Fusion protein

Abstract

One embodiment of the present invention provides a fusion protein which contains a conjugate of an IdeZ variant and an Fc region variant, and the fusion protein contains: (a) a sequence that is 90% or more identical to the amino acid sequence from position 1 to position 315 of the amino acid sequence represented by SEQ ID NO: 11; and (b) a sequence that is 90% or more identical to the amino acid sequence from position 316 to position 541 of the amino acid sequence represented by SEQ ID NO: 11.

Description

Fusion protein

[0001] The present invention relates to a fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant, a method for producing the fusion protein, and the like.

[0002] IdeS derived from Streptococcus is a cysteine protease that sequence-specifically cleaves the hinge region connecting Fab and Fc of human IgG. Due to its high cleavage efficiency and substrate specificity, it is used in characterization of antibody drug candidates, among other applications. In recent years, its applications have been expanding, and for example, it is expected to be used in high-quality recombinant protein production systems, and expected to exert effects against transplant rejection, and autoimmune diseases and diseases in which pathogenic IgG is directly or indirectly involved as a pathogenetic factor (Non-Patent Documents 1 and 2).

[0003] However, humans who have previously been infected with Streptococcus have immunity against IdeS, and the high immunogenicity of IdeS can cause problems, for example, administration of IdeS to such humans leads to production of anti-drug antibodies (Non-Patent Document 3). In order to solve such problems of IdeS, research is also progressing on IdeZ derived from non-human-infecting Streptococcus, and IdeZ variants with amino acid modifications that suppress the production of anti-drug antibodies (Patent Documents 1 and 2). Further improvement in properties of such IdeZ and IdeZ variants is desired.

[0004] As a method for reducing the immunogenicity of antibodies, a means of adding sialic acid to the variable region of antibodies is known (Patent Document 3). As a method for increasing the amount of sialic acid added to the Fc region, a method is known in which an antibody having modifications within the Fc domain (e.g., at F243, V264 and D265) is expressed in a host cell that expresses β-galactosyltransferase and sialyltransferase activities (Patent Document 4). A method for increasing the amount of sialic acid addition by modifying specific amino acids in the Fc region (e.g., amino acids at positions 240 to 243, 258 to 267, and 290 to 305) using HEK293 and YB2 / 0 cells has also been studied (Patent Document 5).

[0005] However, there are various challenges in applying sialic acid conferrants to pharmaceuticals. For example, it is known that the sialic acid content in interferon-γ decreases as neuraminidase activity in cell culture medium increases over time (Non-Patent Literature 4). It is also known that the sialic acid content of antibodies decreases as the number of cell culture days increases (Non-Patent Literature 5).

[0006] International Publication No. 2020 / 159970, International Publication No. 2021 / 233911, International Publication No. 2022 / 053673, International Publication No. 2012 / 113863, JP 2017-522040

[0007] 1st Academic Conference Commemorating the Establishment of the Japanese Antibody Society, November 2022, P-74Drugs, October 2020, Vol. 80, p. 1859-1864Am J Transplant, November 2018, Vol. 18(11), p. 2752-2762Biotechnol Bioeng, July 1997, Vol. 55(2), p. 390-398mAbs, 2015, Vol. 7(3), p571-583

[0008] The present invention aims to provide a fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant that has desirable properties such as suppression of immunogenicity.

[0009] As a result of diligent research, the present inventors have found that by combining a predetermined IdeZ variant with a predetermined Fc region variant to form a fusion protein, desirable properties such as suppression of immunogenicity can be obtained. The present invention is, for example, as follows: [1] A fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant, wherein the fusion protein comprises: (a) a sequence that is 90% or more identical to the amino acid sequence from position 1 to 315 of the amino acid sequence represented by SEQ ID NO: 11; and (b) a sequence that is 90% or more identical to the amino acid sequence from position 316 to 541 of the amino acid sequence represented by SEQ ID NO: 11. [2] The fusion protein described in [1], wherein the IdeZ variant includes the following amino acid modification (a'), and the Fc region variant includes the following amino acid modifications (b') and (b''): (a') The amino acids at positions 120, 171, 269, and 298 of the amino acid sequence represented by Sequence ID No. 1 are replaced with glutamic acid, glycine, arginine, and aspartic acid, respectively; (b') The amino acids at positions 234, 235, 236, 237, and 253 of the Fc region are replaced with alanine, glutamic acid, glutamic acid, valine, and alanine, respectively; (b'') The amino acid at position 293 or 294 of the Fc region is deleted (wherein the amino acid residue numbers of the Fc region are amino acid positions according to the EU index). [3] The Fc domain variant further comprises the following amino acid modifications: (c) the amino acid at position 265 of the Fc domain is replaced with alanine; (d) the amino acid at position 331 of the Fc domain is replaced with glycine.[4] The Fc domain variant further comprises the following amino acid modifications: (e) and / or (f) the fusion protein according to [2] or [3]: (e) one or more amino acids at the positions corresponding to positions 361, 371, 375, 418 and 438 of the Fc domain are substituted with aspartic acid or glutamic acid; (f) one or more amino acids at the positions corresponding to positions 255, 274, 288, 326, 370 and 416 of the Fc domain are substituted with a neutral amino acid. [5] The Fc region variant comprises the following amino acid modifications: (g) the fusion protein described in [4]: ​​(g) the amino acid at position 361 of the Fc region is substituted with aspartic acid, the amino acid at position 370 is substituted with threonine, the amino acid at position 371 is substituted with aspartic acid, the amino acid at position 375 is substituted with glutamic acid, the amino acid at position 416 is substituted with asparagine, the amino acid at position 418 is substituted with glutamic acid, and the amino acid at position 438 is substituted with glutamic acid; (h) the amino acid at position 255 of the Fc region is substituted with leucine, the amino acid at position 274 is substituted with glutamine, the amino acid at position 288 is substituted with glutamine, the amino acid at position 326 is substituted with alanine, the amino acid at position 370 is substituted with threonine, and the amino acid at position 416 is substituted with asparagine. [6] A fusion protein according to any one of [1] to [5], comprising a conjugate formed by the IdeZ variant being bound to the N-terminus of the Fc region variant (first Fc region variant), and the Fc region variant (second Fc region variant) to which the IdeZ variant is not bound.[7] The fusion protein according to [6], wherein the first Fc region variant and the second Fc region variant further include a knobs-into-holes mutation, the knobs-into-holes mutation being either (1) or (2) below: (1) The first Fc region has an amino acid at position 366 of the Fc region substituted with tryptophan; the second Fc region has an amino acid at position 366 of the Fc region substituted with serine, an amino acid at position 368 of the Fc region substituted with alanine, and an amino acid at position 407 of the Fc region substituted with valine; The second Fc region is characterized in that the amino acid at the position corresponding to position 366 of the Fc region is substituted with tryptophan. [8] The fusion protein according to [7], comprising the amino acid sequence represented by SEQ ID NO: 11 and the amino acid sequence represented by SEQ ID NO: 12. [9] The fusion protein according to [7], comprising the amino acid sequence represented by SEQ ID NO: 13 and the amino acid sequence represented by SEQ ID NO: 14.

[10] A polynucleotide encoding the fusion protein according to any one of [1] to [9].

[11] An expression vector comprising the polynucleotide according to

[10] .

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

[11] .

[13] A method for producing the fusion protein, comprising the step of culturing the host cell according to

[12] .

[14] The method according to

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

[15] A pharmaceutical composition comprising the fusion protein according to any one of [1] to [9] and a pharmaceutically acceptable carrier.

[16] The pharmaceutical composition according to

[15] for use in the prevention or treatment of a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

[17] The pharmaceutical composition according to

[16] , wherein the disease is IgG-mediated rapidly progressive glomerulonephritis.

[18] The pharmaceutical composition according to

[17] , wherein IgG-mediated rapidly progressive glomerulonephritis is selected from the group consisting of ANCA-associated vasculitis, immune complex type III hypersensitivity disease, and type II hypersensitivity disease.

[19] The pharmaceutical composition according to

[18] , wherein immune complex type III hypersensitivity disease is selected from the group consisting of lupus nephritis, IgA nephropathy, IgA vasculitis, and cryoglobulinemia glomerulonephritis.

[20] The pharmaceutical composition according to

[18] , wherein type II hypersensitivity disease is anti-GBM antibody disease (Goodpasture syndrome).

[21] A method for preventing or treating a disease selected from autoimmune diseases and diseases involving pathogenic IgG, comprising administering a therapeutically effective amount of the fusion protein according to any one of [1] to [9].

[22] The fusion protein according to any one of [1] to [9] for use in preventing or treating a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

[23] Use of the fusion protein described in any of [1] to [9] in the manufacture of a pharmaceutical composition for the prevention or treatment of a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

[0010] According to the present invention, it is possible to provide a fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant that has desirable properties such as suppression of immunogenicity.

[0011] Figure 1 shows the amount of anti-drug antibody against IdeZ produced by IdeZ variant A-mouse Fc. The vertical axis represents the intensity of the luminescence signal. Figure 2 shows the amount of anti-drug antibody against IdeZ produced by IdeZ variant A-human Fc variant 19. The vertical axis represents the intensity of the luminescence signal. Figure 3 shows the amount of anti-drug antibody against IdeZ produced by IdeZ variant A-human Fc variant 7. The vertical axis represents the intensity of the luminescence signal. Figure 4 shows the amount of anti-drug antibody against IdeZ produced by various IdeZ variant A-human Fc variants. The vertical axis represents the intensity of the luminescence signal. Figure 5 shows the endocytosis activity of various IdeZ variant A-Fc fusion proteins in MUTZ-3 cells. The vertical axis represents the fluorescence intensity. Figure 6 shows the amount of anti-drug antibody against IdeZ produced by various IdeZ variant A-human Fc variants. The vertical axis shows the intensity of the luminescence signal. Figure 7 shows the results of immunogenicity evaluation of various IdeZ variants, A-human Fc variants, and immunogenic proteins in human PBMCs. The vertical axis shows the percentage of donors in which CD4-positive T cells were activated in response to the protein. Figure 8 shows the results of measuring immune complexes in the kidneys of lupus nephritis model mice. The vertical axis shows the IgG2a+C1q positivity rate. Figure 9 shows the results of histopathological evaluation of the kidneys of lupus nephritis model mice. The vertical axis shows the percentage of classified disease types. Type I: almost normal under a light microscope, Type II: mesangial proliferation type, Type III: focal segmental nephritis type (intracapsular pleocellular lesions less than 50% of all glomeruli), Type IV: diffuse nephritis type (intracapsular pleocellular lesions more than 50% of all glomeruli), Type V: membranous nephropathy type, Type VI: progressive sclerosis type.

