Fc region modifiers

Abstract

According to one embodiment, provided is an amino acid-modified sialylated Fc region modified body containing the following (1) and (2): (1) the 234-th position and / or the 235-th position is substituted with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine; and (2) the 293rd site or the 294th site is deleted.

Description

Technical Field

[0001] This invention relates to Fc region modifiers, sialylated Fc region modifiers, polypeptides containing the sialylated Fc region modifiers, and pharmaceutical compositions containing the polypeptides. Background Technology

[0002] In recent years, the development of antibody drugs has been active and has attracted considerable attention. For example, intravenous immunoglobulin therapy (IVIG) using IgG antibodies has been used to treat autoimmune and inflammatory diseases such as idiopathic thrombocytopenic purpura (ITP), Kawasaki disease, and Guillain-Barré syndrome (GBS) (Non-Patent Literature 1). In IVIG, for example, monoclonal IgG antibodies are isolated from mixed plasma from thousands of healthy individuals and prepared into a drug for therapeutic use. However, antibody drugs still face various problems, including high dosage, high manufacturing costs, and the potential for the formation of anti-drug antibodies. If antibody drugs with high therapeutic efficacy can be developed, it is expected that the same or even higher efficacy can be achieved even with reduced dosages.

[0003] Sialic acid is one of the factors that contribute to the therapeutic effects of various antibodies. Sialic acid, also known as N-acetylneuraminic acid (Neu5Ac), binds to galactose residues in the Fc region, forming the non-reducing terminus of the IgG Fc N-glycan. It is known that anti-inflammatory effects are achieved through sialylation of the Fc region of IgG; a high sialylation rate yields good results (Non-Patent Literature 2); sialic acid in intravenously injected immunoglobulins, when combined with sugar chains, inhibits B cells via CD22 (also known as Siglec-2) (Non-Patent Literature 3), etc.

[0004] Therefore, sialylation of the Fc region of immunoglobulins has attracted attention. It is known that peptides containing Fc region modifiers with increased sialic acid content have increased half-lives and satisfactory anti-inflammatory properties (Patent Document 1). As a method to increase the sialic acid content in the Fc region, it is known to express antibodies with mutations in the Fc domain (e.g., F243, V264, and D265) in host cells expressing β-galactosyltransferase and sialyltransferase activities (Patent Document 1). Furthermore, a method to increase the sialic acid content by mutating specific amino acids in the Fc region (e.g., amino acids at positions 240–243, 258–267, and 290–305) has been studied using HEK293 and YB2 / 0 cells (Patent Document 2).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2012 / 113863

[0008] Patent Document 2: Japanese Patent Publication No. 2017-522040

[0009] Non-patent literature

[0010] Non-patent literature 1: Journal of Allergy and Clinical Immunology, March 2017, Vol. 139 (3), Supplement, pp. S1-S46

[0011] Non-patent literature 2: Science, August 4, 2006, Vol.313 (5787), pp.670-673

[0012] Non-patent literature 3: Blood, September 9, 2010, Vol. 116 (10), pp. 1698-1704 Summary of the Invention

[0013] The problem that the invention aims to solve

[0014] The purpose of this invention is to provide a sialylated Fc region modifier with a further increased amount of sialic acid.

[0015] Methods for solving problems

[0016] The inventors conducted in-depth research and found that by adding specific amino acids to the Fc region of immunoglobulins, more sialic acid is added when they are sialylated.

[0017] The present invention is as described below, for example.

[0018] [1] A sialylated Fc region modifier containing the following amino acid modifications (1) and (2): (1)(a) Positions 234 and / or 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine (here, the amino acids replaced by positions 234 and 235 may be the same or different); (b) Position 234 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine, and position 235 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine; or (c) Position 234 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and position 235 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine. (2) The 293rd or 294th position is missing (here, the amino acid residue is numbered according to the amino acid position following the EU index).

[0019] [2] According to the sialylated Fc region modified body described in [1], wherein positions 234 and 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine or isoleucine (here, the amino acids replaced by positions 234 and 235 may be the same or different).

[0020] [3] According to the sialylated Fc region modifier described in [1] or [2], wherein, Positions 234 and 235 were replaced with alanine, or The 234th position was replaced with alanine, and the 235th position was replaced with glutamic acid.

[0021] [4] The sialylated Fc region modifier according to any one of [1] to [3], wherein the Fc region is derived from human IgG.

[0022] [5] The sialylated Fc region modified according to [4], wherein the Fc region is derived from human Igγ1, human Igγ2, human Igγ3 or human Igγ4.

[0023] [6] The sialylated Fc region modifier according to [4] contains an amino acid sequence that is 90% or more identical to the sequence consisting of amino acids from position 218 to position 448 of the amino acid sequence shown in Serial No. 7 or an amino acid sequence that is 90% or more identical to the sequence consisting of amino acids from position 217 to position 447 of the amino acid sequence shown in Serial No. 13.

[0024] [7] A polypeptide containing any one of [1] to [6] sialylated Fc region modifiers.

[0025] [8] The polypeptide according to [7], wherein the polypeptide containing the above-mentioned sialylated Fc region modified body is an antibody.

[0026] [9] The polypeptide according to [8], wherein the antibody is an IgG antibody.

[0027]

[10] The polypeptide according to [7], wherein the polypeptide containing the above-mentioned sialylated Fc region modified body is an Fc fusion protein molecule.

[0028]

[11] The polypeptide according to

[10] , wherein the above-mentioned sialylated Fc fusion protein molecule contains a protein of non-human origin.

[0029]

[12] A polynucleotide that encodes a polypeptide as described in any one of [7] to

[11] .

[0030]

[13] An expression vector containing the polynucleotide described in

[12] .

[0031]

[14] A host cell that has been transformed with the expression vector described in

[13] .

[0032]

[15] A method for producing a polypeptide containing a sialylated Fc region modifier, comprising the step of culturing the host cells described in

[14] .

[0033]

[16] The method according to

[15] further includes the step of adding or expressing β-galactosidase α-2,6-sialyltransferase 1 (ST6GAL1).

[0034]

[17] An Fc region modified body containing the following amino acid modifications (1) and (2): (1)(a) Positions 234 and / or 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine (here, the amino acids replaced by positions 234 and 235 may be the same or different); (b) Position 234 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine, and position 235 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine; or (c) Position 234 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and position 235 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine. (2) The 293rd or 294th position is missing (here, the amino acid residue is numbered according to the amino acid position following the EU index).

[0035]

[18] A pharmaceutical composition comprising any one of the polypeptides described in [7] to

[11] and a pharmaceutically acceptable carrier.

[0036]

[19] The pharmaceutical composition according to

[18] is used to treat autoimmune diseases.

[0037]

[20] The polypeptide according to any one of [7] to

[11] is used to treat autoimmune diseases.

[0038]

[21] A method for treating an autoimmune disease, comprising administering a therapeutically effective amount of any one of [7] to

[11] a polypeptide to a subject.

[0039] The use of any one of the polypeptides described in

[22] , [7] to

[11] in the manufacture of a pharmaceutical composition for the treatment of autoimmune diseases.

[0040] Invention Effects

[0041] According to the present invention, a sialylated Fc region modifier with a further increased amount of sialic acid can be provided. Attached Figure Description

[0042] Figure 1 This diagram depicts the hinge-to-CH2 dimer portion of the full-length IgG1 structure (PDB number: 1HZH). Multiple spheres represent leucine residues at positions 234 and 235 (L234 / L235), and multiple rods represent the glycan chains.

[0043] Figure 2 This diagram is created by overlaying the model structure of the sialylated E294Del mutant with the wild-type structure and depicting the N297 ring and its surrounding structures. Multiple spheres represent L234 / L235, and multiple rods represent the glycan chains (gray: crystal structure of wild-type IgG1, black: model structure of the sialylated E294Del mutant). In the sialylated E294Del mutant, compared to the wild-type, the N297 ring endowed with glycan chains is shorter and the glycan chains are twisted.

[0044] Figure 3 This is a diagram showing the model structure of the sialylated E294Del mutant, including the peripheral structure of L234 / L235. L234 / L235 is depicted using multiple spheres, and the sugar chains are depicted using multiple rods.

[0045] Figure 4 This diagram shows the peripheral structures at positions 234 and 235 (L234A / L235A) of the model structure of the sialylated E294 deletion and L234A / L235A mutant. Alanine residues at positions 234 and 235 are depicted using multiple spheres, and the glycans are depicted using multiple rods.

[0046] Figure 5 This diagram shows the peripheral structures at positions 234 and 235 (L234A / L235E) of the model structure of the sialylated E294 deletion and L234A / L235E mutant. Multiple spheres depict alanine at position 234 and glutamic acid at position 235, and multiple rods depict the glycan chains.

