Homogeneous mutaine of human IL-27α subunit

Abstract

The present invention relates to muteins of the α-subunit of human interleukin-27 and muteins of heterodimeric human interleukin-27 according to the present invention. The present invention further relates to nucleic acid molecules comprising a nucleotide sequence encoding a mutein of the α-subunit of human interleukin-27 or a mutein of heterodimeric human interleukin-27. The present invention further relates to host cells containing nucleic acid molecules comprising a nucleotide sequence encoding a mutein of the α-subunit of human interleukin-27 or a mutein of heterodimeric human interleukin-27. The present invention also relates to immunomodulators comprising a mutein of the α-subunit of human interleukin-27 or a mutein of heterodimeric human interleukin-27, their respective uses, and methods for producing said muteins. TIFF2023501515000004.tif115131

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to EP Patent Application No. 19 208 453.1, filed on 12 November 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Field of Invention The present invention relates to a mutain (mutant protein) of the α-subunit of human interleukin 27. Furthermore, the present invention relates to a mutain of heterodimer human interleukin 27, wherein the α-subunit is the mutain of the α-subunit of human interleukin 27 described herein. The present invention also relates to a mutain of the α-subunit of human interleukin 27 that contains at least 60% sequence identity with respect to the α-subunit of human interleukin 27. The present invention also relates to a mutain of heterodimer human interleukin 27, wherein the α-subunit contains at least 60% sequence identity with respect to the α-subunit of human interleukin 27. Interleukin 27 is composed of an α-subunit p28 and a β-subunit EBI3. The present invention further relates to a nucleic acid molecule comprising a nucleotide sequence encoding a mutain of the α-subunit of human interleukin 27, or a mutain of heterodimer human interleukin 27, wherein the α-subunit is the mutain of the α-subunit of human interleukin 27 described herein. The present invention further relates to a host cell containing a nucleic acid molecule comprising a nucleotide sequence encoding the α-subunit mutein of human interleukin-27 or the mutein of heterodimer human interleukin-27, wherein the α-subunit is the mutein of human interleukin-27 as described herein. The present invention also relates to an immunomodulator comprising the α-subunit mutein of human interleukin-27 or the mutein of heterodimer human interleukin-27, wherein the α-subunit is the mutein of human interleukin-27 as described herein.The present invention further relates to the use of the mutaine of the α-subunit of human interleukin-27 or the mutaine of heterodimer human interleukin-27 as described herein in the manufacture of a pharmaceutical; a method for treating an interleukin-27-mediated disease, comprising the step of administering a composition containing the mutaine of the present invention to a mammal in need thereof; and a method for producing the mutaine of the α-subunit of human interleukin-27 or the mutaine of heterodimer human interleukin-27, wherein the α-subunit is a mutaine having at least 60% sequence identity to the mutaine of the α-subunit of human interleukin-27 or the mutaine of the α-subunit of human interleukin-27 as described herein. [Background technology]

[0003] Background of the Invention A central concept of the human immune system is the balanced regulation of pro-inflammatory and anti-inflammatory responses. This allows for the rapid elimination of threats while protecting the host. Interleukins (ILs) are structurally diverse, small, secreted proteins that mediate pro-inflammatory and anti-inflammatory responses to maintain this balance. Among them is the interleukin-12 (IL-12) family, which includes four established members (IL-12, IL-23, IL-27, and IL-35). 1 (See Figure 1) This is representative of the concept of balanced immunomodulation. IL-12 and IL-23 are primarily pro-inflammatory cytokines, while IL-35 plays an immunosuppressive role. 1,2 IL-27 is functionally diverse, possessing both immunomodulatory pro-inflammatory and anti-inflammatory functions, and acts on various types of T cells. 3 It promotes pro-inflammatory responses and can act synergistically with IL-12 to induce interferon-gamma (IFNγ) production by naive T cells and natural killer (NK) cells. 4 However, IL-27 induces IL-10 as an anti-inflammatory cytokine. 5-7or T cells, a cell type that has attracted attention for its role in a variety of immune-mediated human diseases H 17 cells 8,9 By suppressing the response of 10 the immune response can also be weakened.

[0004] Interleukin-12 (IL-12) cytokines regulate T cell function and development and have a decisive influence on pro-inflammatory and anti-inflammatory responses. Each family member is a heterodimer, and even their isolated subunits also regulate the immune response 11,12,24 . This not only gives the IL-12 family unparalleled regulatory capabilities, but also places high demands on their biosynthesis.

[0005] However, the functions shared by the IL-12 family do not stop at this central role in linking innate and adaptive immunity. All IL-12 cytokines exhibit structural features that distinguish this family from other interleukins: each member of the IL-12 family consists of an α subunit of a four-helix bundle (IL-12α / p35, IL-23α / p19, and IL-27α / p28, respectively) and a β subunit (IL-12β / p40) consisting of two fibronectin (Fn) domains (EBI3) or two Fn domains and one immunoglobulin (Ig) domain, which is a heterodimer 11,12 . Notably, despite their different roles in regulating the immune response, all heterodimeric IL-12 family members are composed of only these three α subunits and two β subunits, and there may be other members 13IL-12β is shared by the pro-inflammatory family members IL-12 and IL-23, while EBI3 is shared by the immunomodulatory / anti-inflammatory members IL-27 and IL-35. This raises important questions about the structural features that mediate assembly specificity and promiscuity within this family. It also places further demands on the mechanisms of protein folding and quality control in the endoplasmic reticulum (ER) where all IL-12 family members are assembled before secretion. Insights into the folding and assembly of IL-12 family cytokines are currently very limited. All human α subunits have been shown to be retained intracellularly on their own and depend on assembly with their cognitive β subunit for secretion. 4,14,15 In the case of IL-12, a founding member of this family, assembly-induced folding of the IL-12α subunit by IL-12β underlies these processes. 16 However, in other cases, the fundamental mechanism remains unclear.

[0006] In relation to the present invention, the inventors focused on IL-27, a family member that is not structurally characterized but is highly functionally diverse (see Figure 2A). In this regard, in contrast to its human orthologue, the mouse IL-27α subunit (p28) can be secreted independently without its β subunit, but it has already been found that the secretion of human IL-27α is strictly dependent on EBI3. 4 (See Figures 2B and C). The differing secretory behavior of human and mouse IL-27α may stem from the fact that mouse IL-27α can form disulfide crosslinks to stabilize its protein, while human IL-27α cannot, and therefore requires EBI3 for secretion. When a second cysteine ​​is mutagenically inserted into human IL-27α, human IL-27α also forms disulfide bonds and becomes autonomously secreted (see Figure 2D). Furthermore, immunosuppressive / modulatory effects have been described for mouse IL-27α, also known as IL-30.17,20 Furthermore, it has been shown that human IL-27α, which is capable of autonomous secretion, also possesses immunomodulatory activity. However, its activity in the STAT phosphorylation assay is reduced to 1 / 700th of that of heterodimer IL-27. 21 .

[0007] Therefore, there is a need in the art to improve the immunosuppressive / modulatory activity of human IL-27, specifically the human α subunit or heterodimer human IL-27. Thus, the underlying technical challenge of this application is to address this need. [Overview of the project]

[0008] The inventors have developed mutains of the α-subunit of human interleukin-27 modified by specific point mutations or specific deletions; these mutains are more homogeneous while maintaining the functionality of the protein, and in some cases even exhibit improved activity compared to the heterogeneous human α-subunit of human interleukin-27 without mutations. Surprisingly, the inventors have found that targeted and rational modifications of the α-subunit of human IL-27, by single point mutations, and even specific deletions, result in prevention of O-glycosylation of the protein, which is crucial for its homogeneity, while maintaining its functionality, and in some cases even result in enhanced activity compared to the heterogeneous human α-subunit of human interleukin-27 without mutations.

[0009] Accordingly, in the first aspect, the present invention relates to a mutaine of the α-subunit of human interleukin 27, wherein at least one amino acid residue selected from the group consisting of sequence positions 187, 238, and 240 of the α-subunit of human interleukin 27 is mutated. The present invention also provides a mutaine of the α-subunit of human interleukin 27, which has at least 60% sequence identity with respect to the α-subunit of human interleukin 27 as defined herein.

[0010] In yet another aspect, the present invention may also provide a mutant of the α subunit of human interleukin-27 in which the residue at amino acid position 234 is mutated. The present invention may also include a mutant of the α subunit of human interleukin-27 in which the residue at amino acid position 238 is mutated. Preferably, the present invention encompasses a mutant of the α subunit of human interleukin-27 in which the residues at amino acid positions 234 and 238 are mutated.

[0011] In a second aspect, the present invention provides a mutant of human interleukin-27 comprising an α subunit p28 and a β subunit EBI3, wherein the α subunit is a mutant of the α subunit of human interleukin-27 as described herein. In a further embodiment thereof, the α subunit is a mutant comprising at least 60% sequence identity to the α subunit of human interleukin-27 as described herein.

[0012] In a third aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a mutant of human interleukin-27 according to the present invention or a mutant of the α subunit of human interleukin-27. In a further embodiment thereof, the nucleic acid molecule comprises a nucleotide sequence encoding a mutant of the α subunit of human interleukin-27 comprising at least 60% sequence identity to the α subunit of human interleukin-27.

[0013] In a fourth aspect, the present invention also provides a host cell comprising a nucleic acid molecule according to the present invention.

[0014] In a fifth aspect, the present invention provides an immunomodulatory agent comprising a mutant according to the present invention.

[0015] In a sixth aspect, the present invention provides the use of a mutant according to the present invention in the manufacture of a medicament for treating infectious diseases, autoimmune diseases, cancer, transplantation-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma in mammals.

[0016] In related aspects, the invention provides muteins according to the invention for use in therapy. Further, the invention provides muteins according to the invention for use in the treatment of infectious diseases, autoimmune diseases, cancer, transplantation-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma.

[0017] The invention also provides a method of treating an interleukin-27-mediated disease in a mammal, preferably an infectious disease, an autoimmune disease, cancer, a transplantation-related disease such as graft-versus-host disease, a chronic inflammatory disease such as chronic inflammatory bowel disease, an acute inflammatory disease, sepsis, septic shock, diabetes, or asthma, the method comprising administering to a mammal in need thereof a composition comprising a mutein as described herein.

[0018] Furthermore, the invention provides a method of producing a mutein as described herein, introducing into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide, or a polypeptide comprising at least 60% sequence identity to the human interleukin-27α subunit polypeptide, a nucleotide sequence that mutates at least one amino acid residue selected from the group consisting of amino acid residues 187, 238, and 240 of a polypeptide comprising at least 60% sequence identity to human interleukin-27 or the α subunit of human interleukin-27 or the human interleukin-27α subunit polypeptide; and introducing the resulting nucleic acid molecule for expression into a suitable host cell or a suitable cell extract or cell lysate comprising.