[0012] Embodiments of the present invention will be described in detail below. The materials, configurations, etc., described below are not limiting to the present invention and can be modified in various ways within the scope of the spirit of the present invention.

[0013] 1. Definitions Unless otherwise defined below, terms used herein have the meanings commonly used by those skilled in the art. In this specification, “IdeZ variant” means an IdeZ having at least one amino acid modification relative to the wild-type IdeZ (SEQ ID NO: 1), and amino acid modifications include substitution, addition, deletion, insertion, and modification of amino acids. In this specification, the terms “modification” and “mutation” of amino acids are used synonymously.

[0014] In this specification, "Fc region variant" means an Fc region that includes at least one amino acid modification relative to the wild-type Fc region, and amino acid modifications include amino acid substitution, addition, deletion, insertion, and modification. When the terms "Fc region" or "Fc region variant" are used in this specification, it is intended to include complexes (e.g., dimers) consisting of multiple Fc regions.

[0015] In this specification, the "Fc region (modified)" may be derived from any antibody. An antibody (or immunoglobulin) is a glycoprotein consisting of a quadruple chain structure having a symmetrical Y-shape, composed 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 the antibody molecule is common to all classes, with 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 linked by disulfide bonds and non-covalent bonds, forming an antibody molecule consisting of a Y-shaped quadruple chain structure with a molecular weight of 150,000 to 190,000.

[0016] Heavy chains typically consist of polypeptide chains containing approximately 440 amino acids and have characteristic structures for each class, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively, and are called Igγ, Igμ, Igα, Igδ, and Igε. Furthermore, IgG has subclasses IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4. Light chains typically consist of polypeptide chains containing approximately 220 amino acids and are known in two types: L-type and K-type, called Igλ and Igκ, respectively. These two types of light chains can be paired with any type of heavy chain.

[0017] Antibody molecules have four disulfide bonds in the heavy chain (five in Igμ and Igε) and two in the light chain, forming one loop every 100-110 amino acid residues. Their three-dimensional structures are similar across loops and are called structural units or domains. In both the heavy and light chains, the N-terminal domain is called the variable region. Even antibodies produced from the same class (or subclass) of the same animal species exhibit diverse amino acid sequences and are known to be involved in antibody-antigen binding specificity. 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. The heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH) from the N-terminus to the C-terminus. The CH region is further divided into three domains from the N-terminus: the CH1 domain, the CH2 domain, and the CH3 domain (in the case of IgG). The light chain has a variable light chain region (VL) and a constant light chain region (CL) extending from the N-terminus to the C-terminus.

[0018] The amino acid sequences of the three complementarity-determining regions (CDRs) present in the VH and VL chains vary considerably, contributing to the variability of the variable region. 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 the heavy and light chains, respectively, and form the antigen-binding site. On the other hand, the portion of the variable region other than the CDRs is called the framework region (FR), consisting of FR1 to FR4, and its amino acid sequence changes relatively little.

[0019] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two N-terminal fragments are called the Fab (antigen-binding) region, and the C-terminal fragment is called the Fc (crystallizable) region. In this specification, the "Fc region" refers to the polypeptide consisting of the constant region of the antibody excluding the CH1 domain. Therefore, the Fc region of IgA, IgD, and IgG consists of the last two heavy chain constant region domains (CH2 and CH3) at the C-terminus, while the Fc region of IgE and IgM includes the last three heavy chain constant region domains (CH2, CH3, and CH4) at the C-terminus. Furthermore, if present, the hinge region, J chain, etc., are also included in the "Fc region." For example, the Fc region of human IgG1 includes a polypeptide consisting of CH2 and CH3 domains, and part or all of the hinge region, as well as a polypeptide consisting of CH2 and CH3 domains. The Fc region in other classes of antibodies can be determined by sequence alignment with the heavy chain or fragments of human IgG1.

[0020] In this specification, the term "conjugate of IdeZ variant and Fc region variant" refers to both a structure in which one IdeZ variant is bound to the N-terminus of each of the 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. A fusion protein containing a conjugate of IdeZ variant and Fc region variant is also referred to as an "IdeZ-Fc fusion protein."

[0021] Furthermore, terms such as "sialylated IdeZ-Fc fusion protein" and "sialylated fusion protein" refer to fusion products of an IdeZ variant and an Fc domain variant to which sialic acid has been intentionally added by a sialylation process commonly used in the field (for example, treatment with β-galactoside α-2,6-sialyltransferase 1). On the other hand, in this specification, terms such as "non-sialylated IdeZ-Fc fusion protein" and "non-sialylated fusion protein" refer to fusion products of an IdeZ variant and an Fc domain variant that have not undergone the sialylation process described above, and do not prevent such fusion proteins from containing sialic acid.

[0022] In this specification, the "consensus sequence for N-linked glycans" in IdeZ refers to the sequence located at the glycan-binding site required for glycan modification by binding to the asparagine residue of the polypeptide, for example, the sequence asparagine (N)-X-serine (S) / threonine (T) (where X is an amino acid other than proline). In wild-type IdeZ represented by Sequence ID No. 1, the above consensus sequence is present at positions 118-120, 171-173, 267-269, and 298-300.

[0023] The amino acid residue numbers in antibody sequences such as the Fc region used herein 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).

[0024] In this specification, the terms “first” or “second” are used for convenience to distinguish between two or more types of parts. The use of such terms is not intended to assign any particular order or meaning unless explicitly stated.

[0025] In this specification, the presence of a peptide linker is optional. "Peptide linker" means one or more arbitrary amino acid residues that can be introduced by genetic engineering to link two or more parts. The length of the peptide linker used in this invention is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose.

[0026] In this specification, "identity" means the value of Identity obtained using the default parameters provided with EMBOSS Needle (Nucleic Acids Res., 2015, Vol. 43, pW580-W584). The parameters are as follows: Gap Open Penalty = 10 Gap Extended Penalty = 0.5 Matrix = EBLOSUM62 End Gap Penalty = false

[0027] In this specification, “subject” means a human or other animal in need of prevention or treatment. In some embodiments, it refers to a human being in need of prevention or treatment.

[0028] In this specification, “prevention or treatment” means concepts including preventing the onset of a disease or reducing the risk of developing a disease, delaying the onset of a disease, improving or alleviating the symptoms of a disease, slowing its progression, preventing relapse, and improving the prognosis.

[0029] In this specification, "autoimmune disease" means a disease in which the immune system, which should normally exhibit immune tolerance to itself, fails, and an immune response is triggered against antigens of self-derived origin, resulting in inflammation, dysfunction, or tissue damage to the body's own tissues or cells. More specifically, autoimmune diseases include systemic autoimmune diseases, organ-specific autoimmune diseases, autoantibody-mediated diseases, T-cell-mediated diseases, and mixed types thereof. Examples include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, systemic scleroderma, dermatomyositis, polymyositis, antiphospholipid syndrome, mixed connective tissue disease, type 1 diabetes mellitus, Hashimoto's disease, Graves' disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune hepatitis, primary biliary cholangitis, inflammatory bowel disease, multiple sclerosis, neuromyelitis optica, psoriasis, pemphigus, bullous pemphigoid, IgA nephropathy, IgA vasculitis, cryoglobulinemia vasculitis, and lupus nephritis.

[0030] In this specification, "diseases involving pathogenic IgG" means diseases in which immunoglobulin G (IgG) is directly or indirectly involved in the onset, progression, severity, pathogenesis, or manifestation of clinical symptoms of the disease, and includes, but is not limited to, diseases in which IgG functions as an autoantibody, diseases in which immune complexes containing IgG are involved in the pathogenesis, and diseases in which complement activation or antibody-dependent cell-mediated cytotoxicity (ADCC) by IgG is involved. More specifically, diseases involving pathogenic IgG include autoimmune diseases in which the pathogenesis is formed via autoantibodies (IgG), diseases in which tissue damage occurs due to the deposition of immune complexes containing IgG, diseases in which cell damage is induced by IgG-dependent complement activation or antibody-dependent cell-mediated cytotoxicity (ADCC), and diseases in which these mechanisms are involved in combination, such as systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and anti- This includes, but is not limited to, phospholipid antibody syndrome, systemic sclerosis, mixed connective tissue disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune hepatitis, primary biliary cholangitis, lupus nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), ANCA-associated vasculitis, IgG4-related disease, pemphigus, bullous pemphigoid, autoimmune encephalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), anti-glomerular basement membrane nephritis, etc. It may also include non-autoimmune diseases in which IgG is pathogenic to an exogenous antigen (e.g., serum sickness, neonatal hemolytic disease, post-streptococcal glomerulonephritis). In this specification, "IgG-mediated" means that an immune complex containing IgG or an IgG autoantibody causes glomerular damage via Fc binding or complement activation. In this specification, "rapidly progressive glomerulonephritis (RPGN)" refers to a disease characterized by a rapid decline in renal function over several days to several weeks, accompanied by active inflammatory urea sediment and findings of crescent glomerulonephritis. RPGN is primarily classified into three types: anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, type III immune complex hypersensitivity disorders, and type II immune complex hypersensitivity disorders. Type III immune complex hypersensitivity disorders include lupus nephritis, IgA nephropathy, IgA vasculitis, and cryoglobulinemia-associated glomerulonephritis. Type II immune complex hypersensitivity disorders include anti-glomerular basement membrane disease (Goodpasture syndrome).Although these diseases differ in their serological and histopathological characteristics, they ultimately converge on a common pathway: acute IgG-mediated glomerular damage.

[0031] 2. IdeZ-Fc Fusion Protein According to the present invention, a fusion protein (hereinafter also referred to as "IdeZ-Fc fusion protein") is provided which contains a conjugate of an IdeZ variant and an Fc region variant having excellent properties. The IdeZ variant and the Fc region variant may be directly bound together or bound together via a linker or the like. The linker is not particularly limited and can be appropriately selected and used from those commonly used in the field.

