IL-37 fusion proteins and uses thereof

Abstract

The present invention relates to polypeptides, in particular fusion protein variants comprising interleukin-37 (IL-37) and related therapeutic agents and compositions. More specifically, the present invention relates to fusion proteins comprising a mutant IL-37 polypeptide and an antibody Fc region. The biophysical stability, such as thermal stability, of the fusion proteins can be improved compared to reference IL-37 constructs, such as a wild-type IL-37 polypeptide or a mutant variant of an IL-37 polypeptide. Fusion polypeptide variants and compositions for the treatment of inflammatory diseases or disorders are also contemplated. Furthermore, the present invention relates to nucleic acid molecules encoding such fusion proteins, as well as vectors and host cells comprising such nucleic acid molecules.

Description

Technical Field

[0001] This invention relates to peptides, particularly fusion protein variants comprising interleukin-37 (IL-37) and related therapeutic agents and compositions. More specifically, this invention relates to fusion proteins comprising a mutant IL-37 peptide and an antibody Fc region. It also relates to fusion peptide variants and compositions for treating inflammatory diseases or conditions. Furthermore, this invention relates to nucleic acid molecules encoding such fusion proteins, as well as vectors and host cells comprising such nucleic acid molecules. Background Technology

[0002] The IL-1 family of cytokines has unique and broad anti-inflammatory effects in both innate and adaptive immunity, serving as a first line of defense against pathogens and physical damage / stress. To date, eleven members of the IL-1 family have been identified, including seven receptor agonists (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ), three ligands with antagonistic activity (IL-1Ra, IL-36Ra, and IL-38), and a relatively newly renamed anti-inflammatory cytokine, IL-37 (Garlanda C, et al. Immunity. Dec 12, 2013; 39(6):1003-18; Smith DEJ LeukocBiol. Mar 2011; 89(3):383-92; Dinarello CA et al. Blood. 2011; 117:3720-3732v).

[0003] IL-37, originally called IL-1 family member 7 (IL-1F7), is the seventh member of the IL-1 family discovered through computational cloning in 2000 and renamed in 2010 (Dinarello C, et al. Nat Immunol. Nov 2010; 11(11):973). IL-37 has five different isotypes, including IL-37a, IL-37b, IL-37c, IL-37d, and IL-37e (Boraschi D, et al. Eur Cytokine Netw. Sep 2011; 22(3):127-47; Tete S, et al. Int J Immunopathol Pharmacol. Jan–Mar 2012; 25(1):31-8).

[0004] All members of the IL-1 family share a similar barrel-shaped structure and bind to Ig-like receptors. The human IL-37 gene, located on chromosome 2, is 3,617 kb in length and comprises six exons encoding a 17–26 kDa protein. IL-37 has a molecular weight of approximately 17–25 kDa. The structure of IL-37 consists of 12 β-tubular lines (Boraschi D et al., Eur Cytokine Netw., Sep 2011; 22(3):127–47). The IL-37 gene undergoes alternative splicing; five distinct splice variants of IL-37 have been identified, designated IL-37a–e, with IL-37b being the largest (218 amino acids) and most frequently characterized isoform. Each isoform of the immature IL-37 precursor peptide transitions from an inactive precursor peptide state to an active state during expression via cleavage by caspase-1, and all isoforms regulate each other to form a relatively stable state. For example, IL-37b has the largest molecular weight (218 amino acids), the most complex biological functions, and IL-37b is converted from an inactive propeptide to an active mature peptide under the action of caspase-1 (Boraschi D et al. Eur Cytokine Netw. Sep 2011; 22(3):127-47; Busfield SJ, Comrack CA, Yu G. et al. Genomics. 2000; 66(2):213-216). Specifically, the transcriptomorph encoded by IL-37b contains exons 1 and 2 and has an N-terminal pro-domain that includes a potential caspase-1 cleavage site, resulting in the splicing of the IL-37b precursor into mature IL-37b (Boraschi D, et al. Eur Cytokine Netw. Sep 2011; 22(3):127-47; Ye L, Huang Z. Oncotarget. Sep 2015; 6(26):21775-6). In addition, IL-37b encodes exons 4-6, which contain 12 putative β chains necessary for the formation of IL-1-like β-trefoil secondary structures (Boraschi D, et al. Eur Cytokine Netw. Sep 2011; 22(3):127-47; Murzin AG, Lesk AM, Chothia CJ Mol Biol. Jan 20 1992; 223(2):531-43).

[0005] Furthermore, IL-37 can be detected in a variety of normal cells and tissues, particularly human tissues, including natural killer (NK) cells, stimulated B cells, monocytes, skin keratinocytes, epithelial cells, lymph nodes, thymus, lungs, colon, uterus, and bone marrow, but its expression level is low in healthy human tissues (Nold MF et al. Nat Immunol. 2010 Nov; 11(11):1014-22; Boraschi D et al. Eur Cytokine Netw. 2011 Sep; 22(3):127-47). However, some IL-37 isotypes are expressed in tissue-specific ways. The brain expresses only IL-37a, the kidneys only IL-37b, the heart only IL-37c, and the bone marrow and testes only IL-37d. IL-37b was initially found in the bone marrow and is also found primarily in blood cells (such as peripheral blood mononuclear cells (PBMCs)), epithelial cells, dendritic cells, monocytes, and keratinocytes, as well as in the respiratory and gastrointestinal tracts (Chen et al. J. Trans. Med. 2015; Li Y, Wang Y, Liu Y, Wang Y, Zuo X, Li Y, Lu X Mediators Inflamm. 2014; 2014():136329). Furthermore, IL-37 expression has been detected in human cell lines such as A431, THP-1, U937, IMTLH, KG-1, HL60, HPBMC, HFcolitis, PT-4, and NHDC (Gao W. et al. The Journal of Immunology. 2003; 170(1):107-113).

[0006] It is estimated that IL-37 translocates to the cell nucleus and redistributes between intracellular and extracellular sites, thereby affecting cellular responses (Li Y, Wang Y, Liu Y, Wang Y, Zuo X, Li Y, Lu X Mediators Inflamm. 2014; 2014():136329).

[0007] Therefore, IL-37 can be secreted externally to bind to surface receptors, and it can also be secreted intracellularly. In this regard, since IL-37 has been shown to be expressed at low levels in human cells and tissues, but is upregulated by inflammatory stimuli and pro-cytokines, including several toll-like receptor (TLR) agonists, IL-18, interferon (IFN)γ, IL-1β, transforming growth factor β1, and tumor necrosis factor (TNF), there is new evidence that IL-37 can exert its anti-inflammatory effects through both intracellular and extracellular pathways. For example, IL-37 can trigger signal transduction at the cell membrane through interactions with IL-18 receptor α and IL-1R8 (Sigirr), and intracellularly through interactions with Smad3. IL-18 is a pro-inflammatory cytokine that can induce T cell proliferation and differentiation, promote and maintain Th17 cell secretion of IL-17, and promote Th17 cell-based cellular immune responses. IL-37 can bind to IL-18Rα to form a complex with IL-18BP (the natural antagonist of IL-18), thereby enhancing the inhibitory effect of IL-18BP on IL-18 and reducing the activity of IL-18 as well as the levels of Th1 cells, NK cells and IFN-γ (Kumar S. et al. Cytokine. 2002; 18(2):61-71; Moretti S. et al. PLoS Pathogens. 2014; 10(11); Nold M. et al. Biochemical Pharmacology. 2003; 66(3):505-510).

[0008] In light of the above, IL-37 has been shown to be a physiological inhibitor of the immune response mediated by pro-inflammatory cytokines such as IL-1β and TNF via their receptors and Toll-like receptor ligands, and to have broad protective effects in inflammation triggered by infection or other non-infectious attacks (Nold MF et al. Nat Immunol. Nov 2010; 11(11):1014-22; Tete S, et al. Int J Immunopathol Pharmacol. Jan-Mar 2012; 25(1):31-8). Therefore, IL-37 may suppress the innate immune response by reducing the production of pro-inflammatory cytokines induced by Toll-like receptor (TLR) agonists. For example, IL-37 can reduce the production of pro-inflammatory cytokines (such as IL-1α, IL-1β, IL-1Ra, IL-6, IL-8, IL-17, IL-23, TNF-α, and IFN-γ) and chemokines (such as MIP-2 / CXCL2, CCL12 / MCP-5, and BCA-1 / CXCL13). It can also inhibit the expression of M-GSF and GM-CSF, but increase the production of TGF-β1, which is considered an immunosuppressive factor. Thus, in summary, IL-37 works by binding to the IL-18 receptor and requires the IL-1 family decoy receptor IL-1R8 to exert its anti-inflammatory function. It has been shown that the reduction of pro-inflammatory cytokines (TNF-α, IL-1α, IL-1β, IFN-γ) by IL-37 can play a key role in the development of experimental autoimmune thyroiditis, multiple sclerosis, insulin-dependent diabetes mellitus, or experimental autoimmune diabetes, suggesting a biological and potential therapeutic relevance of IL-37 to these diseases (Li S, et al.; Proc Natl Acad Sci USA; 2015; pp. 2497-2502). Therefore, IL-37 has been investigated as a natural inhibitor of immune responses in chronic inflammatory and autoimmune disorders, as well as in cancer. In this regard, IL-37 has been demonstrated to express and exert anti-inflammatory effects in a variety of diseases, including melanoma, rheumatoid arthritis, acute coronary syndrome, morbid obesity, contact hypersensitivity, atopic dermatitis, inflammatory liver injury, systemic lupus erythematosus (SLE), and IBD (Li Y, Wang Z, Yu T, Chen B, Zhang J, Huang K, Huang Z PLoS One. 2014; 9(9):e107183). In vitro, IL-37 has been shown to effectively eliminate the expression of pro-inflammatory cytokines in several cell types, including PBMCs. In vivo, in mouse models, IL-37 reduces inflammatory responses and clinical symptoms of cerebral ischemia, myocardial ischemia / reperfusion injury, psoriasis, and asthma (Chen et al. J. Trans. Med. 2015).In summary, these data demonstrate the complex biological functions of IL-37 in various diseases, with IL-37 expression in autoimmune diseases appearing to reduce excessive inflammatory immune responses.

