Fusion-polypeptide comprising a mutein of the alpha-subunit of human il-27 and at least one pharmaceutically acceptable fusion partner

Abstract

The present invention refers to a fusion-polypeptide comprising a mutein of the α-subunit of human Interleukin (27), wherein at least one of the amino acid residues selected from the group consisting of sequence positions (160, 161, 162, 163, 164, 165, 180, 181) and (182) is / are mutated, and at least one pharmaceutically acceptable fusion partner. Further, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding said fusion-polypeptide, to a host cell containing said nucleic acid molecule and to an immune modulator comprising said fusion-polypeptide. The present invention further refers to the use of said fusion-polypeptide for the manufacture of a medicament for treating a disease, the fusion-polypeptide for use as a medicament or for use in the treatment or prevention of a disease. Further, the present invention relates to a method of treating a disease comprising the step of administering a composition comprising said fusion-polypeptide.

Description

FUSION-POLYPEPTIDE COMPRISING A MUTEIN OF THE ALPHA-SUBUNIT OF HUMAN IL-27 AND AT LEAST ONE PHARMACEUTICALLY ACCEPTABLE FUSION PARTNERCROSS-REFERENCE TO RELATED APPLICATIONS AND CLAIM TO PRIORITYThis application claims priority to European patent application with Application No. 24 179 865.1 , filed 4 June 2024 at the European Patent Office, wherein the disclosure thereof is incorporated herein by reference.FIELD OF THE INVENTION

[0001] The present invention refers to a fusion-polypeptide comprising a mutein of the a- subunit of human Interleukin 27, wherein at least one of the amino acid residues of the a- subunit of human Interleukin 27 selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated, and at least one pharmaceutically acceptable fusion partner. Further, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding said fusion-polypeptide, to a host cell containing said nucleic acid molecule and to an immune modulator comprising said fusion-polypeptide. The present invention further refers to the use of the fusion-polypeptide as described herein for the manufacture of a medicament for treating a disease, preferably a disease in a mammal, more preferably a disease in a human. The present invention further refers to the fusion- polypeptide as described herein for use as a medicament or for use in the treatment or prevention of a disease. Further, the present invention relates to a method of treating a disease comprising the step of administering a composition comprising the fusion-polypeptide of the present invention, preferably to a mammal, more preferably to a human, in need thereof.BACKGROUND OF THE INVENTION

[0002] Cytokines can actively control immune reactions and can activate immune cells as well as instruct them to perform certain functions. Like antibodies, cytokines are proteins and therefore belong to the class of biologies, which are also known as biopharmaceuticals. Immune cells use cytokines for cell-cell communication.

[0003] Alternatively, cytokines can also inactivate activated immune cells. An immune activation is crucial in cases of immune paralysis, for example. Here, it is important that not the entire immune system is activated, but rather the impaired immune functions are precisely restored. In the case of autoimmune diseases, on the other hand, a targeted dampening of the immune system is desired. Understanding how immune cells communicate and the molecular mechanisms of diseases has only recently opened the door to the development of cytokine-based therapies. Therefore, the field of immunotherapies is one of the most promising approaches in modern medicine.

[0004] Interleukin-27 (IL-27) is a heterodimeric cytokine that belongs to the interleukin-12 family and plays a crucial role in the immune response. IL-27 has both pro- and an anti-inflammatory effects. On the one hand, IL-27 promotes Type 1 T helper (Th1) immune responses and, on the other hand, IL-27 also modulates the effector functions of CD4+ and CD8+ T cells and induces regulatory T cell functions. It has been described that IL-27 influences the development of Interleukin-17 producing T helper cells (Th17) and is thus relevant in the development of Th17-mediated inflammatory diseases or autoimmune diseases.

[0005] IL-27 consists of an alpha- / a- (also called p28) and a beta- / p-subunit (also called EBI3) and is secreted by activated, antigen-presenting cells. The secretion of human IL-27a is dependent on the beta-subunit EBI3. In contrast, in mice, the alpha-subunit of IL-27 can be secreted independently of the beta-subunit EBI3. Murine IL-27a is an immune signaling molecule on its own in the mouse immune system and is also known as IL-30.

[0006] The inventors of the present invention have now found IL27a-based biopharmaceuticals, which makes them the first that developed such biopharmaceuticals. These can be used in immunotherapies in indications with a dysregulated immune response. An IL-27a-based immunotherapy, also including additive / neoadjuvant or adjuvant therapies, has an unique immunomodulatory mechanism of action. Specifically, in diseases caused by immune dysregulation, this mechanism of action can lead to a therapeutic breakthrough. As the IL-27a-based biopharmaceuticals are immunomodulatory and can be used in indications with immune dysregulation (e.g. sepsis, acute-on-chronic liver failure, oncology, autoimmune diseases), the task of the present invention is to regulate a derailed immune reaction and the resulting pathology. The fusion-polypeptides developed by the inventors of the present invention also address the important aspects of developability.SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention relates to a fusion-polypeptide comprising a mutein of the a-subunit of human Interleukin 27, wherein at least one of the amino acid residues of the a-subunit of human Interleukin 27 selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated, and at least one pharmaceutically acceptable fusion partner.

[0008] The numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 may correspond to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1. The a-subunit of human Interleukin 27 may be the a-subunit of human Interleukin 27 according to SEQ ID NO: 1. Said sequence of SEQ ID NO: 1 corresponds to UniProt-sequence Q8NEV9, which is the human a-subunit of IL-27.

[0009] It is preferred that in the mutein of the a-subunit of human Interleukin 27 one or more of the amino acids of sequence positions 1 to 28 is / are mutated. It is more preferred that in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are muted. It is preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are deleted. The signal sequence as described herein and in the context of the present invention corresponds to aa 1 to 28 of SEQ ID NO: 1 . Thus, it is preferred that in the mutein of the a-subunit of human Interleukin 27 the signal sequence of amino acids sequence positions 1 to 28 are deleted.

[0010] In one aspect of the fusion-polypeptide of the present invention, the at least one pharmaceutically acceptable fusion partner is at least one tag. That tag may not be a V5 tag or a His-tag, specifically not a (His)e-tag. It is preferred that the at least one tag is at least one half-life extending tag. It is preferred that the at least one tag is at least one tag that enhanced expression of the fusion-polypeptide compared to the absence of said tag. It is even more preferred that the at least one pharmaceutically acceptable fusion partner or tag, half-life extending tag or expression-enhancing tag is human serum albumin (SEQ ID NO: 91) or a fragment or mutein thereof. It is also preferred that the at least one pharmaceutically acceptable fusion partner, tag or half-life extending tag or expression-enhancing tag is one or more constant domain of human kappa immunoglobulin light chain (SEQ ID NO. 89), with or without the C-terminal cysteine. It is preferred that the at least one pharmaceutically acceptable fusion partner is a pharmaceutically acceptable, proteinogenic fusion partner.

[0011] In one aspect of the fusion-polypeptide of the present invention, the mutein of the a- subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 1 .

[0012] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated. In one aspect of the fusion-polypeptide of the present invention it is preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated. It is further preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated.

[0013] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 160 is mutated.

[0014] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated.

[0015] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated.

[0016] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated.

[0017] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 164 is mutated.

[0018] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 165 is mutated.

[0019] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated.

[0020] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 181 is mutated.

[0021] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated.

[0022] In one preferred aspect of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated to cysteine. It is preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated to cysteine. It is further preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated to cysteine. It is further preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated to cysteine.

[0023] It is preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 160 is mutated to cysteine (SEQ ID NO: 56). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated to cysteine (SEQ ID NO: 57). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated to cysteine (SEQ ID NO: 2). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated to cysteine (SEQ ID NO: 9). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 164 is mutated to cysteine (SEQ ID NO: 58). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated to cysteine (SEQ ID NO: 59). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acidresidue at sequence position 181 is mutated to cysteine (SEQ ID NO: 60). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated to cysteine (SEQ ID NO: 61).

[0024] It is also preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 comprises one or more salt-bridges.

[0025] It is further preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 is engineered to be devoid of any O-glycosylated residues.

[0026] It is also preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are mutated. It is further preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are deleted. It is further preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 the amino acid residues of sequence positions 229 to 243 are deleted (SEQ ID NO: 62).

[0027] It is also preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 is / are mutated. In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is mutated. In one aspect of the fusion- polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is mutated. It is preferred for the fusion- polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is replaced by alanine (SEQ ID NO: 63). It is also preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is replaced by alanine (SEQ ID NO: 64). It is further preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residues at sequence positions 238 and 240 are replaced by alanine (SEQ ID NO: 65).

[0028] In one further aspect of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 mutations at one or more sequence position(s) selected from the group consisting of sequence positions 229, 230, 231 , 232, 233, 234, 235, 236, 237, 239, 241 , 242 and 243.

[0029] It is also preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 comprises one or more disulfide-bridge(s).

[0030] In one aspect of the fusion-polypeptide of the present invention the mutein of the a- subunit of human Interleukin 27 is fused at its N-terminus to the at least one pharmaceutically acceptable fusion partner. In another aspect of the fusion-polypeptide of the present invention the mutein of the a-subunit of human Interleukin 27 is fused at its C-terminus to the at least one pharmaceutically acceptable fusion partner.

[0031] In one further aspect, in the fusion-polypeptide of the present invention the mutein of the a-subunit of human Interleukin 27 may be fused to at least one pharmaceutically acceptable fusion partner via a linker. For the fusion-polypeptide of the present invention that linker may be a linker comprising at least 3 glycines and a serine, preferably a GGGS-linker (SEQ ID NO: 78), a linker comprising the sequence PAPAP (SEQ ID NO: 79), or a helical linker. For example, the linker may be selected from the group consisting of SEQ ID NOs: 78 to 88. It may also be for the fusion-polypeptide of the present invention that the linker has a length of 0 to 30 residues. In cases, wherein the mutein of the a-subunit of human Interleukin 27 is fused to SEQ ID NO: 89 (so-called “i-Tag” herein), it is preferred that the linker is PAPAP (SEQ ID NO: 79).

[0032] In one aspect of the fusion-polypeptide of the present invention, the fusion-polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to SEQ ID NO: 54, SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74. In one aspect of the present invention, the fusion-polypeptide may be a fusion-polypeptide as depicted in any one of Figures 1 to 13.

[0033] In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion-polypeptide according to the present invention. It is preferred for said nucleic acid molecule that it is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule. It is further preferred that theregulatory sequence comprises a promoter sequence. In one further aspect, the nucleic acid molecule according to the present invention is comprised in a vector.

[0034] In one further aspect, the present invention also provides a host cell containing the nucleic acid molecule according to the present invention.

[0035] In yet another aspect, the present invention provides an immune modulator comprising the fusion-polypeptide according to the present invention.

[0036] The present invention further provides in another aspect the use of a fusion- polypeptide for the manufacture of a medicament for treating a disease, preferably a disease in a mammal, more preferably a disease in a human. It is further preferred in this regard that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0037] Another aspect of the present invention provides the fusion-polypeptide as described herein for use as a medicament.

[0038] A further aspect of the present invention relates to the fusion-polypeptide as described herein for use in the treatment or prevention of a disease. In this regard, it is preferred that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0039] In another aspect, the present invention provides a method of treating a disease comprising the step of administering a composition comprising a fusion-polypeptide as described herein to a subject, preferably to a mammal, more preferably to a human, in need thereof. It is further preferred for the method of treating that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0040] These aspects of the invention will be more fully understood in view of the following drawings, detailed description and non-limiting examples.DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are included to further an understanding of the embodiments that are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and together with the description serve to explain principles of embodiments. Other embodiments and many of the intended advantages of embodiments will be readily appreciated, as they become better understood by reference to the detailed description. The elements of the drawings are not necessarily to scale relative to each other.[00421 Figure 1 shows the structural outline of muteins of human IL-27a (SEQ ID NO: 1 corresponds to UniProt-sequence Q8NEV9, in Fig. 1 , top: SEQ ID NO: 2 which corresponds to IL-27aL162Cis shown without the signal sequence of aa 1 to 28, corresponding to SEQ ID NO: 55). Key residues that are known to be O-glycosylated (Thr238 and Ser240) are highlighted. IL-27a of species that have IL-27a as an endogenous immune signalling protein is N-glycosylated. The orthologous position of the murine IL-27a glycosylation site corresponding to the D89N position within human IL-27a is highlighted as well. Our studies have demonstrated that the unstructured region at the N-terminus (corresponding to the residues Phe29 until Leu40, see SEQ ID NO: 66 without signal sequence / aa 1-28) as well as at the C-terminus (corresponding to the residues His229 until Pro243, SEQ ID NO: 62) can be deleted while maintaining complete functionality (see Fig. 3B and Fig. 9B).