[0032] An IdeZ-Fc fusion protein according to one embodiment of the present invention is a fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant, wherein the fusion protein includes (a) a sequence that is 90% or more identical to the amino acid sequence from position 1 to 315 of the amino acid sequence represented by SEQ ID NO: 11; and (b) a sequence that is 90% or more identical to the amino acid sequence from position 316 to 541 of the amino acid sequence represented by SEQ ID NO: 11.

[0033] [1] IdeZ Modified The IdeZ modified in the IdeZ-Fc fusion protein according to the IdeZ Modified Embodiment is an IdeZ modified having IgG cysteine ​​protease activity and includes an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence of the IdeZ modified portion (positions 1 to 315) of the IdeZ-Fc fusion protein represented by SEQ ID NO: 11.

[0034] Furthermore, the IdeZ variant preferably contains (a) a sequence that is 90% or more identical to the amino acid sequence from position 35 to 349 of the amino acid sequence represented by Sequence ID No. 1 (wild-type IdeZ); and (b) no sugar chains are attached to the amino acids at positions corresponding to position 118 and 267 of the amino acid sequence represented by Sequence ID No. 1. The inventors have found that when IdeZ without sugar chains attached to the amino acids at positions 118 and 267 is expressed in mammalian cells, the attenuation of protease activity is suppressed. At the same time, the inventors have also found that when wild-type IdeZ is produced using mammalian cells, there is a problem of decreased expression levels, and that when the above-mentioned IdeZ variant is produced using mammalian cells, the decrease in expression levels is suppressed.

[0035] To prevent the attachment of glycans at the aforementioned positions, for example, the amino acids at positions 118 and 267 can be modified to amino acids other than asparagine, or amino acid modifications can be made to the consensus sequence of the N-linked glycan that includes these positions. By making at least one amino acid modification to the consensus sequence of the N-linked glycan containing asparagine, glycan attachment will be prevented. However, even if the sequence matches the consensus sequence, glycan attachment may not occur 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 (where X is the same amino acid and both sequences are located 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).

[0036] According to one embodiment, the IdeZ variant may include the following amino acid modifications (o) and (p). Such amino acid modifications make it possible to obtain an IdeZ variant in which no sugar chains are attached to the amino acids at the positions corresponding to position 118 and 267 of the amino acid sequence represented by SEQ ID NO: 1. Each of (o) and (p) may have at least one of the modifications (i) to (iii).In case (o): (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; In case (p): (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 position 269 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.

[0037] More preferably, the IdeZ variant has the amino acid at position 120 of the amino acid sequence represented by SEQ ID NO: 1 substituted with glutamic acid, and the amino acid at position 269 of the amino acid sequence represented by SEQ ID NO: 1 substituted with arginine.

[0038] It is preferable that the IdeZ variant further has no glycan attached to the amino acid at the position corresponding to position 171 of the amino acid sequence represented by SEQ ID NO: 1, and / or no glycan attached to the amino acid at the position corresponding to position 298 of the amino acid sequence represented by SEQ ID NO: 1. That is, as described above, in addition to the absence of glycans attached to the amino acids at positions 118 and 267, it is preferable that no glycans are attached to the amino acids at positions 171 and / or 298. This is because reducing the amount of glycan attachment further improves protease activity. To prevent glycan attachment, for example, the amino acids at the positions corresponding to position 171 and / or 298 can be modified to amino acids other than asparagine, or amino acid modifications can be made to the consensus sequence of the N-linked glycan that includes these positions. The specific details of the amino acid modifications to prevent glycan attachment are the same as those described above for the amino acid modifications at positions 118 and 267.

[0039] Such modifications are, for example, as follows: According to one embodiment, the IdeZ variant comprises the following amino acid modifications (q) and / or (r): (q) (i) the amino acid at 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) the amino acid at position 172 of the amino acid sequence represented by SEQ ID NO: 1 is replaced with proline, and / or (iii) The amino acid at 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; (r) (i) The amino acid at 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 position 299 of the amino acid sequence represented by SEQ ID NO: 1 is replaced with proline; and / or (iii) The amino acid at position 300 of the amino acid sequence represented by Sequence 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.

[0040] Preferably, in the IdeZ variant according to 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.

[0041] More preferably, the IdeZ variant according to the embodiment has 2 to 4 amino acid modifications selected from the following (A) to (D): (A) the amino acid at the position corresponding to position 120 of 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 of 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 of 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 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid.

[0042] The IdeZ variant may have 2 to 4 amino acid modifications, more preferably 3 or 4 amino acid modifications, and particularly preferably 4 amino acid modifications selected from the above (A) to (D) with respect to the amino acid sequence represented by SEQ ID NO: 1. When 2 or 3 amino acid modifications are selected from the above (A) to (D), any combination is acceptable, and the combination preferably includes the amino acid modifications of (A) and (C), (A), (B) and (C), or (A), (C) and (D).

[0043] The IdeZ variant according to the embodiment may contain amino acid modifications other than those described above. When produced using mammalian cells, the IdeZ variant according to the embodiment preferably has 10 to 100-fold, 10 to 70-fold, or 10 to 40-fold protease activity as compared to wild-type IdeZ. Note that since the IdeZ portion contributes to protease activity, the IdeZ-Fc fusion protein described below also has protease activity.

[0044] [2] Fc region variant The Fc region variant in the IdeZ-Fc fusion protein according to the embodiment comprises an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence of the Fc region variant portion (positions 316 to 541) in the IdeZ-Fc fusion protein represented by SEQ ID NO: 11. 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 type Fc regions. The Fc region to be used may be derived from any of these, but is preferably derived from IgG (IgG1, IgG2, IgG3 or IgG4), more preferably derived from human IgG, further preferably derived from human Igγ1, human Igγ2, human Igγ3, or human Igγ4, and particularly preferably derived from human Igγ1.

[0045] Furthermore, the present inventors have found that by using an Fc region variant obtained by modifying predetermined amino acids in the Fc region, autolysis of the fusion protein can be suppressed when the fusion protein with the IdeZ variant described above is produced. Since IdeZ is a cysteine protease that acts on and cleaves the hinge region, in a fusion protein in which IdeZ and an Fc region are bound, autolysis in which IdeZ acts on and cleaves the hinge region of its own fusion protein can be a problem. Accordingly, the present inventors focused on the property of IdeZ and the like that it has an action of cleaving between Fab and Fc of human IgG, but does not act on mouse IgG (IgG1, IgG2b), and constructed a fusion protein by combining an Fc region approximated to the structure of mouse IgG with the aforementioned IdeZ variant. As a result, it was found that autolysis is suppressed.

[0046] Specifically, the Fc region used in a fusion protein of an IdeZ variant and an Fc region variant is preferably modified as follows. In one embodiment, the Fc region variant in the fusion protein has glutamic acid substituted at position 236 and valine substituted at position 237. Preferably, the Fc region variant has alanine substituted at position 234 and glutamic acid substituted at position 235. More preferably, in addition to the above, the Fc region variant includes one or more of the following amino acid modifications: histidine substituted at position 227, lysine substituted at position 228, asparagine substituted at position 233, and / or serine substituted at position 238. Even more preferably, in addition to the above, the Fc region variant includes one or more of the following amino acid modifications: alanine substituted at position 253, deletion at position 293 or 294, and / or alanine substituted at position 296.

[0047] Particularly preferred, the Fc region variant is 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 with the 293rd or 294th position missing.

[0048] In particular, amino acid modifications of the human Igγ1 constant region L234A and L235A are called "LALA mutations." Here, L234A is a substitution of leucine at position 234 to alanine according to the EU index in the human Igγ1 constant region. L235A is a substitution of leucine at position 235 to alanine according to the EU index in the human Igγ1 constant region. Furthermore, amino acid modifications of L234A and L235E are called "LALE mutations." Here, L235E is a substitution of leucine at position 235 to glutamic acid according to the EU index in the human Igγ1 constant region. As will be described later, it is preferable to improve the sialylation rate of the Fc region in order to suppress the production of anti-drug antibodies, and the 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 mutations and LALE mutations. An IdeZ-Fc fusion protein according to one embodiment of the present invention comprises the following amino acid modification (a'), and the Fc region variant comprises the following amino acid modifications (b') and (b''): (a') The amino acids at positions 120, 171, 269, and 298 of the amino acid sequence represented by Sequence ID No. 1 are replaced with glutamic acid, glycine, arginine, and aspartic acid, respectively; (b') The amino acids at positions 234, 235, 236, 237, and 253 of the Fc region are replaced with alanine, glutamic acid, glutamic acid, valine, and alanine, respectively; (b'') The amino acid at position 293 or 294 of the Fc region is deleted.

[0049] Furthermore, the inventors have found that the production of anti-drug antibodies can be further suppressed by modifying the Fc region to reduce its binding activity to the Fcγ receptor. Examples of such modifications include the following modifications (c) and (d). According to one embodiment of the present invention, the Fc region variant further comprises the following amino acid modifications (c) and / or (d): (c) the amino acid at the position corresponding to position 265 of the Fc region is substituted with alanine; (d) the amino acid at the position corresponding to position 331 of the Fc region is substituted with glycine.

[0050] Furthermore, the inventors have also found that endocytosis can be suppressed by adding a modification to the Fc region that imparts a negative charge. More specifically, the above effect can be achieved by replacing amino acids at predetermined positions with acidic amino acids (aspartic acid, glutamic acid) or neutral amino acids. For example, the Fc region variant includes the following amino acid modifications: (e) one or more amino acids at positions corresponding to 361, 371, 375, 418 and 438 of the Fc region are replaced with aspartic acid or glutamic acid; (f) one or more amino acids at positions corresponding to 255, 274, 288, 326, 370 and 416 of the Fc region are replaced with neutral amino acids.

[0051] More preferably, the Fc region variant comprises the following amino acid modifications: (g) the amino acid at position 361 of the Fc region is substituted with aspartic acid, the amino acid at position 370 is substituted with threonine, the amino acid at position 371 is substituted with aspartic acid, the amino acid at position 375 is substituted with glutamic acid, the amino acid at position 416 is substituted with asparagine, the amino acid at position 418 is substituted with glutamic acid, and the amino acid at position 438 is substituted with glutamic acid; (h) the amino acid at position 255 of the Fc region is substituted with leucine, the amino acid at position 274 is substituted with glutamine, the amino acid at position 288 is substituted with glutamine, the amino acid at position 326 is substituted with alanine, the amino acid at position 370 is substituted with threonine, and the amino acid at position 416 is substituted with asparagine.