[0009] Furthermore, IL-37 plays a role in protecting the body from endotoxic shock, ischemia-reperfusion injury, and cardiovascular disease. In addition, IL-37 has potential anti-tumor effects. Therefore, IL-37 can serve as a target for tumor research, diagnosis, and treatment. In this context, IL-37 has shown anti-tumor effects in a mouse model of non-small cell lung cancer (Ge G., Wang A., Yang J., et al., Journal of Experimental & Clinical Cancer Research. 2016; 35). Compared with the control group, transfected mice stably expressing IL-37 showed significantly reduced tumor growth rate, microvessel density, and expression levels of VGEF and CD34. Furthermore, IL-37 treatment significantly inhibited the growth and angiogenesis of human umbilical vein endothelial cells (HUVECs). It is speculated that IL-37 can inhibit tumor angiogenesis and thus play a role in tumor suppression. IL-37 has also been shown to inhibit tumor cells in renal cell carcinoma and cervical cancer (Jiang Y, et al. Med Oncol. Nov 2015; 32(11):250; Wang S., An W., Yao Y., et al. Journal of Cancer. 2015; 6(10):962-969). These studies indicate that IL-37 can inhibit tumor cell migration and proliferation and induce apoptosis by inhibiting STAT3 expression and phosphorylation.

[0010] In summary, as a novel anti-inflammatory inhibitor, IL-37 plays an important role in the immune response, combating inflammatory diseases and autoimmune diseases, making this cytokine a new target for the diagnosis and treatment of cancer, inflammatory diseases, and autoimmune diseases. Summary of the Invention

[0011] The inventors have developed a novel IL-37 fusion protein comprising a mutant IL-37 peptide and an antibody Fc region. Furthermore, the inventors have unexpectedly discovered that the IL-37 fusion protein can be generated, wherein the amino acid sequence of the IL-37 peptide includes an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 181, preferably serine; an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 183, preferably serine; and an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 207, preferably serine. Preferably, the amino acid sequence of the IL-37 peptide includes a serine residue at amino acid positions 181, 183, and 207. The IL-37 fusion protein can be stably expressed in mammalian cell lines such as HEK293 cells and can be produced in high yields. For example, the amino acid sequence of the IL-37 fusion protein may comprise 46 to 218 amino acids. Furthermore, the fusion proteins described herein and illustrated in the appended examples have improved properties, such as increased anti-inflammatory activity, compared to known mutant variants of wild-type IL-37 and / or IL-37 peptides. As another example, the fusion proteins described herein have the ability to be produced in higher product yields while maintaining the potency of known IL-37 constructs (such as wild-type IL-37 and / or known mutant variants of IL-37 peptides). This potency may include anti-inflammatory activity and / or biophysical stability (such as thermal stability) compared to reference IL-37 peptides (such as wild-type IL-37 or known mutant variants of IL-37 peptides). Reference IL-37 constructs and / or peptides described herein and known in the art may include, but are not limited to, those described in WO 2016 / 201503 and WO 2018 / 175403. In particular, these reference IL-37 constructs and / or peptides may contain mutant variants or wild-type peptides and / or constructs, such as fusion constructs.

[0012] In particular, the inventors have discovered that the fusion protein described herein can induce an anti-inflammatory response in subjects in need, such as patients with inflammatory diseases or conditions. In other words, the fusion protein described herein can induce an anti-inflammatory response sufficient to treat subjects in need (such as patients with inflammatory diseases or conditions). They have further discovered that the fusion protein can be produced in a high yield compared to proteins containing IL-37, such as wild-type IL-37 peptides. Therefore, the fusion protein described herein can be used as a therapeutic treatment for diseases (such as inflammatory diseases or conditions) and can be produced in a high yield. For example, a higher product yield of the fusion protein of the present invention can be achieved compared to the product yield of known proteins containing IL-37, such as wild-type IL-37 peptides.

[0013] Therefore, the present invention relates to a fusion protein comprising an IL-37 peptide and an antibody Fc region, wherein the amino acid sequence of the IL-37 peptide comprises an amino acid residue selected from serine, threonine, glycine, alanine, glutamine and asparagine at amino acid position 181, preferably serine;

[0014] An amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 183, preferably serine; and

[0015] The amino acid residue at amino acid position 207 is selected from serine, threonine, glycine, alanine, glutamine, and asparagine, with serine being preferred.

[0016] Preferably, the fusion protein contains serine at amino acid positions 181, 183, and 207.

[0017] Preferably, the fusion protein is an anti-inflammatory fusion protein.

[0018] Preferably, the IL-37 polypeptide of the fusion protein is isotype A or isotype B, more preferably isotype B.

[0019] Preferably, the amino acid sequence of the IL-37 polypeptide of the fusion protein comprises amino acids 46 to 218.

[0020] Preferably, the IL-37 polypeptide of the fusion protein has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% or 94% sequence identity with SEQ ID NO.1, preferably at least 95%, 96%, 97% or 98%, more preferably at least 95% or 96%, and even more preferably at least 96%.

[0021] Preferably, the IL-37 peptide of the fusion protein also contains an additional disulfide bridge compared to the wild-type IL-37 peptide. More preferably, the additional disulfide bridge of the fusion protein is formed between two cysteine ​​residues of the IL-37 peptide, and one of said cysteine ​​residues is a cysteine ​​substitution mutation, preferably at position 169.

[0022] Preferably, the antibody Fc region of the fusion protein is the Fc region of IgG, more preferably IgG1.

[0023] Preferably, the IL-37 peptide of the fusion protein is fused to the Fc region at the C-terminus. Alternatively, the IL-37 peptide of the fusion protein is fused to the Fc region at the C-terminus via a linker.

[0024] Preferably, the Fc region of the fusion protein contains two heavy chain segments, more preferably the CH2 and CH3 domains of the heavy chains.

[0025] Preferably, the IL-37 polypeptide of the fusion protein further includes isoleucine at amino acid position 119 and / or an amino acid selected from threonine, alanine, tyrosine, lysine, phenylalanine, histidine, and glycine at amino acid position 178.

[0026] Preferably, the IL-37 polypeptide of the fusion protein does not contain any mutations in amino acids located at positions 71 to 74, 78, 80, 83 to 88, and 184. Alternatively, the IL-37 polypeptide of the fusion protein contains mutations in one or more amino acids located at positions selected from 71 to 74, 78, 80, 83 to 88, and 184.

[0027] Preferably, the amino acid sequence of the IL-37 polypeptide of the fusion protein contains alanine at amino acid position 85.

[0028] The present invention also relates to pharmaceutical compositions comprising fusion proteins.

[0029] Preferably, the fusion protein or pharmaceutical composition containing the fusion protein further comprises a pharmaceutically acceptable diluent, excipient, or carrier.

[0030] Preferably, the fusion protein or pharmaceutical composition containing the fusion protein is used to treat or prevent inflammatory diseases or conditions. More preferably, the inflammatory disease or condition is selected from inflammatory bowel disease; lupus erythematosus; arthritis; preferably rheumatoid arthritis; psoriasis, allergic rhinitis; Graves' disease; ankylosing spondylitis; ischemia-reperfusion injury, preferably selected from myocardial infarction and stroke; asthma; periodontitis; diabetes and metabolic syndrome; cancer, preferably solid carcinoma types; and atherosclerosis and Behcet's disease. Even more preferably, the inflammatory disease or condition is inflammatory bowel disease.

[0031] The present invention also relates to nucleic acid molecules, vectors and cells containing nucleic acid molecules encoding fusion proteins.

[0032] Similarly, the present invention relates to a method for treating or preventing an inflammatory disease or condition in a patient, comprising administering to the patient a therapeutically effective amount of a fusion protein or a pharmaceutical composition comprising a fusion protein. Detailed Implementation

[0033] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0034] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include plural indicators. Throughout this specification and embodiments, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the said integer or group of integers, but not to exclude any other integer or group of integers.