[0019] [Invention 1001] A mutain of the α-subunit of human interleukin-27, wherein at least one amino acid residue selected from the group consisting of sequence positions 238 and 240 of the α-subunit of human interleukin-27 is mutated. [Invention 1002] The mutain of the present invention 1001, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is mutated. [Invention 1003] A mutain according to the present invention 1001 or 1002, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is mutated. [Invention 1004] A mutain according to any of the invention 1001 to 1003, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are mutated. [Invention 1005] A mutain according to the present invention, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 3). [Invention 1006] A mutain according to the present invention, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 4). [Invention 1007] A mutain according to the present invention, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are substituted with alanine (SEQ ID NO: 8). [Invention 1008] Any of the mutaines of the present invention further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243. [Invention 1009] Any of the mutaines of the present invention, wherein at least one residue at amino acid positions 234-238, at least one residue at amino acid positions 234-239, at least one residue at amino acid positions 234-240, at least one residue at amino acid positions 234-241, at least one residue at amino acid positions 234-242, or at least one residue at amino acid positions 234-243 is mutated. [Invention 1010] A mutaine according to the present invention, wherein the amino acid residues at positions 229-243 are mutated (SEQ ID NO: 11). [Invention 1011] A mutain according to the present invention, comprising at least 60% sequence identity with respect to the α-subunit of human interleukin 27. [Invention 1012] Any of the mutaines of the present invention, further comprising one or more salt crosslinks. [Invention 1013] Any of the mutaines of the present invention, further comprising one or more disulfide crosslinks. [Invention 1014] A mutaine of human interleukin 27, comprising an α-subunit p28 and a β-subunit Ebi3, wherein the α-subunit is a mutaine of the α-subunit of human interleukin 27 according to any of the present inventions 1001 to 1013. [Invention 1015] The mutain of the present invention 1014, wherein at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 of the α-subunit of human interleukin 27 is mutated. [Invention 1016] A mutain according to the present invention 1014 or 1015, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is mutated. [Invention 1017] A mutain according to any of the invention 1014 to 1016, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is mutated. [Invention 1018] A mutain according to any of the present invention 1014 to 1017, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are mutated. [Invention 1019] A mutain according to any of the inventions 1014 to 1018, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 3). [Invention 1020] A mutein according to any of the invention 1014-1019, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 4). [Invention 1021] A mutain according to any of the inventions 1014 to 1020, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are substituted with alanine (SEQ ID NO: 8). [Invention 1022] A mutaine according to any of the inventions 1014 to 1021, further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243. [Invention 1023] A mutaine according to any of the invention 1014 to 1022, wherein at least one residue at amino acid positions 234-238, at least one residue at amino acid positions 234-239, at least one residue at amino acid positions 234-240, at least one residue at amino acid positions 234-241, at least one residue at amino acid positions 234-242, or at least one residue at amino acid positions 234-243 is mutated. [Invention 1024] A mutain according to any of the present invention 1014 to 1023, wherein the residue at amino acid positions 229 to 243 is mutated. [Invention 1025] A mutain according to any one of the present invention 1014 to 1024, wherein the α-subunit has at least 60% sequence identity with respect to the α-subunit of human interleukin 27. [Invention 1026] A mutaine according to any one of invention 1014 to 1025, further comprising one or more salt crosslinks. [Invention 1027] A mutaine according to any one of the present invention 1014 to 1026, further comprising one or more disulfide crosslinks. [Invention 1028] A nucleic acid molecule comprising a nucleotide sequence encoding the mutaine of human interleukin-27 or the mutaine of the α-subunit of human interleukin-27 according to any of invention 1001 to 1027. [Invention 1029] The nucleic acid molecule of the present invention 1028, wherein the nucleotide sequence encodes the mutain of the α-subunit of human interleukin 27, and the mutain has at least 60% sequence identity with respect to the α-subunit of human interleukin 27. [Invention 1030] A nucleic acid molecule of the present invention 1028 or 1029, comprising a nucleotide sequence encoding mutain with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 11. [Invention 1031] A nucleic acid molecule according to any of the present invention 1028 to 1030, which is functionally linked to a regulatory sequence that enables the expression of the nucleic acid molecule. [Invention 1032] A nucleic acid molecule according to the present invention 1031, wherein the regulatory sequence includes a promoter sequence. [Invention 1033] A nucleic acid molecule contained in the vector, which is one of the nucleic acid molecules described in items 1028 to 1032 of the present invention. [Invention 1034] A host cell containing any nucleic acid molecule according to invention 1028 to 1033. [Invention 1035] An immunomodulator comprising any of the mutaine described in invention 1001 to 1027. [Invention 1036] A mutaine according to any of invention 1001 to 1027 for use in treatment. [Invention 1037] A mutaine according to any one of the present invention 1001 to 1027 for use in the treatment of infectious diseases, autoimmune diseases, multiple sclerosis, cancer, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma in mammals. [Invention 1038] A method for producing any of the mutaines described in invention 1001 to 1027, (a) The step of introducing a nucleotide sequence into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide or human interleukin-27α subunit polypeptide, which mutates at least one amino acid residue selected from the group consisting of sequence positions 238 and 240 of a polypeptide having at least 60% sequence identity with human interleukin-27 or human interleukin-27α subunit polypeptide; and (b) The process of introducing the nucleic acid molecule for expression obtained in step (a) or (b) into a suitable host cell or a suitable cell extract or cell lysate. Methods that include... [Invention 1039] The method of the present invention 1038, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with respect to human interleukin-27 polypeptide, human interleukin-27α subunit polypeptide, or human interleukin-27α subunit polypeptide, which mutates the amino acid residue at sequence position 238 of the α subunit of human interleukin-27. [Invention 1040] The method of the present invention 1038 or 1039, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with respect to human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide or human interleukin-27α subunit polypeptide, which mutates the amino acid residue at sequence position 240 of the α subunit of human interleukin-27. [Invention 1041] A method according to any one of the present invention 1038 to 1040, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide or a polypeptide having at least 60% sequence identity with respect to the human interleukin-27α subunit polypeptide, which mutates the amino acid residues at sequence positions 238 and 240 of the α subunit of human interleukin-27. [Invention 1042] A method according to any one of the present invention 1038 to 1041, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide or a polypeptide having at least 60% sequence identity with respect to the human interleukin-27α subunit polypeptide, causing the amino acid residue at sequence position 238 of the α subunit of human interleukin-27 to be mutated to alanine. [Invention 1043] A method according to any one of the present invention 1038 to 1042, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide or a polypeptide having at least 60% sequence identity with respect to the human interleukin-27α subunit polypeptide, causing the amino acid residue at sequence position 240 of the α subunit of human interleukin-27 to be mutated to alanine. [Invention 1044] A method according to any one of the present invention 1038 to 1043, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide or a polypeptide having at least 60% sequence identity with respect to the human interleukin-27α subunit polypeptide, causing the amino acid residues at sequence positions 238 and 240 of the α subunit of human interleukin-27 to be mutated to alanine. [Invention 1045] A method according to any one of the 1038 to 1044 of the present invention, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with respect to human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide or human interleukin-27α subunit polypeptide, which is further mutated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 times at one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243. [Invention 1046] A method according to any one of the present invention 1038 to 1045, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with respect to human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide or human interleukin-27α subunit polypeptide, which mutates at least residues at amino acid positions 234 to 238, at least residues at amino acid positions 234 to 239, at least residues at amino acid positions 234 to 240, at least residues at amino acid positions 234 to 241, at least residues at amino acid positions 234 to 242, or at least residues at amino acid positions 234 to 243. [Invention 1047] In step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide having at least 60% sequence identity with human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide or human interleukin-27α subunit polypeptide, which mutates the residues at amino acid positions 229-243 (SEQ ID NO: 11), any method according to any one of the present invention 1038-1046. [Brief explanation of the drawing]

[0020] The accompanying drawings are included for further understanding of the embodiments incorporated herein and constituting part of this specification. These drawings illustrate embodiments and, together with the detailed description, are useful in illustrating the principles of those embodiments. Many of the advantages of other embodiments and intended embodiments will be readily apparent, as they will be better understood by referring to the detailed description. The elements of the drawings do not necessarily adhere to relative scales with respect to one another. [Figure 1] Figure 1 shows schematic diagrams of the IL-12 family members, their structures, and immunological activities. [Figure 2] Figure 2(a) shows a schematic diagram of the IL-27 structure. Figure 2(b) shows the secretory capacity of the IL-27 subunit in humans, and Figure 2(c) shows the secretory capacity of the IL-27 subunit in mice. Figure 2(d) shows a genetically modified, autonomously secreted human IL-27αL162C subunit (SEQ ID NO: 1). [Figure 3]Figure 3 shows an analysis of the glycosylation status of human IL-27α. Secreted IL-27α shows two species resulting from glycolysis (in (a) and (b)). Figure 3(a) shows that secreted V5-tagged IL-27α was treated with N-glycosidase (E: EndoH and P: PNGaseF) and O-glycosidase (O). From this analysis, it can be seen that IL-27α is not N-glycosylated but is O-glycosylated. The protein moves more rapidly after cleavage of sugar residues. Figure 3(a) shows that untagged IL-27α is also O-glycosylated. [Figure 4] Figure 4(a) shows the analysis of the N-glycosylation status of mouse IL-27α (SEQ ID NO: 10). Secreted mouse IL-27α was treated with N-glycosidase PNGaseF (+). From this analysis, it can be seen that IL-27α is N-glycosylated because the protein moves more rapidly after cleavage of sugar residues by PNGase. Figure 4(b) shows the O-glycosidase analysis, which shows that mouse IL-27α is not O-glycosylated. [Figure 5] Figure 5 shows a sequence comparison of human IL-27αL162C (O-glycosylated form) (SEQ ID NO: 1) and mouse IL-27α (N-glycosylated form) (SEQ ID NO: 10). Threonine and serine, which are present in humans but not in mice, are printed in italics. Threonine and serine exposed in the human protein structure prediction are further marked with arrows. [Figure 6] Figure 6 shows a structural model of human IL-27αL162C (SEQ ID NO: 1). Threonine and serine present in humans but not in mice are shown as Ser110, Ser202, Ser187, Thr238, and Ser240. Threonine and serine exposed to solvents are further marked with arrows. [Figure 7]Figure 7 shows mutations in the potential O-glycosylation sites of human IL-27αL162C. The potentially O-glycosylable threonine and serine were mutated to alanine. The mutant IL-27αL162C,T238A,S240A (SEQ ID NO: 8) exhibits the only protein species that migrates faster than the other mutants due to its lower molecular weight. [Figure 8] Figure 8 shows that IL-27αL162C,T238A,S240A (SEQ ID NO: 8) is not O-glycosylated. Although the secreted IL-27αL162C,T238A,S240A (SEQ ID NO: 8) is treated with O-glycosidase, it does not migrate faster than the negative control after treatment. This indicates that IL-27αL162C,T238A,S240A (SEQ ID NO: 8) is not O-glycosylated. [Figure 9] Figure 9 shows the expression of IL-27αL162C,T238A,S240A (SEQ ID NO: 8) in mammalian cells at a concentration of 4.1 μg / mL. [Figure 10] Figure 10 shows that non-glycosylated IL-27αL162C,T238A,S240A (SEQ ID NO: 8) is 8.5 times more active than O-glycosylated IL-27αL162C (SEQ ID NO: 1). BL-2 cells expressing the IL-27 receptor were incubated with 1000 ng / mL of IL-27αL162C (SEQ ID NO: 1) or IL-27αL162C,T238A,S240A (SEQ ID NO: 8) for 60 minutes, and STAT1 activation was measured by immunoblotting against phosphorylated STAT1. [Figure 11] Figure 11(a) shows a structural model of hIL-27αL162C (SEQ ID NO: 1), where Leu234, Thr238, and Ser240 are indicated. Figure 11(b) shows the amino acid sequence of hIL-27αL162C (SEQ ID NO: 1). Leu234, Thr238, and Ser240 are marked with arrows. This structural model does not include the ER signal sequence, which is underlined in the amino acid sequence. [Figure 12] Figure 12 shows various IL-27α pairs (without cysteine ​​substitution at leucine 162 (L162C)) tested for functionality on BL-2 cells. These consisted of WT pairs (O- / N-glycosylated), pairs where only hIL-27α lacked O-glycosylation, pairs where only hEBI3 lacked N-glycosylation, or pairs where both subunits lacked O- / N-glycosylation (hIL-27αT238A,S240A (=ΔO) and hEBI3N55QN105Q (=ΔN)). These data indicate that O-glycosylation is not required for the function of heterodimer IL-27. [Figure 13] Figure 13 shows that IL-27α (including leucine 162 substitution with cysteine ​​(L162C)) was cleaved after Gly228 to delete its C-terminal O-glycosylation site. This indicates that the C-terminus is not important for the function of IL-27α(L162C). [Modes for carrying out the invention]

