments, polyols and sugars also serve to protect peptides from freeze-drying-induced damage and enhance stability during storage in the dry state. In some embodiments, sugars are effective both during freeze-drying and during storage. Other classes of molecules, including monosaccharides and disaccharides, as well as polymers such as PVP, have also been reported as stabilizers for freeze-dried products.

[0207] For injection, in some embodiments, the pharmaceutical compositions disclosed herein are powders suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, lyophilized powders, rotary-dried powders or spray-dried powders, amorphous powders, granules, precipitates, or microparticles. For injection, the compositions optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0208] In some embodiments, sustained-release articles are prepared. Suitable examples of sustained-release articles include semi-permeable matrices of solid hydrophobic polymers containing peptides, in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid:glycolic acid copolymers, such as injectable microspheres comprising lactic acid:glycolic acid copolymers and leuprolide acetate, and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid:glycolic acid enable molecular release to continue for more than 100 days, some hydrogels result in shorter durations of protein release. Although encapsulated peptides are retained in vivo for extended periods, they may denature or aggregate due to exposure to moisture at 37°C, leading to loss of biological activity and potential changes in immunogenicity. In some cases, a suitable strategy for stabilization depends on the mechanism involved. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds through thio-disulfide exchange, stabilization can be achieved in some cases by modifying thiol residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0209] In some embodiments, the pharmaceutical compositions disclosed herein are designed for short-acting, rapid-release, long-acting, or sustained-release as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or for slow release.

[0210] Pharmaceutical compositions are administered, for example, by injection, including but not limited to subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, intracerebrospinal, intraosseous, or intramuscular injection. This document contemplates excipients and carriers for formulating compositions for each type of injection. The following descriptions are merely illustrative and are not intended to limit the scope of the compositions. Compositions for injection include, but are not limited to, aqueous solutions (in the case of water solubility) or dispersions, and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, 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, etc.), and suitable mixtures thereof. For example, flowability is maintained by maintaining a required particle size in the case of dispersions through the use of coatings such as lecithin, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In some embodiments, the composition includes isotonic agents such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. In some embodiments, the resulting solution is packaged for use as is or lyophilized; in some embodiments, the lyophilized product is subsequently combined with a sterile solution prior to administration. For intravenous or site-specific injection, the active ingredient will be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Any suitable solution using, for example, isotonic media such as sodium chloride injection, Ringer's solution, and lactated Ringer's solution. In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. In some embodiments, the sterile injectable solution is prepared by incorporating the required amount of the active ingredient, as needed, with one or a combination of the ingredients listed above into a suitable solvent, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the active ingredient into a sterile medium comprising a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, which produce powders containing the active ingredient plus any other desired components from the previously sterile filtered solution.

[0211] 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 parenteral acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability. In some embodiments, suitable solutions are prepared using, for example, isotonic media (such as sodium chloride injection, Ringer's solution, lactated Ringer's solution). In some embodiments, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed. Additionally, in some embodiments, the composition is administered via aerosolization.

[0212] For parenteral administration, the peptide is formulated with a pharmaceutically acceptable parenteral medium into a unit-dose injectable form (e.g., solution, suspension, or emulsion). Examples of such media include water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Non-aqueous media such as non-volatile oils and ethyl oleate are also used. In some embodiments, liposomes are used as the delivery vehicle. The medium contains small amounts of additives, such as substances that enhance isotonicity and chemical stability, for example, buffers and preservatives. The peptide is typically formulated in such media at concentrations from about 1 mg / mL to 10 mg / mL.

[0213] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, for example, to extend shelf life. When considering the use of the compositions in the pharmaceuticals or any methods provided herein, in some embodiments, it is envisioned that the compositions are substantially pyrogen-free, such that they will not cause inflammatory reactions or unsafe allergic reactions when administered to human subjects.

[0214] In some embodiments, the acceptable carrier contains compounds that stabilize, increase, or delay absorption or clearance. Such compounds include, for example, sugars 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, detergents are also used to stabilize or increase or decrease the absorption of the pharmaceutical composition, including the liposome carrier. To prevent digestion, in some embodiments, the compound is conjugated to the composition to make it resistant to acid and enzymatic hydrolysis, or the compound is conjugated in a suitable resistant carrier such as a liposome. Protecting the compound from digestion can be achieved using any suitable method.

[0215] Packaging, reagent kits and pre-filled containers This document also provides kits comprising one or more of the therapeutic agents described above. In some embodiments, the kit includes a modified and highly sialylated Fc peptide of this disclosure or a composition comprising therein in a suitable container.

[0216] In some embodiments, a container device comprising the composition described herein is provided. In some embodiments, the container device is any suitable container that holds, for example, a liquid or lyophilized composition, including but not limited to vials, syringes, bottles, and intravenous (IV) bags or ampoules. The syringe holds any volume of liquid suitable for injection into a subject, including but not limited to 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc or more.

[0217] This document provides kits comprising one or more compositions described herein. In some embodiments, this document provides kits for treating subjects suffering from autoimmune disorders, said kits comprising the compositions described herein and optionally additional therapeutic agents.

[0218] In some embodiments, this document provides a kit for treating autoimmune disorders, the kit comprising the composition described herein and a label attached to or packaged with a container describing the use of the composition, optionally in combination with other therapeutic agents.

[0219] In some embodiments, the container device of the kit will typically include at least one vial, test tube, flask, bottle, ampoule, syringe, intravenous (IV) bag and / or other container device in which at least one modified Fc polypeptide of the present disclosure or a composition containing the thereof is placed and / or suitably aliquoted.

[0220] In some embodiments, the kit includes means for containing at least one modified Fc peptide and / or a composition containing the same within a closed enclosure for commercial sale. In some embodiments, such a container includes an injection-molded plastic container and / or a blow-molded plastic container in which a desired vial is stored. In some embodiments, the kit also includes printed material for use with materials in the kit.

[0221] In some embodiments, the packaging and kit additionally include buffers, preservatives, and / or stabilizers from the pharmaceutical formulation. In some embodiments, each component of the kit is encapsulated in a separate container, and all the different containers are contained in a single package. In some embodiments, the disclosed kit is designed for refrigerated or room temperature storage.

[0222] Additionally, in some embodiments, the product contains stabilizers to increase the shelf life of the kit and includes, for example, bovine serum albumin (BSA). In some embodiments, where the composition is lyophilized, the kit contains additional solution preparations to reconstitute the lyophilized formulation. Acceptable reconstituted solutions include, for example, pharmaceutically acceptable phosphate-buffered saline (PBS).

[0223] In some embodiments, the packaging and kit further include one or more components for assays such as ELISA, HPLC, or mass spectrometry. Samples to be tested in this application include, for example, blood, plasma, serum, tissue sections and secretions, urine, lymph, and their products. In some embodiments, the packaging and kit also include one or more components for collecting samples (e.g., syringes, cups, swabs, etc.).

[0224] In some embodiments, the packaging and kits also include labels specifying information required by the U.S. FDA or similar regulatory agencies, such as product description, dosage and method of administration, and / or therapeutic indications. In some embodiments, the packaging provided herein includes any composition as described herein.

[0225] The term "packaging material" refers to the physical structure that contains the components of the kit. In some embodiments, the packaging material maintains the sterility of the components and is made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). In some embodiments, the label or packaging insert includes appropriate written instructions (e.g., instructing the user of the kit to perform one or more of the methods disclosed herein). In some embodiments, the kit additionally includes a label or instruction manual for using the kit components in any of the methods described herein. In some embodiments, the kit contains a compound in a package or dispenser along with instructions for administering the compound as described herein.

[0226] In another embodiment, the kit also includes a container for one or more additional therapeutic agents for autoimmune disorders.

[0227] In some embodiments, the instructions for use include instructions for practicing any of the methods (including treatments) described herein. In some embodiments, the instructions for use also include indications of satisfactory clinical endpoints or any adverse symptoms that occur, or additional information for human subjects as required by regulatory agencies (such as the Food and Drug Administration).