[0035] Compared to previous methods for producing IL-37 fusion proteins, the present invention is characterized by minor modifications to the IL-37 peptide, resulting in IL-37 fusion proteins with improved properties. For example, the fusion proteins described below exhibit increased anti-inflammatory activity compared to wild-type IL-37 (i.e., naturally occurring or endogenously expressed IL-37). Furthermore, the fusion proteins of the present invention can be produced in higher yields compared to wild-type IL-37 (i.e., naturally occurring or endogenously expressed IL-37). In particular, the fusion proteins of the present invention can be produced in higher yields while at least maintaining the anti-inflammatory activity of wild-type IL-37. In other words, the fusion proteins of the present invention can be proteins possessing at least one biochemical or biophysical activity of wild-type IL-37; for example, they can bind to the interleukin-18 receptor (IL-18R1 / IL-1Rrp) and can be ligands of said interleukin-18 receptor. It can also bind to interleukin-18 binding protein (IL-18BP) (an inhibitory binding protein of interleukin-18 (IL-18)) and subsequently form a complex with the β chain of the IL-18 receptor, thereby inhibiting the activity of IL-18. Other biochemical or biophysical activities of IL-37 include binding to IL-1R8 (Sigirr), blocking the production of pro-inflammatory but non-anti-inflammatory cytokines (including TLR ligands, IFNγ, TNF, and IL-1β) triggered by broad-spectrum inflammatory attack in human or mouse immune cells, inhibiting dendritic cell activation (reducing the surface expression of CD86 and MHC II), triggering specific patterns of intracellular kinase regulation (including blocking the mTOR, MAPK, and NF-κB pathways), and inducing anti-inflammatory kinases (such as Mer and PTEN). As explained above, the fusion proteins described herein exhibit improved biophysical stability compared to reference IL-37 constructs such as wild-type IL-37 peptides or mutant variants of IL-37 peptides. Biophysical stability can include the stability of biophysical properties, such as, but not limited to, thermal stability, heat capacity, aggregation state, solubility, pH stability, resistance to chemical denaturation, and / or resistance to protein hydrolytic degradation. Therefore, as described herein and demonstrated in the accompanying examples, the fusion protein exhibits improved biophysical stability, such as improved thermal stability, compared to a reference IL-37 construct such as the wild-type IL-37 peptide or a mutant variant of the IL-37 peptide as described in the context. In other words, the fusion protein exhibits increased thermal stability compared to a reference IL-37 construct such as the wild-type or mutant variant of the IL-37 peptide. Thermal stability as used herein can refer to increased resistance to denaturant-induced unfolding and increased resistance to heat-induced unfolding.

[0036] High biophysical stability, such as thermal stability, of fusion proteins can lead to high expression yields in recombinant systems and may be important because it is associated with high solubility, thus enabling the fusion protein to be efficiently formulated into a drug at high concentrations. Also for therapeutic purposes, high biophysical stability, such as thermal stability, is likely important to prevent the aggregation of fusion proteins during various manufacturing steps (including expression and purification) and during storage. Therefore, avoiding aggregation is important for maximizing the economic feasibility of drug manufacturing processes for fusion proteins. Finally, also for therapeutic purposes, high biophysical stability, such as thermal stability, is important for obtaining long half-lives of fusion proteins in both patients and disease models.

[0037] For example, the aggregation of fusion proteins as described herein can be reduced compared to wild-type or mutant variants of a reference IL-37 construct such as the IL-37 peptide. As another example, the aggregation of fusion proteins as described herein can be reduced while maintaining the thermal stability of the reference IL-37 construct (such as the wild-type or mutant variant of the IL-37 peptide). As yet another example, the aggregation of fusion proteins as described herein can be reduced while increasing thermal stability compared to wild-type or mutant variants of a reference IL-37 construct such as the IL-37 peptide.

[0038] Therefore, this invention relates to a fusion protein comprising an IL-37 peptide and an antibody Fc region, wherein the amino acid sequence of the IL-37 peptide includes an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 181, preferably serine; an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 183, preferably serine; and an amino acid residue selected from serine, threonine, glycine, alanine, glutamine, and asparagine at amino acid position 207. Most preferably, the amino acid sequence of the IL-37 peptide includes a serine residue at amino acid positions 181, 183, and 207. This invention targets the anti-inflammatory response induced by the fusion protein or a composition thereof, and therefore targets fusion proteins or compositions thereof for treating inflammatory diseases or conditions in subjects requiring such treatment. Therefore, in the context of this invention, it is preferred that, in the case of currently treating subjects with a fusion protein or a composition thereof, the fusion protein or composition thereof induces an anti-inflammatory response in the subject.

[0039] The method of the present invention can be used to treat diseases in subjects who require such treatment. For example, the method can be used to administer a therapeutically effective amount of a fusion protein or a composition thereof to a subject. The method can also be used to prevent diseases, for example by administering a therapeutically effective amount of a fusion protein or a composition thereof to a subject. An "effective amount" or "therapeutically effective amount" of a pharmaceutical preparation (e.g., a pharmaceutical formulation) refers to an amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dose and for the necessary duration.

[0040] In the context of this invention, "fusion protein" refers to a protein composed of polypeptide moieties from different sources. Therefore, it can also be understood as a "chimeric protein." The fusion protein of this invention refers to a fusion protein in which the IL-37 polypeptide is directly or indirectly linked to an antibody Fc region (such as the IgG Fc region). For example, the fusion protein comprises a human IL-37 polypeptide linked to a human IgG Fc region. As another example, the fusion protein comprises a human IL-37 polypeptide linked to a human IgG1 Fc region. The fusion protein of any one of the preceding claims, wherein the IL-37 polypeptide is fused to the Fc region at the C-terminus.

[0041] Typically, fusion proteins are proteins produced by linking two or more genes (or preferably cDNAs) that originally encode a single protein. Translation of the fusion gene (or fusion cDNA) yields a single polypeptide, preferably having functional properties derived from each of the original proteins. Recombinant fusion proteins are artificially produced using recombinant DNA technology for use in biological research or therapy. The fusion proteins of the present invention can also be modified by conjugation or fusion with another portion to facilitate purification or increase the in vivo half-life of the fusion protein. Modifications considered herein include, but are not limited to, side-chain modifications, incorporation of non-natural amino acids and / or their derivatives during polypeptide synthesis, and other methods using cross-linking agents and imposing conformational constraints on the polypeptides of the present invention. For example, the fusion proteins of the present invention can be modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc.

[0042] The fusion proteins of this invention are generally anti-inflammatory fusion proteins. In this context, "interleukin-37" or "IL-37" refers to a member of the interleukin-1 cytokine (IL-1) family and is primarily an anti-inflammatory cytokine that reduces systemic and local inflammation. Expression is highly induced by several Toll-like receptor (TLR) ligands and pro-inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ. IL-37 is also known as interleukin-37 (FIL1ζ; IL-1ζ; IL-1F7b (IL-1H4, IL-1H, IL-1RP1); IL-1X protein; IL1F7 (standard product IL-1F7b); interleukin-1 family member 7; interleukin-1ζ; interleukin-1 homolog 4; interleukin-1 superfamily 2; interleukin-1 related protein and interleukin-23). ​​The anti-inflammatory properties of the peptides of this invention can be determined by any of the methods described herein, particularly those described in the examples.

[0043] IL-37 exhibits significant sequence similarity to IL-18 and is highly expressed in inflamed tissues, suppressing excessive inflammatory responses. In other words, IL-37 is an effective innate immunosuppressant by diverting cytokine balance from excessive inflammation. In this context, IL-37 can bind to the interleukin-18 receptor (IL-18R) and can be a ligand for IL-18R. This cytokine also binds to interleukin-18 binding protein (IL18BP) (an inhibitory binding protein of interleukin-18 (IL18)) and subsequently forms a complex with the β subunit of the IL18 receptor, thereby inhibiting the activity of IL18. The fusion protein of the present invention can bind to IL-18R, leading to downstream signaling of IL-18R. The fusion protein used in the context of this invention is capable of binding to IL-18R and leading to downstream signaling of IL-18R. The function and / or activity of the fusion protein of the present invention can be determined by methods known in the art, including but not limited to ELISA and ligand-receptor binding assays. Unless otherwise stated, the terms “IL-37” or “IL-37 polypeptide” or “IL-37 protein” as used herein broadly refer to any naturally occurring IL-37 from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats). The terms cover “full-length”, unprocessed IL-37, and any form of IL-37 produced by cellular processing. For example, the invention covers both full-length IL-37 and mature forms, wherein IL-37 is converted from an inactive precursor state to an active state by cleavage of caspase-1 during expression. The terms also cover naturally occurring IL-37 variants, such as splice variants or allelic variants. An exemplary amino acid sequence of IL-37 is shown in SEQ ID NO. 1. As used herein, the IL-37 polypeptide comprising SEQ ID NO.: 1, such as the amino acid sequence of an IL-37 polypeptide comprising amino acids 46 to 218, may refer to a wild-type IL-37 polypeptide. The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region as defined herein.

[0044] Five alternatively spliced ​​transcript variants encoding five different isoforms of IL-37 have been reported. Notably, amino acid sequences 53 to 206 of isoform B of IL-37 are identical to amino acid sequences 27 to 181 of isoform A of IL-37. As used herein, IL-37 can be any isoform of IL-37, such as any isoform of IL37a-e. In the context of this invention, IL-37 is isoform A or B of IL-37, preferably isoform B of IL-37.

[0045] In the context of this invention, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers containing amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics or corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. Therefore, in the context of this invention, the term "polypeptide" refers to a chain comprising or composed of amino acid monomers linked by peptide (amide) bonds. A peptide bond is a covalent chemical bond formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. "Polypeptide" herein is not limited to molecules of a defined length. Therefore, the term "polypeptide" herein refers to peptides, oligopeptides, proteins, or polypeptides comprising chains of amino acids, wherein the amino acid residues are linked by covalent peptide bonds. However, the term "polypeptide" herein also encompasses peptide mimics of such proteins / polypeptides, wherein one or more amino acids and / or one or more peptide bonds have been replaced by functional analogs. The term "polypeptide" also refers to, but does not exclude, modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well described in the art.

