IdeZ VARIANT

Abstract

According to one embodiment of the present invention, provided is an IdeZ variant having IgG cysteine protease activity, the IdeZ variant comprising (a) a sequence that is 90% or more identical to the amino acid sequence from position 35 to position 349 of the amino acid sequence represented by SEQ ID NO: 1, wherein (b) no sugar chain is added to an amino acid at a position corresponding to position 118 and at a position corresponding to position 267 of the amino acid sequence represented by SEQ ID NO: 1.

Description

IdeZ variant

[0001] The present invention relates to IdeZ variants and polypeptides containing the IdeZ variants.

[0002] IdeS, derived from streptococci, is a cysteine ​​protease that sequence-specifically cleaves the hinge region connecting the Fab and Fc regions of human IgG. Due to its high cleavage efficiency and substrate specificity, it has been utilized in the characterization of antibody drug candidates. In recent years, its applications have expanded, including its use in high-quality recombinant protein production systems and its potential effectiveness in treating transplant rejection and autoimmune diseases (Non-Patent Documents 1 and 2). Furthermore, research has been conducted on fusion proteins consisting of IdeS and a human Fc region (Non-Patent Document 1). However, humans with a history of streptococcal infection are immune to IdeS, and administration of IdeS to such humans can lead to the production of anti-drug antibodies (ADA), which can pose a problem due to the high antigenicity of IdeS (Non-Patent Document 3). To address these issues with IdeS, research is also underway on streptococcal-derived IdeZ that does not infect humans, as well as on IdeZ variants modified to suppress ADA production (Patent Documents 1 and 2). Further improvements in the properties of such IdeZ and IdeZ variants are desired.

[0003] International Publication No. 2020 / 159970 International Publication No. 2021 / 233911

[0004] The 1st Anniversary Academic Meeting of the Japanese Society for Antibody Research, November 2022, P-74 Drugs, October 2020, Vol. 80, pp. 1859-1864 Am J Transplant, November 2018, Vol. 18(11), pp. 2752-2762

[0005] The present inventors have conducted extensive research to obtain an IgG cysteine ​​protease with even better properties, and in the process have discovered a problem in that protease activity is attenuated when wild-type IdeZ is produced using mammalian cells (e.g., CHO cells).An object of the present invention is to provide IdeZ variants and polypeptides containing the IdeZ variants that have good protease activity even when produced using mammalian cells.

[0006] In light of the above-mentioned problems, the present inventors conducted further intensive research and found that by modifying amino acids at specific positions in IdeZ (e.g., the consensus sequence for N-glycans), good protease activity can be obtained even when produced using mammalian cells.

[0007] The present invention provides, for example, the following: [1] An IdeZ variant having IgG cysteine ​​protease activity, the variant comprising: (a) a sequence that is 90% or more identical to the amino acid sequence from positions 35 to 349 of the amino acid sequence represented by SEQ ID NO: 1; and (b) an IdeZ variant in which no glycosylation is performed on the amino acids at positions 118 and 267 of the amino acid sequence represented by SEQ ID NO: 1.[2] The IdeZ variant according to [1], comprising the following amino acid modifications (b') and (b''): (b') (i) the amino acid at position 118 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 119 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) the amino acid at position 120 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (b'') (i) the amino acid at position 267 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 268 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) The amino acid at the position corresponding to position 269 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.[3] The IdeZ variant according to [2], in which the amino acid at position 120 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glutamic acid and the amino acid at position 269 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with arginine. [4] The IdeZ variant according to any of [1] to [3], in which (c) the amino acid at position 171 in the amino acid sequence represented by SEQ ID NO: 1 does not have a glycosylated amino acid, and / or (d) the amino acid at position 298 in the amino acid sequence represented by SEQ ID NO: 1 does not have a glycosylated amino acid.[5] The IdeZ variant according to [4], comprising the following amino acid modifications (c') and / or (d'): (c') (i) the amino acid at position 171 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 172 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with proline; and / or (iii) the amino acid at position 173 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (d') (i) the amino acid at position 298 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 299 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) The amino acid at the position corresponding to position 300 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.[6] The IdeZ variant according to [5], wherein the amino acid at position 171 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine and / or the amino acid at position 298 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid. [7] The IdeZ variant according to [6], wherein the amino acid at position 120 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with glutamic acid, the amino acid at position 171 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, the amino acid at position 269 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with arginine, and the amino acid at position 298 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid. [8] The IdeZ variant according to [7], wherein the IdeZ variant comprises an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 5. [9] A polypeptide comprising the IdeZ variant according to any of [1] to [8].

[10] The polypeptide according to [9], wherein the polypeptide is a fusion protein of the IdeZ variant and an Fc region.

[11] The polypeptide according to

[10] , wherein the Fc region is derived from human IgG.

[12] The polypeptide according to

[11] , wherein the Fc region is derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4.

[13] The polypeptide according to any of

[10] to

[12] , wherein the Fc region comprises a substitution with glutamic acid at position 236 and a substitution with valine at position 237 (wherein the amino acid residue numbers are amino acid positions according to the EU index).

[14] The polypeptide according to

[13] , wherein the Fc region further comprises a substitution with alanine at position 234 and a substitution with glutamic acid at position 235.

[15] The polypeptide of

[14] , wherein the Fc region further comprises a substitution at position 227 with histidine, a substitution at position 228 with lysine, a substitution at position 233 with asparagine, and / or a substitution at position 238 with serine.

[16] The polypeptide of

[14] or

[15] , wherein the Fc region further comprises a substitution at position 253 with alanine, a deletion at position 293 or 294, and / or a substitution at position 296 with alanine.

[17] The polypeptide according to

[16] , wherein the Fc region is selected from the following (1) and (2): (1) an Fc region having a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with glutamic acid at position 237, a substitution with valine at position 237, a substitution with alanine at position 253, and a deletion at position 293 or 294; (2) an Fc region having a substitution with histidine at position 227, a substitution with lysine at position 228, a substitution with asparagine at position 233, a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with valine at position 237, a substitution with serine at position 238, and a substitution with alanine at position 253; An Fc region lacking positions 293 or 294.

[18] The polypeptide of

[17] , wherein the Fc region comprises an amino acid sequence that is 90% or more identical to the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 16, the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 17, the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 21, or the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 22.

[19] The polypeptide of any of

[10] to

[18] , wherein the fusion protein is a fusion protein in which the IdeZ variant is linked to the N-terminus of the Fc region, and two of the fusion proteins form a dimer.

[20] The polypeptide of any of

[10] to

[18] , wherein the polypeptide comprises a fusion protein in which the IdeZ variant is linked to the N-terminus of a first Fc region, and a second Fc region to which the IdeZ variant is not linked.

[21] The polypeptide of

[20] , wherein the first Fc region and the second Fc region comprise knobs-into-holes mutations, and the knobs-into-holes mutations are either of the following (1) or (2): (1) in the first Fc region, a substitution with tryptophan at position 366; and in the second Fc region, a substitution with serine at position 366, a substitution with alanine at position 368, and a substitution with valine at position 407; (2) in the first Fc region, a substitution with serine at position 366, a substitution with alanine at position 368, and a substitution with valine at position 407; and in the second Fc region, a substitution with tryptophan at position 366.

[22] The polypeptide of any of

[19] to

[21] , wherein the Fc region or the first Fc region and the second Fc region are sialylated.

[23] A polynucleotide encoding the polypeptide according to any one of [9] to

[22] .

[24] An expression vector comprising the polynucleotide according to

[23] .

[25] A host cell transformed with the expression vector according to

[24] .

[26] A method for producing a polypeptide comprising an IdeZ variant, comprising the step of culturing the host cell according to

[25] .

[27] The method according to

[26] , further comprising the step of adding or expressing β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1).

[28] A variant Fc region in which position 236 is substituted with glutamic acid and position 237 is substituted with valine (wherein the amino acid residue numbers are amino acid positions according to the EU index).

[29] The variant Fc region according to

[28] , further comprising a substitution with alanine at position 234 and a substitution with glutamic acid at position 235.

[30] The Fc region variant of

[29] , further comprising the following amino acid alterations: a substitution of histidine at position 227, a substitution of lysine at position 228, a substitution of asparagine at position 233, and / or a substitution of serine at position 238.

[31] The Fc region variant of

[29] or

[30] , further comprising the following amino acid modifications: a substitution of alanine at position 253, a deletion of position 293 or 294, and / or a substitution of alanine at position 296.

[32] The Fc region variant according to

[31] , selected from the following (1) and (2): (1) an Fc region variant having a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with glutamic acid at position 237, a substitution with valine at position 237, a substitution with alanine at position 253, and a deletion at position 293 or 294; (2) an Fc region variant having a substitution with histidine at position 227, a substitution with lysine at position 228, a substitution with asparagine at position 233, a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with valine at position 237, a substitution with serine at position 238, and a substitution with alanine at position 253; An Fc region variant having a deletion at position 293 or 294.

[33] The Fc region variant according to any of

[28] to

[32] , wherein the Fc region is derived from human IgG.

[34] The Fc region variant according to

[33] , wherein the Fc region is derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4.

[35] A polypeptide comprising the Fc region variant according to any of

[28] to

[34] , which is not cleaved by an IgG cysteine ​​protease.

[36] A fusion protein of an IgG cysteine ​​protease and the Fc region variant according to any of

[28] to

[34] .

[37] The fusion protein according to

[36] , wherein the IgG cysteine ​​protease is IdeS, IdeZ, IdeZ2, IdeE, IdeE2, IdeP, IdeSORK, or IgdE, or a variant thereof.

[38] The fusion protein according to

[37] , comprising an amino acid sequence that is 90% or more identical to a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 16, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 17, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 21, or a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 22.

[0008] According to the present invention, it is possible to provide IdeZ variants that have good protease activity even when produced using mammalian cells, and polypeptides containing the IdeZ variants.