[0021] Detailed explanation The following language and description of certain preferred embodiments of the invention are provided for further understanding of the principles of the invention. However, it will be understood that the invention is not intended to be limited, and further modifications, modifications, and applications of the principles of the invention are also included.

[0022] The present invention is directed to a mutain of the α-subunit of human interleukin 27 (IL-27), wherein at least one amino acid residue selected from the group consisting of sequence positions 187, 238, and 240 of the α-subunit of human interleukin 27 is mutated.

[0023] Secreted proteins such as interleukins are often glycosylated. This modification involves N-glycosylation of asparagine residues. 22 , or O-glycosylation, often more heterogeneous, on serine and threonine residues. 23This includes [specific compounds]. However, human IL-27α is not N-glycosylated but is O-glycosylated (see Figure 3). On the other hand, mouse IL-27α is N-glycosylated but not O-glycosylated (see Figure 4).

[0024] IL-27α is a secreted protein that folds in the endoplasmic reticulum (ER) and is O-glycosylated in the Golgi apparatus on its way to the extracellular space. The formation of the three-dimensional protein structure usually takes place in the ER before the protein reaches the Golgi apparatus. Therefore, O-glycosylation in the Golgi apparatus is only possible at surface-exposed serine and threonine residues. By aligning the sequences and structures of mouse and human interleukin-27, we identified surface-exposed serine and threonine residues involved in O-glycosylation in the α subunit of human interleukin-27 (see Figures 5 and 6).

[0025] According to the present invention, the inventors subsequently replaced the detected serine and threonine residues with alanine. By replacing the serine and threonine residues exposed on the surface of the protein with alanine residues, O-glycosylation of the protein in the Golgi apparatus is prevented. In particular, this analysis revealed that mutating specific amino acid residues selected from the group consisting of sequence positions 187, 238, and 240 of the α-subunit of human IL-27 prevents O-glycosylation at at least one of the mutated amino acid residues, resulting in a more homogeneous protein while maintaining the full functionality of the protein. The same effect is achieved when specific deletions of the α-subunit of human IL-27 are made, as described elsewhere in this specification.

[0026] Proteins with secretory potential (IL-27α L162CComparison with the mobility of the mutant on an SDS-PAGE gel shows that replacing threonine 238 (Thr 238) and serine 240 (Ser 240) with alanine residues results in a single species that moves faster on the immunoblot (see Figure 7). This indicates the presence of a lower molecular weight due to a lack of O-glycosylation. Treatment with O-glycosidase shows that the mutant in which Thr238 and Ser240 are replaced with alanine lacks O-glycosylation (see Figure 8).

[0027] Since O-glycans are known to be highly heterogeneous, the glycosylated protein according to the present invention is highly homogeneous. This is particularly beneficial with respect to medical usability and the resulting biopharmaceutical requirements. This new homogeneous O-glycosylation-free protein was subsequently produced in mammalian cell lines (see Figure 9) and its activity was tested in an immunoassay. This homogeneous O-glycosylation-free human interleukin-27 α-subunit exhibited approximately 10 times higher activity than the unmutated heterogeneous human interleukin-27 α-subunit in the aforementioned immunoassay (see Figure 10). This data further demonstrates that O-glycosylation in the human interleukin-27 α-subunit without cysteine ​​substitution at leucine 162 (L162C) does not affect the function of the heterodimer IL-27. Thus, when at least one amino acid residue selected from the group consisting of sequence positions 238 and 240 of the α subunit of human interleukin-27 is mutated, a more homogeneous IL-27 is obtained while maintaining full functionality (see Figure 12). Furthermore, it has been demonstrated that even a complete deletion of the C-terminus of human IL-27α does not impair its functionality and simultaneously results in a homogeneous species (see Figure 13).

[0028] Due to its homogeneity without compromising functionality, it has potential as a novel immunomodulator, for example, in the treatment of sepsis. Its suitability as a novel immunomodulator may be further enhanced by its additionally enhanced activity compared to the wild-type currently in use. It could also lead to the availability of lower doses of human interleukin-27α as a biopharmaceutical, or heterodimerized interleukin-27 as a biopharmaceutical containing interleukin-27α, potentially making the production of such biopharmaceuticals more cost-effective. Thus, in the development of human IL-27α subunits as biopharmaceuticals, and in the development of heterodimerized IL-27 as a biopharmaceutical containing IL-27α subunits, removing the O-glycosylation site has proven beneficial in obtaining homogeneous products.

[0029] Finally, the present invention makes it possible to design an autonomously folding human IL-27α subunit that acts as a homogeneous and improved immunomodulator. In this regard, it should be noted that the terms “human interleukin 27(IL-27)α subunit” or “human interleukin 27(IL-27) alpha subunit” as used herein refer, in particular, to the polypeptide sequence SEQ ID NO: 1 deposited under UniProtKB accession number Q8NEV9. The terms “human interleukin 27(IL-27)β subunit” or “hEBI3” (SEQ ID NO: 9) as used herein refer to the polypeptide sequence deposited under UniProtKB accession number Q14213, which associates with the human IL-27α subunit to form interleukin 27, a heterodimeric cytokine that functions in immune responses. As used herein, the term “mouse interleukin-27 (IL-27) α subunit” or “mIL-27□” (SEQ ID NO: 10) refers to the polypeptide sequence deposited under genbank identifier NP 663611.1. As used herein, the term “mouse interleukin-27 (IL-27) β subunit” or “mEBI3” refers to the polypeptide sequence deposited under UniProtKB accession number O35228. As used herein, the term “human interleukin-27 (IL-27) β subunit” or “hEBI3” refers to the polypeptide sequence deposited under UniProtKB accession number Q14213 (SEQ ID NO: 12). Therefore, the term “IL-27” or “interleukin-27” refers to the heterodimer cytokine formed by the IL-27 α subunit and the IL-27 β subunit. However, the β subunit, EBI3, is not O-glycosylated but is N-glycosylated. EBI3 can be N-glycosylated at multiple sites. hEBI3 can be N-glycosylated at at least one amino acid residue selected from the group consisting of sequence positions 55 and 105, which correspond to the sequence position of SEQ ID NO: 12.If at least one of the amino acid residues at sequence positions 55 and 105, corresponding to sequence position 12 of human interleukin-27's human β-subunit EBI3, is mutated, it refers to an hEBI3 mutant lacking N-glycosylation. In this context, the mutation means replacing at least one of the amino acid residues at sequence positions 55 and 105, corresponding to sequence position 12 of human interleukin-27's β-subunit EBI3, with glutamine (Gln / Q).

[0030] As used herein, when the terms “human interleukin 27(IL-27)α subunit” or “human interleukin 27(IL-27) alpha subunit” refer to the polypeptide sequence with SEQ ID NO: 1, SEQ ID NO: 1 refers to either the wild-type (WT) human interleukin 27(IL-27)α subunit / human interleukin 27(IL-27) alpha subunit (Uniprot accession number Q8NEV9) containing leucine at position 162, or a variant of the WT human interleukin 27(IL-27)α subunit / human interleukin 27(IL-27) alpha subunit containing cysteine ​​substitution at leucine 162 (L162C). In the present invention, the latter is preferred. The variant of the present invention, i.e., the muteins of the α subunit of human interleukin 27 as defined elsewhere herein, are derived from the WT human interleukin 27(IL-27)α subunit having SEQ ID NO: 1 as defined herein. As used herein, when the terms "human interleukin-27 (IL-27) α subunit" or "human interleukin-27 (IL-27) alpha subunit" refer to a polypeptide sequence with SEQ ID NO: 1 that includes a cysteine ​​substitution (L162C) at leucine 162, the mutein is secretory.Such terms also refer to the SEQ ID NO of the α-subunit of human interleukin-27: This can refer to a polypeptide sequence containing an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with respect to amino acid sequence 1, where SEQ ID NO: The amino acid residue (leucine) at sequence position 162, corresponding to sequence position 1, is replaced with cysteine ​​(L162C) compared to the α-subunit of human interleukin 27, yet the mutein still retains secretory activity. Therefore, the mutein of human interleukin 27 that is the α-subunit of human interleukin 27, which contains α-subunit p28 and β-subunit EBI3, and whose α-subunit has the cysteine ​​substitution of leucine 162 (L162C), is secretory activity. Thus, the present disclosure, which refers to the sequence identity described above and the fact that the mutein still retains secretory activity, is also applicable to the mutein of human interleukin 27 containing α-subunit p28 and β-subunit EBI3. Therefore, if there is a disclosure of SEQ ID NO: 2 to 8 in the present invention, these specific sequences are also based on SEQ ID NO: 1, either having leucine at position 162 or including a cysteine ​​substitution (L162C) of leucine 162. In some cases, a cysteine ​​substitution (L162C) of leucine 162 may be preferred for SEQ ID NO: 2 to 8.

[0031] The terms "secrete" or "secretion" are used in the present invention in their ordinary sense, meaning the active transport of proteins from a cell (such as a human cell) to the extracellular environment. Generally, secretion occurs through intracellular secretory pathways, in which, for example, the endoplasmic reticulum and Golgi apparatus are involved in eukaryotic cells.