[0043] Figure 2 shows a schematic of human IL-27a based biopharmaceuticals according to the present invention that comprise an IL-27a part, optionally a linker and a fusion protein or tag at either N- or C-terminus. IL-27aL162C AH229'P243corresponds to SEQ ID NO: 3.

[0044] Figure 3 shows the fusion of muteins of IL-27a to HSA (Human Serum Albumin, SEQ ID NO: 91 ; PDB: 5Z0B). That constructs, regardless of a linker, shows significantly increased expression titers in mammalian cells while the functionality is maintained. Figure 3A shows the fusion of HSA at the N- or C-terminus of IL-27a, which results in significantly higher expression titer in mammalian cells compared to IL-27a constructs without fusion. In contrast, a significant effect of the various linkers on expression levels was not detected. Figure 3B shows a HeLa STAT1 bioluminescence assay, which confirms the preserved activity of untagged versus IL-27a fusion proteins from Expi293F™ supernatants. Mock, supernatantsof untransfected Expi293F™ cells, as well as supernatants of Expi293F™ cells transfected with HSA (SEQ ID NO: 91) only served as negative control in this activity assay. HeLa cells were stimulated with a constant volume of 25 pl of Expi293F™ supernatants in a total volume of 75 pl per well. For Figure 3A and Figure 3B the following legend applies: Lane 2 corresponds to SEQ ID NO: 2 and without signal sequence: SEQ ID NO: 55, lane 3 corresponds to SEQ ID NO: 3, lane 4 corresponds to SEQ ID NO: 14, lane 5 corresponds to SEQ ID NO: 15, lane 6 corresponds to SEQ ID NO: 16, lane 7 corresponds to SEQ ID NO: 17, lane 8 corresponds to SEQ ID NO: 18, lane 9 corresponds SEQ ID NO: 19, lane 10 corresponds to SEQ ID NO: 20, lane 11 corresponds to SEQ ID NO: 21 , lane 12 corresponds to SEQ ID NO: 22, lane 13 corresponds to SEQ ID NO: 23, lane 14 corresponds to SEQ ID NO: 24, lane 15 corresponds to SEQ ID NO: 25 and lane 16 corresponds to SEQ ID NO: 91.

[0045] Figure 4 shows the fusion of muteins of IL-27a to i-Tagi, i-Tags, i-Tags or i-Tag?, which significantly increases the expression titer in mammalian cells. Figure 4A shows that CL domain-based platform biotechnology can be utilized for a standardized protein production workflow, starting with the CL domain fusion on DNA level, protein expression, and a one-step CL domain-based affinity purification. Figure 4B demonstrates that increased secretion titers were achieved via fusion of IL-27aL162C’AH229'P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) to the kappa (K) constant light chain (CL) domain (SEQ ID NOs: 95 and 96) via a PAPAP linker, while no increase in secretion levels was achieved with the lambda ( ) CL fusion constructs (SEQ ID NO: 94). Expi293™ cells were transiently transfected with constructs of five K or CL domain repeats, alone (SEQ ID NOs: 95 and 96) or fused to IL-27aL162C’AH229‘P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76). Protein levels in medium samples were examined by reducing SDS-PAGE and Coomassie stain. Non-reducing samples confirmed proper formation of disulfide bonds by mobility shifts (see dotted line). Figure 4C shows that the number of K CL domains within the immunoglobulin domain-based tag (i-Tag, SEQ ID NO: 89) can be varied and fused to muteins of IL-27a as described herein. Muteins of IL-27a, K CL fusions of 1 , 3, 5, or 7 K CL domains connected by a PAPAP-linker were tested. After one-step affinity chromatography purification of Expi293F™ supernatants with the CaptureSelect™ KappaXP column, i-Tag (SEQ ID NO: 89) fusion samples were analyzed by reducing and non-reducing SDS-PAGE. Relative protein levels of the reduced samples were quantified, divided by the number of CL domains, and normalized to the i-Tagi (SEQ ID NO: 89) fusion signal. Figure 4D shows that expression levels increased up to 240- fold for the mutein of IL-27a when fused to i-Tags (SEQ ID NO: 90) via a PAPAP-linker (SEQ ID NO: 79) (Iane2, corresponding to SEQ ID NO: 37) compared to untagged mutein of IL-27a (lane 1 , corresponding to SEQ ID NO: 3, without signal sequence SEQ ID NO: 76). Expressionlevels were measured by ELISA of Expi293F™ supernatants. Figure 4D: Lane 1 corresponds to SEQ ID NO: 3 and without signal sequence corresponds to SEQ ID NO: 76, lane 2 corresponds to SEQ ID NO: 37.

[0046] Figure 5 shows the fusion of muteins of IL-27a to i-Tag (SEQ ID NO: 89). Regardless of a linker, the constructs significantly increase the expression titer in mammalian cells while the functionality is maintained. Figure 5A shows that secretion levels of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) by transfected Expi293F™ cells were increased by i-Tagi (SEQ ID NO: 89) or i-Tags (SEQ ID NO: 90) fusion (fusions correspond to SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35, 36, 37, 30, 38, 39 and 49), regardless of whether or which linker between IL-27alphaL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) and i-Tag (SEQ ID NO: 90) was used. Expression levels were measured by ELISA of Expi293F™ supernatants, n = 6, ± SD. Figure 5B shows that functionality of i-Tagged IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) with all linkers tested was preserved. A HeLa STAT1 bioluminescence assay was used to determine functionality of i-Tags-fused IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) (fusions: SEQ ID NOs: 27, 29, 32, 33 and 34), K CL protein, without or with linker, from Expi293F™ supernatants compared to mock, which served as negative control in this activity assay. HeLa cells were stimulated with adjusted volumes of Expi293F™ supernatants, for which protein concentrations were measured by ELISA and normalized to 104 nM before HeLa STAT1 cell stimulation, n = 2, ± SD. For Figure 5A the following legend applies: Lane 2 corresponds to SEQ ID NO: 3 (and without signal sequence: SEQ ID NO: 76), lane 3 corresponds to SEQ ID NO: 27, line 4 corresponds to SEQ ID NO: 28, lane 5 corresponds to SEQ ID NO: 29, lane 6 corresponds to SEQ ID NO: 32, lane 7 corresponds to SEQ ID NO: 33, lane 8 corresponds to SEQ ID NO: 34, lane 9 corresponds to SEQ ID NO: 35, lane 10 corresponds to SEQ ID NO: 36, lane 11 corresponds to SEQ ID NO: 37, lane 12 corresponds to SEQ ID NO: 30, lane 13 corresponds to SEQ ID NO: 38, lane 14 corresponds to SEQ ID NO: 39 and lane 15 corresponds to SEQ ID NO: 40. For Figure 5B the following legend applies: Lane 2 corresponds to SEQ ID NO: 27, lane 3 corresponds to SEQ ID NO: 29, line 4 corresponds to SEQ ID NO: 32, lane 5 corresponds to SEQ ID NO: 33 and lane 6 corresponds to SEQ ID NO: 34.

[0047] Figure 6 shows that N- as well as C-terminal i-Tags fusion to IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76, fusion: N-terminal: SEQ ID NO: 99 and C-terminal: SEQ ID NO: 37) enables a standardized protein production in mammalian cells. Figure 6A shows that secretion levels of N- as well as C-terminal i-Tagged via a PAPAP-linker(SEQ ID NO: 79) IL-27aL162C AH229’P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) were assessed by Coomassie stained SDS-PAGE. N- as well as C-terminal i-Tags fusion to IL-27aL162C AH229-p243corresponds to SEQ ID NO: 99 and SEQ ID NO: 37). Supernatants were harvested after transfection of Expi293F™ cells (load) and purified with the CaptureSelect™ KappaXP column via i-Tags (SEQ ID NO: 90) (purified protein). A bovine serum albumin (BSA) dilution series served as concentration standard to quantify protein levels. MW, molecular weight. Figure 6B shows that expression levels were significantly increased when fused to i-Tags (SEQ ID NO: 90) via a PAPAP-linker (SEQ ID NO: 79) with no detectable IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) expression when untagged. Figure 6C presents quantification of the Coomassie gel (A) and shows that the standardized purification via CaptureSelect™ KappaXP column resulted in a very high purity of above 95% for N- as well as C-terminally i-Tagged IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76). N- as well as C-terminal i-Tags fusion to IL-27aL162C AH229-p243corresponds to SEQ ID NO: 99 and SEQ ID NO: 37. Figure 6D shows THP-1 cell stimulation and subsequent CXCL10 ELISA proved that the functionality of i-Tagged via a PAPAP-linker (SEQ ID NO: 79) IL-27alphaL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) proteins is preserved.[00481 Figure 7 shows that IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion to i-Tags via a PAPAP linker with and without an interdomain linker between the K CL domains can be produced via standard procedures while protein functionality is maintained. Figure 7A shows that protein expression as well as purification of i-Tags IL-27aL162C AH229-p243(N- as well as C-terminal i-Tags fusion to IL-27aL162C AH229-p243corresponds to SEQ ID NO: 99 and SEQ ID NO: 37) via a PAPAP (SEQ ID NO: 79)-linker fusion constructs (IL-27aL162C AH229'P243-PAPAP-i-Tags corresponds to SEQ ID NO: 38) are comparable with and without GAGAG (SEQ ID NO: 92)-interdomain linkers between the K CL domains of the i-Tags (SEQ ID NO: 90) fusion protein. Figure 7B shows that functionality of i- Tagged IL-27aL162C AH229-p243(SEQ ID NO: 37) protein is unaffected of interdomain linkers, as proven by THP-1 assay and CXCL10 readout.

[0049] Figure 8 shows that the specifity of the functionality of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins was confirmed via competition assays with human Interleukin 27 receptor alpha (in each case, the construct with SEQ ID NO: 75 was used). THP-1 cell stimulation and subsequent CXCL10 ELISA proved that the functionality of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ IDNO: 76) fusion constructs (see SEQ ID NOs: 14, 15, 16 and 38) is given. Addition of human interleukin 27 receptor alpha (SEQ ID NO: 75) completely inhibited the function of IL-27aL162CAH229-P243 (gEQ |p |\| Q . 3wjthout signal sequence: SEQ ID NO: 76) fusion proteins (see SEQ ID NOs: 14, 15, 16 and 38). Thus, the specificity of the functionality was confirmed. For Figure 8 the following legend applies: Lane 1 corresponds to SEQ ID NO: 14, lane 2 corresponds to SEQ ID NO: 15, lane 3 corresponds to SEQ ID NO: 16, line 4 corresponds to SEQ ID NO: 38, lane 5 corresponds to a specific buffer (Phosphate-buffered saline, PBS) + SEQ ID NO: 75, lane 6 corresponds to SEQ ID NOs: 14 + 75, lane 7 corresponds to SEQ ID NOs: 15 + 75, lane 8 corresponds to SEQ ID NOs: 16 + 75, lane 9 corresponds to SEQ ID NOs: 39 + 75 and lane 10 corresponds to the specific buffer (Phosphate-buffered saline, PBS; used as negative control).