[0047] Figure 6 This diagram shows the model structure of the sialylated E294 deletion and L234F / L235Q mutant, including the peripheral structures at positions 234 and 235 (L234F / L235Q). Multiple spheres represent phenylalanine at position 234 and glutamine at position 235, while multiple rods represent the glycan chains. Detailed Implementation

[0048] The embodiments of the present invention will be described in detail below. It should be noted that the materials, structures, etc., described below are not intended to limit the present invention, and various modifications can be made within the scope of the spirit of the present invention.

[0049] 1. Definition

[0050] Unless otherwise defined herein, the terms used herein shall be used in the sense commonly understood by those skilled in the art.

[0051] Antibodies (or immunoglobulins) are glycoproteins composed of four chains forming a Y-shaped structure with bilateral symmetry, consisting of two heavy chains with a single sequence and two light chains with a single sequence. There are five types of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of antibody molecules is common across all types: two heavy chains with a molecular weight of 50,000–70,000 and two light chains with a molecular weight of 20,000–30,000 are linked by disulfide bonds and non-covalent bonds to form an antibody molecule with a molecular weight of 150,000–190,000, consisting of four Y-shaped chains. The heavy chains are typically composed of polypeptide chains containing approximately 440 amino acids and have characteristic structures in each type, corresponding to IgG, IgM, IgA, IgD, and IgE as Igγ, Igμ, Igα, Igδ, and Igε, respectively. In addition, IgG exists in subclasses: IgG1, IgG2, IgG3, and IgG4, with their corresponding heavy chains designated as Igγ1, Igγ2, Igγ3, and Igγ4, respectively. Light chains typically consist of polypeptide chains containing approximately 220 amino acids, and are known to exist in two forms: L-type and K-type, designated Igλ and Igκ, respectively. These two types of light chains can pair with any type of heavy chain.

[0052] Regarding intrachain disulfide bonds in antibody molecules, there are 4 in the heavy chain (5 in Igμ and Igε) and 2 in the light chain, forming a ring every 100-110 amino acid residues. Their three-dimensional structures are similar between the rings and are called structural units or domains. In both the heavy and light chains, the N-terminal domain is called the variable region. It is known that even antibodies produced from the same class (or subclass) of the same animal species have diverse amino acid sequences, participating in the specific binding of antibodies to antigens. The amino acid sequence of the C-terminal domain downstream of the variable region is roughly constant across classes or subclasses and is called the constant region. In the heavy chain, from the N-terminus to the C-terminus, there are heavy chain variable regions (VH) and heavy chain constant regions (CH). Within the CH, from the N-terminus, there are three domains: CH1, CH2, and CH3 (in the case of IgG). In the light chain, from the N-terminus to the C-terminus, there are light chain variable regions (VL) and light chain constant regions (CL).

[0053] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL are highly variable, contributing to the variability of the variable region. CDRs are regions of approximately 5–10 amino acid residues located at the N-terminus of the heavy and light chains, in the order CDR1, CDR2, and CDR3, respectively, forming the antigen-binding site. On the other hand, the portion of the variable region outside the CDRs is called the framework region (FR), composed of FR1–4, and its amino acid sequence varies less.

[0054] When the antibody is treated with papain, a proteolytic enzyme, three antibody fragments are obtained. The two fragments at the N-terminus are called the Fab (antigen-binding) region. In this specification, the "Fab region" refers to the region composed of the VH and CH1 domains of the heavy chain and the light chains (VL and CL), through which the antigen-binding site at the front end of the Fab region binds to the antigen. In this specification, the "heavy chain fragment" refers to the fragment composed of the VH and CH1 domains of the heavy chain that constitute the Fab region.

[0055] The C-terminal fragment is referred to as the Fc (crystallizable) region. In this specification, the "Fc region" refers to a polypeptide composed of the constant regions of the antibody, excluding the CH1 domain. Therefore, the Fc regions of IgA, IgD, and IgG are the last two heavy chain constant regions (CH2 and CH3) on the C-terminal side, while the Fc regions of IgE and IgM contain the last three heavy chain constant regions (CH2, CH3, and CH4) on the C-terminal side. Additionally, where present, the hinge region, J chain, etc., may also be included and referred to as the "Fc region." For example, the Fc region of human IgG1 contains polypeptides composed of CH2 and CH3 domains, as well as polypeptides composed of part or all of the hinge region, and CH2 and CH3 domains. The Fc regions of other antibody classes can be determined by sequence alignment with the heavy chain or fragments of human IgG1.

[0056] In this specification, "Fc region modifier" refers to an Fc region modified with at least one amino acid relative to the wild-type Fc region. Amino acid modification includes substitution, addition, deletion, insertion, and alteration of amino acids. In this specification, the terms "modification" and "mutation" are used interchangeably for amino acids. Additionally, in this specification, "sialylated Fc region modifier" refers to an Fc region modifier obtained by sialylation (i.e., sialic acid addition) using methods known in the art. In this specification, unless the context specifically limits it, the term "Fc region modifier" is used to encompass both unsialylated and sialylated Fc region modifiers. The use of the terms "Fc region" or "Fc region modifier" in this specification is also intended to include complexes (e.g., dimers) composed of two or more Fc regions.

[0057] One-armed antibodies and one-armed Fc fusion proteins are also included within the scope of this invention. For example, in the case of a one-armed IgG antibody, a complex comprising one Fab region and two Fc regions has a structure in which a heavy chain fragment of the Fab region is attached to one of the two Fc regions. In one embodiment, the one-armed antibody comprises one heavy chain (VH, CH1 domains, and a sialylated Fc region modifier (hinge region + CH2 domain + CH3 domain)), one light chain (VL and CL), and a sialylated Fc region modifier. In one embodiment, the one-armed Fc fusion protein has a structure in which another protein or peptide is bound only to one of the complexes comprising the two sialylated Fc region modifiers.

[0058] In this specification, "multispecific antibody" refers to an antibody that can specifically bind to two or more different antigens, and is called, for example, a bispecific antibody or a trispecific antibody, depending on the number of antigens bound. Multispecific antibodies include complexes of two or more antibodies and / or antigen-binding fragments that can bind to different antigens respectively. The term "antibody" as used in this specification includes multispecific antibodies unless otherwise specified in the context.

[0059] In this specification, "bispecific antibody" refers to an antibody that can specifically bind to two different antigens.

[0060] In this specification, "human antibody" refers to an antibody having the amino acid sequence of human immunoglobulins. In this specification, "humanized antibody" refers to an antibody in which some, most, or all of the amino acid residues other than the CDR are replaced by amino acid residues derived from human immunoglobulin molecules. There are no particular limitations on the method of humanization; for example, humanized antibodies can be prepared with reference to U.S. Patent No. 5,225,539 and U.S. Patent No. 6,180,370.

[0061] The Fc region or the amino acid residue numbering of the antibody used in this specification follows the EU index proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, NIH Publication No. 91-3242).

[0062] In this specification, "linked" or "linked" means that multiple components (e.g., Fab and Fc regions) are bound directly or by means of one or more mediators (e.g., peptide linkers). In this specification, a "peptide linker" refers to one or more arbitrary amino acid residues that can be introduced through genetic engineering for linking two or more parts (e.g., between the heavy chain variable region and the light chain variable region of an antibody). The length of the peptide linkers used in this invention is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose.

[0063] In this specification, "consistency" refers to the Identity value obtained using the EMBOSS Needle (Nucleic Acids Res., 2015, Vol. 43, pW580-W584) with the default settings. These parameters are described below.

[0064] Gap Open Penalty=10

[0065] Gap Extend Penalty=0.5

[0066] Matrix=EBLOSUM62

[0067] End Gap Penalty=false

[0068] In this specification, "object" refers to a person or other animal that requires the prevention or treatment. In one approach, it refers to a person who requires the prevention or treatment.

[0069] 2. Sialidized Fc region modifier

[0070] One embodiment of the present invention comprises a sialylated Fc region modifier containing the following amino acid modifications (1) and (2): (1)(a) Positions 234 and / or 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine (here, the amino acids replaced by positions 234 and 235 may be the same or different); (b) Position 234 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine, and position 235 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine; or (c) Position 234 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and position 235 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine. (2) The 293rd or 294th position is missing (here, the amino acid residue is numbered according to the amino acid position following the EU index).

[0071] That is, the sialylated Fc region modifier of one embodiment of the present invention contains the amino acid modifications of both (1) and (2) above. For (1), it is sufficient to contain any of the amino acid modifications from (a) to (c). Compared with the sialylation of the wild-type Fc region and the sialylation of the Fc region modifier that only lacks the amino acid at position 294, the sialylated Fc region modifier has an increased amount of sialic acid added (i.e., the degree of sialylation). In particular, the sialylated Fc region modifier of the embodiment has an increased amount of sialic acid added to the sugar chain bound to asparagine at position 297.