[0052] The desired effect can be fully obtained if the Fc domain variant includes either the modification that reduces the binding activity to the Fcγ receptor or the modification that imparts a negative charge. However, it is more preferable that the Fc domain variant includes both the modification that reduces the binding activity to the Fcγ receptor and the modification that imparts a negative charge.

[0053] Fc domain variants may include modifications other than those described above. They are not particularly limited as long as the desired effect is achieved, but for example, Fc domain variants may include modifications based on the Knobs-into-Holes technique (hereinafter also referred to as "Knobs-into-Holes mutations"). Knobs-into-Holes mutations are modifications in which, for example, in an Fc domain dimer, the amino acid at position 366 of one Fc domain is replaced with tryptophan, and the amino acid at position 366 of the other Fc domain is replaced with serine, the amino acid at position 368 is replaced with alanine, and the amino acid at position 407 is replaced with valine (International Publication No. 1998 / 050431).

[0054] More specifically, preferred structures of Fc variants include sequences that are 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence of the Fc region in the amino acid sequences represented by Sequence ID No. 4 to 14.

[0055] The IdeZ-Fc fusion protein may have a structure in which one IdeZ variant is bound to the N-terminus of each of two Fc-domain variants in a complex, or it may have a one-armed structure in which an IdeZ variant is bound to only one of the Fc-domain variants. In one embodiment, the IdeZ-Fc fusion protein is a fusion protein in which an IdeZ variant is bound to the N-terminus of an Fc-domain variant, and two such fusion proteins form a dimer. In another embodiment, the IdeZ-Fc fusion protein is a one-armed fusion protein containing a conjugate in which an IdeZ variant is bound to the N-terminus of a first Fc-domain variant, and a second Fc-domain variant to which an IdeZ variant is not bound. As will be described later, it is preferable that the Fc-domain variants in the fusion protein are sialylated, but the one-armed fusion protein containing the Fc-domain variant can achieve a relatively high sialylation rate without using glycosyltransferase for sialylation. Furthermore, while IdeZ-Fc region fusion proteins can be prone to aggregation, this aggregation problem is less likely to occur with single-arm fusion proteins.

[0056] In the case of a single-arm fusion protein, the first Fc region variant and the second Fc region variant may contain the knobs-into-holes mutation described above. According to embodiments of the present invention, it is preferable that the first Fc region variant and the second Fc region variant contain the knobs-into-holes mutation described above.

[0057] Therefore, according to one embodiment, an IdeZ-Fc fusion protein is provided in which the first Fc region variant and the second Fc region variant further include a knobs-into-holes mutation, the knobs-into-holes mutation being either (1) or (2) below: (1) The first Fc region has an amino acid at the position corresponding to position 366 of the Fc region substituted with tryptophan; The second Fc region has an amino acid at the position corresponding to position 366 of the Fc region substituted with serine, an amino acid at the position corresponding to position 368 substituted with alanine, and an amino acid at the position corresponding to position 407 substituted with valine; In the second Fc region, the amino acid at position 366 of the Fc region is substituted with tryptophan.

[0058] More specific examples of one-arm fusion protein structures include structures containing the amino acid sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10; structures containing the amino acid sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12; or structures containing the amino acid sequence represented by SEQ ID NO: 13 and SEQ ID NO: 14; and structures that are 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to these.

[0059] IdeZ-Fc fusion proteins may contain other components besides the IdeZ variant and the Fc region variant. These other components may include proteins, more specifically antibodies, antibody antigen-binding fragments (single-stranded variable region fragments (scFv), Fab fragments, Fab' fragments, F(ab')). ２ Examples include fragments, bioactive peptides (e.g., receptors, adhesion molecules, ligands, enzymes, etc.), but are not limited to these.

[0060] The IdeZ-Fc fusion protein described above can be used in conventionally known applications of IgG cysteine ​​proteases. Such applications include, for example, use in high-quality recombinant protein production systems, use for suppressing rejection during transplantation, and use for the treatment of autoimmune diseases and diseases involving pathogenic IgG (particularly autoimmune diseases involving pathogenic IgG), as described in the background art section above (Patent Documents 1 and 2, WO2006 / 131347, WO2016 / 012285, etc.). Other possible applications include pretreatment for gene therapy using AAV (Patent Document 1).

[0061] 3. Polynucleotide and Expression Vector - According to one embodiment, a polynucleotide encoding the IdeZ-Fc fusion protein described above is provided. The polynucleotide according to the embodiment can be prepared by those skilled in the art using methods known in the art based on its base sequence. For example, the polynucleotide according to the embodiment can be synthesized using gene synthesis methods known in the art. Various methods known to those skilled in the art can be used as such gene synthesis methods, such as the method for synthesizing antibody genes described in International Publication No. 90 / 07861.

[0062] In a further embodiment, an expression vector containing the polynucleotide described above is provided. In the case of a one-armed fusion protein, the polynucleotide encoding the fusion protein, which includes an IdeZ variant bound to the N-terminus of a first Fc region, and the polynucleotide encoding the one-armed fusion protein, which includes a second Fc region to which the IdeZ variant is not bound, may each be contained in separate vectors, or multiple polynucleotides may be contained in a single vector.

[0063] The expression vector according to the embodiment is not particularly limited as long as it can produce 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 include the pcDNA series (Thermo Fisher Scientific), pALTER®-MAX (Promega), PHEK293 Ultra Expression Vector (Takara Bio), pEE6.4 or pEE12.4 (Lonza Biologics). Examples of viral vectors include lentiviruses, adenoviruses, retroviruses, and adeno-associated viruses. For example, when using a lentivirus to introduce polynucleotides according to the embodiment into cells, the lentivirus can be a pLVSIN-CMV / EF1α vector (Takara Bio Inc.), a pLenti vector (Thermo Fisher Scientific Inc.), or the like. In one embodiment, the vectors used in the expression vector according to the embodiment are pcDNA® 3.4-TOPO® (Thermo Fisher Scientific Inc.) and pcDNA® 3.1 (Thermo Fisher Scientific Inc.).

[0064] The expression vector according to the embodiment may include a promoter ligated to the IdeZ-Fc fusion protein according to the embodiment in an operable manner. Examples of promoters for expressing the polynucleotide according to the embodiment in animal cells include virus-derived promoters such as CMV, RSV, and SV40, actin promoters, EF (elogenation factor) 1α promoters, and heat shock promoters. Examples of promoters for expressing the polynucleotide according to the embodiment in bacteria (e.g., Escherichia species) include trp promoter, lac promoter, λPL promoter, and tac promoter. Examples of promoters for expressing the polynucleotide according to the embodiment in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.

[0065] When using animal cells, insect cells, or yeast as the host cell, the expression vector according to the embodiment may include a start codon and a stop codon. In this case, it may also include an enhancer sequence, the 5' and 3' untranslated regions of the gene encoding the IdeZ-Fc fusion protein according to the embodiment, a secretory signal sequence, a splicing junction, a polyadenylation site, or a repeatable unit. When using Escherichia coli as the host cell, the expression vector according to the embodiment may include a start codon, a stop codon, a terminator region, and a repeatable unit. The expression vector according to the embodiment may also include commonly used drug selection marker genes (e.g., tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) depending on the purpose.

[0066] 4. Host Cells According to one embodiment, host cells transformed with the expression vector described above are provided. The host cells according to the embodiment may contain one or more polynucleotides according to the embodiment as a result of transformation with the expression vector according to the embodiment.

[0067] The host cells to be transformed are not particularly limited, as long as they are compatible with the expression vector used and can be transformed with the expression vector to express the IdeZ-Fc fusion protein according to the embodiment. Various cells can be used as the host cells to be transformed, such as conventional cells or artificially established cells commonly used in the field. Examples include animal cells (CHO-K1 cells, ExpiCHO-S™ cells, CHOK1SV cells, CHO-DG44 cells, HEK293 cells, NS0 cells, etc.), insect cells (Sf9, etc.), bacteria (Escherichia species, etc.), and yeasts (Saccharomyces, Pichia species, etc.). In one embodiment, the host cells are CHO-K1 cells or ExpiCHO-S cells.

[0068] The method for transforming host cells is not particularly limited, but methods commonly used by those skilled in the art, such as calcium phosphate, electroporation, or lipofection, can be used.

[0069] The selection of transformed host cells can be carried out by methods commonly used by those skilled in the art. Selection methods may include, for example, drug selection methods using drug selection marker genes and drugs such as tetracycline, ampicillin, neomycin, or hygromycin, as well as cell isolation methods such as ultradilution, single-cell sorting, and colony pickup.

[0070] 5. Production Method According to one embodiment, a method for producing an IdeZ-Fc fusion protein according to the embodiment is provided. The method according to the embodiment may include steps such as culturing the transformed host cells described above and expressing the IdeZ-Fc fusion protein in the host cells or culture supernatant, and recovering, isolating, and purifying the IdeZ-Fc fusion protein, but is not limited to these methods as long as the target IdeZ-Fc fusion protein is produced. Modification of the IdeZ and Fc regions can be appropriately carried out by a person skilled in the art using methods known in the art based on the sequence information disclosed herein. For example, it can be obtained by making amino acid modifications to the wild-type IdeZ or Fc region using methods commonly used in the art.

[0071] Transformed host cells can be cultured by known methods. Culture conditions, such as temperature, pH of the culture medium, and culture time, can be appropriately selected by those skilled in the art. When the host cells are animal cells, suitable culture media can be, for example, MEM medium containing approximately 5-20% fetal bovine serum (Science, 1959, Vol. 130, pp. 432-437), DMEM medium (Virol., 1959, Vol. 8, pp. 396), RPMI-1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, pp. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, pp. 1-8), etc. The pH of the culture medium is, for example, about 6 to 8, and the culture is usually carried out at about 30 to 40°C for about 15 to 336 hours, with aeration and stirring as needed. When the host cells are insect cells, a culture medium such as Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA., 1985, Vol. 82, p. 8404) can be used. The pH of the culture medium is, for example, about 5 to 8, and the culture is usually carried out at about 20 to 40°C for about 15 to 100 hours, with aeration and stirring as needed.