[0050] Figure 9 shows that IL-27aL162CAH229'P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (SEQ ID NOs: 43, 37, 44, 38 and 45) have an improved thermal stability compared to unfused IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) proteins. Figure 9A shows a HeLa STAT1 bioluminescence assay that was used to determine functionality of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) protein with and without an HSA fusion, from Expi293F™ supernatants compared to mock, which served as negative control in this activity assay. HeLa cells were stimulated with 25 pl of Expi293F™ supernatants in a total volume of 75 pl. n = 2, ± SD. Figure 9B shows that the remaining activity of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (see SEQ ID NOs: 43, 37, 44, 38 and 45) after incubation at 50 °C for 30 min was assessed by the HeLa STAT1 bioluminescence assay. In contrast to IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (see SEQ ID NOs: 43, 37, 44, 38 and 45), no functionality was measured for the untagged IL-27aL162C AH229-p243protein (SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) after incubation at 50 °C for 30 min. For Figure 9 the following legend applies: Lane 1 corresponds to SEQ ID NO: 3 (without signal sequence SEQ ID NO: 76), lane 2 corresponds to SEQ ID NO: 43, lane 3 corresponds to SEQ ID NO: 37, line 4 corresponds to SEQ ID NO: 44, lane 5 corresponds to SEQ ID NO: 38 and lane 6 corresponds to SEQ ID NO: 45.[00511 Figure 10 shows that IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins with an N-linked glycosylation site at an orthologous location to murine IL-27a N-glycosylation site are functional. THP-1 cell stimulation and subsequent CXCL10 ELISA proved that the functionality of IL-27aL162C AH229-P243(SEQ ID NO: 3, withoutsignal sequence: SEQ ID NO: 76) fusion proteins with and without the N-linked glycosylation site, D89N (IL-27alphaD89Nshown in SEQ ID NO: 70) is given. Addition of human interleukin 27 receptor alpha (SEQ ID NO: 75) completely inhibited the function of all IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (SEQ ID NOs: 14, 15, 16, 32, 51 , 52, 53 and 54). Thus, the specificity of the functionality in this assay was confirmed. Phosphate-buffered saline (PBS) was used as a negative control. For Figure 10 the following legend applies: Lane 1 (PBS) corresponds to a buffer, lane 2 corresponds to SEQ ID NO: 14, lane 3 corresponds to SEQ ID NO: 15, line 4 corresponds to SEQ ID NO: 16, lane 5 corresponds to SEQ ID NO: 32, lane 6 corresponds to SEQ ID NO: 51 , lane 7 corresponds to SEQ ID NO: 52, lane 8 corresponds to SEQ ID NO: 53, lane 9 corresponds to SEQ ID NO: 54, lane 10 corresponds to SEQ ID NOs: 14 + 75, lane 11 corresponds to SEQ ID NOs: 15 + 75, lane 12 corresponds to SEQ ID NOs: 16 + 75, lane 13 corresponds to SEQ ID NOs: 32 + 75, lane 14 corresponds to SEQ ID NOs: 51 + 75, lane 15 corresponds to SEQ ID NOs: 52 + 75, lane 16 corresponds to SEQ ID NOs: 53 + 75 and lane 17 corresponds to SEQ ID NOs: 54 + 75.[00521 Figure 11 shows that IL-27a Fc fusion proteins can be produced by standardized procedure and are functional. Figure 11 A shows that secretion levels of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) by transfected Expi293F™ cells were increased by tested fusion proteins. The expression levels of the IL-27aL162C AH229-p243Fc fusion proteins were lower compared to HSA fusions. Figure 11 B shows that BL-2 cells expressing the IL-27 receptor were incubated for 60 minutes with 1000 ng / mL IL-27aL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55), IL-27a LI62C, T238A, S24OA(S EQI D N 0 :7, without signal sequence SEQ ID NO: 77), IL-27aL162C-Fc fusion (SEQ ID NO: 72) or IL- 27aL162C AH229-P243-Fc fusion (SEQ ID NO: 73) and STAT1 activation was determined by immunoblotting against phosphorylated STAT 1 . For Figure 11 A the following legend applies: Lane 1 corresponds to SEQ ID NO: 2 (without signal sequence SEQ ID NO: 55), lane 2 corresponds to SEQ ID NO: 3 (without signal sequence SEQ ID NO: 76), lane 3 corresponds to SEQ ID NO: 14, lane 4 corresponds to SEQ ID NO: 15, lane 5 corresponds to SEQ ID NO: 23, lane 6 corresponds to SEQ ID NO: 72 and lane 7 corresponds to SEQ ID NO: 73. For Figure 11B the following legend applies: Lane 2 corresponds to SEQ ID NO: 2 (without signal sequence SEQ ID NO: 55), lane 3 corresponds to SEQ ID NO: 7 (without signal sequence SEQ ID NO: 77), lane 4 corresponds to SEQ ID NO: 72 and lane 5 corresponds to SEQ ID NO: 73.r00531 Figure 12 compares expression levels of various IL-27a-mutein fusion proteins by transfected Expi293F™ cells. After cell culture and protein expression, the protein concentrations in the supernatant were measured via the IL-27 ELISA. It shows that expression levels of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) by transfected Expi293F™ cells were increased by tested fusion proteins of said mutein with Fc, i-Tags or HSA. However, expression levels were not increased by fusion of said mutein with Hiss or V5-tag. The expression level of the fusion of IL-27aL162C AH229-p243with HSA were highest here. For Figure 12, the following legend applies: Lane 2 corresponds to SEQ ID NO: 3 (without signal sequence: SEQ ID NO: 76), lane 3 corresponds to SEQ ID NO:101 , lane 4 corresponds to SEQ ID NO: 102, lane 5 corresponds to SEQ ID NO: 73, lane 6 corresponds to SEQ ID NO: 37 and lane 7 corresponds to SEQ ID NO: 14.[00541 Figure 13 demonstrates that IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (SEQ ID NOs: 37 and 73) have an improved thermal stability compared to unfused IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) and compared to fusion of IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) with Hise or V5-tag proteins (SEQ ID NOs: 101 and 102). Figure 13A displays CXCL10 levels after THP-1 cells have been stimulated with Expi293F™ supernatant, containing either IL-27aL162C AH229'P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) or IL-27aL162C AH229-p243fusion proteins (SEQ ID NOs: 37, 73, 101 and 102). Supernatants were treated at 30°C or 50°C for 30 min alongside a mock sample, which served as negative control in this activity assay. THP-1 cells were stimulated with 25 nM of polypeptides and the CXCL10 concentration in the supernatant was measured with an ELISA as mentioned above n = 2, ± SD. Figure 13B shows that the remaining activity of IL- 27aL162C AH229’P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) or IL-27aL162CAH229-P243 fusjonproteins (see SEQ ID NOs: 43, 37, 44, 38 and 45) after incubation at 50 °C for 30 min. Activity was assessed by measuring CXCL10 level as described in Figure 13A. While IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) and its Hiss and V5-tagged fusion proteins (see SEQ ID NOs: 101 and 102) had reduced activity after treatment at 50°C, minor loss of functionality was measured for IL-27aL162C AH229-P243fusion proteins with Fc or i-Tags (SEQ ID NOs: 37 and 73) after incubation at 50°C for 30 min. For Figure 13A, the following legend applies: Lane 2 corresponds to SEQ ID NO: 3 (without signal sequence: SEQ ID NO: 76), lane 3 corresponds to SEQ ID NO: 101 , lane 4 corresponds to SEQ ID NO:102, line 5 corresponds to SEQ ID NO: 73, lane 6 corresponds to SEQ ID NO: 37. For Figure 13B the following legend applies: Lane 1 corresponds to SEQ ID NO: 3 (without signal sequence: SEQ ID NO: 76), lane 2 corresponds to SEQ ID NO: 101 , lane 3 corresponds toSEQ ID NO: 102, lane 4 corresponds to SEQ ID NO: 73 and lane 5 corresponds to SEQ ID NO: 37.DETAILED DESCRIPTION OF THE INVENTION

[0055] The following language and descriptions of certain preferred embodiments of the present invention are provided in order to further an understanding of the principles of the present invention. However, it will be understood that no limitations of the present invention are intended, and that further alterations, modifications, and applications of the principles of the present invention are also included.

[0056] In general, the present invention is directed to a fusion-polypeptide comprising a mutein of the a-subunit of human Interleukin 27, wherein at least one of the amino acid residues of the a-subunit of human Interleukin 27 selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated, and at least one pharmaceutically acceptable fusion partner.

[0057] In this context, it is noted that the term “human IL-27 alpha-subunit” or“p28” or “human IL-27 a-subunit” or “a-subunit of human Interleukin 27” or “IL-27a” as used herein refers to the polypeptide sequence of SEQ ID NO: 1 that has been deposited under UniProtKB accession number Q8NEV9. The a-subunit of human Interleukin 27 may be the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0058] In the context of the present invention, it may be that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1. Specifically, the numbering of sequence positions of said mutein of the a- subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0059] In the context of the present invention, when referring to the mature protein of human IL-27 a-subunit according to SEQ ID NO: 1 , wherein the signal sequence is cleaved, the numbering of sequence positions changes by 28 residues, i.e. position 162 then becomes position 134, respectively.

[0060] In one aspect, the present invention is directed to a fusion-polypeptide comprising a mutein of the a-subunit of human Interleukin 27 (SEQ ID NO: 1), wherein at least one of the amino acid residues of the a-subunit of human Interleukin 27 (SEQ ID NO: 1) selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated and wherein the numbering of sequence positions of said mutein of the a- subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1 , and at least one pharmaceutically acceptable fusion partner.

[0061] In some embodiments of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 one or more of the amino acids of sequence positions 1 to 28 is / are mutated. In some embodiments of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are mutated. It is preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are deleted. In this regard, it may be that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1. In one further aspect of the fusion- polypeptide as described herein, it may be that in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 29 to 40 are mutated. It is preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 29 to 40 are deleted (SEQ ID NO: 66). In this regard, it may be that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0062] In some embodiments of the fusion-polypeptide of the present invention, at least one pharmaceutically acceptable fusion partner is at least one tag. The tag is preferably not a V5- tag or a His-tag, specifically not a (His)e-tag. It is preferred in this regard that the at least one tag is at least one half-life extending tag. It is preferred that the at least one tag is at least one tag that enhances expression of the fusion-polypeptide compared to the absence of said tag. It is preferred in this regard that the at least one pharmaceutically acceptable fusion partner or tag or half-life extending tag or expression-enhancing tag is human serum albumin, a human serum albumin mutein or a fragment thereof. It is preferred in this regard that the at least one pharmaceutically acceptable fusion partner or tag or half-life extending tag is one or more of the constant domain of human kappa immunoglobulin light chain (also called i-Tagherein), preferably w / o the C-terminal cysteine. It is preferred that the at least one pharmaceutically acceptable fusion partner is a pharmaceutically acceptable, proteinogenic fusion partner. It is preferred that the at least one pharmaceutically acceptable fusion partner is not a pharmaceutically acceptable, non-proteinogenic fusion partner.

[0063] In general, a pharmaceutically acceptable fusion partner is a molecule that can be combined with e.g. a therapeutic protein to improve its properties in a drug formulation. These partners are typically peptides, proteins, or other molecules that enhance the stability, halflife, or bioavailability of the therapeutic protein. As used herein, the term “pharmaceutically acceptable fusion partner” means any protein or peptide that can be linked with or attached to the mutein as described herein, such that it can serve as a drug and fulfils the requirements of pharmaceutical acceptability. The phrase "pharmaceutically acceptable" generally means those compounds, materials, compositions, and herein fusion partner, which are, using safe medical judgment, and following all applicable governmental regulations, safe and suitable for use and administration to a human or animal.

[0064] In the context of the present invention, the term “tag” may refer to a sub-domain or peptide sequence of a fusion-polypeptide linked to e.g. a protein of interest, which is in the context of the present invention a mutein of the a-subunit of human Interleukin 27.

[0065] The term “half-life extending tag” as used herein means any tag as defined herein for extending the half-life of biological therapeutics, wherein the protein's lifespan is measured in terms of its half-life.

[0066] The term “expression-enhancing tag” as used herein means any tag as defined herein for enhancing the expression of the fusion-polypeptide compared to the expression of the mutein of the a-subunit of human Interleukin 27 without the pharmaceutically acceptable fusion partner / without the respective tag.

[0067] “Human serum albumin” as used in the context of the present invention is a highly water-soluble globular monomeric plasma protein with a relative molecular weight of 67 KDa, consists of 585 amino acids and it is the most abundant of all plasma proteins (see SEQ ID NO: 91).

[0068] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 1 .

[0069] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 2.

[0070] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 7.

[0071] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 55.

[0072] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 76.

[0073] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 77.

[0074] By "identity" or “sequence identity” as used herein is meant a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present invention means the percentage of pair-wise identical residues - following (homology) alignment of a sequence of a polypeptide of the present invention with a sequence in question - with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

[0075] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf.Altschul, S. F. 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 sequences (matrix: BLOSLIM 62; gap costs: 11.1 ; cutoff value set to 10'3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0076] It is preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated. It is further preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated. It is further preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated. In this regard, it may be that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0077] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 160 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1 .

[0078] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1 .

[0079] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunitof human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0080] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0081] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 164 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0082] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 165 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0083] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0084] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 181 is mutated. Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.