[0009] FIG. 1 shows electrophoretic images of each fusion protein produced in Example 1 (left panel shows results under non-reducing conditions, right panel shows results under reducing conditions). Lane 5 shows molecular weight markers, lane 7 shows wild-type IdeZ-mouse Fc fusion protein, and lane 9 shows IdeZ variant A-mouse Fc fusion protein. FIG. 2 shows the cleavage activity of each fusion protein produced in Example 1. Arrow 1 indicates the H chain. Arrow 2 indicates the Fab′ fragment. Lanes 3 to 7 show electrophoretic images of cleaved antibodies at antibody concentration ratios of 10, 33, 100, 333, and 1000 times that of wild-type IdeZ-mouse Fc fusion protein, respectively. Lanes 8 to 12 show electrophoretic images of antibodies obtained in a similar test using IdeZ variant A-mouse Fc fusion protein. Lane 13 shows the electrophoretic image of anti-KLH_hIgG1 alone. Figure 3 shows electrophoretic patterns of each fusion protein produced in Example 2 (left panel shows non-reducing conditions, right panel shows reducing conditions). Lane 5 shows the IdeZ variant 2-mouse Fc fusion protein, lane 7 shows the IdeZ variant 3-mouse Fc fusion protein, and lane 9 shows the IdeZ variant 4-mouse Fc fusion protein. Figure 4 shows the cleavage activity of each fusion protein produced in Example 2. Arrow 1 indicates the H chain. Arrow 2 indicates the Fab' fragment. Lanes 3 to 7 in the left panel show electrophoretic patterns of cleaved antibodies at antibody concentration ratios of 10, 33, 100, 333, and 1000 times that of the IdeZ variant 2-mouse Fc fusion protein, respectively. Lanes 8 to 12 show electrophoretic patterns of antibodies obtained in a similar test using the IdeZ variant 3-mouse Fc fusion protein. Lanes 3 to 7 in the right panel show electrophoretic patterns of antibodies obtained in a similar test using IdeZ variant 4-mouse Fc fusion protein. Figure 5 shows electrophoretic patterns of each fusion protein produced in Example 3-1 (the left panel shows electrophoretic patterns under reducing conditions, and the right panel shows electrophoretic patterns under non-reducing conditions). Lane 1 is a molecular weight marker, and lanes 2 to 6 show sialylated IdeZ variant A-human Fc variant 5, sialylated IdeZ variant A-human Fc variant 6, sialylated IdeZ variant A-human Fc variant 9, sialylated IdeZ variant A-human Fc variant 10, and sialylated IdeZ variant A-human Fc variant 11, respectively. Figure 6 shows electrophoretic patterns of each fusion protein produced in Example 3-2 (the left panel shows electrophoretic patterns under reducing conditions, and the right panel shows electrophoretic patterns under non-reducing conditions).Lane 1 shows molecular weight markers, and lanes 2 to 5 show non-sialylated IdeZ variant A-human Fc variants 7, 8, 17, and 18, respectively. Lanes 6 to 9 show sialylated IdeZ variant A-human Fc variants 7, 8, 17, and 18, respectively. Figure 7 shows electrophoretic images of each fusion protein produced in Example 4 (left panel shows images under reducing conditions, right panel shows images under non-reducing conditions). Lane 1 shows molecular weight markers, lane 2 shows non-sialylated IdeZ variant A-human Fc variant 19, and lane 3 shows sialylated IdeZ variant A-human Fc variant 19. Figure 8 shows the cleavage activity of each fusion protein produced in Example 5. Lanes 2 to 5 show electrophoretic images after reaction such that the final concentrations of sialylated IdeZ variant A-human Fc variant 7 relative to the amount of anti-KLH_hIgG1 were 0.2, 0.04, 0.008, and 0.0016. Lanes 8 to 11 show electrophoretic images after reaction such that the final concentrations of sialylated IdeZ variant A-human Fc variant 19 relative to the amount of anti-KLH_hIgG1 were 0.2, 0.04, 0.008, and 0.0016. Lanes 1 and 14 show molecular weight markers, lanes 7 and 13 show anti-KLH_hIgG1, lane 6 shows sialylated IdeZ variant A-human Fc variant 7, and lane 12 shows sialylated IdeZ variant A-human Fc variant 19.

[0010] The following describes in detail the embodiments of the present invention. Note that the materials, configurations, etc. described below do not limit the present invention, and various modifications can be made within the scope of the present invention.

[0011] 1. Definitions Unless otherwise defined below, terms used herein are used in the sense commonly used by those skilled in the art. As used herein, "IdeZ variant" refers to IdeZ containing at least one amino acid modification relative to wild-type IdeZ (SEQ ID NO: 1), and amino acid modifications include amino acid substitutions, additions, deletions, insertions, modifications, etc. As used herein, the terms "modification" and "mutation" of an amino acid are used interchangeably.

[0012] As used herein, "Fc region variant" refers to an Fc region that contains at least one amino acid alteration relative to a wild-type Fc region, and amino acid alterations include amino acid substitutions, additions, deletions, insertions, modifications, etc. When the terms "Fc region" or "Fc region variant" are used herein, they are also intended to include complexes (e.g., dimers) consisting of multiple Fc regions.

[0013] As used herein, the term "Fc region (variant)" may be derived from any antibody. An antibody (or immunoglobulin) refers to a glycoprotein consisting of a four-chain structure with a symmetric Y-shaped configuration, consisting of two heavy chains with a single sequence and two light chains with a single sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of antibody molecules is common to all classes: two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000 are bound by disulfide bonds and non-covalent bonds to form an antibody molecule with a molecular weight of 150,000 to 190,000, consisting of a Y-shaped four-chain structure. The heavy chain is typically a polypeptide chain containing approximately 440 amino acids, and has a characteristic structure for each class; it is called Igγ, Igμ, Igα, Igδ, and Igε for IgG, IgM, IgA, IgD, and IgE, respectively. Furthermore, IgG exists as subclasses, IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains are typically polypeptide chains containing approximately 220 amino acids, and two types, L-type and K-type, are known, and are called Igλ and Igκ, respectively. The two types of light chains can pair with any type of heavy chain.

[0014] Antibody molecules have four intrachain disulfide bonds in heavy chains (five in Igμ and Igε) and two in light chains, forming a loop every 100-110 amino acid residues. The three-dimensional structures of these loops are similar and are called structural units or domains. The domain located at the N-terminus of both heavy and light chains is called the variable region, which has diverse amino acid sequences even among antibodies produced from the same class (or subclass) of the same animal species and is known to be involved in the specific binding between the antibody and the antigen. The amino acid sequence of the C-terminal domain downstream of the variable region is nearly constant for each class or subclass and is called the constant region. From the N-terminus to the C-terminus, the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH). The CH is further divided into three domains from the N-terminus: CH1, CH2, and CH3 (in the case of IgG). Light chains have, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL).

[0015] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL vary greatly, contributing to the variability of the variable regions. The CDRs are regions consisting of approximately 5 to 10 amino acid residues located in the order of CDR1, CDR2, and CDR3 at the N-terminus of each heavy and light chain, and form the antigen-binding site. On the other hand, the portions of the variable regions other than the CDRs are called framework regions (FRs), and consist of FR1 to FR4, and show relatively little variation in amino acid sequence.

[0016] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two N-terminal fragments are called the Fab (Fragment, Antigen Binding) region, and the C-terminal fragment is called the Fc (Fragment, Crystallizable) region. As used herein, "Fc region" refers to a polypeptide consisting of the antibody constant region excluding the CH1 domain. Thus, the Fc region of IgA, IgD, and IgG comprises the last two C-terminal heavy chain constant region domains (CH2 and CH3), while the Fc region of IgE and IgM comprises the last three C-terminal heavy chain constant region domains (CH2, CH3, and CH4). The "Fc region" also includes the hinge region, J chain, and other components, if present. For example, the Fc region of human IgG1 comprises a polypeptide consisting of a CH2 domain and a CH3 domain, and a polypeptide consisting of part or all of the hinge region and the CH2 domain and the CH3 domain. The Fc regions of other classes of antibodies can be determined by sequence alignment with the heavy chain of human IgG1 or a fragment thereof.

[0017] As used herein, the term "IdeZ variant-Fc region (or Fc region variant) fusion" (i.e., fusion protein) refers to both a structure in which one IdeZ variant is bound to the N-terminus of each of two Fc regions of a complex, and a one-armed structure in which an IdeZ variant is bound to only one of the Fc regions.

[0018] Furthermore, terms such as "sialylated IdeZ variant-Fc region (or Fc region variant) fusion," "sialylated fusion protein," and the like refer to a fusion product between an IdeZ variant and an Fc region (or an Fc region variant) to which sialic acid has been intentionally added by a sialylation process commonly used in the art (e.g., treatment with β-galactoside α-2,6-sialyltransferase 1). On the other hand, as used herein, terms such as "non-sialylated IdeZ variant-Fc region (or Fc region variant) fusion product," "non-sialylated fusion protein," and the like refer to a fusion product between an IdeZ variant and an Fc region (or an Fc region variant) that has not been subjected to the above-mentioned sialylation process, and do not preclude the fusion protein from containing sialic acid.

[0019] As used herein, the "consensus sequence for N-glycans" in IdeZ refers to a sequence located at a glycosylation site required for glycosylation of an asparagine residue in a polypeptide, such as the sequence asparagine (N)-X-serine (S) / threonine (T) (X is an amino acid other than proline). In wild-type IdeZ represented by SEQ ID NO: 1, the above consensus sequences are present at positions 118-120, 171-173, 267-269, and 298-300.

[0020] As used herein, amino acid residue numbers in antibody sequences such as the Fc region are defined according to the EU index by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, NIH Publication No. 91-3242).

[0021] The terms "first" and "second" are used herein for convenience to distinguish between two or more of each type of moiety. The use of such terms is not intended to imply a particular order or meaning unless expressly stated.

[0022] As used herein, the presence of a peptide linker is optional. The term "peptide linker" refers to one or more amino acid residues that can be introduced by genetic engineering to link two or more moieties. The length of the peptide linker used in the present invention is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose.

[0023] As used herein, "identity" refers to the Identity value obtained using EMBOSS Needle (Nucleic Acids Res., 2015, Vol. 43, pW580-W584) with the default parameters. The parameters are as follows: Gap Open Penalty = 10, Gap Extend Penalty = 0.5, Matrix = EBLOSUM62, End Gap Penalty = false.

[0024] As used herein, the term "subject" refers to a human or other animal in need of such prevention or treatment. In one embodiment, the subject is a human in need of such prevention or treatment.

[0025] 2. IdeZ Variants (1) IdeZ Variants An IdeZ variant according to one embodiment of the present invention has IgG cysteine ​​protease activity, and the variant comprises: (a) a sequence that is 90% or more identical to the amino acid sequence from positions 35 to 349 of the amino acid sequence represented by SEQ ID NO: 1 (wild-type IdeZ); and (b) no glycosylation occurs at the amino acids corresponding to positions 118 and 267 of the amino acid sequence represented by SEQ ID NO: 1. The present inventors have found that IdeZ lacking glycosylation at positions 118 and 267 exhibits suppressed protease activity even when expressed in mammalian cells. Furthermore, the present inventors have also found that the production of wild-type IdeZ using mammalian cells poses a problem of reduced expression levels, and that the production of the above-described IdeZ variants using mammalian cells suppresses this reduction in expression levels.