[0072] When the host cells are E. coli or yeast, a suitable culture medium is, for example, a liquid medium containing nutrients. 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 cells. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose, while examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn slush liquor, peptone, casein, meat extract, soybean meal, and potato extract. Optionally, other nutrients (e.g., inorganic salts (e.g., calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins), and antibiotics (e.g., tetracycline, neomycin, ampicillin, kanamycin) may also be included. The pH of the medium is, for example, about 5 to 8. When the host cell is E. coli, suitable culture media include, for example, LB medium or M9 medium (Molecular Cloning, Cold Spring Harbor Laboratory, Vol. 3, A2.2). Culturing is typically carried out at approximately 14-43°C for about 3-24 hours, with aeration and stirring as needed. When the host cell is yeast, suitable culture media include, for example, Burkholder Minimal Medium (Proc. Natl. Acad. Sci. USA., 1980, Vol. 77, p. 4505). Culturing is typically carried out at approximately 20-35°C for about 14-144 hours, with aeration and stirring as needed. By culturing as described above, the target IdeZ-Fc fusion protein can be expressed.

[0073] The method according to the embodiment may further include, in addition to the step of culturing transformed host cells and expressing IdeZ-Fc fusion protein, a step of recovering, isolating, or purifying the IdeZ-Fc fusion protein from the host cells. Examples of isolation or purification methods include methods utilizing solubility such as salting out and solvent precipitation, methods utilizing differences in molecular weight such as dialysis, ultrafiltration, and gel filtration, methods utilizing charge such as ion exchange chromatography and hydroxyl apatite chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing differences in hydrophobicity such as reverse-phase high-performance liquid chromatography, and methods utilizing differences in isoelectric point such as isoelectric focusing. In one embodiment, polypeptides secreted into the culture supernatant can be purified by various chromatography methods, for example, column chromatography using a protein A column or a protein G column. According to one embodiment, an IdeZ-Fc fusion protein produced by the above-described production method is provided.

[0074] 6. Sialized Fusion Protein The IdeZ-Fc fusion protein described above may have its Fc region sialylated (i.e., sialic acid added). By sialyzing the Fc region, the production of anti-drug antibodies against the fusion protein and / or endocytosis can be further suppressed when the fusion protein is administered to a living organism. It is preferable to modify the Fc region to increase the sialylation rate, and examples of Fc regions including such modifications include LALA mutations or LALE mutations and Fc region modifiers including deletion of the amino acid at position 293 or 294. Normally, in wild-type IgG, both the amino acids at positions 293 and 294 are glutamic acid, so the same modification can be obtained by deleting either one. In one embodiment, the Fc region of the IdeZ-Fc fusion protein has a deletion of glutamic acid at position 293 or 294. According to one embodiment, a sialylated IdeZ-Fc fusion protein is provided.

[0075] The method of sialylation is not particularly limited, but a sialylated fusion protein can be obtained by adding or expressing a sialylate transferase during the host cell culture process 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 sialylate transferase may be simply added, or an expression vector may be created by inserting the DNA encoding it, and the protein may be expressed together with a polypeptide such as an antibody. When adding the sialylate transferase, the timing and method are not particularly limited; for example, it may be added during the polypeptide production process by cell culture, or it may be added and reacted after the polypeptide purification. When expressing the sialylate transferase, for example, a plasmid encoding the sialylate transferase may be co-expressed in cells together with a plasmid encoding the polypeptide to produce a sialylated polypeptide, but this is not limited to this method. When polypeptides are produced using cells containing endogenous glycosyltransferases, such as CHO cells, sialic acid can be added during the polypeptide production process without the need to add sialic acid transferases as described above.

[0076] Fusion proteins containing Fc region variants that include the above-mentioned modifications that increase the sialylation rate exhibit an increased amount of sialic acid added (i.e., degree of sialylation) compared to cases where the wild-type Fc region or an Fc region variant with only the amino acid at position 294 is sialylated. For example, the amount of sialic acid added to the glycan bound 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 "sialylation rate (%)". In this specification, "sialylation rate (%)" means the percentage of glycans to which sialic acid is added out of all glycans present in the Fc region, and the specific measurement and calculation methods are as described in the examples below. The sialylated fusion proteins according to the embodiment have a sialylation rate of, for example, 20-100%, 40-100%, 60-100%, 80-100%, or 90-100%.

[0077] The IdeZ-Fc fusion protein and sialylated fusion protein described above can be used for conventionally known applications of IgG cysteine ​​proteases. Such applications include, for example, use in high-quality recombinant protein production systems, use for suppressing rejection reactions during transplantation, and use for the treatment of autoimmune diseases and diseases involving pathogenic IgG (particularly autoimmune diseases involving pathogenic IgG), as described in the background art section above (Patent Documents 1 and 2, WO2006 / 131347, WO2016 / 012285, etc.). Furthermore, by using fusion proteins, advantages such as easy purification and modification, and the ability to adjust the half-life can be obtained.

[0078] 7. Pharmaceutical Composition According to one embodiment, a pharmaceutical composition is provided comprising the above-described IdeZ-Fc fusion protein and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. Examples of dosage forms of the pharmaceutical composition include, for example, parenteral preparations such as injections and intravenous infusions, which can be administered by intravenous, subcutaneous, or intraperitoneal administration. In formulation, excipients, carriers, additives, etc., can be used according to these dosage forms, within a pharmaceutically acceptable range. The pharmaceutical composition according to the embodiment may further contain one or more additional therapeutic agents. The pharmaceutical composition according to the embodiment may also be used in combination (simultaneously or sequentially) with one or more additional therapeutic agents.

[0079] The amount of the IdeZ-Fc fusion protein according to the embodiment in the pharmaceutical composition can be appropriately set depending on the severity of the patient's symptoms, age, dosage form of the formulation used, etc. The IdeZ-Fc fusion protein according to the embodiment and the pharmaceutical composition containing it can be used for suppressing rejection reactions during organ transplantation, treating autoimmune diseases and diseases involving pathogenic IgG (particularly autoimmune diseases involving pathogenic IgG), and as a pretreatment for gene therapy using AAV. Furthermore, according to another embodiment, a method for preventing or treating autoimmune diseases, diseases involving pathogenic IgG, etc. is provided, which includes administering a therapeutically effective amount of the IdeZ-Fc fusion protein according to the embodiment to a target.Further embodiments provide the IdeZ-Fc fusion protein of the present invention for use in preventing or treating autoimmune diseases, diseases involving pathogenic IgG, etc.Further embodiments provide the use of the IdeZ-Fc fusion protein according to the embodiment in the manufacture of a pharmaceutical composition for preventing or treating autoimmune diseases, diseases involving pathogenic IgG, etc.

[0080] The present invention will be described in detail below with reference to examples, but the content of the present invention is not limited thereto. <Example 1: Preparation of IdeZ Modified A-Mouse Fc and IdeZ Modified A-Human Fc> (1) IdeZ Modified A-Mouse Fc A gene encoding a protein in which four amino acid modifications (T120E / N171G / S269R / N298D according to the numbering in SEQ ID NO: 1) were introduced into the amino acid sequence from position 35 to 349 of the IdeZ protein (UniPro accession number: Q0PIW1, SEQ ID NO: 1) derived from Streptococcus zooepidemicus, and an antibody heavy chain signal (Protein) was introduced on the 5' side. The nucleic acid sequence encoding (Eng. 1987, Vol. 1, pp. 499-505) and the nucleic acid sequence encoding the mouse Igγ1 constant region (from glycine at amino acid position 223 to lysine at position 447, following the EU index) were concatenated. The resulting nucleic acid sequences were inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) to create an expression vector. The designed sequences are shown in Table 1.

[0081] To add α2,6-linked sialic acid, an expression vector encoding β-1,4-galactosyltransferase 1 (B4GALT1) (UniPro accession number: P15291-1) and an expression vector encoding human β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) (UniPro accession number: P15907-1) were transfected into ExpiCHO-S cells (Thermo Fischer Scientific) together with an expression vector encoding IdeZ variant A-mouse Fc, and the cells were cultured. Following standard procedures, the culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns to obtain sialylated IdeZ variant A-mouse Fc. Furthermore, in order to obtain IdeZ-modified A-mouse Fc prepared under non-co-expression conditions of B4GALT1 and ST6GAL1, an expression vector encoding IdeZ-modified A-mouse Fc was transfected into ExpiCHO-S cells and cultured. Following standard procedures, the culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns to obtain non-sialyzed IdeZ-modified A-mouse Fc.

[0082] (2) IdeZ variant A - Human Fc variants 7, 25, 26 To obtain fusion proteins of the bivalent IdeZ variant A and the human IgG1 Fc region variant, modified sequences of human Fc were designed. The names of each variant and the designed sequences are shown in Table 2.

[0083] DNA encoding various IdeZ-modified A-human Fc variants was synthesized according to conventional methods, and nucleic acid sequences encoding antibody heavy chain signals were ligated to the 5' end of each DNA molecule before insertion into a pcDNA3.4 vector to produce expression vectors for each IdeZ-modified A-human Fc variant. Using the same method as above, expression vectors encoding B4GALT1 and ST6GAL1, along with expression vectors encoding various IdeZ-modified A-human Fc variants, were transfected and cultured. The culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns according to conventional methods to obtain sialyzed IdeZ-modified A-human Fc variants 7, 25, and 26. Furthermore, using the same method as above, expression vectors encoding various IdeZ-modified A-human Fc variants were transfected into ExpiCHO-S cells and cultured. Following conventional methods, the culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns to obtain non-sialyzed IdeZ variants A-human Fc variants 7, 25, and 26.

[0084] (3) IdeZ variant A - Human Fc variant 19 In order to obtain a fusion protein of monovalent IdeZ variant A and a variant of the human IgG1 Fc region, a modified sequence of human Fc was designed. The names of each variant and the designed sequences are shown in Table 3.