[0085] In one aspect of the fusion-polypeptide of the present invention, in the mutein of the a- subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated.Here, it is preferred that the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a- subunit of human Interleukin 27 according to SEQ ID NO: 1 .

[0086] In some embodiments of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residue(s) selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated to cysteine. It is preferred for the fusion-polypeptide according to the present invention that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residue(s) selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated to cysteine. It is further preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residue(s) selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated to cysteine. It is further preferred that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residue(s) selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated to cysteine.

[0087] It is preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 160 is mutated to cysteine (SEQ ID NO: 56). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated to cysteine (SEQ ID NO: 57). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated to cysteine (SEQ ID NO: 2). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated to cysteine (SEQ ID NO: 9). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 164 is mutated to cysteine (SEQ ID NO: 58). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated to cysteine (SEQ ID NO: 59). It is further preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 181 is mutated to cysteine (SEQ ID NO: 60). It is also preferred in one aspect for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated to cysteine (SEQ ID NO: 61).

[0088] It is preferred for the fusion-polypeptide of the present invention that said fusion- polypeptide comprises as mutein of the a-subunit of human Interleukin 27 an IL-27a (L162C) (AC) protein, also mentioned as p28L162C AC. Therein, the C-terminus, which may comprise the amino acids 229 to 249 may be deleted (SEQ ID NO: 3). However, it may also be that the C- terminus, which may comprise the amino acids 231 to 249 may be deleted (SEQ ID NO: 10). Further, it may also be that the C-terminus, which may comprise the amino acids 234 to 249 may be deleted (SEQ ID NO: 11). It can also be that the C-terminus, which may comprise the amino acids 238 to 249 may be deleted (SEQ ID NO: 12). This mutein of the a-subunit of human Interleukin 27 may then be fused to the at least one pharmaceutically acceptable fusion partner as described herein, thereby forming a fusion polypeptide of the present invention.

[0089] It is also preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 comprises one or more salt-bridge(s). Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. They contribute to protein structure and to the specificity of interaction of proteins with other biomolecules.

[0090] It is further preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 is further engineered to be devoid of any O- glycosylated residues. This can, for example, be achieved by deleting the C-terminus of the a-subunit of human Interleukin 27 of SEQ ID NO: 1 , which may be amino acid residues 229 to 243 of SEQ ID NO: 1 . However, this can also be achieved by mutating amino acid residues 238 and / or 240 of the a-subunit of human Interleukin 27 of SEQ ID NO: 1.

[0091] It is therefore preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are mutated. It is further preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are deleted. It is more preferred in this regard that in the mutein of the a-subunit of human Interleukin 27 the amino acid residues of sequence positions 229 to 243 are deleted (SEQ ID NO: 62). In this embodiment, the numbering of the amino acid residues may correspond to the amino acid numbering of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1. However, it may also be that the C-terminus isdeleted, such that the amino acids 231 to 249 of the C-terminus are deleted (SEQ ID NO: 67). Further, it may also be that the C-terminus is deleted, such that the amino acids 234 to 249 of the C-terminus are deleted (SEQ ID NO: 68). It can also be that the C-terminus is deleted, such that the amino acids 238 to 249 of the C-terminus are deleted (SEQ ID NO: 69).

[0092] It is also preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 is / are mutated. In some embodiments of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is mutated. In some embodiments of the fusion-polypeptide of the present invention, in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is mutated. It is preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is replaced by alanine (SEQ ID NO: 63). It is also preferred for the fusion-polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is replaced by alanine (SEQ ID NO: 64). It is further preferred for the fusion- polypeptide of the present invention that in the mutein of the a-subunit of human Interleukin 27 of the fusion-polypeptide the amino acid residues at sequence positions 238 and 240 are replaced by alanine (SEQ ID NO: 65).

[0093] In some embodiments of the fusion-polypeptide of the present invention, the mutein of the a-subunit of human Interleukin 27 further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 mutations at one or more sequence positions selected from the group consisting of sequence positions 229, 230, 231 , 232, 233, 234, 235, 236, 237, 239, 241 , 242 and 243.

[0094] The mutein of the a-subunit of human Interleukin 27 of the fusion-polypeptide may further be mutated at the amino acid residue 89. Preferably, the amino acid residue at sequence position may be mutated to asparagine (N). When the amino acid at sequence position 89 of SEQ ID NO: 1 is mutated to N, this results in the amino acid sequence according to SEQ ID NO: 70.

[0095] It is also preferred for the fusion-polypeptide of the present invention that the mutein of the a-subunit of human Interleukin 27 comprises one or more disulfide-bridge(s). Disulfide bridges, or disulfide bonds or S-S bonds, are covalent links between the sulphur atoms of two cysteine amino acids.

[0096] In some embodiments of the fusion-polypeptide of the present invention the mutein of the a-subunit of human Interleukin 27 is fused at its N-terminus to the at least one pharmaceutically acceptable fusion partner.

[0097] In some embodiments of the fusion-polypeptide of the present invention the mutein of the a-subunit of human Interleukin 27 is fused at its C-terminus to at least one pharmaceutically acceptable fusion partner.

[0098] It may be for the fusion-polypeptide of the present invention that the mutein of the a- subunit of human Interleukin 27 is fused to the at least one pharmaceutically acceptable fusion partner via a linker. It may be for the fusion-polypeptide of the present invention that the linker is a linker comprising at least 3 guanosines, preferably a GGGS-linker (SEQ ID NO: 78), a linker comprising the sequence PAPAP (SEQ ID NO: 79), or a helical linker. It may be for the fusion-polypeptide of the present invention that the linker has a length of 0 to 30 residues. In the context of the present invention, the linker may be any one of SEQ ID NOs: 78 to 88.

[0099] The linker used for the fusion-polypeptide according to the present invention may be a cleavable or a non-cleavable linker. A cleavable linker could be used if it is desired to cleave the pharmaceutically acceptable fusion partner and the mutein of the a-subunit of human Interleukin 27 at a defined position after purification of the fusion-polypeptide. A non-cleavable linker may be used, if e.g. the pharmaceutically acceptable fusion partner is further used for detection of the fusion-polypeptide or is necessary to enhance the stability of the fusion- polypeptide or to increase its half-life and / or to increase its expression level compared to the absence of said fusion partner.

[0100] Suitable linkers are known to the skilled person and examples of such linkers are a Ga-linker, such as a GGGS-linker (SEQ ID NO: 78), a G4-linker, such as a GGGGS-linker (SEQ ID NO: 80), a Gs-linker, a PAPAP-linker (SEQ ID NO: 79), a APAPAPA-linker (SEQ ID NO: 85), a APAPAPAPAPKPA-linker (SEQ ID NO: 86), a APAPAPAPAPAPA-linker (SEQ ID NO: 87), and a A(EAAAK)4A-linker (such as SEQ ID NO: 84) or one or more disulfide bridge(s). For example, a suitable linker may be A(EAAAK)iA (SEQ ID NO: 81), A(EAAAK)2A (SEQ ID NO: 82), A(EAAAK)3A (SEQ ID NO: 83), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 84), APAPAPA (SEQ ID NO: 85), APAPAPAPAPKPA (SEQ ID NO: 86), APAPAPAPAPAPA (SEQ ID NO: 87), and A(EAAAK)4A (SEQ ID NO: 88). A further suitable linker may be GAGAG (SEQ ID NO: 92) between each CL domain as interdomain linker (IDL).

[0101] In one aspect, the present invention provides a fusion-polypeptide, wherein the fusion-polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to SEQ ID NO: 54, SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74. In one aspect of the present invention, the fusion-polypeptide may be a fusion-polypeptide as depicted in any one of Figures 1 to 13.

[0102] In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion-polypeptide according to the present invention. It is preferred for said nucleic acid molecule that it is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule. It is further preferred that the regulatory sequence comprises a promoter sequence. The term “promoter” or “promoter sequence” means a DNA sequence which initiates and directs the transcription of a gene into a RNA transcript in cells. In some embodiments, the nucleic acid molecule according to the present invention is comprised in a vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0103] In one further aspect, the present invention also provides a host cell containing the nucleic acid molecule according to the present invention. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g. insect cells, yeast or mammalian cells). Preferably, the host cell is an eukaryotic cell.

[0104] In yet another aspect, the present invention provides an immune modulator comprising the fusion-polypeptide according to the present invention. Immunomodulation is described as regulation and modulation of immunity that might be achieved by reducing or enhancing the immune response. In particular, the fusion-polypeptide according to the present invention may enable restoring impaired immune functions in a disease setting.

[0105] The present invention further provides in another aspect the use of a fusion- polypeptide for the manufacture of a medicament for treating a disease, preferably a disease in a mammal, more preferably a disease in a human. It is preferred in this regard that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0106] Another aspect of the present invention provides the fusion-polypeptide as described herein for use as a medicament.

[0107] A further aspect of the present invention relates to the fusion-polypeptide as described herein for use in the treatment or prevention of a disease. In this regard, it is preferred that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0108] In another aspect, the present invention provides a method of treating a disease comprising the step of administering a composition comprising a fusion-polypeptide as described herein to a subject, preferably to a mammal, more preferably to a human, in need thereof. It is further preferred for the method of treating that the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

[0109] An overview of the SEQ ID NOs and detailed sequences, as used in the context of the present invention, is given in the following Table 1. The description of the sequences, shown in the sequence listing and as used in the context of the present invention, is as follows:Table 1 :It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.The term "and / or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".The terms “polypeptide” and “protein” can be used interchangeable in the context of the present invention.The term “less than” or in turn “more than” does not include the concrete number.For example, “less than 20” means less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. “more than 80 %” means more than or greater than the indicated number of 80 %.Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes, when used herein, with the term “having”. When used herein, “consisting of" excludes any element, step, or ingredient not specified.The term “including” means “including, but not limited to”, “including” and “including, but not limited to” are used interchangeably herein.It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments, only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.The content of all documents and patent documents cited herein is incorporated by reference in their entirety.A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.The invention is further characterized by the following items:ITEMS:1. A fusion-polypeptide comprising- a mutein of the a-subunit of human Interleukin 27, wherein at least one of the amino acid residues of the a-subunit of human Interleukin 27 selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated, and- at least one pharmaceutically acceptable fusion partner.2. The fusion-polypeptide according to item 1 , wherein the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.3. The fusion-polypeptide according to item 1 or 2, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are mutated.4. The fusion-polypeptide according to item 3, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are deleted.5. The fusion-polypeptide according to any one of the preceding items, wherein the at least one pharmaceutically acceptable fusion partner is at least one tag.6. The fusion-polypeptide according to item 5, wherein the at least one tag is at least one halflife extending tag.7. The fusion-polypeptide according to any one of the preceding items, wherein the least one pharmaceutically acceptable fusion partner or tag or half-life extending tag is human serumalbumin or a fragment thereof or one or more constant domains of human kappa immunoglobulin light chain, optionally without the C-terminal cysteine.8. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a- subunit of human Interleukin 27 according to SEQ ID NO: 1.9. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated.10. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated.11 . The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated.12. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated.13. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated.14. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated.15. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected fromthe group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated to cysteine.16. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 180, 181 and 182 is / are mutated to cysteine.17. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated to cysteine.18. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated to cysteine.19. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 160 is mutated to cysteine (SEQ ID NO: 56).20. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 161 is mutated to cysteine (SEQ ID NO: 57).21 . The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 162 is mutated to cysteine (SEQ ID NO: 2).22. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 163 is mutated to cysteine (SEQ ID NO: 9).23. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 164 is mutated to cysteine (SEQ ID NO: 58).24. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 165 is mutated to cysteine (SEQ ID NO: 71).25. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated to cysteine (SEQ ID NO: 59).26. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 181 is mutated to cysteine (SEQ ID NO: 60).27. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated to cysteine (SEQ ID NO: 61).28. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 comprises one or more salt-bridges.29. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 is further engineered to be devoid of any O- glycosylated residues.30. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are mutated.31. The fusion-polypeptide according to item 30, wherein in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus are deleted.32. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residues of sequence positions 229 to 243 are deleted (SEQ ID NO: 62).33. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 is / are mutated.34. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is mutated.35. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is mutated.36. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residues at sequence positions 238 and 240 are mutated.37. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is replaced by alanine (SEQ ID NO: 63).38. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is replaced by alanine (SEQ ID NO: 64).39. The fusion-polypeptide according to any one of the preceding items, wherein in the mutein of the a-subunit of human Interleukin 27 of the fusion-polypeptide the amino acid residues of the a-subunit of human Interleukin 27 at sequence positions 238 and 240 are replaced by alanine (SEQ ID NO: 65).40. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 mutations at one or more sequence positions selected from the group consisting of sequence positions 229, 230, 231 , 232, 233, 234, 235, 236, 237, 239, 241 , 242 and 243.41. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 comprises one or more disulfide-bridges.42. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 is fused at its N-terminus to the at least one pharmaceutically acceptable fusion partner.43. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 is fused at its C-terminus to the at least one pharmaceutically acceptable fusion partner.44. The fusion-polypeptide according to any one of the preceding items, wherein the mutein of the a-subunit of human Interleukin 27 is fused to the at least one pharmaceutically acceptable fusion partner via a linker.45. The fusion-polypeptide according to item 44, wherein the linker is a linker comprising at least 3 guanosines, preferably a GGGS-linker, a linker comprising the sequence PAPAP, or a helical linker, and / or wherein the linker is preferably selected from the group consisting of any one of SEQ ID NOs: 78 to 88.46. The fusion-polypeptide according to item 44 or 45, wherein the linker has a length of 0 to 30 residues.47. The fusion-polypeptide according to any one of the preceding items, wherein the fusion- polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 54, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and / or wherein the fusion-polypeptide is a fusion-polypeptide as depicted in any one of Figures 1 to 11.48. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion- polypeptide according to any one of items 1 to 47.49. The nucleic acid molecule according to item 48, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.50. The nucleic acid molecule according to item 49, wherein the regulatory sequence comprises a promoter sequence.51. The nucleic acid molecule according to any one of items 48 to 50 comprised in a vector.52. A host cell containing a nucleic acid molecule of any one of items 48 to 51.53. An immune modulator comprising the fusion-polypeptide of any one of items 1 to 47.54. Use of a fusion-polypeptide of any one of items 1 to 47 for the manufacture of a medicament for treating a disease, preferably a disease in a mammal, more preferably a disease in a human.55. The use according to item 54, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver or inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis, preferably abdominal sepsis or urosepsis, and septic shock.56. The fusion-polypeptide according to any one of items 1 to 47 for use as a medicament.57. The fusion-polypeptide according to any one of items 1 to 47 for use in the treatment or prevention of a disease.58. The fusion-polypeptide for use according to item 57, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver or inflammatory diseases of the bile ducts; chronic inflammatory diseases or acute inflammatory diseases; sepsis, preferably abdominal sepsis or urosepsis, and septic shock.59. A method of treating a disease comprising the step of administering a composition comprising a fusion-polypeptide of any one of items 1 to 47 to a subject, preferably to a mammal, more preferably to a human, in need thereof, preferably in combination with adjuvants-therapy.60. The method of treating according to item 59, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver or inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis, preferably abdominal sepsis or urosepsis, and septic shock.EXAMPLES