[0026] To prevent the addition of a sugar chain to the above-mentioned positions, for example, the amino acids at positions 118 and 267 can be modified to amino acids other than asparagine, or an amino acid modification can be added to the consensus sequence for N-glycans containing these positions. Adding at least one amino acid modification to the consensus sequence for N-glycans containing asparagine described above prevents the addition of a sugar chain. However, even if the consensus sequence is met, a sugar chain may not be attached depending on the amino acid sequence. For example, it has been reported that in the consensus sequence NXS / T, NXT may have a higher glycosylation efficiency than NXS (when X is the same amino acid and both sequences are at the same position in the protein) (Biochem. J., 1981, Vol. 195, pp. 639-644; Biochemistry, 1999, Vol. 38, pp. 13584-13591; Biochem. J., 2001, Vol. 355, pp. 245-248; Biochem. J., 1997, Vol. 323, pp. 415-419).

[0027] According to one embodiment, the IdeZ variant may contain the following amino acid modifications (b') and (b''). Such amino acid modifications can result in an IdeZ variant in which no glycosylation is performed on the amino acids at positions 118 and 267 of the amino acid sequence represented by SEQ ID NO: 1. Each of (b') and (b'') may contain at least one of the modifications (i) to (iii).(b') (i) the amino acid at position 118 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 119 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) the amino acid at position 120 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (b'') (i) the amino acid at position 267 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 268 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) the amino acid at position 269 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.

[0028] Preferably, the IdeZ variant has the amino acid at the position corresponding to position 120 of the amino acid sequence represented by SEQ ID NO: 1 substituted with glutamic acid, and the amino acid at the position corresponding to position 269 of the amino acid sequence represented by SEQ ID NO: 1 substituted with arginine.

[0029] Preferably, the IdeZ variant further has (c) no glycosylation at the amino acid corresponding to position 171 of the amino acid sequence represented by SEQ ID NO: 1, and / or (d) no glycosylation at the amino acid corresponding to position 298 of the amino acid sequence represented by SEQ ID NO: 1. That is, as described above, in addition to no glycosylation at positions 118 and 267, it is also preferable that no glycosylation be added to amino acids at positions 171 and / or 298. This is because a reduction in the amount of glycosylation further improves protease activity. To prevent glycosylation, for example, the amino acids at positions 171 and / or 298 can be modified to amino acids other than asparagine, or amino acid modifications can be added to the consensus sequence for N-glycans containing these positions. Specific amino acid modifications that prevent glycosylation are the same as those described above for the amino acid modifications at positions 118 and 267.

[0030] According to one embodiment, the IdeZ variant comprises the following amino acid modifications (c') and / or (d'): (c') (i) the amino acid at position 171 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 172 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with proline; and / or (iii) the amino acid at position 173 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (d') (i) the amino acid at position 298 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 299 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with proline; and / or (iii) The amino acid at the position corresponding to position 300 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.

[0031] Preferably, in the IdeZ variant of the embodiment, the amino acid at the position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, and / or the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid.

[0032] More preferably, the IdeZ variant of the embodiment has two to four amino acid modifications selected from the following (a) to (d): (a) the amino acid at the position corresponding to position 120 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glutamic acid, (b) the amino acid at the position corresponding to position 171 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, (c) the amino acid at the position corresponding to position 269 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with arginine, and (d) the amino acid at the position corresponding to position 298 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid.

[0033] The IdeZ variant may have 2 to 4 amino acid alterations selected from (a) to (d) above, more preferably 3 or 4 amino acid alterations, and particularly preferably 4 amino acid alterations, relative to the amino acid sequence represented by SEQ ID NO: 1. When 2 or 3 amino acid alterations are selected from (a) to (d) above, any combination may be used, and preferably includes the amino acid alterations (a) and (c), (a), (b) and (c), or (a), (c) and (d).

[0034] Particularly preferably, the IdeZ variants according to the embodiments comprise an amino acid sequence that is 90% or more, 95% or more, or 100% identical to the amino acid sequence represented by SEQ ID NO: 5, 9, or 13. The IdeZ variants according to the embodiments may also comprise amino acid modifications other than those described above. When produced using mammalian cells, the IdeZ variants according to the embodiments preferably have 10 to 100-fold, 10 to 70-fold, or 10 to 40-fold greater protease activity than wild-type IdeZ. Because it is the IdeZ moiety that contributes to the protease activity, the IdeZ variant-Fc region fusion protein described below also has protease activity.

[0035] (2) Polynucleotides and Expression Vectors According to one embodiment, a polynucleotide encoding the above-described IdeZ variant is provided. The IdeZ variant according to the embodiment can be prepared by a person skilled in the art based on its nucleotide sequence using a method known in the art. For example, the IdeZ variant according to the embodiment can be synthesized using a gene synthesis method known in the art. Various methods known to those skilled in the art, such as the antibody gene synthesis method described in International Publication No. 90 / 07861, can be used for such gene synthesis.

[0036] According to a further embodiment, an expression vector containing the above-described polynucleotide is provided. The expression vector according to the embodiment is not particularly limited, as long as it is capable of producing the above-described polynucleotide in various host cells, such as eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and prokaryotic cells (e.g., Escherichia coli). Examples of such expression vectors include plasmid vectors and viral vectors. Examples of plasmid vectors that can be used include the pcDNA series (Thermo Fisher Scientific), pALTER®-MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio Inc.), pEE6.4, or pEE12.4 (Lonza Biologics). Examples of viral vectors that can be used include lentivirus, adenovirus, retrovirus, and adeno-associated virus. For example, when a lentivirus is used to introduce a polynucleotide according to the embodiment into a cell, the lentivirus may be a pLVSIN-CMV / EF1α vector (Takara Bio Inc.), a pLenti vector (Thermo Fisher Scientific), etc. In one embodiment, vectors used for the expression vector according to the embodiment are pcDNA™ 3.4-TOPO™ (Thermo Fisher Scientific) and pcDNA™ 3.1 (Thermo Fisher Scientific).

[0037] The expression vector according to the embodiment may include a promoter operably linked to the polynucleotide. Examples of promoters for expressing the polynucleotide 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 in bacteria (e.g., Escherichia) include trp promoters, lac promoters, λPL promoters, and tac promoters. Examples of promoters for expressing the polynucleotide in yeast include GAL1 promoters, GAL10 promoters, PH05 promoters, PGK promoters, GAP promoters, and ADH promoters.

[0038] When animal cells, insect cells, or yeast cells are used as host cells, the expression vectors according to the embodiments may contain a start codon and a stop codon. In this case, they may contain an enhancer sequence, untranslated regions on the 5' and 3' sides of the gene encoding the IdeZ variant according to the embodiments, a secretion signal sequence, a splicing junction, a polyadenylation site, or a replicable unit. When Escherichia coli is used as the host cell, the expression vectors according to the embodiments may contain a start codon, a stop codon, a terminator region, and a replicable unit. The expression vectors according to the embodiments may contain a commonly used drug selection marker gene (e.g., a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene) depending on the purpose.

[0039] (3) Host Cells According to one embodiment, a host cell transformed with the above-described expression vector is provided. The host cell according to the embodiment may contain one or more polynucleotides according to the embodiment by transformation with the expression vector according to the embodiment.

[0040] The host cell to be transformed is not particularly limited, as long as it is compatible with the expression vector used and can be transformed with the expression vector to express the polypeptide of the embodiment. Various cells, such as conventional cells commonly used in the art or artificially established cells, can be used as the host cell to be transformed. Examples include animal cells (CHO-K1 cells, ExpiCHO-S™ cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, NS0 cells, etc.), insect cells (Sf9 cells, etc.), bacteria (Escherichia spp., etc.), and yeast (Saccharomyces spp., Pichia spp., etc.). In one embodiment, the host cell is a CHO-K1 cell or an ExpiCHO-S cell.

[0041] The method for transforming host cells is not particularly limited, and methods commonly used by those skilled in the art, such as the calcium phosphate method, electroporation, or lipofection, can be used.

[0042] Transformed host cells can be selected by methods commonly used by those skilled in the art, such as a drug selection method using a drug selection marker gene and a drug such as tetracycline, ampicillin, neomycin, or hygromycin, or a cell isolation method such as limiting dilution, single cell sorting, or colony pick-up.

[0043] (4) Method for Producing IdeZ Variants IdeZ variants according to the embodiments can be produced by those skilled in the art using methods known in the art based on the sequence information and the like disclosed herein. For example, IdeZ variants can be obtained by modifying wild-type IdeZ using methods commonly used in the art. For example, this method may include culturing the transformed host cells described above and expressing the IdeZ variant in the host cells or culture supernatant, and recovering, isolating, and purifying the IdeZ variant. However, the method is not limited to these methods as long as the desired IdeZ variant is produced. Specific methods for culturing, purifying, etc. are as described in the polypeptide production method described below.

[0044] (5) Uses The IdeZ variants according to the embodiments can be used in conventionally known uses of IgG cysteine ​​proteases. Examples of such uses include, for example, use in high-quality recombinant protein production systems, use for suppressing rejection during transplantation, and use for treating autoimmune diseases, as described in the Background Art section above (Patent Documents 1 and 2, WO 2006 / 131347, WO 2016 / 012285, etc.). Another possible use is pretreatment for AAV-based gene therapy (Patent Document 1).

[0045] 3. Polypeptides Comprising IdeZ Variants, Fc Region Variants, and Polypeptides Comprising Fc Region Variants According to one embodiment, a polypeptide comprising the above-described IdeZ variant is provided. The type of polypeptide is not limited as long as it contains the above-described IdeZ variant. In one embodiment, the polypeptide comprising the above-described IdeZ variant is a fusion protein of the above-described IdeZ variant and an Fc region.

[0046] The origin of the Fc region is not particularly limited, and examples of antibody Fc regions include IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM types. The Fc region used may be derived from any of these, but is preferably derived from IgG (IgG1, IgG2, IgG3, or IgG4), more preferably from human IgG, even more preferably from human Igγ1, human Igγ2, human Igγ3, or human Igγ4, and particularly preferably from human Igγ1.