[0228] In some embodiments, the instructions are on "printed material," for example, inside the kit or affixed to the paper or cardboard of the kit, or on a label or packaging material of the kit, or attached to vials or tubes containing the components of the kit. In some embodiments, the instructions are additionally contained on a computer-readable medium, such as, for example, CD-ROMs, DVDs, flash memory devices, solid-state storage, disks and disk drives, magnetic tapes, cloud computing systems, and servers. In some cases, the programs and instructions are permanently, substantially permanently, semi-permanently, or non-transitory encoded on the medium.

[0229] Other implementation plans Various modifications and variations of the disclosure described herein will be apparent to those skilled in the art without departing from its scope and spirit. Although the disclosure has been described in conjunction with specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. Indeed, it will be apparent to those skilled in the art that various modifications to the implementation of the methods described herein are intended within the scope of the disclosure. Other embodiments are described in the claims.

[0230] Example The following examples are provided to provide those skilled in the art with a description of how the compositions and methods described herein can be used, prepared, and evaluated, and are intended to be merely examples of the contents of this disclosure and are not intended to limit the scope of what the inventors consider their invention.

[0231] Example 1: Compared with the wild-type Fc domain, Fc F241A The mutant exhibited increased levels of α(2,3) sialylation. 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) is the site of the biantennary glycan, which is post-translational modified under normal physiological conditions to control the level of sialic acid bonds. These recombinant Fc domains were purified by size exclusion chromatography (SEC) and suspended in phosphate-buffered saline (PBS). N-glycan profiling was performed using known standards by high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) to define the structure of the biantennary glycan molecule attached to Asn297. The N-glycan structures identified using this analysis were... Figure 1 Examples are provided below.

[0232] exist Figures 2A-2BThe percentage of Fc peptides with a defined glycan structure at Asn297 is depicted. WT Fc peptides primarily consist of N-glycans terminated at N-acetylglucosamine (GlcNAc; 49%) or a GlcNAc moiety containing a single galactose (G1F; 39%), but rarely or no SA-containing glycoforms (<1.0%). In contrast, the introduction of the F241A mutation into Fc peptides results in a significant increase in galactose addition, with 22% of the peptides containing mono-2,3 SA (G2FSA) and 19% containing di-2,3 SA (G2FSA2).

[0233] Example 2: Fc in the recombinant expression system F241A The mutant has high levels of α(2,6) sialylation The human IgG1 Fc domain with a phenylalanine (F) to alanine (A) substitution at amino acid position 241 (F241A) was stably expressed in CHO cells in both 10 mL and 1 L cultures. To manipulate the N-glycan structure present on Asn297, the Fc domain was... F241A Expression was performed alone (pool 1), with β-galactosidase α-2,6-sialyltransferase 1 (ST6GAL1) enzyme (pool 2), with ST6GAL1 and β-1,4-galactosidase 1 (B4GALT1) enzymes (pool 3), or with siRNA targeting sialyl transporter, solute carrier family 35 member A1 (SLC35A1 KD; pool 4). These recombinant Fc F241A The glycovariates were purified by size exclusion chromatography (SEC) and suspended in phosphate-buffered saline (PBS). N-glycan profiling was performed using known standards by high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) to define the structure of the biantennary N-glycans attached to Asn297.

[0234] Data are expressed as percentages of Fc polypeptides containing defined glycan structures from 1 L CHO cultures. On the x-axis, glycan structures are defined as G0 (terminated with GlcNAc), G0F (terminated with GlcNAc containing fucose), G1 (terminated with one galactose), G1F (terminated with one galactose and one fucose), G2 (terminated with two galactoses), G2F (terminated with two galactoses and one fucose), G2FSA (terminated with one sialic acid and one fucose), and G2FSA2 (terminated with two sialic acids and one fucose). Sialic acid is added via α(2,3) bonds in pool 1 and α(2,6) bonds in pools 2 and 3. When recombinant human IgG1 Fc domains carrying F241A were expressed alone in CHO cells (pool 1), 18% of the Fc peptides in this pool were mono-α(2,3)-sialylated, 5% were di-α(2,3)-sialylated, and 47% had terminal galactose (…). Figure 3A When co-expressed with ST6GAL1 (pool 2), the Fc peptide was 30% mono-α(2,6) sialylated, 27% di-α(2,6) sialylated, and 7% terminally galactose-containing ( Figure 3B When co-expressed with ST6GAL1 and B4GALT1 (pool 3), the Fc peptide was 3% mono-α(2,6) sialylated, 81% di-α(2,6) sialylated, and 0% terminally galactose-containing ( Figure 3C When co-expressed with siRNA targeting SLC35A1 (pool 4), the Fc domain was completely devoid of sialic acid, with 84% of the Fc N-glycans ending in galactose. Figure 3D ).

[0235] To independently verify the glycan structures associated with pools 1-4 described above, generated from 1 L of stable CHO culture, recombinant Fc F241A The glycovariates were purified by size exclusion chromatography and suspended in PBS. 5 μg of Fc was added... F241A Each of the glycovariate pools P1, P2, P3, and P4 was distinguished by SDS-PAGE under reducing conditions and then stained with Coomassie stain. Figure 4A ), transferred to a nitrocellulose membrane and incubated with biotinylated elderberry (SNA) lectin to detect α(2,6) sialic acid ( Figure 4B ), or transferred to a nitrocellulose membrane and incubated with biotinylated MAL I lectin to detect terminal galactose ( Figure 4C ALP-conjugated goat anti-biotin antibodies were used to detect lectins. These findings were consistent with HPLC / MS analysis, confirming that pools 2 and 3 uniquely contain 2,6-sialic acid, while pools 1 and 4 do not contain 2,6-sialic acid but instead contain more terminal galactose.

[0236] Example 3: Fc sialylation improves exposure to modified Fc peptides in mice To determine the sialylated Fc F241A In vivo pharmacokinetics of the mutant peptide: Seven- to eight-week-old female JAX-014565 mice (mouse FcRN homozygous knockout, human FcRN hemizygous, Tg32 strain) were randomly divided into three groups of six mice each. Mice received a single intravenous (IV) bolus of 20 mg / kg of recombinant human IgG1 Fc domain containing F241A (stable CHO 10 mL culture) from pool 2 (66% α(2,6) sialylated), pool 3 (93% α(2,6) sialylated), and pool 4 (0% sialylated) via tail vein. Blood was collected at each of the seven time points (three mice per time point) before administration, 0.5 hours, day 1, day 3, day 7, day 21, and day 35. Serum was prepared according to standard procedures and transferred to Eppendorf tubes for storage. Fc concentration in each serum sample was determined by ELISA. F241A Peptide concentration. Briefly, coat a 96-well plate overnight at 4°C with 1 μg of anti-human Fc-specific capture antibody. Wash the plate three times with blocking buffer, then incubate at room temperature with shaking (500 rpm) for 2 hours with blocking buffer (80 μL per well). Wash the plate again and dilute the standards (starting from 300 ng / mL, serially 1:3 for 7 additional concentrations) and apply to the plate to generate a calibration curve. Dilute the samples in dilution buffer (minimum 20x dilution) as needed and apply to the same plate, then incubate at room temperature with shaking (500 rpm) for 2 hours. Wash the plate and incubate at room temperature (shaking at 500 rpm) with a 1:50,000 dilution of the detection antibody for 1 hour. Then develop the washed plate 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 at 450 nm was read using a plate reader. Graphical analysis was performed using Prism software, and the area under the curve (AUC) was determined.