[0110] Materials & Methods

[0111] Constructs

[0112] Constructs for mammalian protein expression were synthesized human codon optimized by GeneArt (Thermo Fisher Scientific) in the pcDNA3.4 TOPO vector (Gibco). For the tag (= i-Tag, SEQ ID NO: 89) the human immunoglobulin kappa constant domain (UniProt accession number: P01834) and repeats thereof were used, the human IL-27a / p28 (UniProt accession number: Q8NEV9, SEQ ID NO: 1) signal sequence (aa 1 to 28 of SEQ ID NO:1) was used as N-terminal signal sequence. An exemplary used DNA sequence for IL-27aL162CA229-243 fusec|toconstant domain of the human immunoglobulin kappa light chain without a codon for the C-terminal cysteine was used, which encodes the amino acid sequence MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLSEV RGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGG LGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNCPEEEEEEEEEEEEERKGLL PGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (signal sequence underlined; i-Tagi sequence highlighted in bold) (SEQ ID NO: 27). Mutants were generated by site-directed mutagenesis. All constructs were sequenced.

[0113] Cell culture and protein expression

[0114] Expi293F™ cells (Gibco) were cultured and transfected according to the manufacturer’s protocol. After expression, medium was centrifuged twice (first, at 5,000 g for 30 min and second, at 20,000 g for 15 min, both at 4 °C). Protein expression and purity were assessed by supplementing 0.2 volumes of 5x Laemmli buffer (0.3125 M Tris / HCI pH 6.8, 10% SDS, 50% glycerol, bromphenol blue) containing either 10% (v / v) p-mercaptoethanol (P-Me) for reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 100 mM / V-ethylmaleimide (NEM) for non-reducing SDS-PAGE. Long-term storage of supernatants directly used in functional assays the storage temperature was -80°C. For purification, the supernatant was supplemented with Roche complete Protease Inhibitor w / o EDTA (Roche Diagnostics) and sterile filtered at 0.22 pm.

[0115] Protein purification

[0116] After expression in Expi293F™ cells (Thermo Fisher, A14527) the proteins were purified via affinity chromatography.

[0117] The i-Tag (SEQ ID NO: 89) fusion constructs were purified by application to a CaptureSelect™ KappaXP pre-packed column (Thermo Fisher, 494321201). Purification was performed according to the manufacturer’s instructions, using 10 mM sodium phosphate (pH 7.4) as equilibration buffer and 100 mM sodium acetate (pH 3.0) for protein elution, with 1 M Tris buffer (pH 8.0) for neutralization of elution fractions. Purified i-Tag (SEQ ID NO: 89) fusion proteins were dialyzed o / n in 20 mM HEPES, 150 mM NaCI (pH 7.4) before further characterization.

[0118] HSA fusion constructs were purified via a YMC EcoPlus TAC10 / 125PE0-AB-2 column with packing adapter packed with CaptureSelect™ Human Albumin Affinity Matrix resin (Thermo Fisher, 191297050). Purification was performed according to the manufacturer’s instructions, using Phosphate-buffered saline (PBS; pH 7.4) as equilibration buffer and 0.1 M Glycine (pH 3.0) for protein elution, with 1 M Tris buffer (pH 8.0) for neutralization of elution fractions. Purified HSA (SEQ ID NO: 91) fusion proteins were dialyzed o / n in 20 mM HEPES, 150 mM NaCI (pH 7.4) before further characterization.

[0119] Fc fusion constructs were purified by application to a CaptureSelect™ FcXP prepacked column (Thermo Fisher, 494371201). The pH of sterile filtered expression medium was adjusted to pH 7.4 with 1 M Tris buffer (pH 8.0) and then loaded on the FcXP column. Purification was performed according to the manufacturer’s instructions, using Phosphate- buffered saline (pH 7.4) as equilibration buffer and 0.1 M sodium acetate (pH 3.0) for protein elution, with 1 M Tris buffer (pH 8.0) for neutralization of elution fractions. The pooled eluate was then dialyzed against Phosphate-buffered saline (pH 7.4) over night, concentrated to 2 mL and then loaded on the Superose 6 column (Sigma-Aldrich, GE29-0915-96) for polishing. The elution fractions containing the Fc-fusion proteins were pooled, sterile filtered via 0.22 .m filter (Corning 431218), aliquoted and frozen at -80°C.

[0120] Activity assays in BL-2 cells

[0121] STAT phosphorylation experiments were performed with 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 incubated in 48-well plates (2x106cells / well) in RPMI- 1640 medium with 0.5% BSA for 60 min with 1000 ng / mL IL-27alphaL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55), IL-27alpha LI62C, T23SA, S24OA (SEQ ID NQ:7 without signal sequence SEQ ID NO: 77), IL-27alphaL162C-Fc fusion (SEQ ID NO: 72) or IL- 27alphaL162C An229-P243_pcfUSjon(SEQ ID NO: 73) Expi293 supernatant or non-transfectedcontrol Expi293 supernatant in 48-well plates (2 x106cells / well). The reaction was stopped by diluting the cells with ice-cold PBS buffer and lysis in NP40 lysis buffer (with protease and phosphatase inhibitors). Phosphorylates and total STAT protein was detected by immunoblotting. Rabbit antibodies from Cell Signaling Technology were used (P-STATI, #9167; STAT1 , #9172) (see Fig. 11 B).

[0122] HeLa STAT1 assayHeLa STAT1 cells (Signosis, SL-0004-FP) were used to measure STAT1 phosphorylation by bioluminescence. For one 96-well plate (Sigma-Aldrich, SIAL0596), 1 x 106HeLa cells or 1 x 104cells / well were seeded and incubated over night at 37 °C and 5% CO2. Thereafter, the cells were stimulated with supernatants containing IL-27a proteins and incubated for 18 h. After incubation, the cells were lysed using lysis buffer (Promega, E1500). Then, Luciferase Assay reagent (Promega, E1500) was added, and the bioluminescence signal was measured at 560 nm in a plate reader (BMG Labtech CLARIOstar®). To assess stability of IL-27a proteins, HeLa STAT1 activation tests were performed after incubation of the protein at 4°C, 30°C, 40°C, 50°C or 60°C as indicated for 30 min.

[0123] THP-1 assay

[0124] 2 x 105THP-1 cells / ml were seeded in RPMI medium containing 10% heat-inactivated FCS, 1% antibiotic / antimycotic, and 25 nM PMA for differentiation. After 48 h incubation, cells were washed with PBS and fresh RPMI medium without PMA was added. The next day, before adding IL-27a-fusion variants, the medium was aspirated and 100 pL fresh RPMI without PMA was added. Cells were then stimulated with IL-27a-fusion variants (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM and 0.01 nM: see Figure 6D and 7B; 10 nM: see Figure 8A and 10A) for 24 h and then harvested at 300 x g for 15 min at 4 °C. For specificity control experiments by receptor inhibition, soluble IL-27Ra-Fc-6xHIS (SEQ ID NO: 75) in PBS was added to each well subsequent to the RPMI medium. After waiting for 10 minutes, suspension containing IL- 27a-variants were added and the 24-well plates were incubated in the cell culture incubator for 24 h and then harvested at 300 x g for 15 min at 4 °C. The supernatants were transferred to fresh tubes, frozen at -80°C until quantification of CXCL10 levels via enzyme-linked immunosorbent assay (ELISA).[001251 ELISA

[0126] Concentrations of IL-27a (fusion) proteins were measured by IL-27 ELISA (abeam, ab2667812) according to the manufacturer’s protocol. The CXCL10 ELISA (R&D, DIP100)was performed according to the manufacturer's instructions. Absorption signals were measured in a plate reader (BMG Labtech CLARIOstar®).

[0127] Quantification, statistics, and illustrations

[0128] Coomassie-stained SDS-PAGE gels were imaged with Gel Doc™ EZ Imaging System with Image Lab 5.2.1 (BioRad) and were quantified using Imaged (Fiji). If no bands were visible, the quantification, thus the x-fold increase, was calculated by assuming that the total amount of protein was just below the detection limit of the Coomassie stain (0.1 pg / band) (Brunelle and Green 2014), and thus 0.1 pg / band were assumed. Statistical analyses and graph illustration were performed using Prism (GraphPad Software). Applied statistical test types and experimental sample sizes are stated in the figure legends. Where no statistical data are shown, experiments were performed at least two times.

[0129] Experimental examples

[0130] Example 1 : Design of an IL-27aL162C-based biopharmaceutical (Figure 1 and 2).The inventors used a variant of the human interleukin (IL) 27a / p28 subunit that contains a stabilizing disulfide bond (IL-27L162C) (Muller, Friedl et al. 2019)) and additionally a C-terminal truncation (AH229-P243), which abrogates O-glycosylation at C-terminal sites (Bohnacker, Hildenbrand et al. 2020) (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76). Other key features of the human IL-27a molecule, which the inventors analyzed, comprised the unstructured region at the N-terminus (AF29-L40) (SEQ ID NO: 66) and the endogenous O- glycosylation sites via point mutation at T238A and S240A (SEQ ID NO: 65) (Figure 1). IL- 27a has immunomodulatory properties (Min, Kim et al. 2021) and thus is an attractive molecule for biomedical applications (Figure 1). However, like many proteins in a non-antibody format IL-27a is difficult to produce (Muller, Friedl et al. 2019). Therefore, the inventors developed a strategy to develop IL-27aL162C-based proteins that fulfill the criteria of biopharmaceuticals comprising i.a. developability, e.g. expression titer, efficient purification and stability, as well as functionality. For this, the inventors developed various IL-27aL162C- based fusion constructs comprising established fusion technologies such as human serum albumin (HSA) (SEQ ID NO: 91), fragment crystallizable region (Fc) derived from antibodies, a novel enabling technology based on the human kappa immunoglobulin light chain that was named i-Tag (SEQ ID NO: 90). The inventors also analyzed various linker options during the development of IL-27a-based biopharmaceuticals. The strategy of development of IL-27a- based biopharmaceuticals is outlined in Figure 2 depicting the alpha subunit of IL-27 in blue derived from the IL-27 quaternary receptor signaling complex (PDB: 7U7N).