[0047] Furthermore, the present inventors have found that the use of an Fc region variant in which specific amino acids in the Fc region have been modified can suppress autolysis of the fusion protein when the fusion protein is formed with the above-mentioned IdeZ variant. Thus, according to one embodiment, a fusion protein of an IdeZ variant and an Fc region variant is provided. IdeZ is a cysteine ​​protease that acts on and cleaves the hinge region. Therefore, in a fusion protein in which IdeZ is bound to an Fc region, autolysis can become a problem, as IdeZ acts on and cleaves the hinge region of the fusion protein itself. Therefore, the present inventors focused on the property of IdeZ, which has the ability to cleave the Fab-Fc region of human IgG but does not act on mouse IgG (IgG1, IgG2b), and constructed a fusion protein by combining an Fc region with the above-mentioned IdeZ variant, which has a structure similar to that of mouse IgG. As a result, it was found that autolysis was suppressed.

[0048] Specifically, the following modifications are preferably made to the Fc region used in the fusion protein of an IdeZ variant and an Fc region variant: According to one embodiment, the Fc region variant used in the fusion protein comprises a substitution with glutamic acid at position 236 and a substitution with valine at position 237. Preferably, the Fc region variant further comprises a substitution with alanine at position 234 and a substitution with glutamic acid at position 235. More preferably, the Fc region variant further comprises any one or more of the following amino acid modifications: a substitution with histidine at position 227, a substitution with lysine at position 228, a substitution with asparagine at position 233, and / or a substitution with serine at position 238. Even more preferably, the Fc region variant further comprises any one or more of the following amino acid modifications: a substitution with alanine at position 253, a deletion at position 293 or 294, and / or a substitution with alanine at position 296.

[0049] Particularly preferably, the Fc region variant is selected from the following (1) and (2): (1) an Fc region variant having a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with glutamic acid at position 237, a substitution with valine at position 237, a substitution with alanine at position 253, and a deletion at position 293 or 294; (2) an Fc region variant having a substitution with histidine at position 227, a substitution with lysine at position 228, a substitution with asparagine at position 233, a substitution with alanine at position 234, a substitution with glutamic acid at position 235, a substitution with glutamic acid at position 236, a substitution with valine at position 237, a substitution with serine at position 238, a substitution with alanine at position 253, and An Fc region variant in which position 293 or 294 is deleted.

[0050] More specifically, Fc region variants include sequences that are 90% or more identical, more preferably 95% or more identical, and even more preferably 100% identical to the amino acid sequence of the Fc region portion of the amino acid sequence of SEQ ID NO: 16, 17, 21, or 22, i.e., the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence of SEQ ID NO: 16, the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence of SEQ ID NO: 17, the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence of SEQ ID NO: 21, and the sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence of SEQ ID NO: 22. Fc region variants having these sequences are preferred in terms of their autolysis inhibitory effect. Furthermore, Fc region variants having the sequence of the Fc region portion of SEQ ID NO: 16 or 21, or sequences that are 90% or more identical, more preferably 95% or more identical to these sequences, are particularly preferred in terms of the reduced amount of contaminants during production. Furthermore, an Fc region variant having the sequence of the Fc region portion in SEQ ID NO: 16 or a sequence that is 90% or more identical thereto, more preferably 95% or more identical thereto, is preferred in that it requires fewer amino acid modifications to be introduced and can achieve a high sialylation rate.

[0051] In particular, the amino acid modifications of L234A and L235A in the human Igγ1 constant region are called "LALA mutations." Here, L234A is a substitution of leucine at position 234 in the human Igγ1 constant region with alanine according to the EU index. L235A is a substitution of leucine at position 235 in the human Igγ1 constant region with alanine according to the EU index. Furthermore, the amino acid modifications of L234A and L235E are called "LALE mutations." Here, L235E is a substitution of leucine at position 235 in the human Igγ1 constant region with glutamic acid according to the EU index. As will be described later, it is preferable to improve the sialylation rate of the Fc region in order to suppress the production of ADA (anti-drug antibodies), and deletion of the amino acid at position 293 or 294 contributes to improving the sialylation rate. In addition to these, it is preferable to include the above-mentioned LALA mutation and LALE mutation.

[0052] The Fc region variant may include modifications other than those described above. While there are no particular limitations as long as the desired effect can be achieved, for example, the Fc region variant may include modifications based on the Knobs-into-Hole technique (hereinafter also referred to as "Knobs-into-Hole mutations"). Knobs-into-Hole mutations are, for example, mutations in which, in an Fc region dimer, the amino acid at position 366 in one Fc region is substituted with tryptophan, and the amino acid at position 366 in the other Fc region is substituted with serine, the amino acid at position 368 with alanine, and the amino acid at position 407 with valine (WO 1998 / 050431).

[0053] An IdeZ variant-Fc region (or Fc region variant) fusion (i.e., a fusion protein) may have a structure in which one IdeZ variant is bound to the N-terminus of each of two Fc regions, or a one-armed structure in which an IdeZ variant is bound to only one of the Fc regions. In one embodiment, the fusion protein is a fusion protein in which an IdeZ variant is bound to the N-terminus of an Fc region (or Fc region variant), and the two fusion proteins form a dimer. In another embodiment, the fusion protein is a one-armed fusion protein comprising a fusion protein in which an IdeZ variant is bound to the N-terminus of a first Fc region and a second Fc region to which no IdeZ variant is bound. As described below, the Fc region in the fusion protein is preferably sialylated, but a one-armed fusion protein comprising such an Fc region variant can achieve a relatively high sialylation rate even without the use of a glycosyltransferase for sialylation. Furthermore, the IdeZ-Fc region fusion protein can have the problem of being prone to aggregation, whereas the single-arm fusion protein is less susceptible to such aggregation problems.

[0054] In the case of a one-arm fusion protein, the first Fc region and the second Fc region can comprise the knobs-into-holes mutation, CRIB, or BEAT described above (Vaccines 2021, 9(7), 724). According to an embodiment of the present invention, the first Fc region and the second Fc region preferably comprise the knobs-into-holes mutation described above.

[0055] According to one embodiment, the Fc region in the one-arm fusion protein comprises, in addition to the amino acid modifications described above, the following knobs-into-holes mutations (1) or (2): (1) in the first Fc region, a substitution of tryptophan at position 366; and in the second Fc region, a substitution of serine at position 366, a substitution of alanine at position 368, and a substitution of valine at position 407; or (2) in the first Fc region, a substitution of serine at position 366, a substitution of alanine at position 368, and a substitution of valine at position 407; and in the second Fc region, a substitution of tryptophan at position 366.

[0056] The fusion proteins containing the above-mentioned IdeZ variants can be used in the conventionally known applications of IgG cysteine ​​proteases, as described above in "2. IdeZ variants (5) Applications."

[0057] According to one embodiment, the Fc region variant itself suitable for the above-mentioned uses is also provided, and the specific amino acid modifications etc. are as described above for the Fc region variant as a component of the fusion protein with the IdeZ variant.

[0058] According to one embodiment, a polypeptide (fusion protein) comprising the above-described Fc region variant with another protein is also provided. Examples of other proteins that can be used include antibodies, antigen-binding fragments of antibodies (single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, F(ab') fragments, etc. ２ Examples of such Fc region variants include, but are not limited to, polypeptides containing Fc regions that are not readily cleaved by IgG cysteine ​​proteases, such as polypeptide fragments, and biologically active peptides (e.g., receptors, adhesion molecules, ligands, enzymes, etc.).

[0059] In one embodiment, a fusion protein of an IgG cysteine ​​protease and the above-described Fc region variant is also provided. As described above, the use of the Fc region variant can suppress autolysis of the fusion protein of the IdeZ variant and the Fc region variant. These effects can also be achieved with fusion proteins with IgG cysteine ​​proteases other than the IdeZ variant. Such IgG cysteine ​​proteases are not limited as long as they have the activity of cleaving IgG, and include IdeS, IdeZ, IdeZ2, IdeE, IdeE2, IdeP, IdeSORK, IgdE, or variants thereof. Of these, IdeZ or the IdeZ variants according to the above-described embodiments are preferred. In one embodiment, the fusion protein is a fusion protein in which the IgG cysteine ​​protease is linked to the N-terminus of the Fc region variant, and two of the fusion proteins form a dimer. In one embodiment, the Fc region variant used comprises a sequence that is 90% or more identical, more preferably 95% or more identical, and even more preferably 100% identical to a sequence selected from the group consisting of the amino acid sequence of positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence of positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 17, the amino acid sequence of positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 21, and the amino acid sequence of positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 22. In another embodiment, the fusion protein is a one-armed fusion protein comprising a fusion protein in which an IgG cysteine ​​protease is linked to the N-terminus of a first Fc region and a second Fc region to which the IgG cysteine ​​protease is not linked. In one embodiment, in the case of a one-armed fusion protein, the first Fc region and the second Fc region comprise the knobs-into-holes mutations described above.

[0060] 4. Polynucleotides and Expression Vectors According to one embodiment, a polynucleotide encoding the above-described polypeptide is provided. A person skilled in the art can prepare the polynucleotide according to the embodiment using a method known in the art based on the base sequence. For example, the polynucleotide according to the embodiment can be synthesized using a gene synthesis method known in the art. Various methods known to those skilled in the art can be used for such gene synthesis, such as the antibody gene synthesis method described in International Publication No. 90 / 07861.

[0061] In a further embodiment, an expression vector is provided comprising the above-described polynucleotide. In the case of a single-arm fusion protein, the polynucleotide encoding the fusion protein in which the IdeZ variant is linked to the N-terminus of a first Fc region and the polynucleotide encoding the single-arm fusion protein comprising a second Fc region to which the IdeZ variant is not linked may each be contained in a separate vector, or multiple polynucleotides may be contained in a single vector.

[0062] The expression vector according to the embodiment is not particularly limited as long as it is capable of producing the polynucleotide according to the embodiment in various host cells, such as eukaryotic cells (e.g., animal cells, insect cells, plant cells, yeast) and prokaryotic cells (e.g., Escherichia coli). Examples of such expression vectors include plasmid vectors and viral vectors. Examples of plasmid vectors that can be used include the pcDNA series (Thermo Fisher Scientific), pALTER (registered trademark)-MAX (Promega), pHEK293 Ultra Expression Vector (Takara Bio Inc.), pEE6.4, or pEE12.4 (Lonza Biologics). Examples of viral vectors that can be used include lentivirus, adenovirus, retrovirus, and adeno-associated virus. For example, when a lentivirus is used to introduce a polynucleotide according to the embodiment into a cell, the lentivirus may be a pLVSIN-CMV / EF1α vector (Takara Bio Inc.), a pLenti vector (Thermo Fisher Scientific), etc. In one embodiment, vectors used for the expression vector according to the embodiment are pcDNA™ 3.4-TOPO™ (Thermo Fisher Scientific) and pcDNA™ 3.1 (Thermo Fisher Scientific).