[0046] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids. Amino acids may be represented herein by their commonly known three-letter symbols or by single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0047] "Antibody" refers to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies (i.e., the antibodies constituting the group are identical and / or bind to the same epitopes, except for antibodies containing, for example, naturally occurring mutations or possible variants generated during the production of the monoclonal antibody formulation, such variants typically exist in small quantities). In contrast to polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein. A "multispecific antibody" is a monoclonal antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic redirection effects to prepare antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004) or crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)). An "antibody fragment" is a molecule that is distinct from the complete antibody, containing a portion of the antigen bound to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0048] The terminology also refers to human antibodies (particularly recombinant human antibodies), heterologous antibodies, and heterologous hybrid antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. In particular, the term "recombinant human antibody" includes antibodies against all human sequences prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from transgenic animals (e.g., mice) for human immunoglobulin genes; antibodies expressed using recombinant expression vectors transfected into host cells; antibodies isolated from recombinant human antibody libraries; or antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Therefore, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, such antibodies can be mutagenized in vitro (or, when using animals that are transgenic for human Ig sequences, in vivo somatic mutagenized), so that although the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and associated with human germline VH and VL sequences, the amino acid sequences of the VH and VL regions of the recombinant antibody may not be sequences naturally present in the in vivo human antibody germline library.

[0049] Generally, antibodies can be derived from immunizing animals or from recombinant antibody libraries, including antibody libraries based on amino acid sequences encoded by nucleic acids that have been designed on a computer and synthesized. For example, antibody sequences can be computer-designed by analyzing a database of human sequences and using the data obtained therefrom to design peptide sequences. For example, methods for designing and obtaining computer-generated sequences are described in, for example, Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001) 254:67; and U.S. Patent No. 6,300,064 to Knappik et al.

[0050] The term "Fc region" in this article is used to define the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region contains the C-terminal region containing antibodies. H 2 and C H The Fc region comprises two heavy chain segments, preferably CH2 and CH3 domains of the heavy chain. In the context of this invention, the Fc region contains two heavy chain segments, preferably CH2 and CH3 domains of the heavy chain. The two heavy chain segments are linked by two or more disulfide bonds and C... H The hydrophobic interactions of the three domains keep them together. The heavy chain constant domains corresponding to different classes of immunoglobulins are named α, δ, ε, γ, and μ, respectively.

[0051] In some respects, fusion proteins do not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to those biological activities attributable to the antibody's Fc region, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a decrease / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays (see, for example, ACTI for flow cytometry) can be used. TM Non-radioactive cytotoxicity assays (Cell Technology, Mountain View, California); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo (e.g., in animal models, such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the antibody does not bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0052] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitutions to alanine residues 265 and 297 (US Patent No. 7,332,581). For example, antibody variants may comprise an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues). For example, the substitutions are L234A and L235A (LALA) (see, for example, WO2012 / 130831). Furthermore, alterations can be made in the Fc region that result in altered (i.e., reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0053] In some aspects, the Fc region includes mutations in the complement (C1q) and / or Fcγ receptor (FcγR) binding sites. In some aspects, such mutations can prevent the fusion protein from producing antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The Fc region, as used in the context of this invention, does not trigger cytotoxicity, such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0054] The term "Fc region" also includes both native Fc regions and variant Fc regions. An Fc region may include the carboxyl terminus of the heavy chain. Antibodies produced by host cells may undergo post-translational cleavage from the C-terminus of the heavy chain, specifically one or two amino acids. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may include the full-length heavy chain or cleaved variants of the full-length heavy chain. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the antibody Fc region may be considered. Amino acid sequence variants of the antibody Fc region can be prepared by introducing appropriate modifications into the nucleotide sequence encoding said antibody or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the amino acid sequence of the antibody Fc region. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as inducing or supporting an anti-inflammatory response.

[0055] Fusing IL-37 to the Fc region of an antibody can produce constructs that have a beneficial effect on IL-37 productivity. Specifically, fusing IL-37 to the Fc region of an antibody can produce constructs with improved productivity compared to references (such as wild-type or mutant IL-37 peptides or constructs). On the other hand, fusing IL-37 to the Fc region of an antibody can produce constructs that have a beneficial effect on IL-37 productivity while maintaining the potency of known IL-37 constructs and fusion proteins known in the art. In other words, fusing IL-37 to the Fc region of an antibody can improve IL-37 productivity compared to references (such as wild-type or mutant IL-37 peptides or constructs) while maintaining the potency of known IL-37 constructs and fusion proteins known in the art. As used herein, the term "potency" with respect to the fusion proteins of the present invention refers to the ability of the fusion protein to bind IL-18R and / or activate downstream IL-18R. Specifically, the potency of the fusion proteins of the present invention refers to anti-inflammatory activity as described in the context herein. Potency can be assessed in receptor binding assays or cell-based binding assays. Its efficacy can be compared with that of reference IL-37 peptides, such as wild-type IL-37 peptides.

[0056] As used herein, the term “productivity” refers to the amount (e.g., mg product / ml) of protein (such as the fusion protein described herein) formed within a given volume. The term “amount” as used herein refers to the yield of protein product, such as the concentration of the fusion protein as described herein. Therefore, “increasing the productivity of a protein (such as the fusion protein described herein)” means an increase in the amount of protein in a given volume compared to a corresponding reference (e.g., wild-type IL-37, mutant IL-37 peptide, or a construct thereof).

[0057] In some respects, the yield of the fusion protein product is at least 43 mg / ml, preferably at least 186 mg / ml.

[0058] The Fc region of an antibody can be the Fc region of any class of antibody, such as IgA, IgD, IgE, IgG, and IgM. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Therefore, as used in the context of this invention, an antibody can be the Fc region of IgG. For example, the Fc region of an antibody can be the Fc region of IgG1, IgG2, IgG2b, IgG3, or IgG4. In some aspects, the fusion protein of this invention comprises IgG in the Fc region of an antibody. In the context of this invention, the Fc region of the antibody is preferably the Fc region of IgG1.

[0059] Furthermore, the fusion proteins provided herein may include a linker (or “spacer”). In the context of this invention, the IL-37 peptide is fused to the Fc region at its C-terminus via a linker. The linker is typically a peptide having a length of up to 20 amino acids. The terms “linked to” or “fused to” refer to a covalent bond, such as a peptide bond, formed between the two portions. Therefore, in the context of this invention, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. For example, the fusion proteins provided herein may include a linker between the IL-37 peptide and the Fc region of an antibody, such as a linker between the N-terminus of the Fc region and the C-terminus of the IL-37 peptide. As another example, the fusion proteins provided herein may include a linker between the IL-37 peptide and the Fc region of an antibody, such as a linker between the C-terminus of the Fc region and the N-terminus of the IL-37 peptide. Specifically, the IL-37 peptide may be fused to the N-terminus of the Fc region at its C-terminus via a linker. The advantage of such linkers is that they allow different peptides of the fusion protein to fold more independently and behave as intended. Therefore, in the context of this invention, the Fc regions of the IL-37 peptide and the antibody can be contained within a single-chain multifunctional peptide. In some aspects, the fusion protein of this invention includes a peptide linker. In some aspects, the peptide linker connects the IL-37 peptide to the Fc region of the antibody. In some aspects, the peptide linker may include the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS). In some aspects, the peptide linker may contain the amino acid sequence GGGGS.

[0060] Several IL-37 expressed in mammalian cells have been sequenced, revealing that it originates at Val46 (Pan et al., 2001, Cytokine 13:1-7). Unprocessed (uncut) sequences have also been detected. In some aspects, the fusion proteins of the present invention comprise fragments of IL-37, such as fragments of the IL-37 polypeptide, or fragments of IL-37 isotypes. For example, such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, when compared to the full-length native protein. Some fragments lack amino acid residues that are not essential for the desired biological activity of the IL-37 polypeptide of the present invention. Therefore, in some aspects, fragments of the IL-37 polypeptide possess biological activity.

[0061] In the context of this invention, the amino acid sequence of the IL-37 polypeptide comprises amino acids 46 to 218. For example, the amino acid sequence of the IL-37 polypeptide may comprise amino acids 46 to 218 of isotype B. As another example, the amino acid sequence of the IL-37 polypeptide may comprise amino acids 46 to 218 of SEQ ID NO. 1. As yet another example, the amino acid sequence of the IL-37 polypeptide may comprise amino acids 21 to 218, as in isotype B. As yet another example, the amino acid sequence of the IL-37 polypeptide may comprise amino acids 21 to 218, as in SEQ ID NO. 1. In some aspects, the amino acid sequence of the IL-37 polypeptide consists of amino acids 46 to 218. In some aspects, the amino acid sequence of the IL-37 polypeptide consists of amino acids 46 to 218 of isotype B. In some aspects, the amino acid sequence of the IL-37 polypeptide consists of amino acids 46 to 218 of SEQ ID NO. 1.