[0131] Example 2: HSA fusion enables standardized production of ll_-27aL162C-based biopharmaceuticals while the functionality of ll_-27aL162C-based proteins is maintained (Figure 3)

[0132] The inventors of the present invention analyzed the impact of HSA fusion to IL- 27aL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55), ||_-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76), and of different linkers on the expression level of the fusion polypeptides (see Figure 3A). Therefore, Expi293F™ cells were transfected with constructs for the expression of fusion polypeptides IL-27aL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55) or IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) or comprising HSA (SEQ ID NO: 91) fused to the C- or N-terminus of the IL-27aL162C AH229’P243(SEQ|D no.3 without signa| sequence SEQ ID NO: 76) polypeptide without (SEQ ID NO: 14) or with a linker having one of the following amino acids sequences: GGGS (SEQ ID NO: 78) or GGGGS (SEQ ID NO: 80), PAPAP (SEQ ID NO: 79), A(EAAAK)iA (SEQ ID NO: 81), A(EAAAK)2A (SEQ ID NO: 82), A(EAAAK)3A (SEQ ID NO: 83). After cell culture and protein expression, the protein concentrations were measured via the IL-27 ELISA. As shown in Figure 3A, the secretion of the fusion polypeptide (lanes No. 4 to 15, SEQ ID NOs: 14 to 25) is generally increased compared to the untagged I L-27a proteins (lanes No. 2 and 3). Further, the secretion level of the fusion IL-27aL162C AH229-P243-protein comprising the HSA fusion at the N-terminus (SEQ ID NOs: 20 to 25) (lanes No. 10 to 15) is generally higher than the secretion level of the fusion IL-27aL162C AH229-P243-protein comprising the HSA-fusion at the C-terminus (SEQ ID NOs: 14 to 19) (lanes No. 4 to 9) (see Figure 3A). For testing the functionality of I L-27aL162C AH229-P243-HSA-fusion proteins (SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 and 25) compared to untagged IL-27aL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55) and I L-27aL162C AH229-P243proteins (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76), the HeLa STAT 1 bioluminescence assay was used (see Figure 3B). For this, a constant volume of 25 pl of the supernatants of Expi293F™ cells transfected with constructs for secretion of fusion polypeptides containing the IL-27aL162(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55), IL-27aL162C AH229’P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) proteins or IL-27aL162C AH229-P243protein fused at the C- or N-terminus to the HSA fusion protein without (lanes No. 4 and 10) (SEQ ID NO: 14 and SEQ ID NO: 20) or a linker having one of the following amino acids sequences: GGGS (lane No. 5, SEQ ID NO: 15) (linker: SEQ ID NO: 78) or GGGGS (lane No. 11 , SEQ ID NO: 21) (linker: SEQ ID NO: 80), PAPAP (linker: SEQ ID NO 79:), A(EAAAK)iA (linker: SEQ ID NO: 81), A(EAAAK)2A (linker: SEQ ID NO: 82), A(EAAAK)3A (linker: SEQ ID NO: 83) were addedto a total stimulation volume of 75 pl. The mock (lane No. 1) and HSA only (lane No. 16, SEQ ID NO: 91) control of the above experiment served as negative controls. The HeLa cells were stimulated with a constant volume of 25 pl of the respective expression medium that were added to 50 pl of DM EM medium containing 0.1 % FCS resulting in a total volume of 75 pl for HeLa STAT1 cell stimulation. After 18 hours the resulting bioluminescence was measured. As shown in Figure 3B, the secreted fusion polypeptides were functional regardless of if a linker was present or not. However, the bioluminescence was generally increased when supernatants containing the fusion polypeptides with the C-terminal HSA fusion were used.

[0133] Example 3: i-Tag is a suitable fusion protein for IL-27aL162C-based proteins (Figure 4)

[0134] A protein-tag should ideally be inert, improve expression, and be amenable to simple purification procedures. An enabling platform biotechnology that in addition enhances the quality attributes of the protein of interest, such as stability, provides further significant advantages for the production process as well as the product per se. Previous studies demonstrate that the constant domain of the antibody light chain (CL) combines these desired features. Most biopharmaceuticals are produced as secreted proteins from the medium of mammalian cells. Previous work has shown that CL does not significantly engage chaperones in the endoplasmic reticulum (ER), where secretory proteins pass through and acquire their structure (Behnke, Mann et al. 2016, Adams, Oster et al. 2019). This argues for a particularly robust folding process of the CL domain in cells. Furthermore, the CL domain is resistant against misfolding (Feige, Groscurth et al. 2008). Antibody light chains, that contain the CL domain, are naturally secreted in high quantities from human B cells without any harm to the human body (Nakano, Miyazaki et al. 2006). Lastly, different e.g. nanobody-based, GMP- compatible purification procedures exist that allow the purification of antibodies via binding of their CL domain. This should allow for the simple purification of any protein where the CL domain is fused to (see Figure 4A). Humans contain two types of antibody light chains, which differ in the CL domain: the K or subtype (Hill, Delaney et al. 1966, Das, Nikolaidis et al. 2008). The inventors thus first compared the effect of fusing either K or X CL domains fused to I L-27alphaL162C AH229'P243protein (SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76). When fused to five sequential K CL domains (SEQ ID NO: 37), strong expression and secretion of to IL-27aL162C AH229-p243fused to 5x K CL domains (SEQ ID NO: 95) from Expi293F™ cells was observed, whereas the corresponding X CL fusion (SEQ ID NO: 94) showed no detectable secretion (see Figure 4B). The same was observed for either five sequential K or X CL domains alone (Figure 4B) (SEQ ID NO: 89 and SEQ ID NO: 90). Theinventors of the present invention thus continued testing different K CL fusion lengths, leveraging the modular character of the CL domain as a building block. Since these data indicated that the K CL protein is potentially suited as a platform enabling biotechnology, the inventors named it i-Tag (SEQ ID NO: 89) (Figure 4C). With one to seven K CL domains (i- Tagi, i-Tag3, i-Tag5, i-Tag7) fused ||_-27aL162C AH229-p243(SEQ ID NO: 32, SEQ ID NO: 97, SEQ ID NO: 38, SEQ ID NO: 98), expression levels of the protein of interest were significantly increased. In each case, the i-Tag fusion enabled a simple one-step purification process of the protein of interest via a commercially available column with immobilized anti-K CL nanobodies (CaptureSelect™ KappaXP) (see Figure 4C). Building on these findings, the inventors further confirmed that the K CL fusion tag (i-Tags, SEQ ID NO: 90) significantly increases the secretion levels of IL-27aL162C AH229'P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) (see Figure 4D). To quantify the expression titer of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) or IL-27aL162C AH229-p243-PAPAP-i-Tag5(SEQ ID NO: 38)-fusion construct, the inventors of the present invention performed an IL-27 ELISA. The results are depicted in Figure 4D demonstrating an increase of expression titer >200-fold upon i-Tag5-fusion via a PAPAP-linker to ||_-27aL162C AH229-p243(SEQ ID NO: 38). The i-Tag (SEQ ID NO: 89) thus warranted further investigations in terms of expression and functionality fused to IL-27aL162C-based proteins.

[0135] Example 4: Increased expression titer and preserved functionality are obtained regardless of a linker that fuses the i-Tag to IL-27aL162C AH229-p243(Figure 5)

[0136] The inventors of the present invention further analyzed the impact of different linkers between IL-27aL162C AH229-p243and i-Tag (see SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35, 36, 37, 30, 38, 39 and 40), and of two different sizes of the tag on the expression level of the fusion polypeptides (see Figure 5A). Therefore, Expi293F™ cells were transfected with constructs for the expression of fusion polypeptides comprising i-Tag (SEQ ID NO: 89) containing one or five constant domain(s) of the human kappa immunoglobulin light chain fused to the C- terminus of the IL-27aL162C AH229-p243polypeptide without (SEQ ID NOs: 27 and 37) or with a linker having one of the following amino acids sequences: GGGS (SEQ ID NO: 78) (only for tag with one constant domain of the human kappa immunoglobulin light chain (SEQ ID NO: 28), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 30, linker: SEQ ID NO: 84), PAPAP (SEQ ID NOs: 32 and 38, linker: SEQ ID NO: 79), APAPAPA (SEQ ID NOs: 33 and 39, linker: SEQ ID NO: 85), APAPAPAPAPKPA (SEQ ID NOs: 34 and 40, linker: SEQ ID NO: 86), APAPAPAPAPAPA (only for tag with one constant domain of the human kappaimmunoglobulin light chain (SEQ ID NO: 35, linker: SEQ ID NO: 87), A(EAAAK)4A (only for tag with one constant domain of the human kappa immunoglobulin light chain (SEQ ID NO: 36, linker: SEQ ID NO. 88). As shown in Figure 5A, the secretion of the fusion polypeptide (lanes No. 3 to 15, SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35, 36, 37, 30, 38, 39 and 40) is generally increased compared to the untagged I L-27aL162C AH229-P243protein (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) (lane No. 2). Further, the secretion level of the fusion IL-27aL162C AH229'P243-protein comprising the i-Tag with five constant domains (SEQ ID NOs: 37, 30, 38, 39 and 40) (lanes No. 11 to 15) is generally higher than the secretion level of the fusion IL-27aL162C AH229'P243-protein comprising the i-Tag with one constant domain (SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35 and 36) (lanes No. 3 to 10) (see Figure 5A). For testing the functionality of IL-27aL162C AH229'P243-i-Tags-fusion protein, the HeLa STAT1 bioluminescence assay was used (see Figure 5B). The supernatants of Expi293F™ cells transfected with constructs for secretion of fusion polypeptides containing the IL-27aL162C AH229-p243protein fused at the C- terminus to the i-Tags fusion protein containing five constant domains of the human kappa immunoglobulin light chain without (SEQ ID NO: 27) or a linker (SEQ ID NOs: 29, 32, 33 and 34) having one of the following amino acid sequences: A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 84), PAPAP (SEQ ID NO: 79), APAPAPA (SEQ ID NO: 85) and APAPAPAPAPKPA (SEQ ID NO: 86). The mock control of the above experiment served as negative control. The HeLa cells were stimulated with adjusted volumes of these supernatants, for which protein concentrations were measured via the IL-27 ELISA and normalized to a concentration of 104 nM before HeLa STAT1 cell stimulation and the resulting bioluminescence was measured. As shown in Figure 5B, the secreted fusion polypeptides were functional regardless of if a linker was present or not. However, the bioluminescence was increased when supernatants containing the fusion polypeptides with the A(EAAAK)4ALEA(EAAAK)4A-linker (SEQ ID NO: 84) or the PAPAP-linker (SEQ ID NO: 79) were used.

[0137] Example 5: N- as well as C-terminal i-Tags fusion to IL-27aL162C AH229'P243enables development IL-27aL162C AH229 P243-based biopharmaceuticals (Figure 6).

[0138] This experiment was done to further analyze, if the fusion of the i-Tags (SEQ ID NO: 90) to the N-terminus or the C-terminus of IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) makes any difference. As tag, a fusion of five constant domains of the human kappa immunoglobulin light chain (SEQ ID NO: 90) was used as mentioned above. The target polypeptide was the I L-27aL162C AH229-P243protein (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) (mentioned above). The inventors of the present invention analyzed the levels of the secretion of the fusion polypeptides of transfected Expi293F™ cellsby Coomassie stained SDS-PAGE. Supernatants were harvested after transfection of Expi293F™ cells (load) and purified with the CaptureSelect™ KappaXP column via i-Tags (purified protein). A bovine serum albumin (BSA) dilution series served as concentration standard to quantify protein levels (see Figure 6A). The expression levels of the N-terminal fusion polypeptide (SEQ ID NO: 99) and the C-terminal fusion polypeptide (SEQ ID NO: 37) are comparable and significantly increased compared to the expression level of the untagged target polypeptide, which was not detectable via SDS-PAGE (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) (see Figure 6B). Quantification of the Coomassie gel (Figure 6A) shows that the standardized purification via CaptureSelect™ KappaXP column resulted in a very high purity of above 95% for both the N-terminally and the C-terminally i-Taggeds IL- 27aL162C AH229-P243(SEQ ID NOs: 99 and 37) (Figure 6C). For analyzing the functionality of the IL-27aL162C AH229-P243fused at the N-terminus or the C-terminus to the i-Tags (SEQ ID NO: 99 and 37), THP-1 cells were stimulated with different concentrations of both fusion polypeptides and the CXCL10 concentration in the supernatant was measured with an ELISA as mentioned above. The ELISA showed that both, the N-terminally and the C-terminally tagged IL-27aL162CAH229-P243 (S£Q ID NOs: 99anc| 37)arefunctional fusion polypeptides (Figure 6D).