[0063] Expression vectors according to embodiments may include a promoter operably linked to a polynucleotide of the present invention. Examples of promoters for expressing polynucleotides according to 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 polynucleotides according to embodiments in bacteria (e.g., Escherichia) include trp promoters, lac promoters, λPL promoters, and tac promoters. Examples of promoters for expressing polynucleotides according to embodiments in yeast include GAL1 promoters, GAL10 promoters, PH05 promoters, PGK promoters, GAP promoters, and ADH promoters.

[0064] When animal cells, insect cells, or yeast cells are used as host cells, expression vectors according to embodiments may contain a start codon and a stop codon. In this case, they may contain an enhancer sequence, untranslated regions on the 5' and 3' sides of the gene encoding the Fc region variant according to embodiments, a secretion signal sequence, a splicing junction, a polyadenylation site, or a replicable unit. When Escherichia coli is used as the host cell, expression vectors according to embodiments may contain a start codon, a stop codon, a terminator region, and a replicable unit. Expression vectors according to embodiments may contain a commonly used drug selection marker gene (e.g., a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene) depending on the purpose.

[0065] 5. Host Cells According to one embodiment, a host cell is provided that has been transformed with the expression vector described above. The host cell according to the embodiment may contain one or more polynucleotides according to the embodiment via transformation with the expression vector according to the embodiment.

[0066] The host cell to be transformed is not particularly limited, as long as it is compatible with the expression vector used and can be transformed with the expression vector to express the polypeptide of the embodiment. Various cells, such as conventional cells commonly used in the art or artificially established cells, can be used as the host cell to be transformed. Examples include animal cells (CHO-K1 cells, ExpiCHO-S™ cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, NS0 cells, etc.), insect cells (Sf9 cells, etc.), bacteria (Escherichia spp., etc.), and yeast (Saccharomyces spp., Pichia spp., etc.). In one embodiment, the host cell is a CHO-K1 cell or an ExpiCHO-S cell.

[0067] The method for transforming host cells is not particularly limited, and methods commonly used by those skilled in the art, such as the calcium phosphate method, electroporation, or lipofection, can be used.

[0068] Transformed host cells can be selected by methods commonly used by those skilled in the art, such as a drug selection method using a drug selection marker gene and a drug such as tetracycline, ampicillin, neomycin, or hygromycin, or a cell isolation method such as limiting dilution, single cell sorting, or colony pick-up.

[0069] 6. Polypeptide Production Method

[0033] One embodiment provides a method for producing a polypeptide comprising an IdeZ variant according to an embodiment. The method according to an embodiment may include steps of culturing the transformed host cells described above and expressing the polypeptide in the host cells or culture supernatant, and steps of recovering, isolating, and purifying the polypeptide. However, the method is not limited to these steps as long as the desired polypeptide is produced.

[0070] Transformed host cells can be cultured by known methods. Culture conditions, such as temperature, medium pH, and culture time, can be appropriately selected by those skilled in the art. When the host cells are animal cells, examples of media that can be used include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, Vol. 130, pp. 432-437), DMEM medium (Virol., 1959, Vol. 8, p. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), and 199 medium (Exp. Biol. Med., 1950, Vol. 73, pp. 1-8). The pH of the medium is, for example, about 6 to 8, and the culture is typically performed at about 30 to 40°C for about 15 to 336 hours, with aeration and stirring as necessary. When the host cells are insect cells, the medium may 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 is typically performed at about 20 to 40°C for about 15 to 100 hours, with aeration and stirring as necessary.

[0071] When the host cell is Escherichia coli or yeast, a suitable medium is, for example, a liquid medium containing nutrient sources. The nutrient medium contains, for example, a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of the transformed host cell. Carbon sources include, for example, glucose, dextran, soluble starch, and sucrose, while inorganic and organic nitrogen sources include, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If desired, the medium may contain other nutrients (e.g., inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins), antibiotics (e.g., tetracycline, neomycin, ampicillin, and kanamycin), etc.). The pH of the medium is, for example, about 5 to 8. When the host cell is Escherichia coli, the medium may be, for example, LB medium or M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2). The culture is typically carried out at about 14 to 43°C for about 3 to 24 hours, with aeration and stirring as necessary. When the host cell is yeast, the medium may be, for example, Burkholder's minimal medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505). The culture is typically carried out at about 20 to 35°C for about 14 to 144 hours, with aeration and stirring as necessary. The target polypeptide can be expressed by the culture described above.

[0072] In addition to culturing transformed host cells and expressing a polypeptide containing an IdeZ variant, the method of the present invention may further include recovering, isolating, or purifying the polypeptide from the host cells. Examples of isolation or purification methods include methods that utilize solubility (e.g., salting out or solvent precipitation); methods that utilize molecular weight differences (e.g., dialysis, ultrafiltration, or gel filtration); methods that utilize charge (e.g., ion exchange chromatography or hydroxylapatite chromatography); methods that utilize specific affinity (e.g., affinity chromatography); methods that utilize hydrophobicity differences (e.g., reverse-phase high-performance liquid chromatography); and methods that utilize isoelectric point differences (e.g., isoelectric focusing). In one embodiment, the polypeptide secreted into the culture supernatant can be purified by various types of chromatography, for example, column chromatography using a protein A column or a protein G column. According to one embodiment, a polypeptide containing an IdeZ variant produced by the above-described production method is provided.

[0073] 7. Sialylated Fusion Proteins The IdeZ variant-Fc region (or Fc region variant) fusion protein described above may have a sialylated Fc region (i.e., sialylated). Sialylation of the Fc region can suppress ADA production in response to the fusion protein when administered to a living body. It is preferable to modify the Fc region to increase the sialylation rate. Examples of Fc regions containing such modifications include Fc region variants containing a LALA or LALE mutation and a deletion of amino acids at positions 293 or 294. When position 294 is deleted and the interaction between positions 234 and 235 is reduced by the LALA or LALE mutation, steric space, such as a gap, is created between these positions, which is thought to allow further twisting of the sugar chain attached to asparagine at position 297, making the fusion protein more susceptible to sialic acid addition. Typically, in wild-type IgG, both amino acids at positions 293 and 294 are glutamic acid, so the same modification can be achieved by deleting either one. In one embodiment, the Fc region variant lacks glutamic acid at position 293 or 294. According to one embodiment, a sialylated IdeZ variant-Fc region (or Fc region variant) fusion protein is provided.

[0074] The sialylation method is not particularly limited, and a sialylated fusion protein can be obtained by adding or expressing a sialyltransferase during the process of culturing the host cells described above. Examples of such enzymes include β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1), sialyltransferase, and β-galactoside α-2,3-sialyltransferase. This further increases the amount of sialic acid added to the Fc region. The sialyltransferase may be simply added, or an expression vector may be constructed by inserting DNA encoding the sialyltransferase and expressing it together with a polypeptide such as an antibody. When adding the sialyltransferase, the timing and method of addition are not particularly limited. For example, the sialyltransferase may be added during the process of producing a polypeptide by cell culture, or may be added and reacted after purifying the polypeptide. When expressing the sialyltransferase, a sialylated polypeptide can be produced by, but not limited to, co-expressing a plasmid encoding the sialyltransferase in cells together with a plasmid encoding the polypeptide. When a polypeptide is produced using cells containing endogenous glycosyltransferases, such as CHO cells, sialic acid can be added during the polypeptide production process without the addition of the above-mentioned sialyltransferase.

[0075] Fusion proteins containing Fc region variants with modifications that increase the sialylation rate as described above have an increased amount of sialic acid added (i.e., the degree of sialylation) compared to sialylated wild-type Fc regions or Fc region variants in which only the amino acid at position 294 has been deleted. For example, the amount of sialic acid added to the glycan linked to asparagine at position 297 increases. The amount of sialic acid added can be measured as described in the Examples below and is expressed as the "sialylation rate (%)." As used herein, "sialylation rate (%)" refers to the proportion of glycans to which sialic acid has been added among all glycans present in the Fc region, and specific measurement and calculation methods are as described in the Examples below. The sialylated fusion proteins of the present embodiments have a sialylation rate of, for example, 20-100%, 40-100%, 60-100%, 80-100%, or 90-100%.

[0076] The above-described fusion proteins and sialylated fusion proteins can be used in conventionally known applications of IgG cysteine ​​proteases. Examples of such applications include, as described in the Background Art section above, use in high-quality recombinant protein production systems, use for suppressing rejection during transplantation, and use for treating autoimmune diseases (Patent Documents 1 and 2, WO 2006 / 131347, WO 2016 / 012285, etc.). Furthermore, the use of fusion proteins offers the advantages of easy purification and modification, and adjustable half-life.

[0077] 8. Pharmaceutical Compositions According to one embodiment, a pharmaceutical composition is provided comprising the above-described IdeZ variant or polypeptide and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by a commonly used method using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. Examples of dosage forms of the pharmaceutical composition include parenteral preparations such as injections and infusions, which can be administered intravenously, subcutaneously, intraperitoneally, etc. When formulating the pharmaceutical composition, excipients, carriers, additives, etc. appropriate for these dosage forms can be used within a pharmaceutically acceptable range. Pharmaceutical compositions according to embodiments may further comprise one or more additional therapeutic agents. Furthermore, pharmaceutical compositions according to embodiments may be used in combination (simultaneously or sequentially) with one or more additional therapeutic agents.

[0078] The amount of the IdeZ variant or polypeptide according to the embodiment in the pharmaceutical composition can be appropriately determined depending on the severity and age of the patient's symptoms, the dosage form of the preparation used, etc. The IdeZ variant or polypeptide according to the embodiment and a pharmaceutical composition containing the same can be used for suppressing rejection during organ transplantation, treating autoimmune diseases, pretreatment for gene therapy using AAV, etc. In addition, according to another embodiment, a method for treating an autoimmune disease is provided, which comprises administering a therapeutically effective amount of the IdeZ variant or polypeptide according to the embodiment to a subject. In addition, according to a further embodiment, the IdeZ variant or polypeptide of the present invention is included for use in treating an autoimmune disease. The present invention also provides use of the IdeZ variant or polypeptide according to the embodiment in the manufacture of a pharmaceutical composition for treating an autoimmune disease. The autoimmune disease to be treated is not particularly limited.