[0237] exist Figures 5A-5B Fc is depicted in F241A Serum concentrations (μg / mL) over time (days) for each of pools 2 (hollow square), 3 (solid circle), and 4 (solid triangle). Data are plotted on a logarithmic scale. Figure 5A ) and linear scaling ( Figure 5B(The values ​​are expressed as concentrations.) These pools were directly compared as a means of assessing the effect of the degree of α(2,6) sialylation on pharmacokinetics in human FcRN mice. Table 1 below summarizes these Fc... F241A The AUC of each glycovariate revealed that the degree of sialylation was proportional to the exposure in these mice. Pool 4, the non-sialylated Fc, had the lowest AUC, followed by Pool 2 with 66% α(2,6) sialylation. Maximum exposure was achieved in Pool 3 with 93% α(2,6) sialylation.

[0238] Table 1: Fc of α(2,6) sialylated F241A Pharmacokinetics of mutant peptides To compare α(2,3) sialylated Fc F241A Mutants and α(2,6)-sialylated Fc F241A To investigate pharmacokinetic differences between mutants, 7-8 week old female JAX-014565 mice were randomly divided into three groups of six mice each. Mice received a single IV bolus injection via tail vein of recombinant human IgG1 Fc domain with F241A (20 mg / kg) from stable 10 mL CHO cultures of pool 1 (35% α(2,3) sialylated) or pool 3 (93% α(2,6) sialylated) material, or human IgG1 Fc domain with Abdeg mutation (M252Y / S254T / T256E / H433K / N434F) (Egamod (EFG)) to enhance FcRn binding transiently expressed by CHO cells (0% sialylated). Blood was collected at each of the following six time points: before administration, 0.5 h after administration, day 1, day 3, day 7, and day 14 (three mice at each time point). Serum was prepared according to standard procedures and transferred to tubes for storage. Fc levels in each serum sample were determined by ELISA as described above. F241A The concentration.

[0239] For Fc F241A Each of pools 1, 3, and EFG Fc shows a concentration-time profile. Figures 5C-5D Data is measured on a logarithmic scale. Figure 5C ) and linear scaling ( Figure 5D Plotting concentrations. At α(2,3) sialylated Fc F241A and α(2,6) sialylated Fc F241AThe observed difference in AUC is likely due to differences in the degree of sialylation, rather than differences in α(2,3) sialylation relative to α(2,6) sialylation, as similar effects were observed when adjusting for levels of α(2,6) sialylation. 93% α(2,6) sialylation Fc F241A The highest exposure was observed in these mice. The EFG Fc domain had the lowest AUC, similar to the unsialylated Fc. F241A (Pool 4), such as Figures 5A-5B As shown elsewhere. The AUC values ​​for each Fc pool are provided in Table 2 below.

[0240] Table 2: Fc of α(2,3) sialylated Fc F241A mutant peptides and α(2,6)-sialylated Fc F241A Pharmacokinetics of mutant peptides Then, stable CHO 1 L culture Fc was used. F241A Materials were used in similar experiments in male and female CD1 mice as follows. Male and female CD1 mice aged 5-7 weeks were divided into two groups (6 males and 6 females per group) and administered 20 mg / kg of Chi1 Fc via a single IV bolus injection. F241A (23% α(2,3) sialylation) or pool 3 Fc F241A (84% α(2,6) sialylation). Blood was collected via retro-orbital sampling at each of the six time points (3 mice per time point) before administration, 1 hour and 4 hours after administration, and at 1, 3, 7, and 14 days after administration. Serum was prepared according to standard procedures and transferred to tubes for storage. Fc in each serum sample was determined by ELISA as described above. F241A The concentration.

[0241] Such as concentration-time summary ( Figure 5E As shown in Table 3 below, Fc receives either α(2,3) sialylation (hollow triangles and solid triangles) or α(2,6) sialylation (hollow circles and solid circles). F241A There was no significant difference in exposure between male and female mice. However, as previously observed, α(2,6)-sialylated materials had greater exposure than α(2,3)-sialylated materials, although this difference may be attributed to the degree of sialylation rather than the α(2,3) bond versus the α(2,6) bond.

[0242] Table 3: Fc of α(2,3) sialylated Fc F241A mutant peptides and α(2,6)-sialylated Fc F241A Pharmacokinetics of mutant peptides Finally, repeated administration of α(2,6) sialylated Fc was tested in CD1 mice. F241A Effects of exposure to Fc-241A. Male and female CD1 mice aged 5–7 weeks were randomly divided into three groups (n=6 males and n=6 females per group) and administered 100 mg / kg of Fc-241A via IV bolus injection four times weekly. F241A (23% α(2,3) sialylation) or 100 mg / kg of pool 3Fc F241A (84% α(2,6) sialylated). Blood was collected immediately before the fourth dose on day 21 and 24 hours after the fourth dose on day 22. 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.

[0243] Fc relative to α(2,3) sialylated F241A In the administration of α(2,6) sialylated Fc F241A Subsequently, in male and female CD1 mice, in C min Greater exposure was achieved at both the time of the fourth dose and at 24 hours after the fourth dose. Figure 5F Notably, 100 mg / kg of α(2,6)-sialylated material achieved a minimum exposure greater than 200 μg / mL, which is twice the concentration required for binding the DC-SIGN mechanism in cell-based assays.

[0244] Example 4: Fc F241A α(2,6) sialylation enhances the potency of anti-inflammatory activity in a mouse model of arthritis. To determine the sialylated Fc F241AThe therapeutic efficacy of mutant peptides in inflammatory diseases was investigated using a mouse model of arthritis. KRN transgenic heterozygous mice on a C57BL / 6 background were bred with NOD / SHiLtJ to produce K / BxN mice. Serum collected from animals with inflamed joints was pooled from mice (6–9 weeks old) that spontaneously developed inflammation. The pooled serum was frozen in aliquots and used in all experiments presented here. Joint inflammation was induced in C57BL / 6 mice by intravenous injection of 4–200 μL of the pooled K / BxN serum. Symptoms of arthritis 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. Scores generated by two independent technicians were averaged to produce a final clinical score. Fc generated by transient transfection of CHO cells was used in these experiments. F241A For treatment conditions, mice were given 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 1 hour before administration of KBxN serum.

[0245] Mice (n=4 per group) were randomly assigned to receive either a single dose of 1 g / kg IVIG or 50 mg / kg F241A. The IVIG treatment group and Fc... F241A Clinical scores in the treatment groups did not differ significantly from each other except on days 6 and 7, as determined by an unpaired t-test. Figure 6A In PBS-treated mice, inflammation peaked on day 7. Therefore, clinical scores on days 7 and 8 were studied. Figures 6B-6C The values ​​are shown in box plots, and are expressed as mean and standard error of the mean. Both 1 g / kg IVIG and 50 mg / kg F241A significantly suppressed inflammation relative to PBS. These data suggest that F241A is approximately 20 times more potent than IVIG in the K / BxN serum metastasis model of arthritis.

[0246] Then, 50 mg / kg (84%) α(2,6) sialylated Fc was used. F241A Repeated use of Fc F241ASimilar experiments were conducted on stable CHO 1 L culture materials. The two different F241A glycoforms evaluated here differed in two aspects. 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, as determined by HPLC, approximately 23% of the α(2,3)-linked material contained SA, while approximately 84% of the α(2,6)-linked material contained SA. Mice (n=5–6 per group) were randomly assigned to receive a single administration of 1 g / kg IVIG, 50 mg / kg of α(2,3)-sialylated Fc. F241A Or 50 mg / kg of α(2,6)-sialylated Fc F241A Treatment was initiated. As determined by an unpaired t-test, from day 5 onwards, IVIG showed significantly greater anti-inflammatory activity than F241A α(2,3) sialylated Fc (p < 0.01). Figure 6D In contrast, from day 8 onwards, IVIG was significantly different from F241A α(2,6)-sialylated Fc only (unpaired t-test; p < 0.05). At the peak of inflammation in PBS-treated animals, namely days 6 and 7, α(2,6)-sialylated Fc... F241A Fc that leads to sialylation relative to α(2,3) F241A Significantly less inflammation Figures 6E-6F ).