[0139] Example 6: Standardized protein production and protein functionality are obtained by i-Tags fusion to IL-27aL162C AH229-p243regardless of an interdomain linker (Figure 7)

[0140] In this example the inventors of the present invention examined, if a linker between the constant domains of the human kappa immunoglobulin light chain comprised in the i-Tags (SEQ ID NO: 90) fusion protein has any impact on the functionality of the IL-27aL162C AH229-p243i-Tags (SEQ ID NO: 37) fusion polypeptide. The inventors of the present invention transfected Expi293F™ cells with constructs for the expression of fusion polypeptides containing the IL- 27aL162C AH229-p243protein fused with the PAPAP-linker (SEQ ID NO: 79) to i-Tag5(SEQ ID NO: 90) which comprised five constant domains of the human kappa immunoglobulin light chain linked together without a linker (SEQ ID NO: 38) or five constant domains of the human kappa immunoglobulin light chain linked together via a GAGAG-linker (SEQ ID NO: 92). The supernatants of the transfected cells were harvested (load) and purified with the CaptureSelect™ KappaXP column via the i-Tags (purified protein) and analyzed on a Coomassie stained SDS-PAGE gel. The expression of the fusion polypeptides with or without a linker between the constant domains of the tag was equal, as shown in Figure 7A. Also, the function of both fusion polypeptides was equal as tested by THP-1 cell stimulation and subsequent CXCL10 ELISA and shown in Figure 7B.

[0141] Example 7: The specificity of the functionality of IL-27aL162C AH229-p243fusion constructs is confirmed via competition assay with human IL-27 receptor alpha proteins (Figure 8)

[0142] In this example the inventors of the present invention examined, if the functionality of IL-27aL162C AH229-p243fusion constructs on THP-1 cells can be inhibited via addition of the human IL-27 receptor alpha proteins. The inventors of the present invention transfected Expi293F™ cells with constructs for the expression of fusion polypeptides containing the IL- 27aL162C AH229'P243protein fused with the PAPAP-linker to i-Tags which comprised five constant domains of the human kappa immunoglobulin light chain (SEQ ID NO: 38) or HSA without a linker (SEQ ID NO: 14), a GGGS linker (SEQ ID NO: 15), or a PAPAP linker (SEQ ID NO: 16). The supernatants of the transfected cells were harvested and purified with the CaptureSelect™ KappaXP column via the i-Tags fusion or the CaptureSelect™ Human Albumin Affinity Matrix via the HSA fusion. For expression of the human IL-27 receptor alpha Fc Fusion polypeptide (SEQ ID NO: 93) the inventors of the present invention transfected Expi293F™ cells. The human IL-27 receptor alpha Fc Fusion polypeptide was purified via CaptureSelect™ FcXP followed by a Superose 6 purification step. The protein levels were quantified via Coomassie stained SDS-PAGE and a bovine serum albumin (BSA) dilution series as concentration standard. The protein levels of IL-27aL162C AH229-p243fusion constructs were adjusted to 10 nM final concentration and of the human IL-27 receptor alpha proteins to 80 nM in the THP-1 assay. The stimulation effect of the IL-27aL162C AH229'P243fusion constructs on the THP-1 cells with or without human IL-27 receptor alpha proteins function of both fusion polypeptides was analyzed via a subsequent CXCL10 ELISA and shown in Figure 8. PBS samples (lanes No. 5 and 10) served as a negative control in this assay. The fragment of IL- 27Ra (full length corresponding to UniProt-ID Q6UWB1) used in this experiment corresponds to SEQ ID NO: 75.

[0143] Example 8: IL-27aL162C AH229 P243-HSA as well as i-Tag fusion constructs have an improved thermal stability compared to IL-27aL162C AH229 P243only (Figure 9).

[0144] Key for any biopharmaceutical is their functionality, developability, i.e. expression titer and efficient purification, as well as their stability. Stability is a prerequisite for storage and transportation. To evaluate the stability of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) the inventors examined the activity of the proteins upon thermal stress. The inventors transfected Expi293F™ cells with constructs for the expression of fusion polypeptides containing the IL-27aL162C AH229'P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) protein or IL-27aL162C AH229'P243protein fused to HSA (SEQ ID NO: 43) (see Figure9A). Mock samples served as a negative control. Prior to HeLa STAT1 assay, the expression medium was incubated at 30°C, 40°C, 50°C or 60°C for 30 min. Subsequently, the HeLa STAT1 reporter cells were stimulated with a constant volume of 25 pl of the respective expression medium that were added to 50 pl of DM EM medium containing 0.1 % FCS resulting in a total volume of 75 pl for HeLa STAT1 cell stimulation. After 18 hours the resulting bioluminescence was measured. As shown in Figure 9A, the IL-27aL162C AH229'P243-HSA fusion polypeptide (SEQ ID NO: 43) was still functional after an incubation at 50°C, while the IL- 27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) unfused protein lost its activity. To examine the stability upon thermal stress at 50°C the inventors of the present invention transfected Expi293F™ cells with constructs for the expression of fusion polypeptides containing the IL-27aL162C AH229-p243protein (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76), the IL-27aL162C AF29-L4°AH229’P243protein fused to HSA (SEQ ID NO: 43), the |L-27aL162C AH229-P243protein fused to i-Tag5(SEQ ID NO: 37), the IL-27aL162C AF29-L4° AH229-P243 j_-|-ag5fusion protein (SEQ ID NO: 44), the IL-27aL162C AH229-p243fused via a PAPAP linker to i-Tags (SEQ ID NO: 38) or IL-27aL162C AF29-L4° AH229-P243 fusec|vja aPAPAP linker to i- Tags (SEQ ID NO: 45). Prior to HeLa STAT1 assay, the expression medium was incubated at 4°C or 50°C for 30 min. Subsequently, the HeLa STAT 1 reporter cells were stimulated with a constant volume of 50 pl of the respective expression medium that were added to 50 pl of DMEM medium containing 0.1 % FCS resulting in a total volume of 100 pl for HeLa STAT1 cell stimulation. After 18 hours the resulting bioluminescence was measured. The signal of the expression medium incubated at 4°C was set to 1 and the signal of the expression medium incubated at 50°C was analyzed relative to that. While the unfused IL-27aL162C AH229'P243(SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) lost its functionality upon incubation at 50°C (lane No. 1), the functionality for the fusion constructs was largely preserved (lanes No. 2-6, SEQ ID NOs: 43, 37, 44, 38 and 45).

[0145] Example 9: The IL-27aL162C AH229'P243fusion constructs with a N-glycosylation site at D89N are functional and the specificity of the functionality is confirmed via competition assay with human IL-27 receptor alpha proteins (Figure 10).

[0146] In this example the inventors of the present invention examined, whether IL-27aL162CAH229-P243 fusjonconstructs with an N-glycosylation site at D89N are functional and the specificity can be confirmed via inhibition by the human IL-27 receptor alpha. The inventors of the present invention transfected Expi293F™ cells with constructs for the expression of fusion polypeptides containing the IL-27aL162C AH229-p243protein fused to HSA without a linker (SEQID NO: 14), a GGGS linker (SEQ ID NO: 15), or a PAPAP linker (SEQ ID NO: 16); or fusion polypeptides containing the IL-27aL162C AH229-p243protein with an N-glycosylation site D89N fused to HSA without a linker (SEQ ID NO: 52), a GGGS linker (SEQ ID NO: 53), or a PAPAP linker (SEQ ID NO: 54); or fusion polypeptides containing the IL-27aL162C AH229-p243protein fused to i-Tags via a PAPAP-linker (SEQ ID NO: 32), or a fusion polypeptide containing the IL-27aL162C AH229'P243protein with an N-glycosylation site at D89N fused to i-Tags via a PAPAP- linker (SEQ ID NO: 51). The supernatants of the transfected cells were harvested and purified with the CaptureSelect™ KappaXP column via the i-Tags fusion or the CaptureSelect™ Human Albumin Affinity Matrix via the HSA fusion. For expression of the human IL-27 receptor alpha Fc Fusion polypeptide (SEQ ID NO: 75; the fragment of IL-27Ra (full length corresponding to UniProt-ID Q6LIWB1) used in this experiment corresponds to SEQ ID NO: 100) the inventors of the present invention transfected Expi293F™ cells. The human IL-27 receptor alpha Fc fusion polypeptide was purified via CaptureSelect™ FcXP followed by a Superose 6 purification step. The protein levels were quantified via Coomassie stained SDS- PAGE and a bovine serum albumin (BSA) dilution series as concentration standard. The protein levels of IL-27aL162C AH229-p243fusion constructs as well as the human IL-27 receptor alpha proteins were adjusted to 10 nM final concentration in the THP-1 assay. The stimulation effect of the IL-27aL162C AH229-p243fusion constructs on the THP-1 cells with or without human IL-27 receptor alpha proteins. The function of the fusion polypeptides was analyzed via a subsequent CXCL10 ELISA and shown in Figure 10. PBS only sample (lane No. 1) served as a negative control in this assay.

[0147] Example 10: IL-27aL162C AH229-p243Fc fusion constructs are functional with the expression titer of IL-27aL162C AH229-p243Fc fusion constructs being higher compared to unfused IL-27aL162C-based proteins, but lower than IL-27aL162C-based HSA-fusion proteins (Figure 11)

[0148] To analyze the effect of Fc fusion, fragment crystallizable region, on developability of IL-27aL162C-based biopharmaceutical constructs containing IL-27aL162Cor IL-27aL162C AH229'P243fused to Fc (SEQ ID NOs: 72 and 73) were generated. To analyze the expression titer of these constructs, Expi293F™ cells were transfected with constructs for the expression containing unfused IL-27aL162C(SEQ ID NO: 2, without signal sequence SEQ ID NO: 55), or IL-27aL162CAH229-P243 (S£Q ID |\|Q:3 without signal sequence SEQ ID NO: 76) polypeptides or fusion constructs containing IL-27aL162C AH229-p243fused to HSA at the C-terminus without a linker (SEQ ID NO: 14) or with a GGGS-linker (SEQ ID NO: 15), or IL-27aL162C AH229-p243fused to HSA at the N-terminus A(EAAAK)iA (SEQ ID NO: 23), or IL-27aL162Cfused to Fe at the C-terminus (SEQ ID NO: 72), IL-27aL162C AH229-p243fused to Fe at the C-terminus (SEQ ID NO: 73). As shown in Figure 3A and Figure 5A, the secretion of the I L-27aL162C AH229-P243fusion polypeptide (Figure 3A: lanes No. 4 to 15, SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 and 25; Figure 5A: lanes No. 3 to 15, SEQ ID NOs: 27, 28, 29, 32, 33, 34, 35, 36, 37, 30, 38, 39 and 40) is generally increased compared to the untagged IL-27aL162C AH229-P243protein (SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) (lane No. 2). Subsequently, the expression levels of IL-27aL162C-based proteins in the cell supernatant were measured via IL- 27 ELISA (see Figure 11A). As demonstrated previously, the expression titer of unfused constructs IL-27aL162C(lane No. 1 , SEQ ID NO: 2, without signal sequence SEQ ID NO: 55) and IL-27aL162C AH229'P243(lane No. 2, SEQ ID NO: 3, without signal sequence SEQ ID NO: 76) were below detection limit, while HSA fusions significantly increased expression titer of fusion constructs (lanes No. 3-5, SEQ ID NOs: 14, 15 and 23). The expression titer of Fc-fusion constructs (lanes No. 6 and 7, SEQ ID NOs: 72 and 73) were increased compared to unfused constructs (lanes No. 1 and 2, SEQ ID NO: 2, without signal sequence SEQ ID NO: 55, SEQ ID NO: 3, without signal sequence SEQ ID NO: 76), but significantly lower compared to HSA fusion constructs (lanes No. 3-5, SEQ ID NOs: 14, 15 and 23). To analyze the functionality of the proteins, the inventors of the present invention utilized an assay with BL-2 cells expressing the IL-27 receptor (see Figure 11 B). Mock was used as a negative control (lane No. 1). For reference the unfused IL-27aL162C(lanes No. 2, SEQ ID NO: 2, without signal sequence SEQ ID NO: 55) and the IL-27aL162C T238A S240A(SEQ ID NO: 7, without signal sequence SEQ ID NO: 77), with the endogenous O-glycosylation sites removed via point mutation (lane No. 3), constructs were used. The Expi293F™ expression supernatants with the respective proteins were adjusted to 1000ng / ml before stimulation of the BL-2 cells. To quantify the protein levels hlL-27aL162C His6purified from inclusion bodies in E. coli was used as a reference to obtain a standard curve with linear fit for quantification of hlL-27aL162Cbased proteins in Expi293 supernatants using immunoblot signals (Muller, Friedl et al. 2019). The obtained functional levels upon BL-2 cells stimulation for 60 min based on STAT1 phosphorylation of the IL- 27aL162C- (SEQ ID NO: 72) (lane No. 4) as well as IL-27aL162C AH229-p243-Fc-fusion (SEQ ID NO: 73) (lane No. 5) constructs were comparable to the IL-27aL162C-protein (SEQ ID NO: 2, without signal sequence SEQ ID NO: 55) (lane No. 2). The unfused IL-27alphaL162C T238A S240Aconstruct (SEQ ID NO: 7, without signal sequence SEQ ID NO: 77) with the endogenous O-glycosylation levels removed exhibited increased STAT1 phosphorylation levels in the BL-2 assay compared to other analyzed constructs.