[0032] The mutein according to the present invention is said to have "secretory ability" or "secretory capacity" if it can completely pass through the cell membrane via the cell's secretory pathway.

[0033] In contrast, the term "non-secretory" mutain, in this invention, refers to mutain that is not naturally secreted from cells into the extracellular environment.

[0034] As disclosed above, the mutain of the human IL-27 α-subunit of the present invention can be mutated in at least one of the amino acid residues at sequence positions 187, 238, and 240 of SEQ ID NO: 1. This means that the mutain of the present invention may contain not only a single mutation at one of these sequence positions, but also mutations at two or all three of these sequence positions. In this context, the term “mutated” as used in the present invention refers to a replacement / substitution by another amino acid, such as a single point mutation as defined elsewhere in this specification, or a deletion of a specific amino acid as defined elsewhere in this specification. Any additional mutations (possible) at SEQ ID NO: 1 that do not affect the protein functionality of the human IL-27 α-subunit or heterodimer human interleukin 27, although not expressly disclosed herein, may also be included herein by the term “mutated.” Also, where applicable, any additional mutations (possible) at SEQ ID NO: 1 that do not impair secretory ability, although not expressly disclosed herein, may also be included herein by the term “mutated.” Whenever the term "mutated" is used, the term "replaced" can be used interchangeably. In some cases, the term "deleted" can be used interchangeably with the term "mutated," and vice versa.

[0035] More specifically, as used herein, the term “mutation” means that experimental conditions are selected such that an amino acid naturally present at a given sequence position in the α-subunit of human IL-27 of the present invention can be replaced with at least one amino acid that is not present at this particular position in the respective natural polypeptide sequence. Thus, the term “mutation” includes the substitution of at least one amino acid that is not present at this particular position in the respective natural polypeptide sequence. The term “mutation” also includes (additional) alterations to the length of a sequence segment due to the deletion or insertion of one or more amino acids. For example, one amino acid at a selected sequence position may be replaced with a stretch of two, three, or more random mutations, resulting in the insertion of one, two, or more amino acid residues compared to the length of each segment of the wild-type protein. The term also includes inversion, which refers to a type of mutation in which the order of amino acids in a portion of an amino acid sequence is reversed relative to the rest of the amino acid sequence. Therefore, any type and number of mutations, including substitutions, deletions, and insertions, are assumed, as long as the provided mutain retains its functionality / secretory ability, the functionality / secretory ability of the α subunit of human IL-27 or heterodimer human interleukin-27, where applicable, and / or has sequence identity that is at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or more identity with the amino acid sequence of reference WT human interleukin-27 (IL-27) α subunit SEQ ID NO.: 1.

[0036] Therefore, in the mutaine of the human IL-27 α-subunit of the present invention, mutating the amino acid residue at sequence position 187, corresponding to sequence position SEQ ID NO: 1, as described herein is included in the present invention. Furthermore, in the mutaine of the human IL-27 α-subunit of the present invention, mutating the amino acid residue at sequence position 238, corresponding to sequence position SEQ ID NO: 1, as described herein is also included in the present invention. In another embodiment included in the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residue at sequence position 240, corresponding to sequence position SEQ ID NO: 1, can also be mutated as described herein.

[0037] In yet another aspect of the present invention, the amino acid residues at sequence positions 187 and 238, corresponding to sequence position SEQ ID NO: 1, can be mutated in the human IL-27 α-subunit mutaine of the present invention as described herein. In yet another aspect of the present invention, the amino acid residues at sequence positions 187 and 240, corresponding to sequence position SEQ ID NO: 1, can be mutated in the human IL-27 α-subunit mutaine of the present invention as described herein. Finally, in yet another aspect of the present invention, the amino acid residues at sequence positions 187, 238, and 240, corresponding to sequence position SEQ ID NO: 1, can be mutated in the human IL-27 α-subunit mutaine of the present invention as described herein.

[0038] Preferably, in the mutain of the human IL-27 α-subunit of the present invention, the amino acid residues at sequence positions 238 and 240, corresponding to the sequence position of SEQ ID NO: 1, are mutated as described herein.

[0039] The aforementioned mutation may be any amino acid that cannot be O-glycosylated, i.e., any amino acid other than serine or threonine.

[0040] In one aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residue at sequence position 187, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 2). In another aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residue at sequence position 238, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 3). In yet another aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residue at sequence position 240, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 4).

[0041] In yet another aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residues at sequence positions 187 and 238, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 5). In yet another aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residues at sequence positions 187 and 240, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 6). In yet another aspect of the present invention, in the mutaine of the human IL-27 α-subunit of the present invention, the amino acid residues at sequence positions 187, 238, and 240, corresponding to sequence position SEQ ID NO: 1, can be replaced with alanine (SEQ ID NO: 7).

[0042] Most preferably, in the mutain of the human IL-27 α-subunit of the present invention, the amino acid residues at sequence positions 238 and 240, corresponding to sequence position SEQ ID NO: 1, are substituted with alanine (SEQ ID NO: 8).

[0043] In accordance with the above, the fact that the aforementioned mutations to alanine at amino acid residues 187, 238, and 240 of the α-subunit of human IL-27 can form a mutein having one, two, or three alanine residues at any of the aforementioned positions 187, 238, and 240, which correspond to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, is within the scope of the present invention.

[0044] In yet another aspect of the present invention, in the mutaine of the α-subunit of human IL-27 of the present invention, at least one amino acid residue at sequence positions 187, 238, and 240, corresponding to the sequence position of SEQ ID NO: 1, can be replaced with any amino acid that cannot be O-glycosylated, i.e., any amino acid other than serine or threonine.

[0045] Furthermore, the mutein of the human IL-27 α-subunit of the present invention may further comprise one or more disulfide crosslinks. Additionally or alternatively, the mutein of the human IL-27 α-subunit of the present invention may further comprise one or more salt crosslinks, such salt crosslinks acting as structural homologs of intrachain disulfide crosslinks formed, for example, between a native cysteine ​​residue located at sequence position 107 corresponding to sequence position SEQ ID NO: 1 of the human IL-27 α-subunit and a cysteine ​​residue introduced at position 162 corresponding to sequence position SEQ ID NO: 1 of the human IL-27 α-subunit. The salt crosslinks may, for example, be anionic carboxylates of aspartic acid or glutamic acid (RCOO - ) group and lysine-derived cationic ammonium (RNH3 + ) or arginine guanidinium (RNHC(NH2)2 + ) can be generated from these. While these are the most common, other residues with ionizable side chains, such as histidine, tyrosine, and serine, can also be involved in the formation of salt bridges.

[0046] In a further aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 We provide a mutain of the α-subunit of human interleukin 27 containing an amino acid sequence with sequence identity of 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, wherein in this mutain, at least one amino acid residue selected from the group consisting of sequence positions 187, 238, and 240 corresponding to the sequence position of SEQ ID NO: 1 is mutated compared to the α-subunit of human interleukin 27. In this regard, it should be noted that the □ subunit of mouse interleukin 27 (SEQ ID NO: 10) has an amino acid length of 234 residues, while the □ subunit of human interleukin 27 has an amino acid length of 243 residues. The sequence identity between SEQ ID NO: 1 and SEQ ID NO: 10 has been determined to be 75%. Therefore, it is preferable that the mutain of the present invention comprises an amino acid sequence having at least 76% sequence identity to the amino acid sequence of SEQ ID NO: 1 of the α-subunit of human interleukin 27, having the mutations defined as mentioned elsewhere in this specification. Thus, the aforementioned preferred sequence identity, such as at least 76% of the amino acid sequence of SEQ ID NO: 1 of the α-subunit of human interleukin 27, for example, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, can be combined with each embodiment of this specification.

[0047] In another aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin-27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin-27, the amino acid residue at sequence position 187, corresponding to sequence position SEQ ID NO: 1, is mutated, preferably substituted with alanine (SEQ ID NO: 2).

[0048] In a more preferred aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin 27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin 27, the amino acid residue at sequence position 238, corresponding to sequence position SEQ ID NO: 1, is mutated, preferably substituted with alanine (SEQ ID NO: 3).

[0049] In another, more preferable aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin 27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin 27, the amino acid residue at sequence position 240, corresponding to sequence position SEQ ID NO: 1, is mutated, preferably substituted with alanine (SEQ ID NO: 4).

[0050] In another aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin-27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin-27, amino acid residues at sequence positions 187 and 238, corresponding to sequence position SEQ ID NO: 1, are mutated, preferably substituted with alanine (SEQ ID NO: 5).

[0051] In another aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin-27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin-27, amino acid residues at sequence positions 187 and 240, corresponding to the sequence position of SEQ ID NO: 1, are mutated, preferably substituted with alanine (SEQ ID NO: 6).

[0052] In another aspect, the present invention relates to the amino acid sequence of SEQ ID NO: 1 of the α subunit of human interleukin 27, with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin-27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin-27, amino acid residues at sequence positions 187, 238, and 240, corresponding to sequence position SEQ ID NO: 1, are mutated, preferably substituted with alanine (SEQ ID NO: 7).

[0053] In its most preferred aspect, the present invention provides at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 The present invention provides a mutain of the α-subunit of human interleukin-27 containing an amino acid sequence having 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, wherein, compared to the α-subunit of human interleukin-27, amino acid residues at sequence positions 238 and 240, corresponding to the sequence position of SEQ ID NO: 1, are mutated, preferably substituted with alanine (SEQ ID NO: 8).

[0054] "Identity" or "sequence identity" refers to the property of sequences that measures the similarity or relationship between sequences. As used in this invention, "sequence identity" or "identity" refers to the percentage of pair-wise identical residues relative to the number of residues in the longer of the two sequences, after the (homological) alignment of the polypeptide sequence of this invention with the sequence in question. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

[0055] The term “homology” is used herein in its ordinary sense and includes identical amino acids at equivalent positions in the linear amino acid sequence of the polypeptides of this disclosure (e.g., any lipocalin mutein of this disclosure), as well as amino acids that are considered to be conserved substitutions (e.g., exchange of a glutamic acid residue with an aspartic acid residue).

[0056] The percentage of sequence homology or sequence identity can be determined herein, for example, using the program BLASTP, version blastp 2.2.5 (November 16, 2002; see Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment, the percentage of homology is based on the alignment of the entire polypeptide sequence containing each sequence (matrix: BLOSUM 62; gap cost: 11.1; cutoff value set to 10⁻³). This is calculated as the percentage obtained by dividing the number of “positive” (homologous amino acids) shown as a result in the output of the BLASTP program by the total number of amino acids selected by the program for alignment.