[0247] Example 5: In a mouse model of idiopathic thrombocytopenic purpura, sialylated Fc F241A Peptides are more effective than IVIG To determine the sialylated Fc F241A The therapeutic efficacy of the mutant peptide in another inflammatory disease was investigated using a mouse model of idiopathic thrombocytopenic purpura (ITP). Mouse 6A6-IgG2a anti-platelet antibody was generated in 293T cells via transient transfection in serum-free medium and then purified with Protein G beads. Six- to eight-week-old female C56BL / 6 mice (n = 5 per group) received a single tail vein injection of 4 μg of 6A6 antibody at time zero. Mice were randomly assigned to receive PBS, 1 g / kg IVIG, or 100 mg / kg of α(2,3)-sialylated Fc via tail vein injection 2 hours prior to 6A6 administration. F241A (F241A α(2,3) sia), Fc with α(2,3) sialylation F241A(F241A α(2,3) sia) was transiently expressed in CHO cells and 41% sialylated. Platelet counts were monitored on day 0 before 6A6 administration and again at 24, 48, and 72 hours post-administration. Box-whisker plots representing the first and third quartiles, median, and minimum and maximum values ​​were generated.

[0248] In this ITP model, baseline platelet counts were similar across all three treatment groups on day 0. Figure 7 Following administration of 6A6 antiplatelet antibody, platelets in PBS control mice were >90% depleted by 24 hours, recovered to ~40% of pre-6A6 levels by 48 hours, and returned to normal by 72 hours. Platelets were also depleted by 1 g / kg IVIG or 100 mg / kg α(2,3) sialylated Fc. F241A Treatment at 24 and 48 hours following 6A6 administration resulted in a statistically significant protective effect against platelet loss (p < 0.01), as determined by unpaired t-tests. These findings suggest that, in this ITP model, α(2,3) sialylated Fc F241A Its potency is about 10 times that of IVIG.

[0249] Example 6: Fc F241A Terminal sialylation enhances in vivo half-life and bioavailability. Full-length IgG is known to have a prolonged serum half-life of up to 21 days due to its interaction with FcRn, which recycles IgG into circulation after cellular uptake. In contrast, the Fc fragment is known to have a shorter serum half-life. Therefore, to test Fc... F241A Does sialylation affect its serum half-life? This was investigated by administering a single intravenous dose of 20 mg / kg of different FcRn molecules to humanized FcRn Tg32 (hFcRn) mice. F241A Each of the glycoform or wild-type (WT) Fc was used, and the amount of human IgG1 Fc in circulation was quantified over time. Mice were randomly assigned to receive 20 mg / kg of Fc. F241A A single intravenous injection of a glucose-based formulation was administered to six mice per group. Blood was collected via retroorbital sampling at 0 min, 30 min, day 1, day 5, day 7, day 14, day 21, and day 35 post-administration for serum separation. Mice were euthanized, and blood was collected via cardiac puncture. Subsequent time points were not plotted if the achieved exposure was below the quantification limit (1 µg / mL). Blood was not collected from all mice at every time point, but each mouse contributed 3–4 times during the study. Fc levels in mouse serum were quantified using ELISA. F241ABefore reaching the required concentration, serum samples should be stored at -80°C.

[0250] Following administration, desialylated WT Fc and Fc produced in cells expressing short interfering RNA (siRNA) targeting the Golgi sialic acid transporter SLC35A1 were observed. F241A (Fc F241A / siSLC Both were rapidly cleared from the cycle and became undetectable after 14 and 10 days, respectively, corresponding to half-lives of 2.71 days and 1.43 days. Figure 8A (and Table 4). These products have a shelf life of 322 days. mg / mL and 118 days The area under the plasma concentration-time curve (AUClast) from mg / mL to the last measurable plasma concentration, and the clearance rates at 61.3 mL / day / kg and 168.3 mL / day / kg. Figure 8A (and Table 4). Fc F241A It has a half-life of 2.96 days and a half-life of 297 days. AUClast of mg / mL and clearance of 42.1 mL / day / kg (mg / mL) Figure 8A (and Table 4). Fc produced in cells expressing α-2,6-sialyltransferase ST6GAL1 F241A (Fc F241A / ST6 It has a serum half-life of 3.72 days and a serum half-life of 483 days. The AUClast was 40.8 mg / mL and the clearance rate was 40.8 mL / day / kg. Fc produced in cells expressing β-1,4-galactosyltransferase B4GALT1 and ST6GAL1 F241A (Fc F241A / B4ST6 It exhibited the longest residence time in serum, with a half-life of 5.2 days, and decreased to concentrations below 1 µg / mL only after day 35 post-administration, with an AUClast of 782 days. mg / mL, and the clearance rate was 24.8 mL / day / kg ( Figure 8A (and Table 4).

[0251] Table 4: In vivo half-life and bioavailability of F241A sugar form Fc Despite Fc F241A The percentage of sialylation of N297 glycans showed no correlation with anti-inflammatory activity (R). 2 =0.0042; Figure 8B However, compared to the half-life (R...), 2 =0.7952; Figure 8C ), AUClast (R 2=0.83; Figure 8D There is a significant positive correlation between ) and a significant negative correlation between ) and clearance rate (R 2 =0.5816; Figure 8E These results are surprising because the half-life of IgG is thought to be primarily regulated by FcRn. Furthermore, it is believed that the Fc glycan is embedded in a pocket at the top of the CH2 domain. Figure 11B Furthermore, it is largely inaccessible. Based on these results, Fc with ~90% α-2,6-sialylation on N297 glycan was selected. F241A / B4ST6 The product is used for further analysis and experiments.

[0252] Example 7: Fc F241A Terminal sialylation enhances in vivo half-life and bioavailability due to its weaker affinity for ASGPR binding. Because sialylation affects Fc F241A The serum half-life was used to evaluate the role of desialyl glycoprotein receptor (ASGPR) in accelerating Fc. F241A / siSLC Possible role in clearance. Administration of Fc to WT mice. F241A / B4ST6 (Sialidized Fc) F241A ) or Fc F241A / siSLC (non-sialylated Fc) F241A Fc was administered in combination with either a control (α(1)-acid glycoprotein (AGP)) or an ASGPR blocker (neuraminidase-treated AGP). Fc administration and serum collection were performed as described in Example 6.

[0253] To determine the binding affinity of Fc to ASGPR, a biolayer interferometry study was conducted. Fc... F241A / B4ST6 or Fc F241A / siSLC Immobilized on an anti-human Fc biosensor. The Fc-captured biosensor was immersed in wells containing commercial human and mouse FcRn, present at a series of 2-fold serial dilutions ranging from 1600 nM to 50 nM at pH 6.0, and the binding response was measured. The biosensor was then immersed in a pH 6.0 buffer to observe the dissociation of FcRn from the captured Fc. The biosensor was regenerated and reused for subsequent reagents.

[0254] A 100 nM histidine-tagged human desialylate glycoprotein receptor 1 (ASGPR1) was immobilized on an anti-pentahistidine (HIS1K) biosensor. Fc was prepared in Octet buffer. F241A / B4ST6 or Fc F241A / siSLC A series of 2x serial dilutions, starting from 171 The experiment started at a concentration of g / ml. The experimental protocol consisted of a 60-second baseline step, followed by a 120-second association step and a subsequent 300-second dissociation step. After each cycle, the sensor was regenerated by immersion in glycine HCl buffer (10 mM, pH 1.5) for 30 seconds. Binding was measured using an Octet K2 system.

[0255] Fc F241A / B4ST6 The serum half-life is not affected by ASGPR1 blockade. Figure 9A However, Fc F241A / siSLC The half-life of Fc is significantly enhanced by ASGPR1 blockade. Fc with ASGPR blockade... F241A / siSLC The half-life of Fc with or without ASGPR1 blocking F241A / B4ST6 They have similar half-lives.

[0256] Research combining interferometry reveals that Fc F241A / siSLC There is a weak interaction with ASGPR1, while Fc F241A / B4ST6 It does not bind to ASGPR1 (Figure 9). These results indicate that sialylation protects Fc. F241A Avoid being cleared by ASGPR1.