[0062] In some aspects, the IL-37 polypeptide comprises the amino acid sequence of SEQ ID NO. 1. The anti-inflammatory fusion protein of any of the preceding claims, wherein the IL-37 polypeptide has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, preferably at least 95%, 96%, 97%, or 98%, more preferably at least 95% or 96%, and even more preferably at least 96% sequence identity with SEQ ID NO. 1. However, it should be understood that the invention also contemplates minor sequence variations, such as insertions, deletions, substitutions, and especially conserved amino acid substitutions of IL-37, which do not adversely affect the function and / or activity of IL-37. For example, variations of the native sequence of IL-37 (such as SEQ ID NO. 1) can be made using any techniques and guidelines for conserved and non-conserved mutations, such as those described in U.S. Patent No. 5,364,934. As used herein, the term "conserved substitution" refers to the replacement of an amino acid present in the native sequence of a peptide with a naturally or non-naturally occurring amino acid or peptide mimic having similar spatial characteristics. When the side chain of the natural amino acid to be replaced is polar or hydrophobic, conservative substitution should be carried out using a naturally occurring amino acid, a non-natural amino acid, or a peptide analog that is also polar or hydrophobic (and has the same spatial properties as the side chain of the amino acid being replaced).

[0063] Variations can be substitutions, deletions, or insertions of one or more codons encoding the native sequence or variant IL-37, resulting in a change in its amino acid sequence compared to the corresponding native sequence or variant IL-37 (e.g., SEQ ID NO 1). Optionally, a variation is achieved by substituting at least one amino acid with any other amino acid in one or more domains of the native sequence IL-22 polypeptide. By comparing the sequence of IL-22 with the sequences of homologous known protein molecules and minimizing the number of amino acid sequence changes occurring in highly homologous regions, guidance can be found regarding which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity. Amino acid substitution can be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the activity of the resulting variant (e.g., in in vitro assays known in the art).

[0064] As used herein, amino acid residues at positions corresponding to those in SEQ ID NO:1 can be determined by any means known to those skilled in the art. For example, comparing one or more sequences with the amino acid sequence of SEQ ID NO:1 enables those skilled in the art to determine amino acids at positions corresponding to those in SEQ ID NO:1. Those skilled in the art can compare the three-dimensional structure of a polypeptide with the three-dimensional structure of a polypeptide having the amino acid sequence of SEQ ID NO:1 and determine amino acid residues at positions equivalent to those in SEQ ID NO:1.

[0065] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum sequence identity, and after any conserved substitutions are not considered part of the sequence identity for alignment purposes. Alignment for determining the percentage of amino acid sequence identity can be performed in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office (Washington DC 20559) and registered with the U.S. Copyright Office under U.S. Copyright Registration No. TXU510087 and described in WO 2001 / 007611.

[0066] Unless otherwise stated, for the purposes of this article, the search procedure using FASTA package version 36.3.8c or later employs a BLOSUM50 comparison matrix to generate the percentage values ​​of amino acid sequence identity. The FASTA package is authored by W.R. Pearson and DJ Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W.R. Pearson (1996), “Effective protein sequence comparison”, Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare sequences using the search (global protein:protein) procedure and default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to ensure that a global rather than a local alignment is performed. The percentage of amino acid identity is given in the output alignment header.

[0067] In the context of this invention, Compared with wild-type IL-37 peptideThe fusion protein of the present invention may further comprise an additional disulfide bridge. In other words, compared to the wild-type IL-37 polypeptide, the fusion protein of the present invention comprises an additional disulfide bond formed between a first cysteine ​​residue of the fusion protein and a second distinct cysteine ​​residue of the fusion protein. In other words, the IL-37 of the fusion protein of the present invention comprises a first cysteine ​​and a second cysteine, the first cysteine ​​being introduced by mutation in the IL-37, the second cysteine ​​being a naturally occurring cysteine ​​residue of the IL-37, and the first and second cysteines forming a disulfide bond. For example, a disulfide bridge may be formed with the cysteine ​​residue at position 122 of the IL-37 polypeptide. For example, the wild-type IL-37 polypeptide may comprise amino acids 46 to 218 of SEQ ID NO.:1, such as SEQ ID NO.:1. The introduction of non-natural disulfide bridges for stabilization techniques is described in references to, for example, WO 94 / 029350, US5,747,654, Rajagopal, V., et al., Prot. Engin. 10 (1997) 1453-1459; Reiter, Y., et al., Nature Biotechnology 14 (1996) 1239-1245; Reiter, Y., et al., Protein Engineering; 8 (1995) 1323-1331; Webber, KO, et al., Molecular Immunology 32 (1995) 249-258; Reiter, Y., et al., Immunity 2 (1995) 281-287; Reiter, Y., et al., JBC 269 (1994) 18327-18331; Reiter, Y., et al., Inter. J. of Cancer 58 (1994) 142-149, or Reiter, Y., Cancer Res. 54 (1994) 2714-2718. In a preferred aspect of the invention, an additional disulfide bridge is formed between two cysteine ​​residues of the IL-37 polypeptide, and one of said cysteine ​​residues is a substitution mutation of the cysteine, preferably at position 169. The native amino acid sequence can be altered to produce the mutant polypeptide by various means known to those skilled in the art, such as by insertion, deletion, and / or substitution. For example, site-specific mutations can be introduced by linking a synthetic oligonucleotide containing the modified site to an expression vector. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as those disclosed in Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Patent Nos. 4,518,584 and 4,737,462.The preferred method for introducing mutations is the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California). Any suitable expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and a corresponding suitable host can be used to produce recombinant peptides. Expression hosts include, but are not limited to, bacterial species within the genera *Escherichia*, *Bacillus*, *Pseudomonas*, and *Salmonella*, mammalian or insect host cell systems (including baculovirus systems) (e.g., as described in Luckow et al., *Bio / Technology 6:47 (1988)), and established cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. Those skilled in the art will recognize that the choice of expression host affects the type of peptide produced. For example, the glycosylation of peptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that of peptides produced in bacterial cells such as *Escherichia coli*. Alternatively, the peptides of the present invention can be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., as outlined in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). In particular, the peptides can be synthesized using solid-phase synthesis procedures (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30:705-739 (1987); and U.S. Patent No. 5,424,398). An automated peptide synthesizer can be used if desired. Removal of the tert-butoxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (Fmoc) amino acid blocking groups and separation of the peptides from the resin can be accomplished, for example, by acid treatment at a reduced temperature. The mixture containing the polypeptide can then be extracted, for example, with dimethyl ether to remove non-peptide organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). After polypeptide synthesis, further purification (e.g., using high-performance liquid chromatography (HPLC)) can optionally be performed to eliminate any incomplete polypeptides or free amino acids. The synthesized polypeptide can be analyzed by amino acid and / or HPLC to verify its identity. For other applications according to the invention, the polypeptide may preferably be generated as part of a larger fusion protein (as described herein or by other genetic methods), or as part of a larger conjugate (e.g., by physical or chemical conjugation), as known to those skilled in the art and described herein.

[0068] In one aspect of the invention, the IL-37 polypeptide may further include isoleucine at amino acid position 119 and / or an amino acid selected from threonine, alanine, tyrosine, lysine, phenylalanine, histidine, and glycine at amino acid position 178.

[0069] In one aspect of the invention, the IL-37 polypeptide does not contain any mutations in amino acids located at positions 71 to 74, 78, 80, 83 to 88, and 184.

[0070] In another aspect of the invention, the IL-37 polypeptide comprises a mutation of one or more amino acids at positions selected from amino acid positions 71 to 74, 78, 80, 83 to 88, and 184. In a preferred aspect of the invention, the amino acid sequence of the IL-37 polypeptide comprises alanine at amino acid position 85.

[0071] For example, the IL-37 polypeptide may contain mutations at one or more amino acids, as described in WO2016 / 201503. For example, amino acid residues at positions 71, 72, 73, 74, 78, 80, 83, 84, 85, 86, 87, 88 and / or 184 in SEQ ID NO:1, or at equivalent positions, may be mutated.

[0072] In this context Located at one or more amino acid positions selected from positions 71 to 74, 78, 80, 83 to 88, and 184. amino acids mutation It may contain Non-conservative substitution mutations relative to the amino acid present at that position, such as in SEQ ID NO.1 at that position.

[0073] As used herein, the phrase "non-conservative substitution" or "non-conservative residue" refers to the replacement of an amino acid present in the parental sequence with another naturally occurring or non-naturally occurring amino acid that has different electrochemical and / or steric properties. Therefore, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the substituted natural amino acid and / or may have functional groups with significantly different electronic properties than the substituted amino acid. Examples of this type of non-conservative substitution include phenylalanine or cyclohexylmethylglycine replacing alanine, isoleucine replacing glycine, or -NH-CH[(-CH2)5-COOH]-CO- replacing aspartic acid. Non-conservative substitution includes any mutation that is not considered conserved.

[0074] Nonconservative amino acid substitutions can be caused by changes in: (a) the structure of the amino acid backbone in the substituted region; (b) the charge or hydrophobicity of the amino acid; or (c) the volume of the amino acid side chain. Substitutions that are generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue substitutes for (or is substituted by) a hydrophobic residue; (b) proline substitutes for (or is substituted by) any other residue; (c) a residue with a large side chain (e.g., phenylalanine) substitutes for (or is substituted by) a residue without a side chain (e.g., glycine); or (d) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidine) substitutes for (or is substituted by) a negatively charged residue (e.g., glutamyl or aspartic acid).

[0075] For example, the mutation could be a substitution with alanine or an amino acid with the opposite charge. As another example, the amino acid at position 85 could be alanine, the amino acid at position 83 could be glutamic acid, the amino acid at position 73 could be alanine, and / or the amino acid at position 73 could be lysine.