[0085] DNA encoding IdeZ variant A-human Fc variant 19_holes and human Fc variant 19_knobs was synthesized according to conventional methods and inserted into pcDNA3.4 vectors to produce expression vectors for each variant's constituent strands. Using the same method as above, expression vectors encoding B4GALT1 and ST6GAL1, the expression vector for IdeZ variant A-human Fc variant 19_holes, and the expression vector for human Fc variant 19_knobs were transfected and cultured. The culture supernatant was purified using affinity chromatography and gel filtration chromatography columns according to conventional methods to obtain sialyzed IdeZ variant A-human Fc variant 19. Furthermore, using the same method as above, the expression vectors for IdeZ variant A-human Fc variant 19_holes and human Fc variant 19_knobs were transfected into ExpiCHO-S cells and cultured. Following conventional methods, the culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns to obtain non-sialyzed IdeZ variant A-human Fc variant 19.

[0086] The sialylation rate was analyzed for the sialylated and non-sialylated IdeZ variant A-mouse Fc and the sialylated and non-sialylated IdeZ variant A-human Fc variants 7, 19, 25, and 26 prepared as described above. For each sample, dithiothreitol (Thermo Fisher Scientific, A39255, hereafter "DTT") was added and incubated at 37°C for 1 hour to reduce the interchain disulfide bonds. The reduced samples were injected into an HPLC system (Waters), separated using a reversed-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 and the measured mass after reduction, and the percentage of each glycan was calculated from the ionic intensity ratio. The percentage of glycans with sialic acid attached among all glycans present in the Fc region was defined as the "sialylation rate (%)". The sialylation rate was calculated using the following formula: Sialization rate (%) = (Total MS intensity of H chains modified with sialic acid-linked glycans) ÷ (Total MS intensity of all H chains) × 100 The results for IdeZ variant A-mouse Fc and IdeZ variant A-human Fc variants 7, 25, and 26 are shown in Table 4.

[0087] 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. Since each fragment was observed to have a different molecular weight in mass spectrometry, the sialylation rate was calculated for each. The results are shown in Table 5.

[0088] <Example 2: Evaluation of immunogenicity of IdeZ-Fc fusion protein in mice> ・Example 2-1: Exposure to IdeZ variant A-mouse Fc and IdeZ variant A-human Fc variant 19 and serum acquisition Eight-week-old female BALB / cAJcl mice (Claire Japan) were used. Four mice / group were used for each type of IdeZ-Fc fusion protein. CpG and IdeZ-Fc fusion protein were co-administered to enhance the immune response to the IdeZ variant. The administration solution was prepared so that 10 μg of IdeZ-Fc fusion protein and 10 μg of CpG (Thermo Fisher Scientific) were administered per mouse, and the drugs were administered intraperitoneally. Intraperitoneal administration was performed twice, on the first day of administration (day 0) and on day 14, to induce the production of anti-drug antibodies. Blood was drawn on the 21st day after the start of administration, and serum was obtained.

[0089] Example 2-2: Exposure to IdeZ Modified A-Human Fc Modified 7 and Plasma Acquisition Plasma was acquired in the same manner as in Example 2-1. However, 6-week-old female BALB / cAJcl mice (Claire Japan) were used, with 6 mice in each group. The administration solution was prepared so that each mouse received 200 μg of IdeZ-Fc fusion protein and 10 μg of CpG. Intraperitoneal administration was performed only on the first day of administration (day 0), and blood was collected and plasma was acquired on the 14th day from the start of administration.

[0090] • Example 2-3: Exposure to IdeZ variant A-human Fc variants 25 and 26 and plasma acquisition. Plasma was acquired in the same manner as in Example 2-1. However, 9-week-old female BALB / cAJcl mice (Claire Japan) were used, with 8 mice in each group. The administration solution was prepared so that 10 μg of IdeZ-Fc fusion protein and 10 μg of CpG were administered to each mouse. Intraperitoneal administration was performed twice, on the first day of administration (day 0) and on day 14. Blood was collected on day 21 from the start of administration, and plasma was obtained.

[0091] • Example 2-4: Evaluation of anti-drug antibody production. To measure the amount of anti-drug antibodies produced in serum / plasma, an electrochemiluminescence bridging assay was performed. The His-IdeZ modified A protein was prepared by modifying four amino acids (T120E / N171G / S269R / N298D according to the numbering in SEQ ID NO: 1) from amino acid positions 35 to 348 of the IdeZ sequence (Uniprot accession number: Q0PIW1), and ligating the antibody heavy chain signal sequence (Protein Eng., 1987, Vol. 1, p. 499-505) and the HisTag sequence (Hisx6) to its N-terminus. It was then obtained using ExpiCHO-S cells according to a standard procedure. The serum / plasma samples obtained in Examples 2-1 to 2-3 were diluted 100-fold with physiological saline (Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795). To the diluted serum / plasma samples, a mixture of 0.125 μg / mL biotinylated His-IdeZ modified A protein and 0.125 μg / mL sulfonated His-IdeZ modified A protein was added in twice the volume, and the mixture was reacted at room temperature for 1 to 2 hours using Micromixer MX-5 (Sanko Junyaku Co., Ltd.). Subsequently, the sample mixture was added at a rate of 50 μL / well to an MSD GOLD 96 well Streptavidin SECTOR plate (Meso Scale Diagnostics, LLC, L15SA) that had been blocked with Blocker Casein in TBS (Thermo Fisher Scientific, 37532). After reacting at room temperature for 1-2 hours using a Micromixer MX-5, the mixture was washed three times with TBS-T (Nippon Gene, 310-07375) washing solution. After washing, Read Buffer (Meso Scale Diagnostics, LLC, R92TC-2) was added at 150 μL / well, and measurements were rapidly performed using SECTOR S600 (Meso Scale Diagnostics, LLC).

[0092] The experimental results are shown as mean ± standard error. Sialized IdeZ variant A-mouse Fc reduced the production of anti-drug antibodies by half compared to non-sialyzed IdeZ variant A-mouse Fc (Figure 1). On the other hand, as shown in Figures 2 and 3, IdeZ variant A-human Fc variant 19 and IdeZ variant A-human Fc variant 7 did not show a decrease in anti-drug antibody production even after sialylation. It was thought that human Fc has higher antigenicity than mouse Fc for mice, and therefore the inhibitory effect of sialylation on anti-drug antibody production was not exerted.

[0093] Therefore, in order to reduce the immunogenicity of the IdeZ modified A-human Fc modified molecule, a modification was introduced to reduce its binding activity to the Fcγ receptor. Sialized IdeZ modified A-human Fc modified molecule 25, which had the D265A modification introduced in the Fc region, and sialyzed IdeZ modified A-human Fc modified molecule 26, which had the P331G modification introduced, were prepared. As shown in Figure 4, sialyzed IdeZ modified A-human Fc modified molecule 25 and sialyzed IdeZ modified A-human Fc modified molecule 26 produced half the amount of anti-drug antibodies compared to non-sialyzed IdeZ modified A-human Fc modified molecule 7.

[0094] <Example 3: Preparation of IdeZ Modified A-Human Fc Modified Protein> (1) IdeZ Modified A-Human Fc Modified Protein 30 In order to obtain a fusion protein of the bivalent IdeZ Modified A and the Fc region of human IgG1, a modified sequence of human Fc was designed. The names of the modified proteins and the designed sequences are shown in Table 6.

[0095] In the manner described in Example 1, expression vectors encoding B4GALT1 and ST6GAL1, and an expression vector encoding IdeZ variant A-human Fc variant 30 were both transfected and cultured. The culture supernatant was purified using an affinity chromatography column and a gel filtration chromatography column according to conventional methods to obtain sialyzed IdeZ variant A-human Fc variant 30.

[0096] Furthermore, using the method described in Example 1, an expression vector encoding IdeZ variant A-human Fc variant 30 was transfected into ExpiCHO-S cells and cultured. The culture supernatant was purified using an affinity chromatography column and a gel filtration chromatography column according to conventional methods to obtain non-sialyzed IdeZ variant A-human Fc variant 30.

[0097] (2) IdeZ variant A - Human Fc variants 77, 93, 97 To obtain a fusion protein of monovalent IdeZ variant A and the Fc region of human IgG1, modified sequences of human Fc were designed. The names of each variant, the names of the constituent chains, and the designed sequences are shown in Table 7.

[0098] DNA encoding various IdeZ variants A-human Fc variants _holes and various human Fc variants _knobs was synthesized according to conventional methods, and each was inserted into a pcDNA3.4 vector to prepare expression vectors for the constituent strands of each variant. Using the method described in Example 1, expression vectors encoding B4GALT1 and ST6GAL1, and expression vectors for various IdeZ variants A-human Fc variants _holes and various human Fc variants _knobs were transfected together and cultured. The culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns according to conventional methods to obtain sialyzed IdeZ variants A-human Fc variants 77, 93, and 97.

[0099] Furthermore, using the method described in Example 1, expression vectors for various IdeZ variants A-human Fc variants _holes and various human Fc variants _knobs were transfected into ExpiCHO-S cells and cultured. Following conventional methods, the culture supernatant was purified using affinity chromatography columns and gel filtration chromatography columns to obtain non-sialyzed IdeZ variants A-human Fc variants 77, 93, and 97.

[0100] The sialylation rate of the IdeZ variant A-human Fc variant 30 prepared as described above was analyzed using the method described in Example 1. The results for IdeZ variant A-human Fc variant 30 are shown in Table 8.

[0101] IdeZ variant A-human Fc variants 77, 93, and 97 were separated into IdeZ variant A-human Fc variant _holes and human Fc variant _knobs by reduction treatment. Since each fragment was observed to have a different molecular weight in mass spectrometry, the sialylation rate was calculated for each. The results are shown in Table 9.

[0102] <Example 4: Evaluation of Endocytosis Activity of IdeZ Modifications in MUTZ-3 Cells> In mice, the introduction of D265A and P331G modifications was investigated to verify the possibility that reduced intracellular uptake activity (endocytosis activity) may be one of the factors contributing to the decreased immune response to IdeZ. A method is known to extend the blood half-life by modifying the surface amino acids of the variable region of an antibody to lower its isoelectric point (Protein Eng. Des. Sel., 2010, Vol. 23(5), p. 385-392). The proposed mechanism is that the negative charge of the antibody repels the negative charge of the cell surface, reducing nonspecific uptake into cells. Therefore, as a modification to enhance the attenuation of endocytosis activity, a negatively charged amino acid was introduced into the Fc region. Furthermore, since sialic acid has a negative charge, the effect of sialic acid on endocytosis activity was also evaluated.