[0057] The present invention also provides a human IL-27 mutaine comprising an α-subunit p28 and a β-subunit EBI3, wherein the α-subunit is the mutaine of the α-subunit of human IL-27 described herein. In a further embodiment, the α-subunit is the SEQ ID NO: of the α-subunit of human interleukin 27 described herein. The mutain contains an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with respect to amino acid sequence 1. The β-subunit EBI3 of human IL-27 mutein disclosed herein is also the SEQ ID NO: of the β-subunit of human interleukin-27. The amino acid sequence may contain at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with respect to the 12 amino acid sequence, preferably with SEQ ID NO: This refers to 12. In such human IL-27 mutaine of the present invention, at least one of the amino acid residues at sequence positions 187, 238, and 240 of the α-subunit corresponding to sequence position SEQ ID NO: 1 can be mutated. Consistent with the above, in the human IL-27 mutaine of the present invention, the mutaine of the corresponding α-subunit may contain a single mutation at one of these sequence positions, or it may contain mutations at two or all three of these sequence positions.Therefore, the disclosure regarding mutations in the α-subunit of human IL-27 at at least one of the amino acid residues at sequence positions 187, 238, and 240, corresponding to the sequence position of SEQ ID NO: 1, is also applicable to the mutain of human IL-27.

[0058] Thus, according to the present invention, in the human IL-27 mutain of the present invention, the amino acid residue at sequence position 187, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, can be replaced with alanine. Additionally or alternatively, in the human IL-27 mutain of the present invention, the amino acid residue at sequence position 238, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, can be replaced with alanine. Additionally or alternatively, in the human IL-27 mutain of the present invention, the amino acid residue at sequence position 240, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, can be replaced with alanine.

[0059] Furthermore, according to the present invention, in the human IL-27 mutain of the present invention, amino acid residues at sequence positions 187 and 238, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, can be replaced with alanine. In yet another embodiment, in the human IL-27 mutain of the present invention, amino acid residues at sequence positions 187 and 240, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, can also be replaced with alanine.

[0060] Most preferably, in the human IL-27 mutain of the present invention, amino acid residues at sequence positions 238 and 240, corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human IL-27, are replaced with alanine.

[0061] It is within the scope of the present invention that the above-mentioned mutations in human IL-27 to alanine at amino acid residues 187, 238, and 240 corresponding to the sequence position of SEQ ID NO: 1 can form a mutein having one, two, or three alanine at any of the above-mentioned positions 187, 238, and 240 of human IL-27.

[0062] In yet another embodiment, in the human interrotekin 27 mutain of the present invention, at least one amino acid residue at sequence positions 187, 238, and 240, corresponding to sequence position SEQ ID NO: 1, can be replaced with any amino acid that cannot be O-glycosylated, i.e., any amino acid other than serine or threonine.

[0063] Furthermore, the human IL-27 mutein of the present invention may further comprise one or more disulfide crosslinks as described above. Additionally or alternatively, the human IL-27 mutein of the present invention may further comprise one or more salt crosslinks as described above.

[0064] The present invention also provides nucleic acid molecules comprising nucleotide sequences encoding the mutain of human IL-27 or the mutain of the α-subunit of human IL-27 according to the present invention. In a further embodiment, the nucleic acid molecule comprises at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, The nucleotide sequence contains the mutain encoding the α-subunit of human interleukin-27, with sequence identity of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0065] The nucleic acid molecule according to the present invention may contain nucleotide sequences encoding mutaines of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 11, preferably mutaines of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 11.

[0066] The nucleic acid molecule of the present invention is preferably functionally linked to a regulatory sequence to enable the expression of the nucleic acid molecule. This regulatory sequence may include a promoter sequence. The terms “promoter” or “promoter sequence” mean a DNA sequence that initiates and directs the transcription of a gene into an RNA transcript within a cell.

[0067] The nucleic acid molecules according to the present invention can be included in a vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid ligated to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector to which additional DNA segments can be ligated to the viral genome.

[0068] In yet another aspect, the present invention provides nucleic acid molecules described herein for use as therapeutic agents.

[0069] Furthermore, the present invention also provides a host cell containing the nucleic acid molecule of the present invention as described above. The host cell may be any prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., an insect cell, yeast, or mammalian cell). Preferably, the host cell is a eukaryotic cell.

[0070] The present invention also provides an immunomodulator containing the mutaine of the present invention. An immunomodulator is any protein, substance, or composition capable of performing immunomodulation; immunomodulation is the adjustment of an immune response to a desired level, for example, in the case of immune enhancement, immunosuppression, or induction of immune tolerance.

[0071] The present invention also provides the use of the mutain of the present invention (mutain of the α-subunit of human IL-27 or mutain of human IL-27 containing the α-subunit) in the manufacture of pharmaceuticals for treating diseases in mammals, preferably humans. Suitable diseases include, but are not limited to, infections, autoimmune diseases, cancer, multiple sclerosis, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma. Preferably, sepsis is considered a suitable disease to be treated with the mutain of the present invention in mammals (preferably humans).

[0072] Furthermore, the present invention also provides mutaine (mutaine of the α subunit of human IL-27 or mutaine of human IL-27 containing the α subunit) for use in the treatment of diseases including the aforementioned infectious diseases, autoimmune diseases, cancer, multiple sclerosis, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma. In this case as well, sepsis is considered a suitable disease to be treated with the mutaine of the present invention in mammals (preferably humans).

[0073] The present invention also provides a method for treating IL-27-mediated diseases (also known as IL-27-related diseases) in mammals, preferably including infectious diseases, autoimmune diseases, cancer, chronic inflammatory diseases such as multiple sclerosis and chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma, comprising the step of administering the human IL-27 α-subunit mutein of the present invention or a composition containing the human IL-27 mutein of the present invention to a mammal in need thereof. Preferably, the mammal is human. In a more preferred embodiment, sepsis is considered a suitable disease to be treated with the mutein of the present invention in a mammal (preferably human).

[0074] Furthermore, the present invention relates to a method for producing mutaine according to the present invention, (a) At least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Nucleic acid molecules encoding polypeptides containing 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, with the SEQ ID NO: human IL-27 or human IL-27 α-subunit or human interleukin-27 α-subunit polypeptide. For amino acid sequence 1, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 A step of introducing a nucleotide sequence that mutates at least one amino acid residue selected from the group consisting of sequence positions 187, 238, and 240 of a polypeptide having sequence identity of 6%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%; and (b) The process of introducing the obtained nucleic acid molecule for expression into a suitable host cell or a suitable cell extract or cell lysate. We also provide methods that include this.

[0075] In the method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. The present invention includes introducing a nucleotide sequence that mutates the amino acid residue at sequence position 187 into a nucleic acid molecule encoding a polypeptide containing %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity.

[0076] Additionally or alternatively, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. The present invention includes introducing a nucleotide sequence that mutates the amino acid residue at sequence position 238 into a nucleic acid molecule encoding a polypeptide containing %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity.

[0077] Additionally or alternatively, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. The present invention includes introducing a nucleotide sequence that mutates the amino acid residue at sequence position 240 into a nucleic acid molecule encoding a polypeptide containing sequence identity of %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0078] In yet another embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, A nucleic acid molecule encoding a polypeptide with 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity is introduced with nucleotide sequences that mutate amino acid residues at sequence positions 187 and 238.

[0079] In yet another embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, A nucleic acid molecule encoding a polypeptide with 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity is introduced with nucleotide sequences that mutate amino acid residues at sequence positions 187 and 240.

[0080] In a preferred embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, A nucleic acid molecule encoding a polypeptide with 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity is introduced with nucleotide sequences that mutate amino acid residues at sequence positions 238 and 240.

[0081] In a more preferred embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide A nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide containing 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, which mutates the amino acid residue at sequence position 187 to alanine.

[0082] In addition or alternatively, in yet another preferred embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide A nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide containing 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, which mutates the amino acid residue at sequence position 238 to alanine.

[0083] In addition or alternatively, in yet another preferred embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide A nucleotide sequence is introduced into a nucleic acid molecule encoding a polypeptide containing 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity, which mutates the amino acid residue at sequence position 240 to alanine.

[0084] In another embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. A nucleic acid molecule encoding a polypeptide with sequence identity of %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% is introduced with a nucleotide sequence that mutates amino acid residues at sequence positions 187 and 238 to alanine.

[0085] In another embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. A nucleic acid molecule encoding a polypeptide with sequence identity of %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% is introduced with a nucleotide sequence that mutates amino acid residues at sequence positions 187 and 240 to alanine.

[0086] In the most preferred embodiment of the present invention, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide, A nucleic acid molecule encoding a polypeptide with sequence identity of %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% is introduced with a nucleotide sequence that mutates the amino acid residues at sequence positions 238 and 240 to alanine.

[0087] The present invention also provides a method for producing mutein according to the present invention, wherein in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 1 of human IL-27 polypeptide or human IL-27α subunit polypeptide or human interleukin 27α subunit polypeptide. This involves introducing nucleotide sequences into nucleic acid molecules encoding polypeptides with sequence identity of %, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, which mutate one, two, or all three amino acid residues at sequence positions 187, 238, and 240 to alanine.

[0088] In yet another embodiment, a method for producing mutein according to the present invention, in step (a), at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, A nucleic acid molecule encoding a polypeptide with 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity is modified by introducing a nucleotide sequence that mutates at least one amino acid residue at sequence positions 187, 238, and 240 to any amino acid that cannot be O-glycosylated, i.e., any amino acid other than serine or threonine.

[0089] The present invention also provides a mutain of the α-subunit of human interleukin 27 (also called a “deletion mutant”) as defined elsewhere in this specification, wherein at least one amino acid residue of the α-subunit of human interleukin 27 is mutated, selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1, and further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to the sequence position of SEQ ID NO: 1. Therefore, the present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residue of the α-subunit of human interleukin 27 at sequence position 238 corresponding to sequence position SEQ ID NO: 1 is mutated, and further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more of positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to sequence position SEQ ID NO: 1. Therefore, the present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residue of the α-subunit of human interleukin 27 at sequence position 240 corresponding to sequence position SEQ ID NO: 1 is mutated, and further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to sequence position SEQ ID NO: 1.Therefore, the present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residues of the α-subunit of human interleukin 27 are mutated at sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1, and further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to the sequence position of SEQ ID NO: 1. The variants defined below, in this case as well, are derived from the WT human interleukin 27 (IL-27) α-subunit having SEQ ID NO: 1 as defined by the present invention. Accordingly, the present invention provides a mutain of the α-subunit of human interleukin 27 as defined elsewhere herein, further comprising mutated amino acid residues at one or more positions corresponding to positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 of SEQ ID NO: 1. Each combination of mutated amino acids, which refers to an additional mutation at one or more of the following positions corresponding to sequence position 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243, is defined herein as a mutaine in which at least one amino acid residue of the α-subunit of human interleukin 27 is mutated, selected from the group consisting of sequence positions 238 and 240 corresponding to sequence position 238 and 240, or SEQ ID NO: This disclosure can be combined with the present disclosure of a human interleukin 27 in which the amino acid residues of the α-subunit at sequence positions 238 and 240, corresponding to sequence position 1, are mutated.