[0076] One aspect of this invention relates to nucleic acid molecules encoding fusion proteins as provided herein. The term "nucleic acid molecule" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of: a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)); a sugar (i.e., deoxyribose or ribose); and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, by which the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5' to 3'. Hereinafter, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having a derived sugar or phosphate backbone linked or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable for direct expression of the antibodies of the present invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823B1). Thus, a nucleic acid sequence “encoding” a selected polypeptide is a nucleic acid molecule that, when placed under the control of an appropriate regulatory sequence, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end.

[0077] One aspect of this invention relates to a vector comprising a nucleic acid molecule encoding a fusion protein as provided herein. Therefore, this invention provides a vector for the prevention or treatment of inflammatory diseases or conditions, comprising a polynucleotide sequence encoding a polypeptide of the invention. As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” The techniques used can be found in any of the following well-known references: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, California), and Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, New York). Expression vectors are routinely constructed in the field of molecular biology and can, for example, involve the use of plasmid DNA and appropriate starters, promoters, enhancers, and other elements, such as polyadenylation signals that may be necessary and correctly oriented, to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. In this regard, as a further example, we refer to Sambrook et al.

[0078] One aspect of this invention relates to a cell comprising a nucleic acid molecule encoding a fusion protein as provided herein, or a vector comprising a nucleic acid molecule encoding a fusion protein as provided herein. As used herein, a cell may refer to a host cell. The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to a cell in which a foreign nucleic acid has been introduced, including progeny of such cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. Transformed cells include transiently or stably transformed cells. The nucleic acid content of progeny cells may not be exactly the same as that of parent cells, but may contain mutations. This includes mutant progeny cells that have the same function or biological activity as screened or selected in the initial transformed cells. In some aspects, host cells are transiently transfected with a foreign nucleic acid. In other aspects, host cells are stably transfected with a foreign nucleic acid. An “isolated” fusion protein is a fusion protein isolated from the environment of a host cell from which the fusion protein was recombinantly produced. In some respects, the fusion proteins of the present invention are purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0079] In the context of this invention, pharmaceutical compositions comprising fusion proteins as described herein are provided. The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to an preparation which is in a form that makes the biological activity of the active ingredient contained therein effective and does not contain any additional components that would have unacceptable toxicity to a subject administering the pharmaceutical composition. Furthermore, in the context of this invention, methods for treating or preventing inflammatory diseases or conditions in patients are provided, comprising administering to the patient a therapeutically effective amount of a fusion protein or pharmaceutical composition as used in the context of this invention. Therefore, the methods or uses of this invention, or the fusion proteins or pharmaceutical compositions of this invention, can be used to reduce, inhibit, or prevent inflammation induced by other mediators such as other cytokines (IL-1, IFNγ, etc.), other mediators (e.g., complement, leukotrienes, etc.), and chemical and physical damage.

[0080] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be directed at prevention or at the clinicopathological stage. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, slowing of disease progression, improvement or mitigation of the disease state, and relief or improvement of prognosis. For example, in the context of IBD, “treatment” could refer to a reduced likelihood of developing IBD, a reduced rate of IBD occurrence, and a reduction in disease severity. “Alleviation”, “alleviating”, or their equivalents refer to therapeutic treatments and preventative or preventative measures aimed at improving, preventing, slowing (reducing), reducing, or inhibiting the formation of a disease or condition, such as atherosclerotic plaques. Patients requiring treatment include those who already have a disease or condition, those who are susceptible to a disease or condition, or those whose disease or condition requires prevention.

[0081] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some respects, the individual or subject is a human.

[0082] In one aspect of the invention, the pharmaceutical composition comprises a pharmaceutically acceptable diluent, excipient, or carrier. The term "pharmaceutically acceptable," used in conjunction with the compositions of the invention, means the molecular entity and other components of such a composition that are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). The term "pharmaceutically acceptable" may also mean a component approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, more particularly in humans. "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or formulation that is non-toxic to a subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Such pharmaceutically acceptable carriers can be sterile liquids such as water, saline solutions, dextran solutions, glycerol solutions, and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, ARGennaro, 20th edition.

[0083] In one aspect of the invention, the pharmaceutical composition is used to treat or prevent inflammatory diseases or conditions.

[0084] As used interchangeably herein, “disorder,” “disease,” or “symptom” refers to any symptom that would benefit from treatment with the compositions described herein (e.g., pharmaceutical compositions), such as compositions comprising the fusion protein of the present invention (e.g., pharmaceutical compositions). This includes chronic and acute disorders or diseases, including those pathological conditions that make mammals susceptible to the disorders discussed. In some aspects, the disorder is an IL-37-related disorder. In some aspects, the inflammatory disease or symptom is selected from inflammatory bowel disease; lupus erythematosus; arthritis; preferably rheumatoid arthritis; psoriasis; allergic rhinitis; Graves' disease; ankylosing spondylitis; ischemia-reperfusion injury, preferably selected from myocardial infarction and stroke; asthma; periodontitis; diabetes and metabolic syndrome; cancer, preferably solid carcinoma types; and atherosclerosis and Behcet's disease. In a preferred aspect of the invention, the inflammatory disease or symptom is inflammatory bowel disease.

[0085] The terms “inflammatory bowel disorder,” “inflammatory bowel disease,” and “IBD,” which are used interchangeably in this document, are used in the broadest sense and encompass all diseases and pathological conditions whose pathogenesis involves recurrent inflammation in the intestines (including the small and colon). IBD includes, for example, ulcerative colitis and Crohn's disease. IBD is not limited to UC and CD. The manifestations of this disease include, but are not limited to, inflammation and decreased intestinal epithelial integrity.

[0086] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments can be implemented based on the general description provided above.

[0087] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity, the description and examples should not be construed as limiting the scope of the invention. All disclosures in patent documents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

[0088] Example

[0089] To produce the desired gene / protein (e.g., an IL-1 containing one or more amino acid substitution mutations). 37 peptide variants, and fusion proteins containing IL-37 peptide and antibody Fc region)

[0090] Molecular biology methods

[0091] As described in Sambrook, J. et al., Molecular cloning: A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), DNA should be handled using standard methods. Molecular biology reagents should be used according to the manufacturer's instructions.

[0092] The desired gene segment was prepared chemically at Geneart (Regensburg, Germany). The synthesized gene fragment was cloned into an *E. coli* plasmid for proliferation / amplification. Alternatively, chemically synthesized oligonucleotides were prepared by annealing or short synthetic DNA fragments were assembled by PCR. The corresponding oligonucleotides were prepared at metabion (Planegg-Martinsried, Germany). Alternatively, a complete expression plasmid containing the desired cDNA was prepared at Twist Bioscience (San Francisco, California, USA). The complete DNA sequence of the cloned and / or subcloned gene fragment was verified by DNA sequencing.

[0093] To express the desired gene / protein (e.g., IL-37 polypeptide, wild-type or mutant variant, such as one or more substitution mutations of one or more amino acids; or IL-37 polypeptide fused to, for example, the human Fc region (Fc fragment) of IgG-Fc (wild-type or mutant variant)) in HEK293 cells, a transcription unit containing the following functional elements is used:

[0094] - From the immediate early enhancer and promoter of human cytomegalovirus (P-CMV), including intron A.

[0095] -human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0096] -Mouse immunoglobulin heavy chain variable region signal sequence.

[0097] - The gene / protein to be expressed (e.g., the IL-37 peptide, or a fusion protein containing the human Fc region of the IL-37 peptide and human IgG, or a mutant variant thereof), and

[0098] - Bovine growth hormone polyadenylated sequence (BGH pA).

[0099] In addition to the expression unit / cassette containing the desired gene to be expressed, basic / standard mammalian expression plasmids also contain

[0100] - The replication origin from the vector pUC18, which allows the plasmid to replicate in E. coli, and

[0101] - β-lactamase gene that confers ampicillin resistance in Escherichia coli.

[0102] To express a desired gene / protein (e.g., an IL-37 polypeptide, either wild-type or mutant, with one or more substitution mutations of one or more amino acids; or an IL-37 polypeptide fused to, for example, the human Fc region (Fc fragment) of IgG-Fc (either wild-type or mutant)) in E. coli cells, a transcription unit containing the following functional elements is used:

[0103] -T5 starter

[0104] -lac operator

[0105] - The gene / protein to be expressed (e.g., the IL-37 peptide, or a fusion protein containing the human Fc region of the IL-37 peptide and human IgG, or a mutant variant thereof).

[0106] -Terminator

[0107] In addition to the expression unit / cassette containing the desired gene to be expressed, the basic / standard E. coli expression plasmid also contains

[0108] - The replication origin from the pUC18 vector, which allows the plasmid to replicate in E. coli.

[0109] -lacI expression cassette to allow inducible expression of the target gene.

[0110] - β-lactamase expression cassette conferring ampicillin resistance in E. coli

[0111] -URA3 expression cassette, which allows for antibiotic-free expression using complementation in appropriate E. coli strains.

[0112] Production of IL-37 peptide and fusion proteins containing IL-37 peptide and antibody Fc region

[0113] The recombinant IL-37 polypeptide consists of: i) a wild-type human IL-37 polypeptide or a mutant variant of the human IL-37 polypeptide (the mutant variant containing specific amino acid exchanges), and optionally a protein tag for purification (e.g., His-tag, C-tag, etc.) or for enzymatic conjugation purposes (localizing enzyme-tag). Alternatively, the IL-37 polypeptide (wild-type or mutant variant) is genetically fused to the human Fc region (Fc fragment) of IgG or a mutant variant thereof via an adapter sequence.