[0103] The endocytosis activity of the IdeZ variant in MUTZ-3 cells was observed in Gibco cells containing 20% ​​HyClone FBS (HyClone Healthcare, SH30084.03) and 10 ng / mL Recombinant Human GM-CSF Protein (Carrier Free) (R&D, 215-GM-010 / CF). ＴＭ MEM α, nucleosides, GlutaMAX ＴＭ Measurements were performed in the presence of Supplement (Thermo Fisher Scientific, 32571-036). IdeZ variants were labeled with the pH-sensitive fluorescent dye Hydrodo, and evaluated by measuring the fluorescence signal emitted upon lysosome translocation.

[0104] Example 4-1: Preparation of pHrodo-labeled IdeZ variants Various IdeZ-Fc fusion proteins were prepared at a concentration of 1 mg / mL or higher using 100 mM sodium bicarbonate (Thermo Fisher Scientific, 25080-094). pHrodo® Red succinimidyl (NHS) ester (Thermo Fisher Scientific, P36600) was dissolved in DMSO, and an amount equivalent to approximately 10 times the number of moles of protein of each IdeZ-Fc fusion protein was added to a reaction tube and mixed. The mixture was then allowed to react at room temperature for 60 minutes. The reaction mixture was purified by centrifugation using a PD mini G25 desalting column (Gehealthcare, 28918007) and an Amicon Ultra-0.5 mL, 30 K column (Amicon, UFC503024) to obtain pHo-labeled IdeZ. The absorbance at 280 nm and 560 nm was measured using a NanoDrop (trademark registered) 8000 Spectrophotometer (Scrum Co., Ltd.), and the protein concentration was calculated.

[0105] Example 4-2: Evaluation of endocytosis activity in cultured cells. MUTZ-3 cells were 1 x 10⁶ ５ Cells were seeded in a cell suspension of 50 μL / well into a 96 microwell plate (Corning, 353072). Subsequently, 50 μL / well of a 2x concentrated solution of various pHo-labeled IdeZ-Fc fusion proteins, prepared to a final concentration of 25 μg / mL, was added. After incubation overnight at 37°C, the fluorescence signal was measured using an IncuCyte S3 (Sartorius). Measurements were performed in 2 wells per condition. The acquired images were quantitatively analyzed using the image analysis software IncuCyte 2021C (Sartorius).

[0106] The results are shown in Figure 5. Siallylated IdeZ variant A-human Fc variant 7 showed lower endocytosis activity compared to non-sialylated IdeZ variant A-human Fc variant 7. It was thought that the negative charge of sialic acid repelled the negatively charged cell surface, reducing nonspecific uptake into cells. However, as shown in Example 2, sialylated IdeZ variant A-human Fc variant 7 did not suppress the production of anti-drug antibodies compared to non-sialylated IdeZ variant A-human Fc variant 7 under CpG combination conditions using BALB / cAJcl mice. From this, it was considered that the effect of reduced endocytosis activity on the decrease in anti-antibody production was not very large.

[0107] The non-sialylated IdeZ variant A-human Fc variant 77 exhibited lower endocytosis activity compared to the non-sialylated IdeZ variant A-human Fc variant 7 and the sialylated IdeZ variant A-human Fc variant 7. This suggests that the reduction in endocytosis activity due to the introduction of the P331G mutation is related to the reduced immunogenicity in Balb / c mice. The lower endocytosis activity of the sialylated IdeZ variant A-human Fc variant 77 compared to the non-sialylated IdeZ variant A-human Fc variant 77 indicates that the P331G mutation and sialic acid have an additive effect in reducing endocytosis activity.

[0108] The non-sialylated IdeZ variant A-human Fc variant 30 exhibited lower endocytosis activity compared to the non-sialylated IdeZ variant A-human Fc variant 7. This suggests that introducing negatively charged amino acids into the Fc region repelled the negatively charged cell surface, thereby suppressing endocytosis activity. The lower endocytosis activity of the sialylated IdeZ variant A-human Fc variant 30 compared to the non-sialylated IdeZ variant A-human Fc variant 30 indicates that negatively charged amino acids and sialic acid additively contribute to the reduction of endocytosis activity. Siallylated IdeZ variant A-human Fc variant 93 and sialylated IdeZ variant A-human Fc variant 97, which possess the P331G mutation, negative charge, and sialic acid, exhibited lower endocytosis activity compared to sialylated IdeZ variant A-human Fc variant 7.

[0109] <Example 5: Evaluation of immunogenicity of IdeZ-Fc fusion protein in mice> ・Example 5-1: Exposure to various IdeZ variants A-human Fc variants and acquisition of blood Six- or seven-week-old female BALB / cAJcl mice (Claire Japan) were used. Eight mice / group were used for each type of IdeZ-Fc fusion protein. A dosage solution was prepared so that each mouse received 10 μg of IdeZ-Fc fusion protein and 10 μg of CpG, and administered intraperitoneally on the first day of administration (day 0) and on day 14 to induce the production of anti-drug antibodies. For the third intraperitoneal administration on day 21 of administration, only IdeZ-Fc fusion protein was administered, without the use of CpG. Blood was collected on day 28 from the start of administration and diluted with 198 μL of PBS in 2 μL of blood.

[0110] Example 5-2: Evaluation of Antidrug Antibody Production To measure the amount of antidrug antibodies produced in the diluted blood sample obtained in Example 5-1, the analysis was performed using the same method as in Example 2-4. Since the sample had already been diluted 100-fold in Example 5-1, no further dilution was performed during this analysis.

[0111] Experimental results are shown as mean ± standard error. Significant difference testing between the group administered sialyzed ideZ variant A-human Fc variant 77 and the groups administered various ideZ-Fc fusion proteins was performed using the unpaired Dunnett test, with a p-value of less than 0.05 considered statistically significant. These tests were performed using Graphpad Prism (version 10.1.2). Sialyzed ideZ variant A-human Fc variants 93 and 97 significantly reduced the production of anti-drug antibodies compared to sialyzed ideZ variant A-human Fc variant 77 (Figure 6). In mice, it was considered that the negatively charged amino acids in the Fc region of sialyzed ideZ variant A-human Fc variants 93 and 97 enhanced the inhibitory effect on anti-drug antibody production.

[0112] <Example 6: Evaluation of IdeZ-Fc Fusion Protein Immunogenicity in Human PBMCs> To evaluate the immunogenicity of each fusion protein produced in the human immune system, evaluation was performed using PBMCs (Peripheral Blood Mononuclear Cells) from 20 donors. Frozen PBMCs (Cellular Technology Limited, # CTL-UP1) were thawed at 37°C and washed twice to remove the cell preservation solution from the cell suspension. After washing, the cells were counted using NovoCyte Penteon (Agilent Technologies), and 1 × 10 CD4-positive T cells were found. ６ Cell suspensions were prepared to a concentration of 100 μg / mL. The cells were then seeded onto 96-well flat-bottom plates (AGC Technoglass, #3860-096, 3861-096), and 2 or 3 wells each of the test protein, an immunogenic protein, or phosphate buffer were added to achieve a final concentration of 30 μg / mL. The cells were then cultured at 37°C for 42 to 72 hours. Table 10 summarizes the SEC purity and endotoxin levels of the proteins evaluated in this example.

[0113] The SEC purity of the target fraction was analyzed using ACQUITY UPLC BEH200 SEC column (Waters #186005225). Endotoxin levels were measured using Limulus Amebocyte Lysate (LAL) Test Cartridges (Veritas #CES-PTS2001F). Subsequent procedures were performed at room temperature unless otherwise specified. After culturing, each well was suspended and the cells were transferred to a 96-well V-bottom plate (Sumitomo Bakelite #MS-3396P) and washed twice. An antibody mixture (anti-CD4 antibody (BioLegend #344604), anti-CD134 antibody (BioLegend #350004), anti-CD137 antibody (BioLegend #309810), and Zombie violet (BioLegend #423114)) was added to the cells, and after suspension, the cells were reacted at 37°C for 20 minutes. After washing the cells twice, 4% paraformaldehyde-phosphate buffer (Nacalai Tesque #09154-85) was added to PBS containing 1% Pluronic-F68 (Thermo Fisher Scientific #24040032) to a final concentration of 2%, and the cells were fixed after suspension for 20 minutes. After washing the cells three times, the total volume was measured using a Novocyte Penteon at a flow rate of 100 μL / min.

[0114] Data was analyzed using the data analysis software Flowjo (Becton, Dickinson and Company). Cell fractions were gated by FSC-SSC, followed by gated CD4-positive cells. Zombie-negative viable cells were further gated, and the proportion of CD134 / CD137 co-positive cells among CD4-positive viable cells was calculated. To standardize the strength of the response between donors, the proportion of CD134 / CD137 co-positive cells among CD4-positive viable cells was divided by the proportion of CD134 / CD137 co-positive cells among CD4-positive viable cells under PBS-added conditions to calculate the Stimulation Index. A cutoff value was set at the point where 95% of the donors' Stimulation Index for the low immunogenic antibody Bevacizumab (Chugai Pharmaceutical Co., Ltd., approval number #21900AMX00910) from 20 donors were included. Donors showing a Stimulation Index above the cutoff value were considered to have responded to the protein.

[0115] Among proteins with known immunogenicity, the low-immunogenic Trastuzumab (Chugai Pharmaceutical Co., Ltd., approval number #21600AMY00065) showed a 0% donor response rate, the high-immunogenic Bococizumab (KEGG: D10621) showed a 90% donor response rate, and the positive control keyhole limpet hemocyanin (KLH) (Merck, Inc., #H7017-20mg) showed a 100% donor response rate. On the other hand, sialylated IdeZ variant A-human Fc variant 77 showed a low donor response rate of 15%, sialylated IdeZ variant A-human Fc variant 93 showed a low response rate of 10%, and sialylated IdeZ variant A-human Fc variant 97 showed a low response rate of 15% (Figure 7). Therefore, all of the IdeZ-Fc fusion proteins we evaluated showed low immunogenicity in the human immune system.