[0257] Example 8: Fc F241A Anti-inflammatory activity does not require sialylation The experiments described in this embodiment investigated the effects of sialylated bonds and sialylation levels on Fc. F241A Effects on anti-inflammatory activity.

[0258] The four preparations (Fc) described in Example 2 F241A 、Fc F241A / ST6 、Fc F241A / B4ST6 and Fc F241A / siSLC Each differentially glycosylated Fc in ) F241A Its ability to prevent joint inflammation was tested in a K / BxN mouse model of arthritis at a working concentration of 50 mg / kg. Figure 10A Regardless of the salivation state, Fc F241A All four glycoforms were equally protected throughout the 10-day timeframe. Furthermore, at the peak of score separation on day 8, each Fc... F241A There was no significant difference in the mean clinical score between the treatment group and the IVIG treatment group. Figure 10B Therefore, although α-2,6 bond sialylation is essential for the anti-inflammatory activity of sialylated IgG Fc, Fc F241A Immunomodulatory effects do not require sialylation.

[0259] Example 9: Preferred receptor binding distinguishes Fc F241A and Fc Abdeg Fc Abdeg (Egamod) is a recently approved FDA therapeutic that accelerates the depletion of circulating total IgG by saturating FcRn and is currently approved for the treatment of myasthenia gravis. Abdeg The Fc region that binds to FcRn contains "Abdeg" mutations (M252Y, S254T, T256E, H433K, and N434F), thus allowing it to outperform natural IgG. Figure 11A and Figure 11B The following experiment was conducted to confirm Fc. Abdeg and Fc F241A / B4ST6 Whether it works through similar receptors and cellular pathways.

[0260] First, surface plasmon resonance (SPR) was used to test WT Fc and Fc using mouse FcRn and human FcRn. F241A / B4ST6 and Fc Abdeg The interaction between WT, Fc, and Fc. F241A / B4ST6 Similarly, with mouse FcRn ( Figure 11C ) and human FcRn ( Figure 11D ) binding. In contrast, for mouse FcRn ( Figure 11C ) and human FcRn ( Figure 11D In terms of Fc Abdeg The dissociation constant (KD) compared to Fc F241A / B4ST6 The levels were 267-fold and 39-fold lower, respectively. Subsequently, the effects of the two mutant Fc on circulating mouse IgG via FcRn were examined. C57BL / 6 and hFcRn (Tg32) mice were administered Fc... F241A / B4ST6 or Fc Abdeg A single intravenous injection was administered, and serum mIgG was determined by ELISA after administration. In wild-type C57BL / 6 mice with mouse FcRn, Fc F241A / B4ST6 Treatment did not reduce serum IgG titers ( Figure 11E In comparison, Fc Abdeg It induced a significant, immediate, and persistent decrease in IgG titers. Figure 11E ). Fc F241A / B4ST6 It does not affect the serum IgG titer in hFcRn (Tg32) mice. Figure 11F However, Fc Abdeg A transient decrease in IgG titer was induced on day 1. These results are consistent with Fc Abdeg The high affinity for mFcRn and the relatively low affinity for hFcRn necessitate multiple administrations in the human body to trigger a long-term decrease in serum IgG.

[0261] Both IVIG and sialylated IgG Fc require mouse C-type lectin-specific ICAM-3-grabbing non-integrin-associated-1 (SIGN-R1) to mediate intracellular anti-inflammatory activity. This requirement can be circumvented by introducing the human ortholog of SIGN-R1, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN). Therefore, Fc was examined via cell binding assays. F241A / B4ST6 and Fc Abdeg The two combine the capabilities of DC-SIGN. From SIGN-R1 - / - and hDC-SIGN + / SIGN-R1 - / - Immortalized bone marrow-derived macrophages (BMDM) were generated in mice, and the cells were then treated with PBS and Fc. F241A / B4ST6 or Fc Abdeg The cells were incubated together. The presence of the receptor was confirmed by flow cytometry using staining with Alexa Fluor 647 anti-hCD209, specifically clone 9E9A8 antibody. Cells were cultured overnight at 37°C and 5% CO2 in high-glucose DMEM medium supplemented with 1X antibiotic-antifungal agent and 1X FBS. The next day, the supernatant was discarded, and cells were separated with enzyme-free dissociation buffer. The suspended cells were collected and centrifuged at 400 rpm for 10 minutes. The pellet was resuspended and washed in binding buffer (1X TBS, 1 nM CaCl2, 2.5% FBS, and 0.05% sodium azide). Fc receptor blockade was achieved by adding anti-mouse CD16 / 32 antibody clone 93. After blocking, cells were washed in binding buffer, followed by the addition of 100 µg / mL Fc receptor. Abdeg and Fc F241A / B4ST6 Then incubate on ice for 1 hour. Afterwards, wash the cells again with binding buffer. Because Fc Abdeg and Fc F241A / B4ST6 Both are based on IgG Fc constructs, and therefore cells were stained with either an APC-conjugated antibody or a PE-conjugated antibody targeting hIgG Fc, i.e., clone M1310G05. Staining was performed in the dark on ice for 30 minutes. After staining, cells were washed with binding buffer and fixed in 2% formaldehyde before analysis. BMDM was analyzed by flow cytometry to detect surface-bound IgG. F241A / B4ST6 or Fc Abdeg SIGN-R1 incubated together - / - No bound IgG was detected on BMDM. Figure 11G However, when using Fc F241A / B4ST6 hDC-SIGN processing + / SIGN-R1 - / - IgG was detected on BMDM, while with Fc Abdeg hDC-SIGN processing + / SIGN-R1 - / - No IgG was detected on BMDM ( Figure 11G Peritoneal macrophages (pMAC) were also isolated to detect surface-bound IgG. Figure 11H and Figure 11I ). with Fc WT In comparison, the significant Fc F241A / B4ST6 With SIGN-R1 + / + pMAC combination ( Figure 11H ). From SIGN-R1 - / - Fc was not observed on the pMAC of mice. F241A / B4ST6 This strong combination.

[0262] To evaluate Fc in vivo F241A / B4ST6 and Fc Abdeg The mechanism of activity difference between C57BL / 6 and SIGN-R1 - / - Mice were administered PBS, high-dose IVIG, and Fc. F241A / B4ST6 or Fc Abdeg K / BxN serum was then administered, and claw swelling was tracked for 10 days. Figures 11J-11L Clinical scores of 0-3 were given to each paw, where a score of 0 represented no inflammation in the paw and a score of 3 represented severe inflammation in all joints of the paw. The mean scores for all four paws of each mouse in the treatment group are shown. In WT mice, significant paw swelling was observed only in mice treated with PBS. Figure 11J and Figure 11L ). In SIGN-R1 - / - In mice, in PBS, IVIG and Fc F241A / B4ST6 Swelling was observed in the treated mice. Figure 11K and Figure 11L However, as measured by clinical scoring, the application of Fc Abdeg SIGN-R1 - / - Mice exhibited significantly less inflammation, including at the peak of the disease (day 7); Figure 11L In summary, these results indicate that F241A / B4ST6 Fc and Fc Abdeg Inhibit autoantibody-induced inflammation through different molecular pathways.

[0263] Example 10: Fc F241A / B4ST6 and Fc Abdeg Combined anti-inflammatory activity To check Fc F241A / B4ST6 and FcAbdeg Whether co-administration in vivo can effectively reduce autoantibody-induced inflammation, in a prophylactic manner in the K / BxN model ( Figure 12A (see above) and therapeutic methods ( Figure 12A (See figure below) Both evaluated Fc F241A / B4ST6 and Fc Abdeg The effects of both mutant Fc cells were significant in preventing inflammation in a manner comparable to high-dose IVIG, regardless of the timing of administration. Figure 11L , Figure 12B and Figure 12C ).