[0114] Expression plasmids used for transient expression of soluble IL-37 peptides or fusion proteins containing IL-37 peptides and antibody Fc regions in HEK293 cells contain, in addition to the IL-37 peptide expression cassette, an origin of replication from the vector pUC18 (which allows the plasmid to replicate in E. coli) and a β-lactamase gene that confers ampicillin resistance in E. coli.

[0115] The transcriptional unit of the IL-37 polypeptide contains the following functional elements:

[0116] - From the immediate early enhancer and promoter of human cytomegalovirus (P-CMV), including intron A.

[0117] -human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0118] -Mouse immunoglobulin heavy chain signal sequence

[0119] The -N-terminated truncated Staphylococcus aureus localization enzyme A encodes nucleic acid, and

[0120] - Bovine growth hormone polyadenylated sequence (BGH pA).

[0121] The expression plasmid for expressing the IL-37 peptide in *E. coli* cells contains an IL-37 peptide expression cassette as well as an origin of replication from the pUC18 vector. This allows the plasmid to replicate in *E. coli*. Furthermore, it contains a β-lactamase expression cassette conferring ampicillin resistance in *E. coli*, a lacI expression cassette allowing inducible expression of the target gene, and a URA3 expression cassette allowing antibiotic-free expression using complementation in appropriate *E. coli* strains.

[0122] Expression of fusion proteins containing mutant variants of the IL-37 peptide and the Fc region of an antibody in mammalian cells

[0123] Recombinant production was achieved by transient transfection of HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen, Carlsbad, California, USA). Alternatively, the Expi293 expression system (Invitrogen) was used. To produce an IL-37 fusion protein containing the antibody Fc region and only a single IL-37 polypeptide (i.e., a single IL-37 moiety), cells were co-transfected using a plasmid containing the corresponding IL-37 polypeptide and antibody Fc unit (i.e., the IL-37-Fc unit) and a second plasmid encoding a non-fusion Fc fragment (i.e., the non-fusion Fc unit) using a mortar and pestle technique. Transfection was performed using methods known in the art and as specified in the respective manufacturers' instructions. Cell culture supernatants were harvested three to seven (3–7) days after transfection. The supernatants were stored at a reduced temperature (e.g., -80°C).

[0124] Purification of IL-37 peptide from mammalian cultures

[0125] Culture supernatant containing IL-37 peptide was captured by affinity chromatography using a HiTrap complete His-Tag (Roche, Basel, Switzerland) equilibrated with 50 mM NaH2PO4 and 300 mM NaCl (pH 7.0). Unbound protein was removed by washing with equilibration buffer. IL-37 peptide was eluted with 10 CV in a gradient up to 50 mM NaH2PO4, 300 mM NaCl, and 500 mM imidazole (pH 7.0). Superdex 75 was used. TMSize exclusion chromatography (SUC) was performed as a second purification step on a GE Healthcare (Chicago, Illinois, USA) sample, and monomeric protein fractions were combined. SUC was performed in 20 mM M stidin, 140 mM NaCl, pH 6.0. The concentration of purified IL-37 peptide was determined by measuring the optical density (OD) at 280 nm, with OD at 320 nm used as background correction, and the molar extinction coefficient calculated based on the amino acid sequence. The purity of the IL-37 peptide was analyzed by CE-SDS under denaturing and reducing conditions. BioSuite was used. TM HR analysis of size exclusion columns (Waters, Milford, MA, USA) was performed using SEC to determine aggregate content. The purified protein was stored at -80°C.

[0126] Purification of IL-37-Fc fusion protein from mammalian cultures

[0127] Culture supernatants containing the fusion protein (containing the IL-37 peptide and the antibody Fc region) were captured by affinity chromatography using a HiTrap MabSelectSuRe (GE Healthcare) equilibrated with 1x PBS (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were removed by washing with equilibration buffer, and the IL-37-Fc fusion protein was eluted with 0.1 M citrate buffer (pH 2.8) and immediately neutralized to pH 6.0 with 2 M Tris-base (pH 9.0). Superdex 200 was used. TM Size exclusion chromatography (SUC) on a GE Healthcare microscope was used as the second purification step, and monomeric protein fractions were combined. SUC was performed in 20 mM Histidin, 140 mM NaCl, pH 6.0. The protein concentration of the purified IL-37-Fc fusion protein was determined by measuring the optical density (OD) at 280 nm, with OD at 320 nm used as background correction, and the molar extinction coefficient calculated based on the amino acid sequence. The purity of the IL-37-Fc fusion protein was analyzed by CE-SDS under denaturing and reducing conditions. BioSuite was used. TM HR analysis using size exclusion columns (Waters) was performed to determine aggregate content via SEC. The purified protein was stored at -80°C.

[0128] Expression of mutant variants of the IL-37 peptide in Escherichia coli (inclusion bodies)

[0129] To express a mutant variant (21 kDa) of the IL-37 peptide, an E. coli host / vector system was used, which enabled antibiotic-free plasmid selection via complementation of E. coli auxotrophs (PyrF) (EP 0 972 838 and US6,291,245).

[0130] Escherichia coli strain CSPZ-25 (ΔpyrF) was transformed by electroporation using expression plasmids D1AC9528 or D1AC7778. The transformed E. coli cells were first grown on agar plates at 37°C. Colonies picked from these plates were transferred to 3 mL of rolling culture and grown at 37°C until an optical density of 1–2 (measured at 578 nm) was achieved. Then, 1000 μL of culture was mixed with 1000 μL of sterile 86% glycerol and immediately frozen at -80°C for long-term storage. The correct product expression of the clone was first validated in small-scale shake-flask experiments and analyzed using SDS-Page, before being transferred to a 10 L fermenter.

[0131] Pre-culture:

[0132] For pre-culture, 1.0 ml of the study seed bank ampoule was inoculated into 220 ml of chemically defined medium in a 1000 ml Erlenmeyer flask with four baffles. The culture was carried out on a rotary shaker at 37 °C and 170 rpm for 12 hours until an optical density >5 (578 nm) was obtained. The inoculation volume was calculated as Vinoc. = 1000 mL * 5 / ODPC and therefore depended on the pre-culture optical density, thus inoculating each batch of medium for each bioreactor run with an equal volume of cells.

[0133] Fermentation:

[0134] For fermentation in a 10L Biostat C, DCU3 fermenter (Sartorius, Melsungen, Germany), a chemically defined batch medium was used. Fermentation was initiated from 4.2L of sterile batch medium plus 100ml of inoculum from a pre-culture, at 31°C, pH 6.9±0.2, 800mbar back pressure, and an initial aeration rate of 10L / min. The relative dissolved oxygen (pO2) was maintained at 50% throughout the fermentation process by increasing the agitator speed to 1500rpm. After the initial glucose replenishment was depleted (indicated by a sharp increase in dissolved oxygen), fermentation entered fed-batch mode at the start of feeding. The feed rate was gradually increased from 90g / h to a final 210g / h over 5.5 hours following a predetermined feed profile. When the CO2 exhaust gas concentration exceeded 2%, the aeration rate was continuously increased from 10L / min to 20L / min over 5 hours. The expression of the recombinant protein was induced by adding 2.4 g of IPTG at an optical density of approximately 120. The target protein was expressed in a partially soluble manner in the cytoplasm.

[0135] After 24 hours of cultivation, the optical density reached 250, and the entire broth was cooled to 4°C–8°C. Bacteria were harvested by centrifugation using a flow-through centrifuge (13,000 rpm, 13 L / h), and the resulting biomass was stored at -20°C until further processing (cell disruption). The yield was 67 g stem cells / L.

[0136] Product formation analysis:

[0137] Samples taken from the fermenter were analyzed by SDS-polyacrylamide gel electrophoresis, one before induction and the others at specific time points after protein expression induction. An equal amount of cells (OD target = 10) from each sample were resuspended in 5 mL of PBS buffer and sonicated on ice. 100 μL of each suspension was then centrifuged (15,000 rpm, 5 min), and the supernatant was collected and transferred to individual vials. This was to distinguish between soluble and insoluble expression of the target protein. 100 μL of SDS sample buffer (Laemmli, UK, Nature 227(1970)680-685) was added to each supernatant (= soluble protein fraction) and 200 μL of SDS sample buffer (Laemmli, UK, Nature 227(1970)680-685) was added to each precipitate (= insoluble protein fraction). The samples were heated at 95 °C with vigorous mixing for 15 min to dissolve and reduce all proteins in the samples. After cooling to room temperature, transfer 5 μL of each sample to a 4%–20% TGX standard stain-free polyacrylamide gel (Bio-Rad). Additionally, apply 5 μL of the molecular weight standard (Precision Plus Protein Standard, Bio-Rad).

[0138] Electrophoresis was performed at 200V for 60 minutes, after which the gel was transferred to a GelDOC EZ Imager (Bio-Rad) and treated with UV radiation for 5 minutes. Gel images were analyzed using Image Lab analysis software (Bio-Rad). Relative quantification of protein expression was performed by comparing the volume of the product bands with the volume of the 25 kDa band of the molecular weight standard.