[0072] The reason why the amount of sialic acid added (especially sialic acid added to the sugar chain bound to asparagine at position 297) can be increased by the amino acid modification described above can be inferred as follows. Although it is known that modification (especially deletion) of the amino acid at position 294 of the Fc region helps to increase the amount of sialic acid added (Patent Document 2), the inventors have found that when the amino acid at position 294 is deleted, the starting position of the sugar chain (near position 297) may be distorted. Figure 1 This is a diagram showing the three-dimensional structure of the CH2 domain of wild-type IgG. Additionally, Figure 2 This is a diagram showing the three-dimensional structure of wild-type IgG with the glutamate at position 294 missing. (See diagram for reference.) Figure 1 As shown, in wild-type IgG, the contact surfaces between CH2 dimers are mainly glycan chains and leucine residues at positions 234 and 235. When glutamate at position 294 is absent, as... Figure 2 As shown, the ring containing asparagine at position 297, which serves as the starting position of the sugar chain, becomes shorter, resulting in misalignment near the starting position of the sugar chain and causing the overall sugar chain to twist.

[0073] Figure 3 This diagram illustrates the distortion of the sugar chain caused by the deletion of glutamic acid at position 294, presented in three-dimensional form. As a result of this distortion, the interaction between leucine residues at positions 234 and 235 is primarily involved in the contact between CH2 dimers. These leucine residues are bound together by van der Waals forces, leaving no space between them and preventing further distortion of the sugar chain.

[0074] The inventors attempted to modify the amino acids at positions 234 and / or 235 to reduce their interactions, and found that the amount of sialic acid added to the sugar chain increased. This is believed to be because when the interaction between positions 234 and 235 is reduced, steric leeway is created between them, allowing for further twisting of the sugar chain. By twisting the sugar chain at positions where sialic acid readily adds, the amount of sialic acid added to the sugar chain is increased. Figure 4 This diagram illustrates the three-dimensional structure of wild-type IgG when leucine at positions 234 and 235 are replaced with alanine, respectively, based on the deletion at position 294. According to... Figure 4 When leucine at positions 234 and 235 are replaced with alanine, the van der Waals forces between them weaken, resulting in a space between them. It is believed that this surplus in the molecular structure allows for further twisting of the sugar chains, leading to an increase in the amount of sialic acid added.

[0075] The substitution of amino acids at positions 234 and / or 235 is not limited to the methods described above. The desired effect can be obtained by selecting an amino acid at that position in a way that reduces the interaction between the amino acids at positions 234 and 235. As a modification to reduce the interaction between the amino acids at positions 234 and 235, a modification that weakens the van der Waals forces between the amino acids at positions 234 and 235 is considered. As a modification to weaken the van der Waals forces between the amino acids at positions 234 and 235, a substitution of at least one of the amino acids at positions 234 and 235 with an amino acid that is less hydrophobic or more hydrophilic than the original amino acid and / or a smaller (smaller in size) amino acid than the original amino acid is considered. For example, if the amino acids at positions 234 and 235 before substitution are both leucine, consider replacing one or both of them with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, or methionine, which are less hydrophobic than leucine (J Mol Biol., 1982, Vol. 157(1), p. 105-32). When arranging amino acids in descending order of volume, the order is: tryptophan > tyrosine > arginine > phenylalanine > histidine > methionine > glutamic acid > lysine > glutamine > aspartic acid > asparagine > leucine > isoleucine > cysteine ​​> threonine > valine > proline > serine > alanine > glycine. Therefore, replacing at least one of the amino acids at positions 234 and 235 with an amino acid smaller in volume than the original amino acid means replacing it with an amino acid smaller in volume than the original amino acid, following the volume order described above. For example, if the original amino acids at positions 234 and 235 are both leucine, consider replacing one or both with isoleucine, cysteine, threonine, valine, proline, serine, alanine, or glycine, which are smaller amino acids. Alternatively, when replacing one of the amino acids at positions 234 and 235 with a hydrophilic amino acid (serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine), the other amino acid can be replaced with a hydrophobic amino acid that is larger in volume than the original amino acid. For example, if the amino acids at positions 234 and 235 before the substitution are both leucine, one of them can be replaced with a hydrophilic amino acid, and the other can be replaced with tryptophan, tyrosine, phenylalanine, or methionine, which are larger hydrophobic amino acids. As an example of such amino acid modification that reduces the interaction between the amino acids at positions 234 and 235, the amino acid modification described in (1) above can be cited.

[0076] As a combination of modifications that weaken the van der Waals interaction between the amino acids at positions 234 and 235, when either leucine is present at position 234 or 235, the other can be replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine.

[0077] In addition, the following combinations of amino acids at positions 234 and 235, which weaken van der Waals interactions, can also be listed: (1) Isoleucine-lysine, isoleucine-arginine, isoleucine-asparagine, isoleucine-aspartic acid, isoleucine-glutamine, isoleucine-glutamic acid, isoleucine-histidine, isoleucine-serine, isoleucine-threonine, isoleucine-methionine, isoleucine-glycine, isoleucine-alanine, isoleucine-cysteine, isoleucine-proline, isoleucine-valine, isoleucine-isoleucine; (2) Valine-lysine, valine-arginine, valine-asparagine, valine-aspartic acid, valine-glutamine, valine-glutamic acid, valine-histidine, valine-serine, valine-threonine, valine-methionine, valine-glycine, valine-alanine, valine-cysteine, valine-proline, valine-valine, valine-isoleucine; (3) Methionine-lysine, methionine-arginine, methionine-asparagine, methionine-aspartic acid, methionine-glutamine, methionine-glutamic acid, methionine-histidine, methionine-serine, methionine-threonine, methionine-methionine, methionine-glycine, methionine-alanine, methionine-cysteine, methionine-proline, methionine-valine, methionine-isoleucine; (4) Proline-lysine, proline-arginine, proline-asparagine, proline-aspartic acid, proline-glutamine, proline-glutamic acid, proline-histidine, proline-serine, proline-threonine, proline-methionine, proline-glycine, proline-alanine, proline-cysteine, proline-proline, proline-valine, proline-isoleucine; (5) Cysteine-lysine, cysteine-arginine, cysteine-asparagine, cysteine-aspartic acid, cysteine-glutamine, cysteine-glutamic acid, cysteine-histidine, cysteine-serine, cysteine-threonine, cysteine-methionine, cysteine-glycine, cysteine-alanine, cysteine-proline, cysteine-valine, cysteine-isoleucine; (6) Alanine-lysine, alanine-arginine, alanine-asparagine, alanine-aspartic acid, alanine-glutamine, alanine-glutamic acid, alanine-histidine, alanine-serine, alanine-threonine, alanine-methionine, alanine-glycine, alanine-alanine, alanine-cysteine, alanine-proline, alanine-valine, alanine-isoleucine; (7) Glycine-lysine, glycine-arginine, glycine-asparagine, glycine-aspartic acid, glycine-glutamine, glycine-glutamic acid, glycine-histidine, glycine-serine, glycine-threonine, glycine-methionine, glycine-glycine, glycine-alanine, glycine-cysteine, glycine-proline, glycine-valine, glycine-isoleucine; (8) Threonine-lysine, threonine-arginine, threonine-asparagine, threonine-aspartic acid, threonine-glutamine, threonine-glutamic acid, threonine-histidine, threonine-serine, threonine-threonine, threonine-methionine, threonine-glycine, threonine-alanine, threonine-cysteine, threonine-proline, threonine-valine, threonine-isoleucine; (9) Serine-lysine, Serine-arginine, Serine-asparagine, Serine-aspartic acid, Serine-glutamine, Serine-glutamic acid, Serine-histidine, Serine-serine, Serine-threonine, Serine-methionine, Serine-glycine, Serine-alanine, Serine-cysteine, Serine-proline, Serine-valine, Serine-isoleucine; (10) Lysine-lysine, lysine-arginine, lysine-asparagine, lysine-aspartic acid, lysine-glutamine, lysine-glutamic acid, lysine-histidine, lysine-serine, lysine-threonine, lysine-methionine, lysine-glycine, lysine-alanine, lysine-cysteine, lysine-proline, lysine-valine, lysine-isoleucine; (11) Histidine-lysine, histidine-arginine, histidine-asparagine, histidine-aspartic acid, histidine-glutamine, histidine-glutamic acid, histidine-histidine, histidine-serine, histidine-threonine, histidine-methionine, histidine-glycine, histidine-alanine, histidine-cysteine, histidine-proline, histidine-valine, histidine-isoleucine; (12) Glutamine-lysine, glutamine-arginine, glutamine-asparagine, glutamine-aspartic acid, glutamine-glutamine, glutamine-glutamic acid, glutamine-histidine, glutamine-serine, glutamine-threonine, glutamine-methionine, glutamine-glycine, glutamine-alanine, glutamine-cysteine, glutamine-proline, glutamine-valine, glutamine-isoleucine; (13) Glutamic acid-lysine, glutamic acid-arginine, glutamic acid-asparagine, glutamic acid-aspartic acid, glutamic acid-glutamine, glutamic acid-glutamic acid, glutamic acid-histidine, glutamic acid-serine, glutamic acid-threonine, glutamic acid-methionine, glutamic acid-glycine, glutamic acid-alanine, glutamic acid-cysteine, glutamic acid-proline, glutamic acid-valine, glutamic acid-isoleucine; (14) Asparagine-lysine, asparagine-arginine, asparagine-asparagine, asparagine-aspartic acid, asparagine-glutamine, asparagine-glutamic acid, asparagine-histidine, asparagine-serine, asparagine-threonine, asparagine-methionine, asparagine-glycine, asparagine-alanine, asparagine-cysteine, asparagine-proline, asparagine-valine, asparagine-isoleucine; (15) Aspartic acid-lysine, aspartic acid-arginine, aspartic acid-asparagine, aspartic acid-aspartic acid, aspartic acid-glutamine, aspartic acid-glutamic acid, aspartic acid-histidine, aspartic acid-serine, aspartic acid-threonine, aspartic acid-methionine, aspartic acid-glycine, aspartic acid-alanine, aspartic acid-cysteine, aspartic acid-proline, aspartic acid-valine, aspartic acid-isoleucine; (16) Arginine-lysine, arginine-arginine, arginine-asparagine, arginine-aspartic acid, arginine-glutamine, arginine-glutamic acid, arginine-histidine, arginine-serine, arginine-threonine, arginine-methionine, arginine-glycine, arginine-alanine, arginine-cysteine, arginine-proline, arginine-valine, arginine-isoleucine; (17) Tryptophan-lysine, tryptophan-arginine, tryptophan-asparagine, tryptophan-aspartic acid, tryptophan-glutamine, tryptophan-glutamic acid, tryptophan-histidine, tryptophan-serine, tryptophan-threonine, tryptophan-cysteine; (18) Tyrosine-lysine, tyrosine-arginine, tyrosine-asparagine, tyrosine-aspartic acid, tyrosine-glutamine, tyrosine-glutamic acid, tyrosine-histidine, tyrosine-serine, tyrosine-threonine, tyrosine-cysteine. (19) Phenylalanine-lysine, phenylalanine-arginine, phenylalanine-asparagine, phenylalanine-aspartic acid, phenylalanine-glutamine, phenylalanine-glutamic acid, phenylalanine-histidine, phenylalanine-serine, phenylalanine-threonine, phenylalanine-cysteine.