[0264] Due to Fc Abdeg By saturating FcRn to inhibit IgG-mediated inflammation, thereby reducing total IgG titers and significantly enhancing their affinity for mFcRn, FcRn was tested. Abdeg Cause Fc F241A / B4ST6 The ability to clear from the loop more quickly. To determine Fc Abdeg The clearing effect will not interfere with Fc F241A / B4ST6 A combination of active or half-life doses of Fc was administered to C57BL / 6 mice with decreasing doses. Abdeg Combined 50 mg / kg Fc F241A / B4ST6 Human Fc titers were measured on day 10 after administration. Figure 12D ). For example, Fc as measured by ELISA Abdeg Circulating Fc occurs in a dose-dependent manner F241A / B4ST6 The reduction. However, 1 mg / kg, which is one-tenth of the clinical dose, of Fc Abdeg Induced cyclic Fc F241A / B4ST6 Minimal clearance. This was also determined by measuring Fc levels in serum on days 1, 3, and 7. F241A / B4ST6 The level was determined to be 1 mg / kg Fc Abdeg For Fc F241A / B4ST6 The effects of clearing ( Figure 12E A slight decrease in total serum mouse IgG was observed at this dose, with a significant decrease only occurring on day 3 (p = 0.0500).

[0265] Because compared to human FcRn, Fc Abdeg It showed at least a 10-fold enhanced affinity for mouse FcRn, therefore it is inferred that a 10-fold lower dose would be more representative of the human system and would not deplete Fc. F241A / B4ST6 Titer. Therefore, C57BL / 6 mice were administered PBS, high-dose IVIG, and 50 mg / kg Fc. F241A / B4ST6 1 mg / kg Fc Abdeg or 50 mg / kg Fc F241A / B4ST6and 1 mg / kg Fc Abdeg First, followed by K / BxN serum. In a prophylactic model, compared with PBS-treated mice, IVIG, 50 mg / kg Fc F241A / B4ST6 and 1 mg / kg Fc Abdeg Both effectively reduced inflammation. Figure 12F and Figure 12G Surprisingly, compared to either Fc alone, the co-administration of Fc... F241A / B4ST6 and Fc Abdeg Mice showed a greater reduction in inflammation.

[0266] The combined effects of the aforementioned Fc mutant as an anti-inflammatory biologic were further tested in a therapeutic K / BxN model. As described above, mice were administered arthritis-inducing K / BxN serum on day 0 and PBS, high-dose IVIG, and 50 mg / kg Fc on day 2. F241A / B4ST6 1 mg / kg Fc Abdeg or 50 mg / kg Fc F241A / B4ST6 and 1 mg / kg Fc Abdeg And monitor foot swelling over the next few days. Figure 12H and Figure 12I Four days after treatment, i.e., by day 6, co-administration reduced inflammation. Surprisingly, this was significantly different from treatment with either Fc alone. These data indicate that Fc is significantly more effective than either Fc alone. F241A / B4ST6 and Fc Abdeg The combined administration of these drugs led to an enhanced anti-inflammatory response in the body.

[0267] Example 11 Fc F241A Efficacy in an antibody transfer model of pemphigoid disease Autoantibody-mediated pemphigoid disease was mimicked in mice by repeated administration of rabbit anti-mouse type VII collagen polyclonal antibody (a pathogenic autoantibody found in acquired epidermal bullae). Treatment was administered on day 0 (3 hours before the first administration of the pathogenic autoantibody) and again on day 3.

[0268] Using this pemphigus disease model, we evaluated the efficacy of 100 mg / kg Fc. F241A Compared to the FcRN inhibitor egamod (10 mg / kg), this study assessed its potential in preventing autoantibody-mediated vesicle formation. Figure 13A The study demonstrated protection against autoantibody-mediated skin blistering over time. As shown, when treated with the PBS control (hollow circles), the percentage of total skin area affected by blisters increased from ~1% on day 4 to 3%-4% on days 9-10. When treated with Fc...F241A At treatment, 1%–2% of maximum vesicles were observed on days 9–10, indicating a significant reduction in vesicle formation compared to the PBS control. Aigamod resulted in a more moderate degree of disease control, with 2%–3% of maximum vesicles observed on days 9–10.

[0269] In this model, immune complexes form together with type VII collagen antigens in the basement membrane region, leading to FcγRIV receptor cross-linking on the surface of infiltrating neutrophils, which results in a key inflammatory response that induces bullous vesicles. Importantly, inhibitory FcγRIV receptors are essential for protective action (Kasperkiewicz, Nimmerjahn et al., 2012). F241A The therapeutic mechanism is thought to involve upregulation of FcγRIIB on B cells and bone marrow cells (including neutrophils, a key mediator of disease in this model). Therefore, we evaluated treatment-mediated changes in FcγRIIB expression on the cell surface of circulating B cells and bone marrow cells. Figure 13B As shown, Fc, relative to the PBS control, F241A It uniquely resulted in a statistically significant increase in the expression of FcγRIIB on the surface of circulating peripheral blood neutrophils. Egamod did not induce any changes in FcγRIIB expression.

[0270] exist Figures 14A-14C The figure shows the Fc of all responding white blood cell populations in the blood. F241A The dynamics of induced FcγRIIB cell surface expression. These include naïve B cells ( Figure 14A ), activated mature B cells ( Figure 14B ) and neutrophils ( Figure 14C Data indicate that 7–9 days after the last dose (day 3 of the study), Fc F241A It has the potential to maintain elevated FcγRIIB cell surface expression.

[0271] At the end of the study (day 13), biopsies of ear skin affected by the disease were collected from mice and subjected to H&E staining, where images were obtained. Figure 15 Presented in the middle. Showing data from the PBS control group (top row), Fc... F241A Two representative images from the treatment group (middle row) and the Egamod treatment group (bottom row). Images are labeled to highlight areas of cartilage, affected by blisters, blood pooling, and dead skin. (via Fc) F241A The treatment's effectiveness results in near-complete protection against skin damage caused by pathogenic autoantibodies. Importantly, Fc F241A Both Fc and Egamod lead to an early reduction in neutrophil infiltration into skin tissues, with Fc being the most effective. F241AThis resulted in a significant reduction in the number of patients compared to the PBS control on days 3-5 of the study. Figure 16A ). Fc F241A Both iodine and iodine resulted in a significant reduction in infiltrative CD62-ligand-positive monocytes in skin tissue on days 3–13. Figure 16B ).

[0272] Fc F241A It is effective in a mouse model of pemphigoid disease and is associated with increased surface expression of FcγRIIB on circulating neutrophils and reduced skin infiltration by neutrophils (the pathogenic immune cells in this model). These data suggest that Fc F241A Upregulation of the immune checkpoint receptor FcγRIIB mediated by RIIB may have therapeutic implications in patients with pemphigoid diseases, including bullous pemphigoid (BP) and acquired epidermolysis bullosa (EBA).

[0273] Example 12: Fc F241A It has a protective effect in a mouse T cell-mediated neuroinflammation EAE model induced by MOG 35-55. In this embodiment, Fc was examined in experimental autoimmune encephalomyelitis (EAE) (a mouse model of multiple sclerosis). F241A Its anti-inflammatory activity.

[0274] EAE was induced in mice, and then mice were randomly assigned to treatment groups (n = 7 per group). Body weight was recorded daily from day 0 to day 21 of the study, and the data were normalized relative to baseline body weight on day 0. Figure 17A and Figure 17B As shown, in the first few days after EAE induction, all treatment groups exhibited an initial decrease in body weight (~10%), which subsequently recovered. As the disease progressed, the mean body weight of the vector-treated mice compared to the treatment intervention groups (FTY-720, high-dose IVIg, and Fc) increased. F241A To a greater extent reduce ( Figure 17A This was largely not statistically significant; however, overall, the results indicate that Fc F241A It showed a non-significant trend in preventing weight loss. At different concentrations of Fc... F241A Similar studies were conducted at concentrations of 20 mg / kg, 50 mg / kg, and 100 mg / kg. Figure 17B ).and Figure 17A Similar to what was observed in the media group, at any concentration of Fc F241A The treated mice maintained their average body weight. Figure 17B ).