[0079] Example 1: Preparation of IdeZ-Fc fusion protein in mammalian cells and evaluation of its activity A gene encoding the amino acid sequence from amino acids 35 to 349 of the IdeZ protein (UniProt accession number: Q0PIW1, SEQ ID NO: 1) derived from Streptococcus equi subsp. zooepidemicus was ligated to a nucleic acid sequence encoding an antibody heavy chain signal (Protein Eng., 1987, Vol. 1, pp. 499-505) on the 5' side and a nucleic acid sequence encoding a mouse Igγ1 constant region (from glycine at amino acid position 223 to lysine at amino acid position 447 according to the EU index) into which the amino acid mutations D265A and F328C had been introduced on the 3' side. The resulting nucleic acid sequence was inserted into the pcDNA3.4 vector (Thermo Fisher). The DNA was inserted into a vector (Chemical Science) to prepare an expression vector.

[0080] The resulting expression vector containing the nucleic acid sequence encoding the IdeZ-Fc fusion protein was transfected into ExpiCHO-S cells (Thermo Fisher Scientific), and the cells were incubated at 37°C, 8% CO ２After 4 days of rotational culture under these conditions, purified IdeZ-Fc fusion protein was obtained according to a standard method. The obtained IdeZ-Fc fusion protein was named wild-type IdeZ-mouse Fc fusion protein.

[0081] Using a similar technique, we constructed an expression vector containing a nucleic acid sequence encoding a variant of the IdeZ protein with four amino acid alterations (T120E / N171G / S269R / N298D according to the numbering in SEQ ID NO: 1) to obtain an IdeZ variant-Fc fusion protein, which was designated IdeZ variant A-mouse Fc fusion protein.

[0082] The nucleotide sequence of IdeZ in the wild-type IdeZ-mouse Fc fusion protein is shown in SEQ ID NO: 2, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 3. The nucleotide sequence of the IdeZ variant in the IdeZ variant A-mouse Fc fusion protein is shown in SEQ ID NO: 4, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 5.

[0083] Each fusion protein was heated at 70°C for 10 minutes in NuPAGE LDS Sample Buffer (Life Technologies, NP0008), and the degree of purification was assessed by SDS-PAGE using NuPAGE 4-12% Bis-Tris Gel (Life Technologies, NP0322BOX). Reduction was performed by adding NuPAGE Sample Reducing Agent (Life Technologies, NP0009) during heating. Precision Plus Protein Unstained Standards (BIO-RAD, 161-0363) were used as molecular weight markers. The electrophoretic patterns of each fusion protein are shown in Figure 1. The results under non-reducing conditions indicate the molecular weight of the homodimer and the presence of aggregates, while the results under reducing conditions indicate the molecular weight of the monomer.

[0084] In Figure 1 , lane 5 shows molecular weight markers, lane 7 shows the wild-type IdeZ-mouse Fc fusion protein, and lane 9 shows the IdeZ variant A-mouse Fc fusion protein. Compared with the wild-type IdeZ-mouse Fc fusion protein, a stronger and clearer band was detected at a lower molecular weight in the IdeZ variant A-mouse Fc fusion protein in which the consensus sequence for the N-glycan had been mutated. This suggests that an N-linked glycan has been added to the wild-type IdeZ-mouse Fc fusion protein, and confirms that the IdeZ variant A-mouse Fc fusion protein has been deglycosylated by mutating the consensus sequence for the N-glycan.

[0085] Next, the IgG protease activity of each fusion protein was evaluated by assessing the cleavage of a human IgG1 antibody. To 10 μg of the cleaved antibody, a human IgG1 antibody (WO 2013 / 094723) (anti-KLH_hIgG1) against KLH (keyhole limpet hemocyanin), an antigen not present in vivo, was added 1.0, 0.3, 0.1, 0.03, or 0.01 μg of each fusion protein (the final antibody to fusion protein concentration ratio was 10, 33, 100, 333, and 1000 times, respectively). The final volume was adjusted to 20 μL, and the mixture was treated at 37°C for 2 hours. The SDS-PAGE method described above was performed under reducing conditions. The electrophoresis image is shown in Figure 2.

[0086] In Figure 2, arrow 1 indicates the H chain. Arrow 2 indicates the Fab' fragment. Lanes 3 to 7 show electrophoretic images of cleaved antibodies at antibody concentration ratios of 10, 33, 100, 333, and 1000 times higher than the wild-type IdeZ-mouse Fc fusion protein, respectively. Lanes 8 to 12 show electrophoretic images of antibodies obtained in a similar test using IdeZ variant A-mouse Fc fusion protein. Lane 13 shows the electrophoretic image of anti-KLH_hIgG1 alone.

[0087] Surprisingly, the wild-type IdeZ-mouse Fc fusion protein was only able to cleave the antibody up to a concentration ratio of about 33 times that of human IgG1 antibody to the fusion protein, whereas the IdeZ variant A-mouse Fc fusion protein was able to cleave the antibody up to a concentration ratio of about 1000 times that of the fusion protein. It was confirmed that N-glycosylation of IdeZ during expression in mammalian cells attenuates the protease activity, and that mutation of the consensus sequence of the N-glycan improves the protease activity.

[0088] Example 2: Preparation of IdeZ-Fc fusion protein with three amino acid substitutions and evaluation of its activity Next, to confirm the influence of the consensus sequence of each N-glycan on the expression and activity of the IdeZ-Fc fusion protein in mammalian cells, IdeZ-Fc fusion proteins with three amino acid modifications each were prepared in the same manner as in Example 1: variant 1 (N171G / S269R / N298D) (hereinafter referred to as "IdeZ variant 1-mouse Fc fusion protein"), variant 2 (T120E / S269R / N298D) (hereinafter referred to as "IdeZ variant 2-mouse Fc fusion protein"), variant 3 (T120E / N171G / N298D) (hereinafter referred to as "IdeZ variant 3-mouse Fc fusion protein"), and variant 4 (T120E / N171G / S269R) (hereinafter referred to as "IdeZ variant 4-mouse Fc fusion protein").

[0089] The nucleotide sequence of the IdeZ variant in the IdeZ variant 1-mouse Fc fusion protein is shown in SEQ ID NO: 6, the amino acid sequence encoded thereby is shown in SEQ ID NO: 7, the nucleotide sequence of the IdeZ variant in the IdeZ variant 2-mouse Fc fusion protein is shown in SEQ ID NO: 8, the amino acid sequence encoded thereby is shown in SEQ ID NO: 9, the nucleotide sequence of the IdeZ variant in the IdeZ variant 3-mouse Fc fusion protein is shown in SEQ ID NO: 10, the amino acid sequence encoded thereby is shown in SEQ ID NO: 11, and the nucleotide sequence of the IdeZ variant in the IdeZ variant 4-mouse Fc fusion protein is shown in SEQ ID NO: 12, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 13.

[0090] Purified proteins could be prepared from the IdeZ variant 2-mouse Fc fusion protein, the IdeZ variant 3-mouse Fc fusion protein, and the IdeZ variant 4-mouse Fc fusion protein, but not from the IdeZ variant 1-mouse Fc fusion protein.

[0091] The purity of the obtained proteins was then evaluated by SDS-PAGE in the same manner as in Example 1. The electrophoretic patterns of the IdeZ variant 2-mouse Fc fusion protein, IdeZ variant 3-mouse Fc fusion protein, and IdeZ variant 4-mouse Fc fusion protein are shown in Figure 3. In Figure 3, lane 5 shows the IdeZ variant 2-mouse Fc fusion protein, lane 7 shows the IdeZ variant 3-mouse Fc fusion protein, and lane 9 shows the IdeZ variant 4-mouse Fc fusion protein. The relatively large molecular weight of the IdeZ variant 3-mouse Fc fusion protein suggested glycosylation at asparagine 267.

[0092] Next, the protease activity of each protein was evaluated by assessing the cleavage of a human IgG1 antibody using the same method as in Example 1. The electrophoretic patterns are shown in Figure 4. In Figure 4, arrow 1 indicates the H chain, and arrow 2 indicates the Fab' fragment. Lanes 3 to 7 in the left panel show electrophoretic patterns of cleaved antibodies when the antibody concentration ratio to the IdeZ variant 2-mouse Fc fusion protein was 10, 33, 100, 333, and 1000 times, respectively. Lanes 8 to 12 show electrophoretic patterns of antibodies obtained in a similar test using the IdeZ variant 3-mouse Fc fusion protein. Lanes 3 to 7 in the right panel show electrophoretic patterns of antibodies obtained in a similar test using the IdeZ variant 4-mouse Fc fusion protein.

[0093] Interestingly, antibody cleavage was confirmed for the IdeZ variant 2-mouse Fc fusion protein and the IdeZ variant 4-mouse Fc fusion protein even at concentration ratios of human IgG1 antibody to fusion protein of approximately 333 to 1000, whereas for the IdeZ variant 3-mouse Fc fusion protein, antibody cleavage was confirmed only at concentration ratios of human IgG1 antibody to fusion protein of approximately 33. These results confirmed that mutation of the N-glycan consensus sequence for asparagine 118 and asparagine 267 of SEQ ID NO: 1 is effective for expressing IdeZ in mammalian cells and maintaining protease activity.

[0094] Example 3-1: Preparation of Bivalent IdeZ Variant A-Human Fc Variant-1> To obtain a fusion of IdeZ variant A with suppressed autolysis and the Fc region of human IgG1, a modified human Fc sequence was designed. Since mouse IgG1 and IgG2b are not cleaved by IdeZ, their sequences were referenced in the design. The Y296N and Y296R mutations, which have been reported as mutations that prevent IdeS binding, were also referenced. Furthermore, the E294del mutation (J. Immunol. 2019, Vol. 202, pp. 1582-1594, WO2016 / 016586) and the I253A mutation (Intl. Immunol., 2006, Vol. 18, pp. 1759-1769) were introduced into each variant. The names of each variant and the designed sequences are shown in Table 1.

[0095] DNA encoding the IdeZ variant A-human Fc variant was synthesized according to standard methods, and a nucleic acid sequence encoding an antibody heavy chain signal (Protein Eng., 1987, Vol. 1, pp. 499-505) was ligated to the 5' end thereof, followed by insertion into the pcDNA3.4 vector (Thermo Fischer Scientific) to prepare expression vectors for each IdeZ variant A-human Fc variant. To add α2,6-linked sialic acid, an expression vector encoding β-1,4-galactosyltransferase 1 (B4GALT1) (UniProt accession number: P15291-1) and an expression vector encoding human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (UniProt accession number: P15907-1) were transfected into ExpiCHO-S cells (Thermo Fischer Scientific) together with expression vectors encoding various IdeZ variant A-human Fc variants, and the cells were incubated at 37°C, 8% CO ２ The cells were cultured under rotational conditions for 4 days. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02) according to standard methods to obtain IdeZ variant A-human Fc variants. The obtained IdeZ variant A-human Fc variants were designated sialylated IdeZ variant A-human Fc variants. Each of the obtained sialylated IdeZ variant A-human Fc variants was subjected to SDS-PAGE analysis by the method described in Example 1. The electrophoresis patterns of each sialylated IdeZ variant A-human Fc variant are shown in Figure 5.