[0078] (Here, in the combination of "amino acid I-amino acid II", the 234th position can be amino acid I and the 235th position can be amino acid II, or vice versa.)

[0079] As a preferred specific example, leucine at positions 234 and 235 can be replaced with alanine. As another preferred specific example, leucine at position 234 can be replaced with alanine, and leucine at position 235 can be replaced with glutamic acid. The structural analysis results of the model in this case are shown below. Figure 5 . Figure 5 This diagram illustrates the three-dimensional structure of wild-type IgG when leucine at position 234 is replaced with alanine and leucine at position 235 is replaced with glutamic acid, based on the deletion at position 294. According to... Figure 5 The side chains of alanine at position 234 and glutamic acid at position 235 face each other, weakening their van der Waals interactions and creating space between them. This excess space in the molecular structure allows for further twisting of the sugar chain, which is thought to result in an increased amount of sialic acid.

[0080] As another preferred example, leucine at position 234 can be replaced with phenylalanine, and leucine at position 235 can be replaced with glutamine. The structural analysis results in this case are shown below. Figure 6 . Figure 6 This is a schematic diagram illustrating the replacement of leucine at position 234 with phenylalanine and leucine at position 235 with glutamine, based on the deletion at position 294 of wild-type IgG. According to... Figure 6 The side chains of phenylalanine at position 234 and glutamine at position 235 face each other, and the van der Waals forces between them weaken, resulting in a space between them. This excess in the molecular structure allows for further twisting of the sugar chain, which is thought to result in an increased amount of sialic acid.

[0081] Figures 1-6 In the structural analysis, modeling was performed using WinCoot (Coot 0.9.6 Marina Bay), plotting was performed using PyMOL (open source Version 2.60a0), and interaction evaluation was performed using SchrodingerMaestro (Maestro Version 12.4.072).

[0082] The sialylated Fc region modifier of the embodiment has an increased amount of sialic acid, thus exhibiting excellent pharmacokinetics such as an increased half-life and exerting excellent anti-inflammatory effects. That is, it has high therapeutic efficacy and therefore also has the advantage of allowing for reduced dosage when used as a drug.

[0083] Furthermore, the generation of antidrug antibodies (ADAs) in vivo upon administration of antibody drugs has repeatedly been a problem. However, the sialylated Fc region modifier of the present invention does not readily generate ADAs in vivo after administration. This indicates that the desired effect can be sustained even with continuous use of the drug containing the sialylated Fc region modifier of the present invention, which is a significant advantage.

[0084] Furthermore, by deleting amino acids at positions 293 and / or 294, the binding activity to FcγR1 can be weakened when used as an antibody drug, resulting in reduced side effects. This effect can be enhanced by substituting amino acids at positions 234 and / or 235 as described above.

[0085] As mentioned above, the amino acid modification at position 234 and / or position 235 is not limited to selecting the amino acid at that position in a manner that minimizes the interaction between the amino acids at positions 234 and 235.

[0086] In a preferred embodiment, positions 234 and 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine (here, the amino acids replaced by positions 234 and 235 can be the same or different).

[0087] In a more preferred embodiment, positions 234 and 235 are replaced with alanine, or position 234 is replaced with alanine and position 235 is replaced with glutamic acid.

[0088] In the sialylated Fc region modifier of the present invention, amino acid at position 293 or 294 is deleted. Typically, the amino acids at positions 293 and 294 in wild-type IgG are both glutamic acid, therefore deletion of either one yields the same modification. In a preferred embodiment, glutamic acid at position 293 or 294 is deleted.

[0089] The source of the Fc region is not particularly limited. As an antibody, the Fc region can be of types such as IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. The Fc region modifier in the embodiment can be derived from any of these, preferably from IgG (IgG1, IgG2, IgG3, or IgG4), more preferably from human IgG, further preferably from human Igγ1, human Igγ2, human Igγ3, or human Igγ4, and particularly preferably from human Igγ1.

[0090] Specifically, the amino acid mutations at L234A and L235A in the human Igγ1 constant region are referred to as "LALA mutations." Here, L234A refers to the substitution of leucine to alanine at position 234 of the human Igγ1 constant region, following the EU index. L235A refers to the substitution of leucine to alanine at position 235 of the human Igγ1 constant region, following the EU index. This mutation is known to reduce antibody-dependent cytotoxic activity and complement-dependent cytotoxic activity (Mol. Immunol., 1992, Vol. 29, p. 633-639).

[0091] The sialylated Fc region modifier of the embodiments exhibits a higher amount of sialic acid (i.e., the degree of sialylation) compared to sialylating the wild-type Fc region or sialylating the Fc region modifier lacking only amino acid at position 294. The Fc region modifier of the embodiments shows a particularly increased amount of sialic acid in the sugar chain bound to asparagine at position 297, but the location of sialic acid addition is not limited to this position. The amount of sialic acid added can be measured as described in the examples described later and is expressed as "sialylation rate (%)". In this specification, "sialylation rate (%)" refers to the proportion of N-type sugar chains containing sialic acid present in the test subject, and the specific measurement and calculation methods are as described in the examples described later.

[0092] The sialylated Fc region modifiers of the embodiments have a sialylation rate that is increased by, for example, 2-10%, 3-8%, or 3-6% compared to modifiers that only lack the amino acid at position 294.

[0093] The sialylated Fc region modifier of the embodiment can be obtained by methods commonly used in the art, and the amino acid modification and sialic acid addition can also be carried out by methods commonly used in the art. As a method to increase the amount of sialic acid added in the Fc region, it is known to produce the sialylated Fc region modifier by co-expressing plasmids encoding β-galactosyltransferase and sialyltransferase together with a plasmid encoding the Fc region modifier in cells, but it is not limited to this method.

[0094] The sialylated Fc region modifier of the embodiment may contain modifications other than those described above. There are no particular limitations as long as the desired sialylation rate can be achieved. For example, the sialylated Fc region modifier may contain S239D / I332E modification (Proc.Natl.Acad.Sci.USA, 2006, Vol.103(11), p.4005-4010) or modification based on mortar and pestle technology (hereinafter also referred to as "mortar and pestle modification"). The pestle-and-mortar technique involves replacing the amino acid side chains in the CH3 region of one heavy chain with larger side chains (knobs); and replacing the amino acid side chains in the CH3 region of another heavy chain with smaller side chains (holes). This promotes heterodimerization of the heavy chains by arranging protrusions within the holes, thereby efficiently obtaining the target heterodimerized antibody molecules (Nature Biotech., 1998, Vol.16, p.677-681; J.Mol.Biol., 1997, Vol.270, p.26-35; Proc.Natl.Acad.Sci.USA, 2013, Vol.110, p.E2987-E2996).