[0275] Clinical scores are presented in Figure 18A , Figure 18B and Figure 18C In the middle. For example Figure 18A and Figure 18C As shown, in the vector control group, measurable disease onset occurred on day 13 of the study and rapidly escalated until reaching a plateau on day 19. Throughout the study, mice treated with FTY-720, mice treated with high-dose IVIg, and mice treated with Fc... F241A Significant protective effects were observed in the treated mice. Figure 18B The area under the curve (AUC) of the inflammation score over the entire time course is shown in the figures for each group. All three treatment interventions demonstrated significant protective effects relative to the mediator control (p < 0.001). There were no significant differences in protective effects at any time point among the three treatment intervention groups. Further analysis was conducted by administering Fc at 20 mg / kg, 50 mg / kg, and 100 mg / kg. F241A An escalation dose assessment was conducted. For example... Figure 18C As shown, compared to the mediator group, the Fc of all doses F241A Both showed significant improvement in clinical scores (p<0.05 on day 18 and p<0.01 on days 19-22).

[0276] These data confirm Fc F241A Protective activity against T cell-mediated neuroinflammation in an EAE model was demonstrated, and mechanisms involving the control of effector T cell-mediated inflammation were exhibited. Not wishing to be bound by any particular theory, IVIg and Fc F241A The protective effects of these substances may vary depending on their ability to amplify Tregs, as seen with previous studies targeting sialylated IVIg and Fc. F241A The mechanism reported (Fiebiger BM et al., “Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type IIFcRs.” Proc Natl Acad Sci US A. 2015 May 5; 112(18):E2385-94).

[0277] Clinical scores were also evaluated in EAE-induced mice from similar experiments. Figure 19 As shown, in the vector control group, measurable disease onset occurred on day 13 of the study and escalated rapidly thereafter. A similar trend was observed in mice treated with WT Fc. In contrast, for Fc... F241AThe treated mice showed a significant protective effect during the study.

[0278] These data indicate that the application of Fc F241A It is associated with improvement in symptoms related to T-cell-mediated inflammation.

[0279] Example 13: Fc F241A Induction of Treg amplification in peripheral blood of wild-type naive mice This embodiment describes experiments conducted in wild-type naïve mice to examine IVIG and Fc observed in Example 12. F241A Is the protective effect due to the expansion of Treg cells?

[0280] Expansion of T-reg cells in naïve mice. C57Bl / 6 mice (n=4 per group) received a single dose iv Administer phosphate-buffered saline (PBS) via bolus injection, 1 g / kg IVIG, or 100 mg / kg Fc. F241A Four days later, peripheral blood was collected to analyze Treg frequency as a percentage of total CD4+ cells.

[0281] like Figure 20A As shown, compared with PBS-treated mice, Fc F241A The treated mice showed a higher percentage of CD4. + CD25 + FOXP3 + Treg cells (P<0.05) indicate Fc F241A Treatment induces Treg amplification.

[0282] Clonalness of expanded Treg cells. To determine whether Treg amplification was due to clonal amplification of antigen-specific Tregs, a series of antigen immunization and treatment experiments were conducted. Figure 20B This is a schematic diagram showing the experimental protocol. Mice (n=2-3 per group) were treated with saline, 2 g / kg IVIG, or 33 mg / kg Fc on day -4 of the study. F241A Or 10 mg / kg Fc F241A Mice were then immunized with the 2W antigen (a model antigen used to track specific clonal T-cell responses) on day 0 of the study. The treatment was repeated on day 1 of the study.

[0283] On day 6, spleen and blood were harvested and analyzed by flow cytometry. The expansion of effector T cells (Teff) and regulatory T cells (Treg) in the spleen was assessed. T cells specific to the 2W antigen were detected using an MHC tetramer binding assay. Figure 20C As shown, compared with the saline group, the IVIG treatment group and the two Fc groupsF241A The treatment groups all had a greater number of antigen-specific Tregs (P<0.05).

[0284] To further depict the expansion of Treg cells relative to Teff cells Figure 20D The ratio of antigen-specific Teff cells to Treg cells was shown in all treatment groups. Although saline-treated mice had approximately 130–135 Teff cells per Treg cell, IVG-treated and Fc-treated mice showed significantly higher Teff cell counts. F241A The treated mice had approximately 20-40 Teff cells per Treg cell.

[0285] The activity of Treg cells. To determine the function of Tregs in the above 2-week immunization experiment, IL10 expression in Tregs was assessed by intracellular staining. Figure 20E The percentage of total spleen-derived 2W antigen-specific Treg cells expressing IL10 is shown. IL10+ antigen-specific Tregs were detected in all treatment groups, including Fc. F241A Two dosage levels.

[0286] These results indicate that Fc F241A Treatment induces peripheral blood Treg amplification, including clonal expansion of antigen-specific T cells after exposure to antigens, and such Tregs include functional Tregs.

[0287] Example 14: Fc F241A It has a protective effect in a mouse model of nephrotoxic nephritis (NTN) mediated by autoantibody-induced kidney injury. In order to evaluate Fc F241A The anti-inflammatory and protective properties of Fc in autoantibody-induced inflammation were evaluated in a mouse model of nephrotoxic nephritis (NTN). F241A ( Figure 21A Experimental NTN was induced in mice by first administering 200 mg sheep IgG (BioRad) intraperitoneally in complete Freund's adjuvant (CFA) (day -4). Four days later (day 0), mice were treated with PBS, 1 g / kg IVIg, or 50–200 mg / kg FC. F241A One hour later, at a rate of 2 per gram of body weight... Mice were administered ovine α-GBM serum (Probetex) via intravenous injection at a dose of 1. On day 7, serum was collected from the treated mice for the measurement of mouse α-sheep IgG and blood urea nitrogen (BUN). Mouse α-sheep IgG was determined by ELISA as described in Example 3. BUN was determined using a urea assay kit employing an enzyme-coupled equilibration method. NTN-treated mice were administered PBS, IVIg, or 200 mg / kg, 100 mg / kg, or 50 mg / kg of FC. F241A .

[0288] Anti-sheep IgG titers were similar across all treatment groups, indicating the effectiveness of IVIG and Fc. F241A It does not affect the pathogenic immune response. Figure 21B On day 7, blood urea nitrogen (BUN) levels increased in PBS-treated animals, but IVIG and Fc at 200 mg / kg and 100 mg / kg remained unchanged. F241A This significantly reduced it, while 50 mg / kg of Fc F241A Then it is not possible ( Figure 21C In summary, these results prove Fc F241A It has the effect of protecting against kidney damage mediated by autoantibodies.

[0289] sequence list Other implementation plans Various modifications and variations of the disclosure described herein will be apparent to those skilled in the art without departing from its scope and spirit. Although the disclosure has been described in conjunction with specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. Indeed, it will be apparent to those skilled in the art that various modifications to the implementation of the methods described herein are intended within the scope of the disclosure. Other embodiments are described in the claims.

Claims

1. A method for treating an autoimmune disorder affecting the skin and / or kidneys in a human subject with appropriate need, the method comprising: The subject is given a therapeutically effective amount of a population of modified Fc peptides or a pharmaceutical composition thereof, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), the population comprising: at least 50% of the modified Fc peptide having a sialic acid (SA) moiety attached to the N-glycan of the modified Fc peptide via an α(2,6) bond.

2. The method according to claim 1, wherein the aliphatic amino acid residue at position 241 is alanine (Ala; F241A).

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

4. The method according to any one of claims 1-3, wherein at least 60% of the modified Fc polypeptide comprises an SA moiety of an N-glycan attached to the modified Fc polypeptide via an α(2,6) bond.

5. The method according to any one of claims 1-4, wherein at least 70% of the modified Fc polypeptide comprises an SA moiety of an N-glycan attached to the modified Fc polypeptide via an α(2,6) bond.