[0096] 5 , lane 1 represents a molecular weight marker, and lanes 2 to 6 represent sialylated IdeZ variant A-human Fc variant 5, sialylated IdeZ variant A-human Fc variant 6, sialylated IdeZ variant A-human Fc variant 9, sialylated IdeZ variant A-human Fc variant 10, and sialylated IdeZ variant A-human Fc variant 11. For sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11, bands thought to be derived from the human Fc region were detected between the molecular weight markers of 25 kDa and 37 kDa under reducing conditions, indicating that sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11 were autodigested.

[0097] Example 3-2: Preparation of bivalent IdeZ variant A-human Fc variants - 2> Because the IdeZ variant A-human Fc variants designed in Example 3-1 were all autolyzed, further mutations were introduced into the human Fc sequences, which were designed with reference to the sequences of mouse IgG1 and IgG2b. The names of the variants and the designed sequences are shown in Table 2.

[0098] The sialylated IdeZ variant A-human Fc variants listed in Table 2 were obtained by the method described in Example 3-1. To obtain various IdeZ variant A-human Fc variants prepared without co-expression of B4GALT1 and ST6GAL1, expression vectors encoding the various IdeZ variant A-human Fc variants were transfected into ExpiCHO-S cells (Thermo Fischer Scientific) and incubated at 37°C, 8% CO. ２ The cells were cultured under rotation for 4 days under these conditions. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02) according to standard methods. The obtained IdeZ variant A-human Fc variant was designated non-sialylated IdeZ variant A-human Fc variant.

[0099] The obtained sialylated IdeZ variant A-human Fc variants and non-sialylated IdeZ variant A-human Fc variants (hereinafter sometimes simply referred to as "IdeZ variant A-human Fc variants") were analyzed by SDS-PAGE under reducing and non-reducing conditions. The electrophoretic patterns of each IdeZ variant A-human Fc variant are shown in Figure 6. In Figure 6, lane 1 shows molecular weight markers, lanes 2 to 5 show non-sialylated IdeZ variant A-human Fc variants 7, 8, 17, and 18, respectively. Lanes 6 to 9 show sialylated IdeZ variant A-human Fc variants 7, 8, 17, and 18, respectively.

[0100] The results obtained under reducing conditions indicated that IdeZ variant A-human Fc variants 7, 8, 17, and 18 were not autolyzed. Furthermore, the results obtained under non-reducing conditions indicated that a band derived from the target product was detected near the 150 kDa molecular weight marker for all IdeZ variant A-human Fc variants. Bands other than the target product were detected between the 50 and 75 kDa molecular weight markers for IdeZ variant A-human Fc variants 8 and 18, but not for IdeZ variant A-human Fc variants 7 and 17. This indicates that the target product can be obtained with higher purity from IdeZ variant A-human Fc variants 7 and 17 than from IdeZ variant A-human Fc variants 8 and 18.

[0101] Example 4: Preparation of monovalent IdeZ variant A-human Fc variants As shown in Figure 6, a band was detected at a higher molecular weight than the target product in the results of SDS-PAGE under non-reducing conditions, suggesting aggregation of the IdeZ variant A-human Fc variants. To improve aggregation, a modified human Fc sequence was designed by further introducing mutations into IdeZ variant A-human Fc variant 7 obtained in Example 3-2, and a monovalent IdeZ variant A-human Fc variant having the modified human Fc sequence was designed. The names of the variants, the names of the constituent chains, and the designed sequences are shown in Table 3.

[0102] DNA encoding the IdeZ variant A-human Fc variant 19_holes and human Fc variant 19_knobs was synthesized according to standard methods and inserted into the pcDNA3.4 vector (Thermo Fischer Scientific) to prepare expression vectors for each variant component chain. Expression vectors encoding B4GALT1 and ST6GAL1, an expression vector for the IdeZ variant A-human Fc variant 19_holes, and an expression vector for the human Fc variant 19_knobs were co-transfected and cultured using the method described in Example 3-1. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02) according to standard methods to obtain the IdeZ variant A-human Fc variant. The obtained protein was designated sialylated IdeZ variant A-human Fc variant 19. Furthermore, the expression vector for IdeZ variant A-human Fc variant 19_holes and the expression vector for human Fc variant 19_knobs were transfected into ExpiCHO-S cells (Thermo Fischer Scientific) and cultured using the method described in Example 3-2. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02) according to standard methods to obtain non-sialylated IdeZ variant A-human Fc variant 19.

[0103] The obtained sialylated IdeZ variant A-human Fc variant 19 and non-sialylated IdeZ variant A-human Fc variant 19 were subjected to SDS-PAGE analysis under reducing and non-reducing conditions. The electrophoresis diagram is shown in Figure 7. In Figure 7, lane 1 shows molecular weight markers, lane 2 shows non-sialylated IdeZ variant A-human Fc variant 19, and lane 3 shows sialylated IdeZ variant A-human Fc variant 19. Under reducing conditions, a band likely derived from human Fc variant 19_knobs was detected between the molecular weight markers of 25 and 37 kDa, and a band likely derived from IdeZ variant A-human Fc variant 19_holes was detected between the molecular weight markers of 50 and 75 kDa. Furthermore, a band was detected around the molecular weight marker of 100 kDa under non-reducing conditions, confirming that the target product had been obtained.

[0104] Example 5: Evaluation of cleavage activity of IdeZ variant A-human Fc variants To 10 μg of the anti-KLH_hIgG1 described in Example 1, sialylated IdeZ variant A-human Fc variant 7 or sialylated IdeZ variant A-human Fc variant 19 was added at 2, 0.4, 0.08, or 0.016 μg (the final concentration ratio of sialylated IdeZ variant A-human Fc variant added to anti-KLH_hIgG1 was 0.2, 0.04, 0.008, and 0.0016 times, respectively), and the reaction was carried out at 37°C for 2 hours in a final volume of 20 μL. The reaction product was subjected to SDS-PAGE analysis under reducing conditions. The electrophoresis pattern is shown in FIG. 8. As a result, both the bivalent IdeZ variant A-human Fc variant 7 and the monovalent sialylated IdeZ variant A-human Fc variant 19 exhibited almost the same anti-KLH_hIgG1 cleaving activity.

[0105] Example 6 Size Exclusion Chromatography Analysis To evaluate the purity after affinity chromatography purification (purification using MabSelect SuRe (Cytiva, 17-5438-02)), sialylated IdeZ variant A-human Fc variant 7 prepared in Example 3-2 and sialylated IdeZ variant A-human Fc variant 19 prepared in Example 4 were diluted to 1 mg / mL and analyzed using an ACQUITY UPLC BEH200 SEC column (Waters, #186005225). The results are shown in Table 4.

[0106] The target peak was detected at a retention time of 2.240 minutes for sialylated IdeZ variant A-human Fc variant 7, and at a retention time of 2.393 minutes for sialylated IdeZ variant A-human Fc variant 19. A peak with a total area of ​​20.6% was detected for sialylated IdeZ variant A-human Fc variant 7 at a shorter retention time than the target peak. On the other hand, a peak with a total area of ​​15.4% was detected for sialylated IdeZ variant A-human Fc variant 19 at a shorter retention time than the target peak. This indicates that sialylated IdeZ variant A-human Fc variant 19 has a lower content of high-molecular-weight contaminants than sialylated IdeZ variant A-human Fc variant 7.

[0107] Example 7: Analysis of sialylation rates of various IdeZ variant A-human Fc variants The IdeZ variant A-human Fc variants obtained in Examples 3 and 4 were analyzed for sialylation rate. Example 7-1: Analysis of sialylation rates of sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11 The sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11 obtained in Example 3-1 (hereinafter sometimes referred to as "uncleaved forms") and their fragments generated by autolysis (hereinafter sometimes referred to as "cleaved forms") were analyzed for sialylation rate. Dithiothreitol (Thermo Fisher Scientific, A39255, hereinafter referred to as "DTT") was added to each sample and incubated at 37°C for 1 hour to reduce interchain disulfide bonds. The reduced sample was injected into an HPLC system (Waters), separated on a reverse-phase column (Waters, ACQUITY UPLC Protein BEH C4), and then introduced into a mass spectrometer (Waters). The type of glycan was assigned from the difference between the theoretical molecular weight after reduction and the measured mass, and the proportion (%) of each glycan was calculated from the ion intensity ratio. The proportion of glycans to which sialic acid was attached among all glycans present in the Fc region was defined as the "sialylation rate (%)." The sialylation rate was calculated using the following formula: Sialylation rate (%) = (total MS intensity of H chains modified with sialic acid-bound glycans) ÷ (total MS intensity of all H chains) × 100. The results are shown in Table 5.

[0108] Most of the sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11 and their truncated forms were sialylated. This result and the results of Example 3 suggest that autolysis occurs during expression and purification for sialylated IdeZ variant A-human Fc variants 5, 6, 9, 10, and 11 depending on the sequence, rather than on the presence or absence of sialylation.

[0109] Example 7-2: Analysis of sialylation rates of IdeZ variant A-human Fc variants 7, 8, 17, and 18 The sialylation rates of IdeZ variant A-human Fc variants 7, 8, 17, and 18 obtained in Example 3-2 were analyzed by the method described in Example 7-1. The results are shown in Table 6. As a result, it was found that the majority of IdeZ variant A-human Fc variants 7, 8, 17, and 18 were sialylated by coexpression of glycosyltransferases B4GALT1 and ST6GAL1.

[0110] Example 7-3: Analysis of sialylation rate of IdeZ variant A-human Fc variant 19 The sialylation rate of IdeZ variant A-human Fc variant 19 obtained in Example 4 was analyzed by the method described in Example 7-1. IdeZ variant A-human Fc variant 19 was separated into IdeZ variant A-human Fc variant 19_holes and human Fc variant 19_knobs by reduction treatment, and since each fragment was observed at a different molecular weight in mass spectrometry, the sialylation rate was calculated for each fragment. The results are shown in Table 7.