[0095] Specifically, the sialylated Fc region modifier of the embodiments can be the Fc region portion of the heavy chain amino acid sequence shown in Serial No. 7 or 13 as described in the examples, that is, a sequence consisting of amino acids from position 218 to 448 of the amino acid sequence shown in Serial No. 7, or a sequence consisting of amino acids from position 217 to 447 of the amino acid sequence shown in Serial No. 13. The Fc region modifier of the embodiments may contain portions different from these sequences as long as the desired effect is achieved. For example, the Fc region modifier of the embodiments has an amino acid sequence that is 90% or more, preferably 95% or more, identical to the Fc region portion of the heavy chain amino acid sequence shown in Serial No. 7 or 13.

[0096] 3. Peptides containing sialylated Fc region modifiers

[0097] According to one embodiment, a polypeptide containing the above-described sialylated Fc region modifier is provided. The type of polypeptide is not limited as long as it contains the above-described sialylated Fc region modifier; examples include antibodies. Therefore, according to one embodiment, an antibody containing the above-described sialylated Fc region modifier is provided.

[0098] In this specification, the term "antibody" is used in the broadest sense, encompassing any antibody that exhibits the desired biological activity, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody mutants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, and so on. Additionally, single-arm antibodies may be used. The antibodies used in the embodiments may be antibodies of any class among IgG, IgM, IgA, IgD, and IgE, for example, IgG antibodies.

[0099] Regarding the antibodies used in the implementation method, there are no limitations on the type of antigen or the source of the antibody; any antibody can be used. As for the source of the antibody, there are no particular limitations; examples include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0100] The portion of the antibody outside the Fc region (light chain, heavy chain variable region, etc.) can be any sequence, and can have sequences from any source of antibody, such as mouse antibody, rat antibody, rabbit antibody, goat antibody, camel antibody, humanized antibodies derived from these non-human antibodies, and human antibodies. Furthermore, modification of amino acid residues is permitted as long as its antigen-binding activity is maintained. When modifying the amino acid sequence of the variable region, there are no particular limitations on the modified site or the number of modified amino acids.

[0101] Additionally, peptide linkers can be present to connect two or more portions of the antibody (e.g., the heavy chain variable region and the light chain variable region of the antibody). Their type and length are not particularly limited, and those skilled in the art can choose appropriately. A preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. As peptide linkers for binding the heavy chain variable region and the light chain variable region of the antibody, for example, glycine-serine linkers (GS linkers) and glycine-lysine-proline-glycine-serine linkers (GKPGS linkers) can be used.

[0102] The antibody of the embodiment can be manufactured according to methods known in the art. Specific methods are described in "6. Methods for producing polypeptides" below.

[0103] Furthermore, the polypeptide containing the sialylated Fc region modifier in the embodiments can be an Fc fusion protein molecule formed by combining the sialylated Fc region modifier with other proteins, physiologically active peptides, etc. Examples of other proteins and physiologically active peptides include, but are not limited to, receptors, adhesion molecules, ligands, enzymes, etc.

[0104] Preferred examples of Fc fusion protein molecules include protein molecules formed by fusing an Fc domain (the sialylated Fc region modifier of the embodiment) to a receptor protein that binds to a target. Examples of such protein molecules include TNFR-Fc fusion protein, IL1R-Fc fusion protein, and VEGFR-Fc fusion protein. Further examples of proteins or peptides used for fusion include scFv molecules, single-domain antibody molecules, and antibody-like molecules (e.g., DARPins, Affibody, Avimer, Adnectin, etc.). Fc fusion protein molecules can be multispecific, binding to multiple target molecules or epitopes. When multiple Fc region modifiers form a complex, other proteins or peptides may bind to only a portion of it. For example, when two Fc region modifiers form a complex, other proteins or peptides may bind to only one of them, forming a single-arm Fc fusion protein.

[0105] Fc fusion protein molecules can contain proteins of non-human origin. By binding the sialylated Fc region to a non-human protein, ADA production can be inhibited, thus enabling repeated administration of Fc fusion proteins to the same subject.

[0106] The Fc fusion protein molecules of the embodiments can be manufactured according to methods known in the art. Specific methods are described later in "6. Methods for the Production of Peptides".

[0107] 4. Polynucleotides and expression vectors

[0108] According to one embodiment, a polynucleotide encoding the Fc region modifier of the present invention and a polynucleotide encoding the aforementioned polypeptide containing the sialylated Fc region modifier are provided. The polynucleotides of the embodiments can be prepared by those skilled in the art based on their base sequences using methods known in the art. For example, the polynucleotides of the embodiments can be synthesized using gene synthesis methods known in the art. As such gene synthesis methods, various methods known to those skilled in the art, such as the antibody gene synthesis method disclosed in International Publication No. 90 / 07861, can be used.

[0109] According to a further embodiment, an expression vector containing the above-described polynucleotides is provided. The polynucleotides may be contained in different vectors, or multiple polynucleotides may be contained in one vector.

[0110] There are no particular limitations on the expression vector used in this embodiment, as long as it can produce the polynucleotides described in this embodiment in various host cells, such as eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and prokaryotic cells (e.g., E. coli). Examples of such expression vectors include plasmid vectors and viral vectors. As plasmid vectors, examples include the pcDNA series (Thermo Fisher Scientific), pALTER (registered trademark)-MAX (Promega), pHEK293 superexpression vector (Takara Bio), pEE6.4 or pEE12.4 (Lonza Biologics). As viral vectors, examples include lentiviruses, adenoviruses, retroviruses, and adeno-associated viruses. For example, when using lentiviruses to introduce the polynucleotides described in this embodiment into cells, the lentivirus can be the pLVSIN-CMV / EF1α vector (Takara Bio), the pLenti vector (Thermo Fisher Scientific), etc. In one embodiment, the expression vector used in the embodiment is pcDNA (trademark) 3.4-TOPO (registered trademark) vector (Thermo Fisher Scientific) and pcDNA (trademark) 3.1 vector (Thermo Fisher Scientific).

[0111] The expression vector of the embodiments may contain a promoter operably linked to the polynucleotide of the present invention. Examples of promoters for expressing the polynucleotide of the embodiments in animal cells include viral promoters such as CMV, RSV, and SV40, actin promoters, EF (elongation factor) 1α promoters, and heat shock promoters. Examples of promoters for expressing the polynucleotide of the embodiments in bacteria (e.g., Escherichia coli) include trp promoters, lac promoters, λPL promoters, and tac promoters. Examples of promoters for expressing the polynucleotide of the embodiments in yeast include GAL1 promoters, GAL10 promoters, PH05 promoters, PGK promoters, GAP promoters, and ADH promoters.

[0112] When using animal cells, insect cells, or yeast as host cells, the expression vector of the embodiments may contain a start codon and a stop codon. In this case, it may contain enhancer sequences, untranslated regions on the 5' and 3' sides of the gene encoding the Fc region modified in the embodiments, secretion signal sequences, splice junctions, polyadenylation sites, or reproducible units, etc. When using *E. coli* as host cells, the expression vector of the embodiments may contain a start codon, a stop codon, a stop region, and reproducible units. The expression vector of the embodiments may contain commonly used drug selection marker genes (e.g., tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, dihydrofolate reductase genes) depending on the purpose.

[0113] 5. Host cells

[0114] According to one embodiment, a host cell transformed with the expression vector described above is provided. Through transformation using the expression vector of the embodiment, the host cell of the embodiment can contain one or more polynucleotides of the embodiment.

[0115] The host cell used for transformation is not particularly limited, as long as it is suitable for the expression vector used and can be transformed with that expression vector to express the polypeptide of the embodiment. Various cells commonly used in the art, such as conventional cells or artificially created cells, can be used as the host cell. Examples include animal cells (CHO-K1 cells, ExpiCHO-S (trademark) cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, NSO cells, etc.), insect cells (Sf9, etc.), bacteria (Escherichia coli, etc.), and yeast (Saccharomyces, Pichia pastoris, etc.). In one embodiment, the host cell is a CHO-K1 cell or an ExpiCHO-S cell.

[0116] There are no particular limitations on the method of transforming host cells. For example, methods commonly used by those skilled in the art, such as calcium phosphate method, electroporation method, or liposome transfection method, can be used.

[0117] Screening of transformed host cells can be performed using methods commonly employed by those skilled in the art. Screening methods may include, for example, drug selection using drug selection marker genes and agents such as tetracycline, ampicillin, neomycin, or hygromycin, limiting dilution methods, single-cell sorting, colony picking, and other cell isolation methods.

[0118] 6. Methods for producing polypeptides

[0119] According to one embodiment, a method for producing a sialylated Fc region modifier and a method for producing a polypeptide containing the aforementioned sialylated Fc region modifier are provided. The method may include steps such as culturing the transformed host cells and expressing the polypeptide in the host cells or culture supernatant, recovering, isolating, and purifying the polypeptide, but is not limited to these methods as long as the target polypeptide can be produced.