6. The method according to any one of claims 1-5, wherein at least 80% of the modified Fc polypeptide comprises an SA moiety of an N-glycan attached to the modified Fc polypeptide via an α(2,6) bond.

7. The method according to any one of claims 1-6, wherein at least 90% of the modified Fc polypeptide comprises an SA moiety of an N-glycan attached to the modified Fc polypeptide via an α(2,6) bond.

8. The method according to any one of claims 1-7, wherein the N-glycan of the modified Fc polypeptide is monosialylated or disialylated.

9. The method of claim 8, wherein at least 30% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

10. The method according to any one of claims 8-9, wherein at least 40% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

11. The method according to any one of claims 8-10, wherein at least 50% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

12. The method according to any one of claims 8-11, wherein at least 60% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

13. The method according to any one of claims 8-12, wherein at least 70% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

14. The method according to any one of claims 8-13, wherein at least 80% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

15. The method according to any one of claims 8-14, wherein at least 90% of the modified Fc polypeptide comprises a monosialylated N-glycan, the monosialylated N-glycan comprising an SA moiety attached via the α(2,6) bond.

16. The method of claim 8, wherein at least 30% of the modified Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

17. The method of claim 8 or claim 16, wherein at least 40% of the Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

18. The method of any one of claims 8, 16 and 17, wherein at least 50% of the Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

19. The method according to any one of claims 8 and 16-18, wherein at least 60% of the Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

20. The method according to any one of claims 8 and 16-19, wherein at least 70% of the Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

21. The method according to any one of claims 8 and 16-20, wherein at least 80% of the Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

22. The method according to any one of claims 8 and 16-21, wherein about 90% of the modified Fc polypeptide comprises a disialialylated N-glycan, the disialialylated N-glycan comprising two SA moieties attached via the α(2,6) bond.

23. The method according to any one of claims 1-22, wherein at least about 60% of the modified Fc polypeptide comprises a galactose moiety.

24. The method according to any one of claims 1-23, wherein at least about 70% of the modified Fc polypeptide comprises a galactose moiety.

25. The method according to any one of claims 1-24, wherein at least about 80% of the modified Fc polypeptide comprises a galactose moiety.

26. The method according to any one of claims 1-25, wherein at least about 90% of the modified Fc polypeptide comprises a galactose moiety.

27. The method according to any one of claims 1-26, wherein about 100% of the modified Fc polypeptide comprises a galactose moiety.

28. The method according to any one of claims 23-27, wherein the galactose portion is attached to the α(1,3) arm and / or α(1,6) arm of the N-glycan.

29. The method according to any one of claims 23-28, wherein the galactose portion is a branched galactose portion.

30. A method for treating an autoimmune disorder affecting the skin and / or kidneys in a human subject with a corresponding need, the method comprising: A population of modified Fc peptides or a pharmaceutical composition thereof, administered to the human subject in a therapeutically effective amount, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), the population comprising: about 40% of the modified Fc peptide having a sialic acid (SA) moiety attached to the N-glycan of the modified Fc peptide via an α(2,3) bond.

31. The method of claim 30, wherein the aliphatic amino acid residue at position 241 is alanine (Ala; F241A).

32. The method according to any one of claims 1-31, wherein the modified Fc polypeptide is an IgG1 Fc polypeptide.

33. The method according to any one of claims 1-31, wherein the modified Fc polypeptide is an IgG3 Fc polypeptide.

34. The method according to any one of claims 1-33, wherein the autoimmune disorder affects the skin.

35. The method of claim 34, wherein the autoimmune disorder is acquired epidermal bullae (EBA).

36. The method according to any one of claims 1-35, wherein the autoimmune disorder affects the kidney.

37. The method of claim 36, wherein the autoimmune disorder is immune-mediated glomerulonephritis.

38. The method according to any one of claims 1-37, wherein the population or the pharmaceutical composition has a half-life of at least 3.5 days after administration to the human subject.

39. The method of claim 38, wherein the population or the pharmaceutical composition has a half-life of at least 4 days after administration to the human subject.

40. The method according to any one of claims 1-39, wherein the population or the pharmaceutical composition is cleared from the circulation of the human subject at a rate not greater than 45 mL / day / kg.

41. The method of claim 40, wherein the population or the pharmaceutical composition is cleared from the circulation of the human subject at a rate not exceeding 40 mL / day / kg.

42. The method of claim 40, wherein the population or the pharmaceutical composition is cleared from the circulation of the human subject at a rate not exceeding 35 mL / day / kg.

43. The method of claim 40, wherein the population or the pharmaceutical composition is cleared from the circulation of the human subject at a rate not exceeding 30 mL / day / kg.

44. The method of claim 40, wherein the population or the pharmaceutical composition is cleared from the circulation of the human subject at a rate not exceeding 25 mL / day / kg.

45. The method according to any one of claims 1-44, wherein the concentration (AUC) of the modified Fc peptide over time is at least 480 days × mg / mL between 1 day and 35 days after administration of the population or the pharmaceutical composition to the human subjects.

46. ​​The method of claim 45, 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 day and 35 days after administration of the human subject to the population or the pharmaceutical composition.

47. The method according to any one of claims 1-46, further comprising administering an additional therapeutic agent to the human subject.

48. The method of claim 47, wherein the additional therapeutic agent is administered to the human subject before, during, or after the administration of the population or the pharmaceutical composition.

49. The method of claim 47 or 48, 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.

50. The method of claim 49, wherein the second modified Fc polypeptide comprises amino acid substitutions of M252Y, S254T, T256E, H433K, and N434F.

51. The method according to claim 49 or 50, wherein the second modified Fc polypeptide has the amino acid sequence shown in SEQ ID NO:

5.

52. The method according to any one of claims 49-51, wherein the second modified Fc polypeptide is administered at a dose of 1 mg / kg to 20 mg / kg.

53. The method according to any one of claims 49-51, wherein the second modified Fc polypeptide is administered at a dose of 10 mg / kg.

54. The method according to any one of claims 49-53, wherein the second modified Fc polypeptide is administered once a week for four weeks.

55. The method according to any one of claims 49-54, wherein the additional therapeutic agent is selected from the group consisting of: anti-inflammatory agents, immunosuppressants, analgesics, disease-modifying antirheumatic drugs (DMARDs), anti-irritants, platelet-enhancing drugs, thrombopoietin receptor (TPOR) agonists, physical therapy, and surgery.

56. The method of claim 55, wherein the anti-inflammatory agent is selected from the group consisting of: nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, anti-inflammatory antibodies or antigen-binding fragments thereof, anti-inflammatory cytokines, kinase inhibitors, and intravenous immunoglobulins (IVIG).

57. A population of modified Fc peptides or a pharmaceutical composition thereof, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), said population comprising: at least 60% of said modified Fc peptide having a sialic acid (SA) moiety attached to the N-glycan of said modified Fc peptide via an α(2,6) bond, for use as a medicament for treating autoimmune disorders affecting the skin and / or kidneys in human subjects with corresponding need.

58. A population of modified Fc peptides or a pharmaceutical composition thereof, each modified Fc peptide 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 (according to the Kabat number; corresponding to amino acid residue 32 of SEQ ID NO: 2), said population comprising: at least 60% of said modified Fc peptide having a sialic acid (SA) moiety attached to the N-glycan of said modified Fc peptide via an α(2,6) bond, for treating autoimmune disorders affecting the skin and / or kidneys in human subjects with corresponding need.

59. The group of modified Fc peptides according to claim 57 or 58 or the pharmaceutical composition thereof, wherein the autoimmune disorder affects the skin.

60. The population of modified Fc peptides according to claim 59 or the pharmaceutical composition thereof, wherein the autoimmune disorder is acquired epidermal bullae (EBA).

61. The population of modified Fc peptides according to claim 57 or 58 or the pharmaceutical composition thereof, wherein the autoimmune disorder affects the kidneys.

62. The population of the modified Fc polypeptide according to claim 61 or the pharmaceutical composition thereof, wherein the autoimmune disorder is immune-mediated glomerulonephritis.

Images