[0149] Example 11 : In this example (see Figure 12) the expression levels of various IL-27a mutein fusion proteins by transfected Expi293F™ cells were compared. After cell culture and protein expression, the protein concentrations were measured via the IL-27 ELISA. It shows that expression levels of IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) by transfected Expi293F™ cells were increased by tested fusion proteins of the respective mutein with Fc, i-Tags or HSA (SEQ ID NOs: 14, 37 and 73) . However, expression levels were not increased by fusion of the respective mutein with a HISe- or V5-tag (SEQ ID NOs: 101 and 102). The expression levels of the IL-27aL162C AH229-P243were highest for the HSA fusion in this Example.[001501 Example 12: This Example (see Figure 13) demonstrates that IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) fusion proteins (SEQ ID NOs: 37 and 73) have an improved thermal stability compared to unfused IL-27aL162C AH229'P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) and IL-27aL162C AH229’P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) with HISe- or V5-tag proteins (SEQ ID NOs: 101 and 102). Figure 13A displays that CXCL10 levels after THP-1 cells have been stimulated with Expi293F™ supernatant, containing either IL-27aL162C AH229-P243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) or I L-27aL162C AH229-P243fusion proteins (SEQ ID NOs: 37, 73, 101 and 102). Supernatants were treated at 30°C or 50°C for 30 min alongside a mock sample, which served as a negative control in this activity assay. THP-1 cells were stimulated with 25 nM of polypeptides and the CXCL10 concentration in the supernatant was measured with an ELISA as mentioned above n = 2, ± SD. Figure 13B shows the remaining activity of IL-27aL162CAH229-P243(S EQ i D N 0 :3without signa| sequence: SEQ ID NO: 76) or IL-27aL162C AH229-p243fusion proteins (see SEQ ID NOs: 37 and 73) after incubation at 50 °C for 30 min. Activity was assessed by measuring CXCL10 level as described in Figure 13A. While IL-27aL162C AH229-p243(SEQ ID NO: 3, without signal sequence: SEQ ID NO: 76) and HISe- and V5-tagged fusion proteins (see SEQ ID NOs: 101 and 102) lost activity after treatment at 50°C, minor loss of functionality was measured for IL-27aL162C AH229-P243fusion proteins with Fc or i-Tags (SEQ ID NO: 37 and 73) after incubation at 50°C for 30 min.REFERENCESAdams, B. M., et al. (2019). "Protein Quality Control in the Endoplasmic Reticulum." Protein J 38(3): 317-329.Behnke, J., et al. (2016). "Members of the Hsp70 Family Recognize Distinct Types of Sequences to Execute ER Quality Control." Mol Cell 63(5): 739-752.Bohnacker, S., et al. (2020). "Influence of glycosylation on IL-12 family cytokine biogenesis and function." Mol Immunol 126: 120-128.Brunelle, J. L. and R. Green (2014). "Coomassie blue staining." Methods Enzymol 541 : 161- 167.Das, S., et al. (2008). "Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers." Proc Natl Acad Sci II S A 105(43): 16647-16652.Feige, M. J., et al. (2008). "The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity." Proc Natl Acad Sci II S A 105(36): 13373- 13378.Hill, R. L., et al. (1966). "The evolutionary origins of the immunoglobulins." Proc Natl Acad Sci U S A 56(6): 1762-1769.Min, B., et al. (2021). "IL-30(dagger) (IL-27A): a familiar stranger in immunity, inflammation, and cancer." Exp Mol Med 53(5): 823-834.Muller, S. I., et al. (2019). "A folding switch regulates interleukin 27 biogenesis and secretion of its alpha-subunit as a cytokine." Proc Natl Acad Sci II S A 116(5): 1585-1590.Nakano, T., et al. (2006). "Immunochemical quantification of free immunoglobulin light chains from an analytical perspective." Clin Chem Lab Med 44(5): 522-532.Steglich, M., et al. (2020). "Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34." PLoS One 15(10): e0240580.

Claims

CLAIMS1. A fusion-polypeptide comprising- a mutein of the a-subunit of human Interleukin 27, wherein at least one of the amino acid residues of the a-subunit of human Interleukin 27 selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated, wherein the numbering of sequence positions of said mutein of the a-subunit of human Interleukin 27 corresponds to the numbering of the sequence positions of the a-subunit of human Interleukin 27 according to SEQ ID NO: 1 , and- at least one pharmaceutically acceptable fusion partner.

2. The fusion-polypeptide according to claim 1 , wherein in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are mutated.

3. The fusion-polypeptide according to claim 2, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acids of sequence positions 1 to 28 are deleted.

4. The fusion-polypeptide according to any one of the preceding claims, wherein the at least one pharmaceutically acceptable fusion partner is at least one tag.

5. The fusion-polypeptide according to claim 4, wherein the at least one tag is not a V5 tag and / or not a His-tag.

6. The fusion-polypeptide according to claim 5, wherein the at least one tag is not a (His)e-tag.

7. The fusion-polypeptide according to claim 4, wherein the at least one tag is at least one half-life extending tag.

8. The fusion-polypeptide according to any one of the preceding claims, wherein the least one pharmaceutically acceptable fusion partner or tag or half-life extending tag is human serum albumin or a fragment thereof or one or more constant domains of human kappa immunoglobulin light chain, optionally without the C-terminal cysteine.

9. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 comprises at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %,89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the a-subunit of human Interleukin 27 according to SEQ ID NO: 1.

10. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161, 162, 163, 180, 181 and 182 is / are mutated.

11. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated.

12. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated.

13. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position161 is mutated.

14. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position162 is mutated.

15. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position163 is mutated.

16. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162, 163, 164, 165, 180, 181 and 182 is / are mutated to cysteine.

17. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residuesselected from the group consisting of sequence positions 160, 161, 162, 163, 180, 181 and 182 is / are mutated to cysteine.

18. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 160, 161 , 162 and 163 is / are mutated to cysteine.

19. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 161 , 162 and 163 is / are mutated to cysteine.

20. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position160 is mutated to cysteine (SEQ ID NO: 56).

21. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position161 is mutated to cysteine (SEQ ID NO: 57).

22. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position162 is mutated to cysteine (SEQ ID NO: 2).

23. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position163 is mutated to cysteine (SEQ ID NO: 9).

24. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position164 is mutated to cysteine (SEQ ID NO: 58).

25. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position165 is mutated to cysteine (SEQ ID NO: 71).

26. The fusion-polypeptide according to any one of the preceding claims 1 to 10, 16 or 17, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 180 is mutated to cysteine (SEQ ID NO: 59).

27. The fusion-polypeptide according to any one of the preceding claims 1 to 10, 16 or 17, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 181 is mutated to cysteine (SEQ ID NO: 60).

28. The fusion-polypeptide according to any one of the preceding claims 1 to 10, 16 or 17, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 182 is mutated to cysteine (SEQ ID NO: 61).

29. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 comprises one or more salt-bridge(s).

30. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 is further engineered to be devoid of any O- glycosylated residues.

31. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acids at the C-terminus is / are mutated.

32. The fusion-polypeptide according to claim 31 , wherein in the mutein of the a-subunit of human Interleukin 27 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 amino acid(s) at the C-terminus is / are deleted.

33. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residues of sequence positions 229 to 243 are deleted (SEQ ID NO: 62).

34. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 at least one of the amino acid residues selected from the group consisting of sequence positions 238 and 240 is / are mutated.

35. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is mutated.

36. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is mutated.

37. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residues at sequence positions 238 and 240 are mutated.

38. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 238 is replaced by alanine (SEQ ID NO: 63).

39. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 the amino acid residue at sequence position 240 is replaced by alanine (SEQ ID NO: 64).

40. The fusion-polypeptide according to any one of the preceding claims, wherein in the mutein of the a-subunit of human Interleukin 27 of the fusion-polypeptide the amino acid residues of the a-subunit of human Interleukin 27 at sequence positions 238 and 240 are replaced by alanine (SEQ ID NO: 65).

41. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 mutation(s) at one or more sequence position(s) selected from the group consisting of sequence positions 229, 230, 231 , 232, 233, 234, 235, 236, 237, 239, 241 , 242 and 243.

42. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 comprises one or more disulfide-bridge(s).

43. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 is fused at its N-terminus to the at least one pharmaceutically acceptable fusion partner.

44. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 is fused at its C-terminus to the at least one pharmaceutically acceptable fusion partner.

45. The fusion-polypeptide according to any one of the preceding claims, wherein the mutein of the a-subunit of human Interleukin 27 is fused to the at least one pharmaceutically acceptable fusion partner via a linker.

46. The fusion-polypeptide according to claim 45, wherein the linker is a linker comprising at least 3 guanosines, preferably a GGGS-linker, a linker comprising the sequence PAPAP, or a helical linker, and / or wherein the linker is preferably selected from the group consisting of any one of SEQ ID NOs: 78 to 88.

47. The fusion-polypeptide according to claim 45 or 46, wherein the linker has a length of 0 to 30 residues.

48. The fusion-polypeptide according to any one of the preceding claims, wherein the fusion- polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 54, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and / or wherein the fusion-polypeptide is a fusion-polypeptide as depicted in any one of Figures 1 to 13.

49. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion- polypeptide according to any one of claims 1 to 48.

50. The nucleic acid molecule according to claim 49, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.

51. The nucleic acid molecule according to claim 50, wherein the regulatory sequence comprises a promoter sequence.

52. The nucleic acid molecule according to any one of claims 49 to 51 comprised in a vector.

53. A host cell containing a nucleic acid molecule of any one of claims 49 to 52.

54. An immune modulator comprising the fusion-polypeptide of any one of claims 1 to 48.

55. The fusion-polypeptide according to any one of claims 1 to 48 for use as a medicament.

56. The fusion-polypeptide according to any one of claims 1 to 48 for use in the treatment or prevention of a disease.

57. The fusion-polypeptide for use according to claim 56, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

58. Use of a fusion-polypeptide according to any one of claims 1 to 48 for the manufacture of a medicament for treating a disease, preferably a disease in a mammal, more preferably a disease in a human.

59. The use according to claim 58, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

60. A method of treating a disease comprising the step of administering a composition comprising a fusion-polypeptide according to any one of claims 1 to 48 to a subject, preferably to a mammal, more preferably to a human, in need thereof, preferably in combination with adjuvants-therapy.

61. The method of treating according to claim 60, wherein the disease is selected from the group consisting of infectious diseases, autoimmune diseases, cancer, inflammatory diseases; preferably inflammatory diseases of the liver, inflammatory diseases of the bile ducts, chronic inflammatory diseases or acute inflammatory diseases; sepsis; preferably abdominal sepsis or urosepsis; and septic shock.

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