[0120] The transformed host cells can be cultured using known methods. Culture conditions, such as temperature, pH of the medium, and culture time, can be appropriately selected by those skilled in the art. When the host cells are animal cells, the culture medium can be, for example, MEM medium (Science, 1959, Vol. 130, p. 432-437), DMEM medium (Virol., 1959, Vol. 8, p. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), or 199 medium (Exp. Biol. Med., 1950, Vol. 73, p. 1-8), containing approximately 5-20% fetal bovine serum. The pH of the medium is, for example, approximately 6-8, and culture can be carried out at approximately 30-40°C for approximately 15-336 hours with aeration and stirring as needed. When the host cell is an insect cell, the culture medium can be, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA., 1985, Vol. 82, p. 8404). The pH of the medium is, for example, about 5 to 8, and the culture can be carried out at a temperature of about 20 to 40°C for about 15 to 100 hours while aerating and stirring, as needed.

[0121] When the host cells are *E. coli* or yeast, a liquid culture medium containing nutrients is suitable. The nutrient medium contains, for example, the carbon, inorganic, or organic nitrogen sources required for the growth of the transformed host cells. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose; examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. Other nutrients (e.g., inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins), and antibiotics (e.g., tetracycline, neomycin, ampicillin, kanamycin) may be included as desired. The pH of the medium is, for example, approximately 5–8. When the host cells are *E. coli*, LB medium or M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2) can be used as the culture medium. Culture can be carried out for approximately 3 to 24 hours at a temperature typically between 14 and 43°C, with aeration and stirring as needed. When yeast is used as the host cell, a medium such as Burkholder Minimal Medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505) can be used as the culture medium. Culture can then be carried out for approximately 14 to 144 hours at a temperature typically between 20 and 35°C, with aeration and stirring as needed. Through such culture, the target peptide can be expressed.

[0122] The above method may also include the step of adding or expressing sialyltransferase. Examples of such enzymes include β-galactosidase α-2,6-sialyltransferase 1 (ST6GAL1), sialyltransferase, and β-galactosidase α-2,3-sialyltransferase. This allows for a further increase in the amount of sialic acid added to the Fc region. The sialyltransferase can be added directly, or an expression vector can be prepared by inserting DNA encoding the sialyltransferase and expressed together with a polypeptide containing the Fc region modifier of the present invention, such as an antibody. The timing and method of adding the sialyltransferase are not particularly limited; for example, it can be added during the production of the polypeptide through cell culture, or it can be added after the polypeptide purification to induce a reaction. When expressing the sialyltransferase, for example, a method of co-expressing a plasmid encoding the sialyltransferase and a plasmid encoding the polypeptide together in cells to produce sialylated polypeptides can be listed, but the method is not limited to this. When using cells containing endogenous glycotransferases, such as CHO cells, to produce peptides, sialic acid can be added during peptide production even without adding sialic acid transferases as described above.

[0123] In addition to the steps of culturing transformed host cells and expressing polypeptides containing sialylated Fc region modifiers, the method of implementation may also include steps of recovering, isolating, or purifying the polypeptide from the host cells. Examples of isolating or purifying methods include, for instance, methods utilizing solubility such as salting out and solvent precipitation; methods utilizing molecular weight differences such as dialysis, ultrafiltration, and gel filtration; methods utilizing charged molecules such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing hydrophobic differences such as reversed-phase high-performance liquid chromatography; and methods utilizing isoelectric point differences such as isoelectric point electrophoresis. In one embodiment, the polypeptide secreted into the culture supernatant can be purified by various chromatography methods, such as column chromatography using protein A columns or protein G columns.

[0124] According to one embodiment, a polypeptide containing a sialylated Fc region modifier produced by the above-described production method is provided.

[0125] 7. Pharmaceutical Composition

[0126] According to one embodiment, a pharmaceutical composition is provided comprising the sialylated Fc region modifier of the present invention or the aforementioned polypeptide containing the sialylated Fc region modifier, and a pharmaceutically acceptable carrier. This pharmaceutical composition can be prepared using excipients, i.e., pharmaceutical excipients, pharmaceutical carriers, etc., commonly used in the art, by conventionally used methods. Examples of dosage forms for these pharmaceutical compositions include non-oral preparations such as injections and intravenous infusions, which can be administered via intravenous, subcutaneous, or intraperitoneal administration. During formulation, excipients, carriers, additives, etc., appropriate to these dosage forms can be used within pharmaceutically acceptable limits.

[0127] The pharmaceutical composition of the embodiments may further contain other antibody drugs and / or one or more additional therapeutic agents. Additionally, the pharmaceutical composition of the embodiments may be used in combination (simultaneously or sequentially) with other antibody drugs and / or one or more additional therapeutic agents.

[0128] The amount of peptide in the pharmaceutical composition can be appropriately set according to the severity of the patient's symptoms, age, dosage form of the preparation used, etc.

[0129] The polypeptides of the embodiments and pharmaceutical compositions containing them can be used to treat autoimmune diseases and inflammatory diseases. Additionally, according to another embodiment, a method for treating autoimmune diseases is provided, comprising the step of administering a therapeutically effective amount of the polypeptide of the embodiments to a subject. Furthermore, according to yet another embodiment, the polypeptide of the present invention is included for treating autoimmune diseases. Additionally, the present invention provides the use of the polypeptides of the embodiments in the manufacture of pharmaceutical compositions for treating autoimmune diseases. The autoimmune diseases to which the treatment is intended are not particularly limited.

[0130] Example

[0131] In the following examples, "sialylation rate" refers to the proportion of sialic acid-containing glycans among all N-type glycans present in the system. Regarding the antibodies manufactured in the following examples, since structural analysis revealed that the N-type glycans are only those bound to asparagine at position 297, the sialylation rate refers to the proportion of sialic acid-containing glycans among all N-type glycans present in the system (i.e., glycans bound to asparagine at position 297).

[0132] <Example 1: Preparation of E294Del mutant of anti-lysozyme antibody>

[0133] DNA encoding the heavy and light chain polypeptides of the anti-lysozyme antibody was synthesized using conventional methods and inserted into the pcDNA3.4-TOPO vector (Thermo Fisher Scientific) to create expression vectors for the heavy chain and light chain, respectively. DNA encoding the heavy chain polypeptide, formed by linking the gene encoding the variable region sequence of the heavy chain of the anti-lysozyme antibody with the gene encoding human Igγ1 with an E294Del (Del = deletion) amino acid mutation, was inserted into the pcDNA3.4-TOPO vector to create an expression vector for the heavy chain with the E294Del amino acid mutation. The heavy chain expression vector and the light chain expression vector, or the expression vector for the heavy chain with the E294Del amino acid mutation and the light chain expression vector, were transfected into ExpiCHO-S cells (Thermo Fisher Scientific, A29127) and cultured. The culture supernatant was purified using MabSelect SuRe (GE Healthcare, 17-5438-02) using conventional methods to obtain purified antibodies. The antibody containing the human Igγ1 constant region (Fc unmodified form) was named LYS_1c3-m1_h10. The antibody containing the E294Del amino acid mutation in the human Igγ1 constant region was named LYS_E294Del_h1x. The amino acid and nucleic acid sequences of each antibody are shown in Table 1.

[0134] [Table 1]

[0135] It has been reported that antibodies with the E294Del mutation exhibit reduced binding activity to human FcγRs (WO2012 / 175751, Table 4). Previous reports (e.g., CELL IMMUNOL., 2000, Vol. 200(1), p. 16-26) have suggested that decreased binding activity to FcγRs inhibits T cell activation, which may alleviate the side effects of treatment for certain conditions.

[0136] Therefore, the binding activity of the antibodies obtained above to human FcγRs was evaluated using Biacore (GE Healthcare, Biacore T200) according to its guidelines. Compared with LYS_1c3-m1_h10, LYS_E294Del_h1x showed reduced binding activity to human FcγR1, but still retained some binding activity.

[0137] <Example 2: Preparation of L234A / L235A / E294Del mutant of anti-lysozyme antibody>

[0138] Antibodies combining the aforementioned E294Del, L234A, and L235A mutations were prepared using the following method. DNA encoding a heavy chain polypeptide, formed by linking the gene encoding the variable region sequence of the heavy chain containing the anti-lysozyme antibody and the gene encoding human Igγ1 with the L234A / L235A / E294Del amino acid mutations, was inserted into the pcDNA3.4-TOPO vector to create an expression vector for the heavy chain with the L234A / L235A / E294Del amino acid mutations. The expression vector for the heavy chain with the L234A / L235A / E294Del amino acid mutations and the expression vector for the light chain prepared in Example 1 were transfected into ExpiCHO-S cells and cultured. The antibodies were then purified using the same method as in Example 1. The obtained antibody, namely the antibody with the amino acid mutation L234A / L235A / E294Del in the human Igγ1 constant region, was named LYS_E294Del_LALA_h1x. The amino acid and nucleic acid sequences of the antibody are shown in Table 2.

[0139] [Table 2]

[0140] The binding activity of LYS_E294Del_LALA_h1x against human FcγR1 was evaluated using Biacore in the same manner as in Example 1. The result showed that LYS_E294Del_LALA_h1x lacked binding activity against human FcγR1. Such antibodies with lost binding activity against human FcγR1 are useful for reducing side effects and achieving therapeutic effects in the treatment of certain conditions.