[0090] In a further aspect, the present invention relates to the SEQ ID NO: of the α subunit of human interleukin 27. The amino acid sequence contains at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of the amino acid sequence, and further includes SEQ ID The present invention provides a mutain of the α-subunit of human interleukin 27 as defined elsewhere herein, containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations at one or more of the following positions: 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243, corresponding to sequence position NO: 1.

[0091] Therefore, a mutain as defined herein may also be envisioned, which is characterized by a mutation in at least one amino acid residue of the α-subunit of human interleukin 27, selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1 (for example, a mutation in the amino acid residue of the α-subunit of human interleukin 27 at sequence position 238 or 240 corresponding to the sequence position of SEQ ID NO: 1, or a mutation in the amino acid residue of the α-subunit of human interleukin 27 at sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1), and further includes a mutation (mutated amino acid residue) at sequence position 229 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further including a mutation at position 230 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further including a mutation at position 231 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 232 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 233 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 234 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 235 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 236 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 237 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 239 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 241, which corresponds to the sequence position of SEQ ID NO: 1.The present invention may also include a mutain as defined herein, further comprising a mutation at position 242 corresponding to the sequence position of SEQ ID NO: 1. The present invention may also include a mutain as defined herein, further comprising a mutation at position 243 corresponding to the sequence position of SEQ ID NO: 1. Each embodiment of each additional mutation as defined herein is a mutation of the α subunit of human interleukin 27 SEQ ID NO: This disclosure can be combined with the present disclosure relating to amino acid sequences having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, sequence identity with respect to amino acid sequence 1.

[0092] Furthermore, the present invention may provide a mutain of the α-subunit of human interleukin 27, wherein at least one amino acid residue of the α-subunit of human interleukin 27 is mutated, selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1, and at least the residues at amino acid positions 234-238 corresponding to the sequence position of SEQ ID NO: 1 are mutated. The present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residue of the α-subunit of human interleukin 27 at sequence position 238 corresponding to the sequence position of SEQ ID NO: 1 is mutated, and at least the residues at amino acid positions 234-238 corresponding to the sequence position of SEQ ID NO: 1 are mutated. The present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residue of the α-subunit of human interleukin 27 at sequence position 240 corresponding to sequence position SEQ ID NO: 1 is mutated, and furthermore, the residues at amino acid positions 234-238 corresponding to sequence position SEQ ID NO: 1 are mutated. The present invention may also provide a mutain of the α-subunit of human interleukin 27, wherein the amino acid residues of the α-subunit of human interleukin 27 at sequence positions 238 and 240 corresponding to sequence position SEQ ID NO: 1 are mutated, and furthermore, the residues at amino acid positions 234-238 corresponding to sequence position SEQ ID NO: 1 are mutated. This means that the mutain as defined elsewhere in this specification further includes four mutations at positions 234, 235, 236, 237, and 238 corresponding to sequence position SEQ ID NO: 1.

[0093] In a further aspect, the present invention relates to the SEQ ID NO: of the α subunit of human interleukin 27. The amino acid sequence comprises at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of the sequence identity of the amino acid sequence 1, and has at least SEQ ID This provides a mutain of the α-subunit of human interleukin 27 as defined herein, wherein the residues at amino acid positions 234-238, corresponding to the sequence position NO: 1, are mutated compared to the α-subunit of human interleukin 27. The mutation at least at amino acid positions 234-238 of SEQ ID NO: 1 removes a glycosylation site (e.g., at position 238 of SEQ ID NO: 1), but may also remove a flexible, possibly unstable region. This does not preclude any additional(s) mutations at other amino acid residues corresponding to the sequence position SEQ ID NO: 1, except those mentioned herein, as long as the functionality of the protein or, where applicable, its secretory capacity is not affected. The mutein of the α-subunit of human interleukin 27 as described herein is secretory, provided that it includes a cysteine ​​substitution (L162C) of leucine 162 and an additional mutation (in this case, deletion) at one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to the sequence position of SEQ ID NO: 1, and / or at least at the amino acid positions 234-238 (e.g., positions 234, 235, 236, 237, and 238) corresponding to the sequence position of SEQ ID NO: 1, then the mutein is secretory.The mutein of the α-subunit of human interleukin-27 described herein is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99% relative to the amino acid sequence of SEQ ID NO: 1 of the α-subunit of human interleukin-27. This refers to a polypeptide sequence containing an amino acid sequence having sequence identity of 1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76% (wherein the residue at amino acid position 162 (leucine) is replaced with cysteine ​​compared to the α subunit of human interleukin 27 (L162C)), and SEQ If the mutain also contains mutations (in this case deletions) at one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to the sequence position of ID NO: 1, and / or at least at the amino acid positions 234-238 (e.g., positions 234, 235, 236, 237, and 238) corresponding to the sequence position of SEQ ID NO: 1, then the mutain may also be secretory as defined elsewhere in this specification. These specific mutations (e.g. deletions) result in the prevention of O-glycosylation as described elsewhere in this specification, which is important for improving homogeneity while maintaining full functionality, and in some cases for increasing the activity of the protein, making the folding more compact, or reducing access to proteolysis.In this regard, mutations (e.g., deletions) of residues at amino acid positions 234-238 corresponding to the sequence position of SEQ ID NO: 1, mutations (e.g., deletions) of residues at amino acid positions 234, 235, 236, 237 and 238 corresponding to the sequence position of SEQ ID NO: 1, as well as amino acid positions 234-239 (e.g., positions 234, 235, 236, 237, 238 and 239) corresponding to the sequence position of SEQ ID NO: 1, including Thr238 which constitutes the O-glycosylation site, and amino acid positions 234-240 (e.g., positions 234, 235, 236, 237, 238, 239 and 240), SEQ ID NO: Mutations (in this case deletions) of residues at amino acid positions 234-241 (e.g., positions 234, 235, 236, 237, 238, 239, 240, and 241) corresponding to sequence position 1, amino acid positions 234-242 (e.g., positions 234, 235, 236, 237, 238, 239, 240, 241, and 242) corresponding to sequence position 1, or amino acid positions 234-243 (e.g., positions 234, 235, 236, 237, 238, 239, 240, 241, 242, and 243) corresponding to sequence position 1 are also possible. Therefore, the term "at least" in this context with respect to "at least the residues at amino acid positions 234-238 corresponding to sequence position 1" includes other deletion variations as mentioned.

[0094] Furthermore, the present invention relates to a system in which at least one amino acid residue of the α-subunit of human interleukin 27 is mutated, selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1. For example, the amino acid residue of the α-subunit of human interleukin 27 at sequence position 238 or 240 corresponding to the sequence position of SEQ ID NO: 1 is mutated, or the amino acid residues of the α-subunit of human interleukin 27 at sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1 are mutated, and at least the residues at amino acid positions 234-239, at least the residues at amino acid positions 234-240, at least the residues at amino acid positions 234-241, at least the residues at amino acid positions 234-242, or SEQ ID NO: This also includes mutain, the α-subunit of human interleukin 27 as defined herein, in which the amino acid residues at positions 234-243, corresponding to sequence position 1, are mutated.Thus, the present invention also relates to the SEQ ID NO: of the α subunit of human interleukin 27. A sequence containing an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, sequence identity with respect to amino acid sequence 1, and at least SEQ ID NO: Amino acid positions 234-239 (e.g., positions 234, 235, 236, 237, 238 and 239) corresponding to sequence position NO: 1, amino acid positions 234-240 (e.g., positions 234, 235, 236, 237, 238, 239 and 240) corresponding to sequence position NO: 1, amino acid positions 234-241 (e.g., positions 234, 235, 236, 237, 238, 239, 240 and 241) corresponding to sequence position NO: 1, amino acid positions 234-242 (e.g., positions 234, 235, 236, 237, 238, 239, 240, 241 and 242) corresponding to sequence position NO: 1, SEQ ID NO: This provides a mutain of the α-subunit of human interleukin 27 as defined above, in which the residues at amino acid positions 234-243 (e.g., positions 234, 235, 236, 237, 238, 239, 240, 241, 242, and 243) corresponding to sequence position 1 are mutated.

[0095] The present invention also provides a mutain of the α-subunit of human interleukin 27 as defined above, wherein at least one amino acid residue of the α-subunit of human interleukin 27 is mutated, selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1, and the residues at amino acid positions 229-243 (e.g., positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243) corresponding to the sequence position of SEQ ID NO: 1 are mutated (see Figure 13). The deletion of the residues at amino acid positions 229-243 corresponding to the sequence position of SEQ ID NO: 1 can refer to the C-terminal region of IL-27α. In this case as well, the mutations in the amino acid residues at positions 229-243 corresponding to the sequence position of SEQ ID NO: 1 remove the glycosylation site (for example, at positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1), but at the same time, they may also remove a flexible, possibly unstable region. In another aspect, the present invention also relates to the SEQ ID NO: of the α-subunit of human interleukin 27. The amino acid sequence contains at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of the amino acid sequence, and SEQ ID The amino acid residues at positions 229-243, corresponding to the sequence position of NO: 1, are mutated as defined herein (SEQ ID NO: 11), providing a mutain of the α-subunit of human interleukin 27 as defined above (see Figure 13).

[0096] Each disclosure made with respect to the human interleukin-27 (IL-27) α subunit as defined elsewhere in this specification, or to human interleukin-27 (IL-27) containing the α subunit (including disclosures concerning nucleic acid molecules, host cells, immunomodulators, first and second medical uses, and methods for producing the mutain), may also be applicable to deletion variants.

[0097] In connection therewith, the method for producing the mutein as defined herein also includes, in step (a), a human interleukin 27 polypeptide or human interleukin 27α subunit polypeptide or human interleukin 27α subunit polypeptide SEQ ID NO: A nucleic acid molecule encoding a polypeptide containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of sequence identity, is assigned a SEQ ID. The present invention provides introducing a nucleotide sequence that undergoes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 further mutations at one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243 corresponding to the sequence position NO: 1.

[0098] Furthermore, in this specification, in step (a), the SEQ ID NO of human interleukin 27 polypeptide or human interleukin 27α subunit polypeptide or human interleukin 27α subunit: A nucleic acid molecule encoding a polypeptide containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of sequence identity, with at least SEQ A method for producing the mutaine as defined herein is provided, which involves introducing a nucleotide sequence that mutates the residues at amino acid positions 234-238 corresponding to the sequence position of ID NO: 1, at least the residues at amino acid positions 234-239 corresponding to the sequence position of SEQ ID NO: 1, at least the residues at amino acid positions 234-240 corresponding to the sequence position of SEQ ID NO: 1, at least the residues at amino acid positions 234-241 corresponding to the sequence position of SEQ ID NO: 1, at least the residues at amino acid positions 234-242 corresponding to the sequence position of SEQ ID NO: 1, or at least the residues at amino acid positions 234-243 corresponding to the sequence position of SEQ ID NO: 1.