[0116] <Example 7: Pharmacological evaluation of IdeZ-Fc fusion protein in lupus nephritis model mice> By administering Poly(I:C), a ligand for Toll-like receptor 3, to NZBWF1 / J mice (Jackson Laboratory), the onset of lupus nephritis-like pathology can be accelerated. In this state of lupus nephritis-like pathology, IdeZ-Fc fusion protein was administered, and the immune complexes deposited in the kidneys were measured, and the kidneys were histopathologically evaluated. The IdeZ-Fc fusion protein used was sialyzed IdeZ variant A-human Fc variant 93 prepared in Example 3.

[0117] 23-week-old NZBWF1 / J mice were administered 6.25 μg × average mouse body weight (g) of Poly(I:C) (InvivoGen, trrl-picw) three times a week for four weeks. Afterward, mice with urinary protein levels (creatinine-corrected) between 2 and 55 during the next three weeks were included in the study. Based on urinary protein levels, the mice were divided into groups and intravenously administered ideZ-Fc fusion protein at a dose of 0.6 mg / kg. Kidney samples were collected one and three days after administration. For a comparative example, mice administered a different medium (PBS) instead of ideZ-Fc fusion protein were used in the same manner.

[0118] Example 7-1: Measurement of immune complexes in the kidney. Frozen sections were prepared from frozen kidney blocks and triple-stained with IgG2a, C1q, and CD31 antibodies. After staining, image data of the entire stained specimen was acquired using a virtual slide device (3DHISTECH, Panoramamic 250 FLASH III), and image analysis was performed using a HALO image analysis system (Indica Labs). In the analysis, all glomerular regions in the specimen were selected, and the area of ​​regions showing positivity for both IgG2a and C1q staining was measured. The ratio to the total glomerular region area (IgG2a + C1q positivity rate) was calculated. The experimental results are shown as the mean ± standard error for each group. Furthermore, to determine the statistical significance of the difference between the sialyzed IdeZ variant A-human Fc variant 93 administration group and the vehicle administration group, an unpaired Student's t-test was used, and a p-value of less than 0.05 was considered statistically significant. The above tests were performed using Graphpad Prism (version 10.1.2).

[0119] In lupus nephritis model mice, immune complexes consisting of IgG and complement such as C1q are deposited in the kidneys, leading to the development of the disease. The average IgG2a+C1q positivity rate, measured as an indicator of immune complexes, was 20.17% one day after administration and 21.56% three days after administration in the vehicle administration group, while it was 3.51% and 6.86%, respectively, in the sialyzed IdeZ variant A-human Fc variant 93 administration group, which were significantly lower than the vehicle administration group (Figure 8). This indicates that the IdeZ-Fc fusion protein according to this embodiment has the effect of degrading and removing immune complexes deposited in the kidneys of lupus nephritis model mice.

[0120] Example 7-2: Pathological tissue evaluation of the kidney. The collected kidney was fixed in 10% formalin, dissected, embedded, and sectioned, and then stained with silver methenamine periodate (PAM) to prepare pathological tissue specimens. The prepared specimens were classified into types I to VI according to the morphological classification of lupus nephritis (revised in 2018) by the International Society of Nephrology / Renal Pathology Society (ISN / RPS).

[0121] In the group administered the medium, 3 out of 6 individuals showed type VI lesions with more than 90% glomerular sclerosis one day after administration, and all individuals showed type VI lesions with more than 90% glomerular sclerosis three days after administration. On the other hand, in the group administered sialyzed IdeZ variant A-human Fc variant 93, only 1 out of 6 individuals progressed to type VI both one day and three days after administration (Figure 9). From these results, it became clear that the IdeZ-Fc fusion protein according to the embodiment suppresses the progression of the disease by degrading immune complexes deposited in the kidneys. It showed remarkably rapid therapeutic effects, suppressing nephrosclerosis from the first day of administration, reducing the proportion of severe type III lesions and increasing the proportion of mild type II lesions by the third day after administration.

[0122] The results of this embodiment demonstrate that the IdeZ-Fc fusion protein according to this embodiment contributes to improving the disease state by degrading pathogenic IgG and immune complexes. This action is applicable to diseases in which pathogenic IgG or immune complexes are similarly involved. For example, it can be commonly applied to a diverse group of diseases in which pathogenic IgG or immune complexes cause tissue damage in organs such as the kidneys, such as IgG-mediated rapidly progressive glomerulonephritis.

[0123] The amino acid sequence and DNA sequence of the IdeZ-Fc fusion protein according to the embodiment are shown in the table below.

[0124] While several embodiments of the present invention have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These novel embodiments can be carried out in a variety of other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims of the invention and its equivalents.

[0125] The IdeZ-Fc fusion protein of the present invention possesses desirable properties such as suppressed immunogenicity and is useful as a pretreatment for gene therapy using AAV, as well as for the prevention or treatment of transplant rejection, autoimmune diseases, and diseases involving pathogenic IgG.

Claims

1. A fusion protein comprising a conjugate of an IdeZ variant and an Fc region variant, wherein the fusion protein comprises: (a) a sequence that is 90% or more identical to the amino acid sequence from position 1 to 315 of the amino acid sequence represented by Sequence ID No. 11; and (b) a sequence that is 90% or more identical to the amino acid sequence from position 316 to 541 of the amino acid sequence represented by Sequence ID No.

11.

2. The fusion protein according to claim 1, wherein the IdeZ variant comprises the following amino acid modification (a'), and the Fc region variant comprises the following amino acid modifications (b') and (b''): (a') The amino acids at positions 120, 171, 269, and 298 of the amino acid sequence represented by Sequence ID No. 1 are replaced with glutamic acid, glycine, arginine, and aspartic acid, respectively; (b') The amino acids at positions 234, 235, 236, 237, and 253 of the Fc region are replaced with alanine, glutamic acid, glutamic acid, valine, and alanine, respectively; (b'') The amino acid at position 293 or 294 of the Fc region is deleted (wherein the amino acid residue numbers of the Fc region are amino acid positions according to the EU index).

3. The fusion protein according to claim 2, wherein the Fc region variant further comprises the following amino acid modifications: (c) the amino acid at position 265 of the Fc region is substituted with alanine; (d) the amino acid at position 331 of the Fc region is substituted with glycine.

4. The fusion protein according to claim 2 or 3, wherein the Fc region variant further comprises the following amino acid modifications: (e) one or more amino acids at positions 361, 371, 375, 418 and 438 of the Fc region are substituted with aspartic acid or glutamic acid; (f) one or more amino acids at positions 255, 274, 288, 326, 370 and 416 of the Fc region are substituted with a neutral amino acid.

5. The fusion protein according to claim 4, wherein the Fc region variant comprises the following amino acid modifications: (g) the amino acid at position 361 of the Fc region is substituted with aspartic acid, the amino acid at position 370 is substituted with threonine, the amino acid at position 371 is substituted with aspartic acid, the amino acid at position 375 is substituted with glutamic acid, the amino acid at position 416 is substituted with asparagine, the amino acid at position 418 is substituted with glutamic acid, and the amino acid at position 438 is substituted with glutamic acid; (h) the amino acid at position 255 of the Fc region is substituted with leucine, the amino acid at position 274 is substituted with glutamine, the amino acid at position 288 is substituted with glutamine, the amino acid at position 326 is substituted with alanine, the amino acid at position 370 is substituted with threonine, and the amino acid at position 416 is substituted with asparagine.

6. The fusion protein according to any one of claims 1 to 5, comprising a conjugate formed by the IdeZ variant being bound to the N-terminus of the Fc region variant (first Fc region variant), and the Fc region variant (second Fc region variant) to which the IdeZ variant is not bound.

7. The fusion protein according to claim 6, wherein the first Fc region variant and the second Fc region variant further comprise a knobs-into-holes mutation, the knobs-into-holes mutation being either (1) or (2) below: (1) The first Fc region is characterized by the substitution of tryptophan at the position corresponding to position 366 of the Fc region; the second Fc region is characterized by the substitution of serine at the position corresponding to position 366 of the Fc region, the substitution of alanine at the position corresponding to position 368, and the substitution of valine at the position corresponding to position 407; (2) The first Fc region is characterized by the substitution of serine at the position corresponding to position 366 of the Fc region, the substitution of alanine at the position corresponding to position 368, and the substitution of valine at the position corresponding to position 407; In the second Fc region, the amino acid at position 366 of the Fc region is substituted with tryptophan.

8. The fusion protein according to claim 7, comprising the amino acid sequence represented by SEQ ID NO: 11 and the amino acid sequence represented by SEQ ID NO:

12.

9. The fusion protein according to claim 7, comprising the amino acid sequence represented by SEQ ID NO: 13 and the amino acid sequence represented by SEQ ID NO:

14.

10. A polynucleotide encoding the fusion protein according to any one of claims 1 to 9.

11. An expression vector comprising the polynucleotide described in claim 10.

12. Host cells transformed with the expression vector described in claim 11.

13. A method for producing a fusion protein, comprising the step of culturing the host cells described in claim 12.

14. The method according to claim 13, further comprising the step of adding or expressing β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1).

15. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15, for use in the prevention or treatment of a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

17. The pharmaceutical composition according to claim 16, wherein the disease is IgG-mediated rapidly progressive glomerulonephritis.

18. The pharmaceutical composition according to claim 17, wherein IgG-mediated rapidly progressive glomerulonephritis is selected from the group consisting of ANCA-associated vasculitis, immune complex type III hypersensitivity disorder, and type II hypersensitivity disorder.

19. The pharmaceutical composition according to claim 18, wherein the immune complex type III hypersensitivity disorder is selected from the group consisting of lupus nephritis, IgA nephropathy, IgA vasculitis, and cryoglobulinemia-induced glomerulonephritis.

20. The pharmaceutical composition according to claim 18, wherein the type II hypersensitivity disorder is anti-GBM antibody disease (Goodpasture syndrome).

21. A method for preventing or treating a disease selected from autoimmune diseases and diseases involving pathogenic IgG, comprising administering a therapeutically effective amount of the fusion protein described in any one of claims 1 to 9 to a target.

22. A fusion protein according to any one of claims 1 to 9, for use in the prevention or treatment of a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

23. Use of a fusion protein according to any one of claims 1 to 9 in the manufacture of a pharmaceutical composition for the prevention or treatment of a disease selected from autoimmune diseases and diseases involving pathogenic IgG.

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