[0141] <Example 3: Preparation of mutant anti-lysozyme antibody by sialylation and determination of sialylation rate>

[0142] Using CHO cells, which are frequently used in the industrial production of antibodies, mutants of anti-lysozyme antibodies supplemented with sialic acid were prepared by the following method. CHO cells do not express α2,6-sialyltransferase (MAbs, 2015, Vol. 7(3), p. 571-83). Therefore, in order to confer α2,6-binding sialic acid to the antibody, an expression vector was prepared by inserting DNA encoding a polypeptide of β-galactosyl α-2,6-sialyltransferase 1 (ST6GAL1) (Uniprot: P15907-1) into the pcDNA3.4-TOPO vector.

[0143] The ST6GAL1 expression vector, along with the expression vector of the heavy chain with the E294Del amino acid mutation prepared in Example 1 and the expression vector of the light chain prepared in Example 1, or the expression vector of the heavy chain with the L234A / L235A / E294Del amino acid mutation prepared in Example 2 and the expression vector of the light chain prepared in Example 1, were transfected into ExpiCHO-S cells to co-express ST6GAL1 and the antibodies. The amount of each expression vector added was adjusted relative to the total amount of the heavy chain and light chain expression vectors to achieve a ratio of 2% for the expression vector encoding ST6GAL1. The antibodies were purified using the same method as in Example 1. The antibody containing the human Igγ1 constant region with the E294Del amino acid mutation was named LYS_E294Del_h1x-ST6_2%. The antibody containing the human Igγ1 constant region with amino acid mutations of L234A / L235A / E294Del was named LYS_E294Del_LALA_h1x-ST6_2.

[0144] Dithiothreitol (Thermo Fisher Scientific, A39255) was added to each antibody, and the incubation at 37°C for 1 hour reduced the disulfide bonds between the chains. The reduced samples were injected into an HPLC system (Waters), separated using a reverse-phase column (Waters ACQUITY UPLC Protein BEH C4), and then introduced into a mass spectrometer (Waters). The theoretical molecular weight obtained by adding various glycan molecular weights to the reduced antibody molecular weight was compared with the measured mass to determine the type of glycan modification. The proportion (%) of each glycan was calculated based on the ionic strength ratio. The sum of the proportions of glycans containing sialic acid was taken as the "sialylation rate (%)". It should be noted that the sialylation rate was calculated using the following formula.

[0145] Sialization rate (%) = (Total MS intensity of H chains modified by sialic acid-bound sugar chains) ÷ (Total MS intensity of all H chains) × 100

[0146] The results are shown in Table 3.

[0147] [Table 3]

[0148] Surprisingly, LYS_E294Del_LALA_h1x-ST6_2% exhibited a higher sialylation rate compared to LYS_E294Del_h1x-ST6_2%. That is, by combining the E294Del mutation with the L234A / L235A mutation, the antibody's sialylation rate was increased compared to the E294Del mutation.

[0149] <Example 4: Stereostructural Analysis of Antibodies>

[0150] To investigate the cause of the increased sialylation rate observed in Example 3, model structures were constructed and their stereostructures analyzed for antibodies with the E294Del mutation, antibodies with the L234A / L235A / E294Del mutation, antibodies with the L234A / L235E / E294Del mutation, and antibodies with the L234F / L235Q / E294Del mutation. Regarding the model structure, the full-length structure of IgG1 with observed SS bonds in the IgG hinge region, forming a hydrophobic scaffold of L234 / L235 (PDB number: 1HZH) was selected. For the full-length structure of IgG1, the glycan chain containing sialic acid was removed from the sialylated Fc structure (PDB number: 4BYH), and after being replaced with the glycan chain of IgG1, the following mutations were introduced: E294Del mutation, L234A / L235A / E294Del mutation, L234A / L235E / E294Del mutation, and L234F / L235Q / E294Del mutation.

[0151] (1) IgG1 model structure

[0152] The L234 / L235 dimers of IgG1 form a strong van der Waals interaction. This interaction forms a scaffold and binds to the Fcγ receptor (J. Biol. Chem., 2017, Vol. 292(9), p. 3900-3908) (Nature, 2000, Vol. 406, p. 267-273). In the full-length structure of IgG1, between the CH2 dimers, L234 / L235 is in contact with the sugar chain bound to the amino group at the N297 side chain (…). Figure 1 Therefore, it is believed that the interaction between L234 / L235 and the sugar chain contributes to the structural formation and stability of the CH2 dimer.

[0153] (2) A model structure of sialylated IgG1 with E294Del mutation was introduced.

[0154] Analysis of the IgG1 model structure of the sialylated E294Del mutant revealed that although the mutations that increase sialylation rate, namely the E293 or E294Del mutants, only lack one residue, the loop containing N297 (a band composed of amino acids, hereinafter referred to as the "N297 loop") extending three residues from that site towards the C-terminus is shortened. Due to the shortening of the N297 loop, a spatial misalignment occurs at the N297 position, confirming that the sugar chain bound to N297 is twisted. Figure 2 It is believed that this distortion makes glycotransferases more accessible, or that it increases the rate of sialylation through a secondary conformational change in the overall CH2 group.

[0155] Next, the dimerization interface of L234 / L235 was analyzed. The results showed that, on the one hand, the sugar chains at the contact surface of the CH2 dimer were twisted, and on the other hand, strong van der Waals interactions between L234 and L235 were still confirmed. Figure 3 ).

[0156] (3) A model structure of sialylated IgG1 with L234A / L235A / E294Del mutation was introduced.

[0157] The model structure of the E294 deletion and L234A / L235A mutant was constructed and analyzed. The results showed that the van der Waals interaction between L234A and L235A was weaker than that between L234 and L235, leaving a gap in the middle. Figure 4 It is believed that this change helps to increase the rate of sialylation.

[0158] (4) A model structure of sialylated IgG1 with L234A / L235E / E294Del mutations was introduced.

[0159] The model structure of the E294-deficient L234A / L235E mutant was constructed and analyzed. The results showed that the alanine (A) and glutamic acid (E) in L234A / L235E are opposite to each other, the van der Waals interaction between L234A / L235E is weakened, and a space is left in the middle. Figure 5 It is believed that, similar to antibodies with the L234A / L235A / E294Del mutation, this change contributes to increasing the sialylation rate.

[0160] (5) A model structure of sialylated IgG1 with L234F / L235Q / E294Del mutation was introduced.

[0161] The model structure of the E294-deficient L234F / L235Q mutant was constructed and analyzed. The results showed that phenylalanine (F) and glutamine (Q) in L234F / L235Q are opposite to each other, the van der Waals interaction between L234F / L235Q is weakened, and a space is left in the middle. Figure 6 It is believed that, similar to antibodies with the L234A / L235A / E294Del mutation, this change contributes to increasing the sialylation rate.

[0162] (6) Investigation of mutations that increase sialylation rate

[0163] The rationale behind the increased sialylation rate due to E294 deletion and the L234A / L235A mutation compared to E294 deletion alone is the weakened van der Waals interaction between the L234 / L235 dimers. Therefore, it is believed that by deleting E294 and replacing the L234 / L235 amino acids with amino acids lower than the hydrophobic leucine (L), or by changing the opposing L234 / L235 residues to a hydrophobic / hydrophilic combination, the van der Waals interaction can be weakened, thus increasing the sialylation rate.

[0164] In addition, it is believed that, besides van der Waals interactions, mutations that disrupt the scaffold formation between L234 / L235 by changing the amino acids of L234 / L235 to amino acids smaller than L also increase the sialylation rate.

[0165] <Example 5: Preparation of Antibody Against Fibronectin Additional Domain-A Isotype (Fn-EDA)>

[0166] DNA encoding a heavy chain polypeptide with an amino acid mutation of E294Del or L234A / L235A / E294Del added to the sequence of the heavy chain of an anti-fibronectin extra domain-A isotype (Fn-EDA) antibody was inserted into the pcDNA3.4-TOPO vector to prepare expression vectors for the heavy chain with the E294Del amino acid mutation and those with the L234A / L235A / E294Del amino acid mutation. DNA encoding a light chain polypeptide of an anti-Fn-EDA antibody was synthesized using conventional methods and inserted into the pcDNA3.4-TOPO vector to prepare expression vectors for the light chain.

[0167] The ST6GAL1 expression vector prepared in Example 3 was transfected into ExpiCHO-S cells along with expression vectors for the heavy chain and light chain containing the E294Del amino acid mutation, or expression vectors for the heavy chain and light chain containing the L234A / L235A / E294Del amino acid mutation, to co-express ST6GAL1 and the antibodies. The amount of each expression vector added was adjusted relative to the total amount of the heavy chain and light chain expression vectors to achieve a ratio of 0.5%, 1%, 2%, and 5% for the expression vector encoding ST6GAL1. The antibodies were purified using the same method as in Example 1.