[0099] Furthermore, in this specification, in step (a), the SEQ ID NO of human interleukin 27 polypeptide or human interleukin 27α subunit polypeptide or human interleukin 27α subunit: A nucleic acid molecule encoding a polypeptide containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably at least 76%, of sequence identity, is assigned a SEQ ID. A method for producing the mutaine as defined herein is provided, which involves introducing a nucleotide sequence that mutates the residues at amino acid positions 229-243 corresponding to the sequence position NO: 1 (SEQ ID NO: 11). The present invention is further illustrated by the following experimental examples. [Examples]

[0100] Alignment of sequences and structures: DNA sequence alignment is Clustal Omega 18 I did it. Human IL-27α L162C For structural homology modeling, the i-Tasser program is used. 19 The following was used. Structural alignment and analysis were performed using Yasara Structure (www.yasara.org).

[0101] Creation of IL-27 variants: Human and mouse IL-27α genes were amplified from their cDNA (Origene) by PCR, and after restriction digestion, cloned into pSVL vectors (Amersham). Mutants were generated using site-directed mutagenesis.

[0102] Cell culture experiment: 293T cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM) containing L-Ala-L-Gln (AQmedia, Sigma-Aldrich) and 10% (v / v) fetal bovine serum (biochrome). A 1% (v / v) antibiotic antifungal solution (25 μg / ml amphotericin B, 10 mg / ml streptomycin, and 10,000 units penicillin; Sigma-Aldrich) was mixed with the 293T cell medium. Transient transfection was performed for 24 hours using p35 poly-D-lysine coated shells (Becton Dickinson) with GeneCellin (BioCellChallenge) according to the manufacturer's protocol. Equal volumes of constructs (α, β, or empty vectors) were transfected with a total of 2 μg of DNA (p35 dish). In the secretion experiment, the cells were transfected for 8 hours, washed twice with PBS, and incubated for a further 16 hours in 0.5 ml of fresh medium. Before lysis, the cells were washed twice with ice-cold PBS. Cell lysis was performed using RIPA buffer (50 mM Tris / HCl, pH 7.5, 150 mM NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, EDTA-free 1× Roche complete protease inhibitor; Roche Diagnostics). To analyze the secreted protein, the medium was centrifuged at 300 g at 4°C for 5 minutes. Subsequently, the sample was mixed with 0.1x volume of 500 mM Tris / HCl, pH 7.5, 1.5 M NaCl, and the protease inhibitor, and centrifuged at 20,000 g at 4°C for 15 minutes. The sample was then analyzed for the presence of protein. 5×Laemmli containing β-mercaptoethanol for reduced SDS-PAGE was added to the sample at 0.2 times the volume. Deglycosylation experiments were performed according to the manufacturer's instructions (NEB).

[0103] Recombinant protein production: Human IL-27α cDNA (without ER transport sequence) optimized for expression in E. coli was obtained from GeneArt and cloned into a pET21a vector (Merck Millipore) containing an N-terminal hexahistidine tag and a TEV protease cleavage site following the tag. The L156C mutation (corresponding to the L162C mutation in the human sequence) was inserted by site-directed mutagenesis. The reference protein was expressed as inclusion bodies in selective LB medium. The culture was induced with 1 mM IPTG at OD600=0.6 and collected after 4 hours by centrifugation (5,000 rpm, 15 min, 4°C). To separate the inclusion bodies, cells were lysed on ice using sonication in 100 mM Tris / HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, and SigmaFAST protease inhibitor, and then centrifuged (20,000 g, 20 min, 4°C). The pellet was resuspended and washed twice with 100 mM Tris / HCl, pH 7.5, 500 mM NaCl, 5 mM EDTA, and 1.0% Triton X-100, and then washed again with 100 mM Tris / HCl, pH 7.5, and 100 mM NaCl. The inclusions were then solubilized at 4°C in 50 mM sodium phosphate, pH 7.5, 250 mM NaCl, 6 M GdmCl, and 10 mM β-mercaptoethanol. After overnight solubilization, the solution was centrifuged (20,000 g, 20 min, 20°C). The supernatant was diluted to 1 volume unit of 50 mM sodium phosphate, pH 7.5, 250 mM NaCl, and 5 M GdmCl and loaded onto a Ni-Sepharose HP column (GE Healthcare). The bound protein was washed with 50 mM sodium phosphate, pH 7.5, 250 mM NaCl, 5 M GdmCl, 30 mM imidazole, and 1 mM DTT, and eluted with 50 mM sodium phosphate, pH 3.5, 250 mM NaCl, 5 M GdmCl, and 1 mM DTT. The eluted protein was further purified by gel filtration (HiPrep 16 / 60 Sephacryl S-400 HR column (GE Healthcare)), and the buffer was replaced with 50 mM MES pH 6.0, 6 M urea, and 1 mM EDTA. The protein concentration was measured by spectrophotometer at A280 nm.Human IL-27α optimized for expression in Homo sapiens (H. sapiens). L162C cDNA was ordered from GeneArt (Thermo Fisher Scientific) in the form of a pcDNA3.4 TOPO vector for mammalian cell expression. T238A and S240A mutations were inserted by site-directed mutagenesis. Protein expression was performed using the Expi293 expression system according to the manufacturer's specifications (Thermo Fisher Scientific). 48 hours after transfection, the culture medium was collected by centrifugation (300g, 15 minutes, 4°C), concentrated to 4.1 μg / ml using a Vivaspin 20 10kDa centrifugation unit (VWR), and used for immunoassays. hIL-27αL156CHis6 purified from E. coli inclusion bodies was used as a reference to compare hIL-27α in the Expi293 supernatant. L162C,T238A,S240A A linearly fitted standard curve for quantitative analysis was established using the immunoblot signal.

[0104] Quantification: Western blots were quantified using Bio-1D software (Vilber Lourmat). IL-27α was used for the quantification of P-STAT immunoblots. L162C,T238A,S240A Western blot signals of cells treated with IL-27α L162C Alternatively, the results were normalized to the IL-27α signal. All experiments were performed at least twice, and representative experiments were selected.

[0105] Activity assay: STAT experiments were performed using the human Burkitt lymphoma BL-2 cell line (DKSM). Before use, BL-2 cells were cultured overnight in serum-free RPMI-1640 medium. Cells were then plated in 48-well plates (2 × 10⁶ cells / well) in RPMI-1640 medium containing 0.5% BSA and treated with 1000 ng / mL of hIL-27α L162C hIL-27α, hIL-27α L162C,T238A,S240A hIL-27α T238A,S240A (=ΔO) and hEBI3, hEBI3 N55QN105Q (=ΔN)Cells were incubated for 60 minutes in 48-well plates (2 × 10⁶ cells / well) with either Expi293 supernatant or untransfected control Expi293 supernatant. The reaction was stopped by diluting the cells in ice-cold PBS buffer and lysed with NP40 lysis buffer (containing protease and phosphatase inhibitors). Phosphorylated and total STAT proteins were detected by immunoblotting. Rabbit antibodies from Cell Signaling Technology (P-STAT1, #9167; STAT1, #9172) were used (see Figures 10 and 12).

[0106] C-terminal truncation of IL-27αL162C: 1 x 10 4 Individual STAT1 luciferase reporter HeLa cells (Signosis, SL-0004-NP) were seeded in 100 μl of 0.1% FCS-containing DMEM (Sigma Aldrich) per well in a 96-well plate. After overnight culture, 60 μl of medium was added to the culture medium from mock-transfected Expi293 cells (vehicle control) or hIL-27α L162C (Full-length construct) or hIL-27α L162C,ΔC The supernatant of 60 μl of clarified cells was replaced with that from Expi293 cells transfected to transiently express and secrete a construct lacking the C-terminal region H229-P243. After 22 hours of stimulation, the cells were washed with PBS and a luciferase assay was performed according to the manufacturer's protocol (Promega, E1500). Data were obtained from full-length hIL-27α L162C The data was normalized for (n=3 biological replicates, each as the average of 4 technical replicates) (see Figure 13).

[0107] The present invention is further characterized by the following: item 1. A mutain of the α-subunit (SEQ ID NO: 1) of human interleukin-27, selected from the group consisting of sequence positions 187, 238, and 240, wherein at least one amino acid residue of the α-subunit (SEQ ID NO: 1) of human interleukin-27 is mutated. 2. The mutain described in item 1, in which the amino acid residue at sequence position 187 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) is substituted with alanine (SEQ ID NO: 2). 3. Mutaine as described in item 1 or 2, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) is substituted with alanine (SEQ ID NO: 3). 4. A mutaine as described in any of the above items, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) is substituted with alanine (SEQ ID NO: 4). 5. A mutain according to any of the above items, wherein the amino acid residues at sequence positions 187 and 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine (SEQ ID NO: 5). 6. A mutain according to any of the above items, wherein the amino acid residues at sequence positions 187 and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine (SEQ ID NO: 6). 7. A mutain according to any of the above items, wherein amino acid residues at sequence positions 187, 238, and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine (SEQ ID NO: 7). 8. A mutain according to any of the above items, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine (SEQ ID NO: 8). 9. The mutain described in any of the preceding items, further comprising one or more salt crosslinks. 10. The mutaine according to any of the preceding items, further comprising one or more disulfide crosslinks. 11. A mutaine of human interleukin 27 comprising an α-subunit p28 and a β-subunit Ebi3, wherein the α-subunit is the mutaine of the α-subunit (SEQ ID NO: 1) of human interleukin 27 described in any of items 1 to 10. 12. A mutain as described in item 11, wherein at least one amino acid residue of the α-subunit (SEQ ID NO: 1) of human interleukin 27 is mutated, selected from the group consisting of sequence positions 187, 238, and 240. 13. A mutain as described in any of items 11-12, wherein the amino acid residue at sequence position 187 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) is substituted with alanine. 14. A mutain as described in any of items 11-13, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) is substituted with alanine. 15. A mutain as described in any of items 11-14, wherein the amino acid residue at sequence position 240 of the α-subunit (SEQ ID NO: 1) of human interleukin-27 is substituted with alanine. 16. A mutaine as described in any of items 11-15, wherein amino acid residues at sequence positions 187 and 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine. 17. A mutaine as described in any of items 11-16, wherein amino acid residues at sequence positions 187 and 240 of the α-subunit (SEQ ID NO: 1) of human interleukin-27 are substituted with alanine. 18. A mutain as described in any of items 11-17, wherein amino acid residues at sequence positions 187, 238, and 240 of the α-subunit (SEQ ID NO: 1) of human interleukin-27 are substituted with alanine. 19. A mutain as described in any of items 11-18, wherein amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) are substituted with alanine. 20. Mutaines according to any one of items 11 to 19, further comprising one or more salt bridges. 21. Mutain according to any one of items 11 to 20, further comprising one or more disulfide crosslinks. 22. A nucleic acid molecule comprising a nucleotide sequence encoding the mutaine of human interleukin-27 or the mutaine of the α-subunit of human interleukin-27 as described in any of items 1 to 21. 23. A nucleic acid molecule as described in item 22, comprising a nucleotide sequence encoding mutaine with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. 24. A nucleic acid molecule according to any of items 22-23, which is functionally linked to a regulatory sequence that enables the expression of the nucleic acid molecule. 25. The nucleic acid molecule described in item 24, wherein the regulatory sequence includes a promoter sequence. 26. A nucleic acid molecule contained in the vector, as described in any of items 22-25. 27. A host cell containing any of the nucleic acid molecules described in items 22-26. 28. An immunomodulator containing mutain as described in any of items 1-21. 29. Use of mutaine as described in any of items 1 to 21 in the manufacture of pharmaceuticals for the treatment of infectious diseases, autoimmune diseases, multiple sclerosis, cancer, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma in mammals. 30. Mutain, as described in any of items 1-21, for use in treatment. 31. Mutain as described in any of items 1-21, for use in the treatment of infections, autoimmune diseases, multiple sclerosis, cancer, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma in mammals. 32. A method for treating interleukin-27-mediated diseases in mammals, preferably infectious diseases, autoimmune diseases, multiple sclerosis, cancer, transplant-related diseases such as graft-versus-host disease, chronic inflammatory diseases such as chronic inflammatory bowel disease, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma, comprising the step of administering to a mammal in need thereof a composition containing mutein as described in any of items 1 to 21. 33. A method for producing mutain as described in any of items 1 to 21, (a) The step of introducing a nucleotide sequence into a nucleic acid molecule encoding human interleukin-27 polypeptide or human interleukin-27 α-subunit polypeptide, which mutates at least one amino acid residue selected from the group consisting of human interleukin-27 or the α-subunit sequence positions 187, 238, and 240; and (b) The process of introducing the nucleic acid molecule for expression obtained in step (a) into a suitable host cell or a suitable cell extract or cell lysate. Methods that include... 34. The method according to item 33, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 187 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 35. The method according to item 33 or 34, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 36. The method according to any one of items 33 to 35, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 37. The method according to any one of items 33 to 36, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates amino acid residues at sequence positions 187 and 238 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 38. The method according to any one of items 33 to 37, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates amino acid residues at sequence positions 187 and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 39. The method according to any one of items 33 to 38, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates amino acid residues at sequence positions 187, 238, and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 40. The method according to any one of items 33 to 39, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin-27 (SEQ ID NO: 1) to alanine. 41. A mutain of the α-subunit (SEQ ID NO: 1) of human interleukin-27, with at least one residue at amino acid positions 234-238 deleted.