[0111] The results showed that coexpression of glycosyltransferases B4GALT1 and ST6GAL1 resulted in the sialylation of most of the fragments of sialylated IdeZ variant A-human Fc variant 19. Surprisingly, compared with the bivalent non-sialylated IdeZ variant A-human Fc variant 7, the sialylation rate was relatively high even without coexpression of glycosyltransferases.

[0112] Furthermore, to assess whether there were differences in the sialic acid linkage mode depending on whether or not glycosyltransferases were co-expressed, analysis was performed using the RapiFluor method. Using the GlycoWorks RapiFluor-MS N-Glycan Kit-96 Sample (Waters, 176003606, hereafter referred to as "Kit"), glycans were excised from the sample according to the Kit's protocol and labeled with a fluorescent dye. The labeled sample was injected into an HPLC system (Waters), separated on a HILIC column (Waters, ACQUITY UPLC Glycan BEH Amide), and then introduced into a mass spectrometer (Waters) and a fluorescence detector (Waters).

[0113] The assignment of glycans, including their linkage type, was performed by comparing the theoretical molecular weight with the measured mass, and by comparing the retention time of a RapiFluor-MS Dextran Calibration Ladder (Waters, 186007982) injected separately as a standard with the measured retention time. The linkage type ratio (%) of FA2G2S2 (Oxford notation), a representative sialylated glycan, was calculated from the peak area obtained by the fluorescence detector. The results are shown in Table 8.

[0114] The results showed that when glycosyltransferase was not co-expressed, non-human α-2,3 linkages were predominant, whereas when glycosyltransferase was co-expressed, α-2,6 linkages were predominant, suggesting that co-expression of glycosyltransferases is effective for producing sialylated Fc regions using CHO cells.

[0115] The amino acid and DNA sequences of the polypeptides of the embodiments are shown in the table below.

[0116] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims.

[0117] The IdeZ variants of the present invention and polypeptides containing the IdeZ variants have good protease activity even when produced using mammalian cells, and are useful for pretreatment of gene therapy using AAV, as well as for the prevention or treatment of transplant rejection reactions and autoimmune diseases, etc.

Claims

1. An IdeZ variant having IgG cysteine ​​protease activity, the variant comprising: (a) a sequence that is 90% or more identical to the amino acid sequence from positions 35 to 349 of the amino acid sequence represented by SEQ ID NO:1; and (b) an IdeZ variant in which no glycosylation is added to the amino acids at positions corresponding to positions 118 and 267 of the amino acid sequence represented by SEQ ID NO:

1.

2. The IdeZ variant according to claim 1, comprising the following amino acid modifications (b') and (b''): (b') (i) the amino acid at position 118 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 119 of the amino acid sequence represented by SEQ ID NO:1 is replaced with proline; and / or (iii) the amino acid at position 120 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (b'') (i) the amino acid at position 267 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at position 268 of the amino acid sequence represented by SEQ ID NO:1 is replaced with proline; and / or (iii) The amino acid at the position corresponding to position 269 of the amino acid sequence represented by SEQ ID NO:1 is substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.

3. The IdeZ variant of claim 2, in which the amino acid at the position corresponding to position 120 of the amino acid sequence represented by SEQ ID NO:1 is replaced with glutamic acid, and the amino acid at the position corresponding to position 269 of the amino acid sequence represented by SEQ ID NO:1 is replaced with arginine.

4. The IdeZ variant according to any one of claims 1 to 3, wherein (c) no glycosylation is performed on the amino acid at the position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO:1, and / or (d) no glycosylation is performed on the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO:

1.

5. The IdeZ variant according to claim 4, comprising the following amino acid modifications (c') and / or (d'): (c') (i) an amino acid at a position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) an amino acid at a position corresponding to position 172 of the amino acid sequence represented by SEQ ID NO:1 is replaced with proline; and / or (iii) the amino acid at the position corresponding to position 173 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (d') (i) the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO:1 is replaced with an amino acid selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan; (ii) the amino acid at the position corresponding to position 299 of the amino acid sequence represented by SEQ ID NO:1 is replaced with proline; and / or (iii) The amino acid at the position corresponding to position 300 in the amino acid sequence represented by SEQ ID NO:1 is substituted with an amino acid selected from the group consisting of glycine, alanine, proline, valine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan.

6. The IdeZ variant of claim 5, wherein the amino acid at the position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO:1 is replaced with glycine, and / or the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO:1 is replaced with aspartic acid.

7. The IdeZ variant according to claim 6, in which the amino acid at the position corresponding to position 120 of the amino acid sequence represented by SEQ ID NO:1 is replaced with glutamic acid, the amino acid at the position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO:1 is replaced with glycine, the amino acid at the position corresponding to position 269 of the amino acid sequence represented by SEQ ID NO:1 is replaced with arginine, and the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO:1 is replaced with aspartic acid.

8. The IdeZ variant of claim 7, comprising an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:

5.

9. A polypeptide comprising an IdeZ variant according to any one of claims 1 to 8.

10. The polypeptide of claim 9, wherein the polypeptide is a fusion protein between the IdeZ variant and an Fc region.

11. The polypeptide of claim 10, wherein the Fc region is derived from human IgG.

12. The polypeptide of claim 11, wherein the Fc region is derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4.

13. The polypeptide according to any one of claims 10 to 12, wherein the Fc region is substituted at position 236 with glutamic acid, and substituted at position 237 with valine, wherein the amino acid residue numbers are amino acid positions according to the EU index.

14. The polypeptide of claim 13, wherein the Fc region further comprises a substitution at position 234 with alanine and a substitution at position 235 with glutamic acid.

15. The polypeptide of claim 14, wherein the Fc region further comprises a substitution at position 227 with histidine, a substitution at position 228 with lysine, a substitution at position 233 with asparagine, and / or a substitution at position 238 with serine.

16. The polypeptide of claim 14 or 15, wherein the Fc region further comprises a substitution at position 253 with alanine, a deletion at position 293 or 294, and / or a substitution at position 296 with alanine.

17. The polypeptide according to claim 16, wherein the Fc region is selected from the following (1) and (2); (1) an Fc region in which position 234 is substituted with alanine, position 235 is substituted with glutamic acid, position 236 is substituted with glutamic acid, position 237 is substituted with valine, position 253 is substituted with alanine, and position 293 or 294 is deleted; (2) an Fc region in which position 227 is substituted with histidine, position 228 is substituted with lysine, position 233 is substituted with asparagine, position 234 is substituted with alanine, position 235 is substituted with glutamic acid, position 236 is substituted with glutamic acid, position 237 is substituted with valine, position 238 is substituted with serine, position 253 is substituted with alanine, and An Fc region having a deletion at position 293 or 294.

18. The polypeptide of claim 17, wherein the Fc region comprises an amino acid sequence that is 90% or more identical to a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 16, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 17, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO: 21, or a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO:

22.

19. A polypeptide according to any one of claims 10 to 18, wherein the fusion protein is a fusion protein in which the IdeZ variant is linked to the N-terminus of the Fc region, and two of the fusion proteins form a dimer.

20. A polypeptide according to any one of claims 10 to 18, wherein the polypeptide comprises a fusion protein in which the IdeZ variant is linked to the N-terminus of a first Fc region, and a second Fc region to which the IdeZ variant is not linked.

21. The polypeptide of claim 20, wherein the first Fc region and the second Fc region comprise knobs-into-holes mutations, and the knobs-into-holes mutations are either of the following (1) and (2): (1) in the first Fc region, a substitution at position 366 with tryptophan; and in the second Fc region, a substitution at position 366 with serine, a substitution at position 368 with alanine, and a substitution at position 407 with valine; (2) in the first Fc region, a substitution at position 366 with serine, a substitution at position 368 with alanine, and a substitution at position 407 with valine; and in the second Fc region, a substitution at position 366 with tryptophan.

22. The polypeptide of any one of claims 19 to 21, wherein the Fc region or the first Fc region and the second Fc region are sialylated.

23. A polynucleotide encoding a polypeptide according to any one of claims 9 to 22.

24. An expression vector comprising the polynucleotide of claim 23.

25. A host cell transformed with the expression vector of claim 24.

26. A method for producing a polypeptide comprising an IdeZ variant, comprising culturing the host cell of claim 25.

27. The method of claim 26, further comprising the step of adding or expressing β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1).

28. An Fc region variant comprising a substitution at position 236 with glutamic acid, and a substitution at position 237 with valine, wherein the amino acid residue numbers are the amino acid positions according to the EU index.

29. The Fc region variant according to claim 28, further comprising a substitution at position 234 with alanine and a substitution at position 235 with glutamic acid.

30. The Fc region variant of claim 29, further comprising the following amino acid modifications: a substitution at position 227 with histidine, a substitution at position 228 with lysine, a substitution at position 233 with asparagine, and / or a substitution at position 238 with serine.

31. The Fc region variant of claim 29 or 30, further comprising the following amino acid modifications: a substitution of alanine at position 253, a deletion at position 293 or 294, and / or a substitution of alanine at position 296.

32. The Fc region variant according to claim 31, selected from the following (1) and (2): (1) an Fc region variant in which position 234 is substituted with alanine, position 235 is substituted with glutamic acid, position 236 is substituted with glutamic acid, position 237 is substituted with valine, position 253 is substituted with alanine, and position 293 or 294 is deleted; (2) an Fc region variant in which position 227 is substituted with histidine, position 228 is substituted with lysine, position 233 is substituted with asparagine, position 234 is substituted with alanine, position 235 is substituted with glutamic acid, position 236 is substituted with glutamic acid, position 237 is substituted with valine, position 238 is substituted with serine, position 253 is substituted with alanine, and An Fc region variant having a deletion at position 293 or 294.

33. An Fc region variant according to any one of claims 28 to 32, wherein the Fc region is derived from human IgG.

34. The Fc region variant of claim 33, wherein the Fc region is derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4.

35. A polypeptide comprising an Fc region variant according to any one of claims 28 to 34, which is not cleaved by IgG cysteine ​​protease.

36. A fusion protein of an IgG cysteine ​​protease and an Fc region variant described in any one of claims 28 to 34.

37. The fusion protein of claim 36, wherein the IgG cysteine ​​protease is IdeS, IdeZ, IdeZ2, IdeE, IdeE2, IdeP, IdeSORK, or IgdE, or a variant thereof.

38. The fusion protein described in claim 37, comprising an amino acid sequence that is 90% or more identical to a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO:16, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO:17, a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO:21, or a sequence consisting of amino acids from positions 316 to 541 in the amino acid sequence represented by SEQ ID NO:22.

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