[0168] Antibodies containing the human Igγ1 constant region with the E294Del amino acid mutation were named GLY_F8_E294Del_h1x, and named GLY_F8_E294Del_h1x(0.5%), GLY_F8_E294Del_h1x(1%), GLY_F8_E294Del_h1x(2%), and GLY_F8_E294Del_h1x(5%) according to the co-expression level of ST6GAL1. Antibodies containing the human Igγ1 constant region with the amino acid mutation L234A / L235A / E294Del were named GLY_F8_E294Del_LALA_h1x, and named GLY_F8_E294Del_LALA_h1x(0.5%), GLY_F8_E294Del_LALA_h1x(1%), GLY_F8_E294Del_LALA_h1x(2%), and GLY_F8_E294Del_LALA_h1x(5%) based on their co-expression levels of ST6GAL1. The amino acid and nucleic acid sequences of each antibody are shown in Table 4.

[0169] [Table 4]

[0170] For each antibody, the sialylation rate was analyzed using the method described in Example 3. The results are shown in Table 5.

[0171] [Table 5]

[0172] Under any ST6GAL1 co-expression condition, anti-Fn-EDA antibodies with L234A / L235A / E294Del mutations showed a higher sialylation rate compared to anti-Fn-EDA antibodies with E294Del mutations.

[0173] <Example 6: Preparation of Anti-Fn-EDA Modified Antibody>

[0174] An anti-Fn-EDA modified antibody was fabricated by replacing the heavy and light chain frameworks (FRs) of the anti-Fn-EDA antibody obtained in Example 5 with the sequences of the FRs of other known human antibodies, and then, to mitigate the potential risk of glycosylation, introducing a K94R mutation into the variable region sequence of the heavy chain. The specific fabrication method is described below.

[0175] DNA encoding the heavy and light chain polypeptides of the anti-Fn-EDA modified antibody was synthesized using conventional methods and inserted into the pcDNA3.4-TOPO vector to create expression vectors for the heavy chain and light chain, respectively. Additionally, DNA encoding the heavy chain polypeptide, formed by linking the gene encoding the variable region sequence of the heavy chain of the anti-Fn-EDA modified antibody with the gene encoding human Igγ1 with amino acid mutations of L234A / L235A, E294Del, or L234A / L235A / E294Del, was inserted into the pcDNA3.4-TOPO vector to create expression vectors for the heavy chain with amino acid mutations of L234A / L235A, E294Del, and L234A / L235A / E294Del.

[0176] Then, the expression vector encoding ST6GAL1 prepared in Example 3, along with the heavy chain expression vector and light chain expression vector obtained above, the heavy chain expression vector and light chain expression vector with L234A / L235A amino acid mutation, the heavy chain expression vector and light chain expression vector with E294Del amino acid mutation, or the heavy chain expression vector and light chain expression vector with L234A / L235A / E294Del amino acid mutation, were transfected into ExpiCHO-S (registered trademark) cells to co-express ST6GAL1 and the antibodies. The amount of each expression vector added was adjusted relative to the total amount of the heavy chain and light chain expression vectors to achieve a ratio of 5% for the expression vector encoding ST6GAL1.

[0177] The antibodies were then purified using the same method as in Example 1. The antibody with the human Igγ1 constant region (Fc unmodified form) was named SSA_m3_K94R_h10. The antibody with an amino acid mutation of L234A / L235A in the human Igγ1 constant region was named SSA_m3_K94R_h1g. The antibody with an amino acid mutation of E294Del in the human Igγ1 constant region was named SSA_E294Del_m3_K94R_h1x. The antibody with amino acid mutations of L234A / L235A / E294Del in the human Igγ1 constant region was named SSA_E294Del_LALA_m3_K94R_h1x.

[0178] The amino acid and nucleic acid sequences of each antibody are shown in Table 6.

[0179] [Table 6]

[0180] For each antibody, the sialylation rate was analyzed using the method described in Example 3. The results are shown in Table 7.

[0181] [Table 7]

[0182] Antibodies with the unmodified Fc variant and the L234A / L235A mutation were hardly conjugated with sialic acid, while antibodies with the E294Del mutation showed increased sialylation. Furthermore, antibodies with both the E294Del and L234A / L235A mutations showed increased sialylation compared to antibodies with the E294Del mutation.

[0183] Furthermore, comparing the anti-Fn-EDA antibodies (i.e., GLY_F8_E294Del_h1x(5%) and GLY_F8_E294Del_LALA_h1x(5%)) obtained in Example 5 under the same conditions with the anti-Fn-EDA modified antibodies (i.e., SSA_E294Del_m3_K94R_h1x and SSA_E294Del_LALA_m3_K94R_h1x) obtained in this example, the antibodies with the E294Del mutation, as well as those with both the E294Del mutation and the L234A / L235A mutation, all showed higher sialylation rates compared to the anti-Fn-EDA modified antibodies. This result indicates that altering the FR sequence can increase the sialylation rate of the Fc region.

[0184] The amino acid and DNA sequences of the peptides described in the embodiments are shown in the table below.

[0185]

[0186] Several embodiments of the present invention have been described, but these embodiments are shown by way of example and are not intended to limit the scope of the invention. These new embodiments can be implemented in various other ways, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, and are included in the scope of the invention as set forth in the claims and its equivalents.

[0187] Industrial availability

[0188] The sialylated Fc region modifier of the present invention, with a further increased amount of sialic acid, is useful in the prevention or treatment of various autoimmune and inflammatory diseases.

Claims

1. A sialylated Fc region modifier comprising the following amino acid modifications (1) and (2): (1)(a) Positions 234 and / or 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine, wherein the amino acids replaced by the substitutions at positions 234 and 235 may be the same or different; (b) Position 234 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine, and position 235 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine; or (c) Position 234 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and position 235 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine. (2) The 293rd or 294th bit is missing. Here, amino acid residues are numbered according to their positions following the EU index.

2. The sialylated Fc region modifier according to claim 1, wherein, Positions 234 and 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine. The amino acids replaced by positions 234 and 235 can be the same or different.

3. The sialylated Fc region modifier according to claim 1 or 2, wherein, Positions 234 and 235 were replaced with alanine, or The 234th position was replaced with alanine, and the 235th position was replaced with glutamic acid.

4. The sialylated Fc region modifier according to any one of claims 1 to 3, wherein, The Fc region is derived from human IgG.

5. The sialylated Fc region modifier according to claim 4, wherein, The Fc region is derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4.

6. The sialylated Fc region modifier according to claim 4, wherein, An amino acid sequence that is 90% or more identical to the sequence consisting of amino acids from position 218 to position 448 of the amino acid sequence shown in Serial No. 7, or an amino acid sequence that is 90% or more identical to the sequence consisting of amino acids from position 217 to position 447 of the amino acid sequence shown in Serial No.

13.

7. A polypeptide comprising the sialylated Fc region modifier according to any one of claims 1 to 6.

8. The polypeptide according to claim 7, wherein, The polypeptide containing the sialylated Fc region modifier is an antibody.

9. The polypeptide according to claim 8, wherein, The antibody is an IgG antibody.

10. The polypeptide according to claim 7, wherein, The polypeptide containing the sialylated Fc region modifier is an Fc fusion protein molecule.

11. The polypeptide according to claim 10, wherein, The sialylated Fc fusion protein molecule contains a protein of non-human origin.

12. A polynucleotide encoding a polypeptide according to any one of claims 7 to 11.

13. An expression vector containing the polynucleotide of claim 12.

14. A host cell that has been transformed with the expression vector of claim 13.

15. A method for producing a polypeptide containing a sialylated Fc region modifier, comprising the step of culturing the host cell of claim 14.

16. The method according to claim 15, wherein, It also includes the steps of adding or expressing β-galactosidase α-2,6-sialyltransferase 1 (ST6GAL1).

17. An Fc region modified body comprising the following amino acid modifications (1) and (2): (1)(a) Positions 234 and / or 235 are replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, alanine, proline, methionine, valine, or isoleucine, wherein the amino acids replaced by the substitutions at positions 234 and 235 may be the same or different; (b) Position 234 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine, and position 235 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine; or (c) Position 234 is replaced with serine, threonine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and position 235 is replaced with phenylalanine, tyrosine, tryptophan, isoleucine, valine, methionine, proline, alanine, or glycine. (2) The 293rd or 294th bit is missing. Here, amino acid residues are numbered according to their positions following the EU index.

18. A pharmaceutical composition comprising the polypeptide of any one of claims 7 to 11 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, for the treatment of autoimmune diseases.

20. The polypeptide according to any one of claims 7 to 11, for the treatment of autoimmune diseases.

21. A method for treating an autoimmune disease, comprising the step of administering a therapeutically effective amount of the polypeptide according to any one of claims 7 to 11 to a subject.

22. Use of the polypeptide according to any one of claims 7 to 11 in the manufacture of a pharmaceutical composition for treating autoimmune diseases.

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