[0108] References TIFF0007867280000001.tif237165TIFF0007867280000002.tif121164

Claims

1. A mutain of the α-subunit of human interleukin 27, wherein at least one amino acid residue selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1 of the α-subunit of human interleukin 27 is deleted or mutated to an amino acid residue that cannot be O-glycosylated, the α-subunit of human interleukin 27 contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and amino acid residue 162 of the α-subunit of human interleukin 27 is mutated to cysteine.

2. The mutain according to claim 1, wherein the amino acid residue at sequence position 238 of the α subunit of human interleukin 27 is mutated to an amino acid residue that cannot be O-glycosylated.

3. The mutain according to claim 1 or 2, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is mutated to an amino acid residue that cannot be O-glycosylated.

4. The mutain according to any one of claims 1 to 3, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are mutated to amino acid residues that cannot be O-glycosylated.

5. The mutain according to any one of claims 1 to 4, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 3).

6. The mutain according to any one of claims 1 to 5, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 4).

7. The mutain according to any one of claims 1 to 6, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are substituted with alanine (SEQ ID NO: 8).

8. The mutaine according to any one of claims 1 to 7, further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations or deletions at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243.

9. The mutaine according to any one of claims 1 to 8, wherein at least one residue at amino acid positions 234 to 238, at least one residue at amino acid positions 234 to 239, at least one residue at amino acid positions 234 to 240, at least one residue at amino acid positions 234 to 241, at least one residue at amino acid positions 234 to 242, or at least one residue at amino acid positions 234 to 243 is deleted.

10. The mutain according to any one of claims 1 to 9, wherein the residues at amino acid positions 229 to 243 are deleted (SEQ ID NO: 11).

11. The mutain according to any one of claims 1 to 10, further comprising one or more salt crosslinks.

12. The mutaine according to any one of claims 1 to 11, further comprising one or more disulfide crosslinks.

13. A mutaine of human interleukin 27, comprising an α-subunit p28 and a β-subunit Ebi3, wherein the α-subunit is the mutaine of the α-subunit of human interleukin 27 described in any one of claims 1 to 12.

14. The mutain according to claim 13, wherein at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1 of the α subunit of human interleukin 27 is mutated to an amino acid residue that cannot be O-glycosylated.

15. The mutain according to claim 13 or 14, wherein the amino acid residue at sequence position 238, corresponding to the sequence position of SEQ ID NO: 1 of the α subunit of human interleukin 27, is mutated to an amino acid residue that cannot be O-glycosylated.

16. The mutain according to any one of claims 13 to 15, wherein the amino acid residue at sequence position 240, corresponding to the sequence position of SEQ ID NO: 1 of the α subunit of human interleukin 27, is mutated to an amino acid residue that cannot be O-glycosylated.

17. The mutain according to any one of claims 13 to 16, wherein the amino acid residues at sequence positions 238 and 240, corresponding to the sequence position of SEQ ID NO: 1 of the α subunit of human interleukin 27, are mutated to amino acid residues that cannot be O-glycosylated.

18. The mutain according to any one of claims 13 to 17, wherein the amino acid residue at sequence position 238 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 3).

19. The mutain according to any one of claims 13 to 18, wherein the amino acid residue at sequence position 240 of the α-subunit of human interleukin 27 is substituted with alanine (SEQ ID NO: 4).

20. The mutain according to any one of claims 13 to 19, wherein the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin 27 are substituted with alanine (SEQ ID NO: 8).

21. The mutaine according to any one of claims 13 to 20, further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations or deletions at any one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243.

22. The mutaine according to any one of claims 13 to 21, wherein at least a residue at amino acid positions 234 to 238, at least a residue at amino acid positions 234 to 239, at least a residue at amino acid positions 234 to 240, at least a residue at amino acid positions 234 to 241, at least a residue at amino acid positions 234 to 242, or at least a residue at amino acid positions 234 to 243 is deleted.

23. The mutain according to any one of claims 13 to 22, wherein the residues at amino acid positions 229 to 243 are deleted.

24. The mutain according to any one of claims 13 to 23, further comprising one or more salt crosslinks.

25. The mutaine according to any one of claims 13 to 24, further comprising one or more disulfide crosslinks.

26. A mutain of the α-subunit of human interleukin 27, wherein the mutain is missing the sequence of amino acid positions 1-28 and C-terminal amino acid residues 229-243 shown in SEQ ID NO: 1, the α-subunit of human interleukin 27 has an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the amino acid residue of the α-subunit of human interleukin 27 at sequence position 162 is mutated to cysteine.

27. The mutaine according to claim 26, wherein the amino acid sequence of the mutaine is shown at amino acid positions 29 to 228 of SEQ ID NO:

1.

28. A nucleic acid molecule comprising a nucleotide sequence encoding the mutaine of human interleukin 27 or the mutaine of the α-subunit of human interleukin 27 as described in any one of claims 1 to 27.

29. The nucleic acid molecule according to claim 28, comprising a nucleotide sequence encoding mutain of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO:

11.

30. The nucleic acid molecule according to claim 28 or 29, which is functionally linked to a regulatory sequence that enables the expression of the nucleic acid molecule.

31. The nucleic acid molecule according to claim 30, wherein the regulatory sequence includes a promoter sequence.

32. A nucleic acid molecule contained in a vector, according to any one of claims 28 to 31.

33. A host cell containing a nucleic acid molecule according to any one of claims 28 to 32.

34. An immunomodulator comprising mutain as described in any one of claims 1 to 27.

35. Mutain according to any one of claims 1 to 27, for use in treatment.

36. Mutein according to any one of claims 1 to 27, for use in the treatment of infectious diseases, autoimmune diseases, multiple sclerosis, cancer, transplant-related diseases, chronic inflammatory diseases, acute inflammatory diseases, sepsis, septic shock, diabetes, or asthma in mammals.

37. The mutaine according to claim 36, wherein the transplant-related disease is graft-versus-host disease.

38. The mutaine according to claim 36, wherein the chronic inflammatory disease is chronic inflammatory bowel disease.

39. An in vitro method for producing mutein according to any one of claims 1 to 27, (a) Introducing a nucleotide sequence into a nucleic acid molecule encoding human interleukin-27 polypeptide or human interleukin-27α-subunit polypeptide, wherein the α-subunit of the human interleukin-27 polypeptide comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, such that at least one amino acid residue selected from the group consisting of sequence positions 238 and 240 corresponding to the sequence position of SEQ ID NO: 1 of human interleukin-27 or the α-subunit of human interleukin-27 is mutated to an amino acid residue that cannot be O-glycosylated, and that the amino acid residue of human interleukin-27 or the α-subunit of human interleukin-27 at sequence position 162 is mutated to cysteine; and (b) A step of introducing the nucleic acid molecule for expression obtained in step (a) into a suitable host cell or a suitable cell extract or cell lysate. Methods that include...

40. The in vitro method according to claim 39, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide, which mutates the amino acid residue at sequence position 238 of the α subunit of human interleukin-27 to an amino acid residue that cannot be O-glycosylated.

41. The in vitro method according to claim 39 or 40, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 240 of the α-subunit of human interleukin-27 to an amino acid residue that cannot be O-glycosylated.

42. The in vitro method according to any one of claims 39 to 41, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide, which mutates the amino acid residues at sequence positions 238 and 240 of the α subunit of human interleukin-27 to amino acid residues that cannot be O-glycosylated.

43. The in vitro method according to any one of claims 39 to 42, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 238 of the α-subunit of human interleukin-27 to alanine.

44. The in vitro method according to any one of claims 39 to 43, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residue at sequence position 240 of the α-subunit of human interleukin-27 to alanine.

45. The in vitro method according to any one of claims 39 to 44, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27 α-subunit polypeptide, which mutates the amino acid residues at sequence positions 238 and 240 of the α-subunit of human interleukin-27 to alanine.

46. The in vitro method according to any one of claims 39 to 45, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide to be further mutated or deleted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 times at one or more of the positions 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, or 243.

47. The in vitro method according to any one of claims 39 to 46, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding human interleukin-27 polypeptide or human interleukin-27α subunit polypeptide, which deletes at least a residue at amino acid positions 234 to 238, at least a residue at amino acid positions 234 to 239, at least a residue at amino acid positions 234 to 240, at least a residue at amino acid positions 234 to 241, at least a residue at amino acid positions 234 to 242, or at least a residue at amino acid positions 234 to 243.

48. The in vitro method according to any one of claims 39 to 47, wherein in step (a), a nucleotide sequence is introduced into a nucleic acid molecule encoding a human interleukin-27 polypeptide or a human interleukin-27α subunit polypeptide, which deletes residues at amino acid positions 229 to 243 (SEQ ID NO: 11).

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