[0139] Preparation of supernatant:

[0140] The preparation of supernatant for purifying 10 L of fermentation broth begins with resuspending the harvested bacterial cells in a disruption buffer (50 mM NaP, 300 mM NaCl, 1 mM DTT, pH 7.0, a “complete” protease inhibitor). The buffer volume is 5 times the wet weight of the biomass, and the resuspending is homogenized at 900 bar (APV LAB60, once) to disrupt the bacteria. The suspension is then centrifuged, and the resulting supernatant is immediately transferred to the DSP department for purification.

[0141] Purification of IL-37 peptide from Escherichia coli culture

[0142] The filtered lysate was loaded onto a HiTrap cOmplete His-Tag column (Roche) equilibrated with 50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT, and pH 7.0. Unbound protein was removed by washing with equilibration buffer. The IL-37 peptide was eluted with 10 CV using a gradient to 50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT, and 250 mM imidazole (pH 7.0). A Superdex 75 was used. TM Size exclusion chromatography (SUC) on GE Healthcare was used as a purification step and performed after combining the monomeric protein fractions. SUC was performed in 50 mM Tris-base, 150 mM NaCl, and 5 mM CaCl2 (pH 7.5). The concentration of the purified IL-37 peptide was determined by the molar extinction coefficient calculated based on the amino acid sequence, using the optical density (OD) at 280 nm. The purity of the IL-37 protein (i.e., the IL-37 peptide) was analyzed by CE-SDS under denaturing and reducing conditions. BioSuite was used. TM HR analysis using size exclusion columns (Waters) was performed to determine aggregate content via SEC. The purified protein was stored at -80°C.

[0143] Enzymatic conjugation of IL-37 peptide with the Fc region of antibody

[0144] The purified human Fc was transferred to conjugation buffer (50 mM Tris-base, 150 mM NaCl, 5 mM CaCl2, pH 7.5). For the localization enzyme reaction, a 5-fold molar excess of IL-37 peptide and a 0.8-fold molar excess of localization enzyme (Roche) were used. After thorough mixing, the reaction solution was incubated at 25°C for 90 min. After conjugation, the affinity chromatography step was run using HiTrap complete His-Tag (Roche) equilibrated with 50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT, and pH 7.0. Here, the desired fusion protein containing the IL-37 peptide and the antibody Fc region (i.e., the IL37-Fc conjugate) was collected in the flow-through. The pooled fraction of the IL37-Fc fusion protein was diluted with double-distilled water to approximately 3 mS / cm conductivity and loaded onto a POROS HS 50 column (Thermo Fisher Scientific, Waltham, MIT, USA) equilibrated with 20 mM Histidin (pH 5.6). Elution was performed with 20 CV buffer at a gradient of 20 mM Histidin and 1 M NaCl (pH 5.6). Superdex 75 TM Size exclusion chromatography (SUC) on a GE Healthcare instrument was used as the purification step. SUC was run in 20 mM Histidin and 140 mM NaCl (pH 6.0). The protein concentration of the purified IL-37-Fc conjugate (i.e., the fusion protein) was determined by molar extinction coefficient calculated based on the amino acid sequence, via optical density (OD) at 280 nm. The purity of the IL-37 fusion protein was analyzed by CE-SDS under denaturing and reducing conditions. BioSuite was used. TM HR analysis using size exclusion columns (Waters) was performed to determine aggregate content via SEC. The purified protein was stored at -80°C.

[0145] Mass spectrometry confirmed the C54-C94 internal disulfide bond in IL37.

[0146] To detect the closed disulfide bonds in the fusion protein, disulfide bond mapping experiments were performed. The sample was denatured (6.5 M final concentration guanidine hydrochloride) and S-carboxymethylated to cap free cysteine. The buffer was then replaced with digestion buffer (50 mM Tris, pH 6.9), followed by trypsin digestion (16 h, 37 °C). The reaction was terminated with 10% formic acid. The sample was further analyzed by UHPLC-MS / MS using a nanoAcquity UPLC (Waters) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). For data interpretation and visualization, PMI (ProteinMetrics Inc.) and Xcalibur software packages were used.

[0147] By detecting the corresponding linked trypsin peptides GEFLLYCDK (C54) and AQVGSWNMLECAAHPGWFIR (C94) with a mass of 3356.55 Da, the disulfide bond formed between Cys54 and Cys94 in the IL37 moiety of the fusion protein was shown.

[0148] In vitro stability test

[0149] Thermostability testing of purified proteins (P1AE1164, P1AE1771, P1AE2450, P1AE3281, P1AE4682, and P1AF2874) was performed using an Uncle apparatus (UNCHAINED LABS, Boston, Massachusetts, USA). Static light scattering at 266 nm and synchronous intrinsic fluorescence at 473 nm were used to determine the aggregation temperature (Tagg) and melting temperature (Tm) of the purified proteins. The temperature was increased from 30 °C to 90 °C in steps of 0.1 °C / min. A 9 μl glass cuvette was used for each sample, and the concentration in 20 mM Histidin, 140 mM NaCl (pH 6.0) buffer was 1 mg / mL. Analysis was performed using the Uncle apparatus (UNCHAINED LABS).

[0150] For molecular evaluation, the purified IL-37 peptide and fusion protein, as shown in Table 1, were transferred to two different buffers at a final concentration of 1.0 mg / ml. Buffer A: 1x PBS (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4); Buffer B: 20 ​​mM Histidin, 140 mM NaCl, pH 6.0. For dialysis, a D-Tube was used. TMDialyzer Mini MWCO 6-8kDa (Millipore, Burlington, Massachusetts, USA). Buffer A samples were incubated at 37°C, and Buffer B samples were incubated at 40°C for 14 days each. After incubation, for the IL-37 peptides and fusion proteins shown in Table 1, the results were obtained using BioSuite via SEC. TM HR analysis of waters (size-limited columns) and analysis under denaturing and reducing conditions using CE-SDS.

[0151] Table 1: Biophysical stability

[0152]

[0153] Table 2: Thermal Stability

[0154]

[0155] * Gather immediately after unfolding.

[0156] Bioactivity assay (HK-2 assay)

[0157] To test the inhibitory effect of IL-37 mutant variants (P1AE1164, P1AE1771, P1AE2450, P1AE3281, P1AE4682, and P1AF2874) on Il-8 release from LPS-stimulated HK-2 cells, the IL-37 variants were prepared in assay medium (RPMI 1640 (Anprotec; catalog number AC-LM-0060) + 2 mM L-glutamine (L-glutamine 200 mM, Gibco, catalog number 25030-024) + 1x ITS (ITS 100x, Sigma, I3146) + 0.1% BSA (MACS)). Dilute to a final concentration of 2.5 μM twice using BSA stock solution (Miltenyi, 130-031-376). Prepare serial dilutions in a 1:3 ladder and transfer 50 μL / well of the IL-37 mutant dilution to a 96-well assay plate. Harvest cells by trypsin digestion (trypsin, PAN, P10-023100) and pellet at 300 g for 5 min. After resuspending in assay medium to a concentration of 4.0E+05 cells / mL, transfer 25 μL / well to the assay plate to obtain 1.0E+04 cells / mL. Total number of cells / well. Dilute LPS (Sigma, L2654) to 2 μg / mL in assay medium. Transfer 25 μL to each well into the assay plate (final concentration 0.5 μg / mL) and incubate the plate overnight in a cell culture incubator (37°C, 5% CO2). The next day, centrifuge the plate at 300g for 3 minutes. Collect the supernatant and determine the IL-8 concentration using the Human IL-8 Uncoated ELISA Kit (Human IL-8 Uncoated ELISA Kit, Invitrogen, 88-8086-22) according to the manufacturer's instructions.

[0158] Table 3: Bioactivity in HK-2 cells

[0159]

[0160] Bioactivity assay (human PBMC)

[0161] Peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with different mutant variants of IL-37 (P1AE1164, P1AE2450, P1AE3281, and P1AE1771), as described in Nold M. et al. (2003) [IL-18BPa: Fc cooperates with immunosuppressive drugs in human whole blood. Biochem Pharmacol 66, 505-510] and Ellisdon, Nold-Petry et al. (2017) [Science Immunology Vol. 2, No. 8, eaaj1548]. In short, PBMCs were isolated from peripheral venous blood of healthy volunteers by density gradient centrifugation, as described in Nold M. et al. (2003) [IL-18BPa: Fc cooperates with immunosuppressive drugs in human whole blood. Biochem Pharmacol 66, 505-510]. PBMCs were plated in RPMI medium containing 1% v / v human serum and 1:500 MycoZap PR (Lonza) in triplicate, and pretreated for 30 min with either the medium or the mutant IL-37 (at concentrations between 10 ng / ml and 1 pg / ml), followed by stimulation with 1 ng / ml LPS for 20 h. The supernatant was then analyzed for IL-1β using Alphalisa (Perkin Elmer) according to the manufacturer's instructions. The percentage change in IL-1β inhibition by IL-37 after LPS administration to individual healthy donor responders was calculated.

[0162] Figure 3 The study showed that IL-1β secretion from freshly isolated human PBMCs was inhibited after stimulation with LPS. The amount of IL-1β secreted was reduced by approximately 20% when the IL-37 variant was added at a specified concentration 30 min before the addition of 1 ng / ml LPS and the cytokine was measured 20 h later. This indicates the anti-inflammatory activity of the tested variant.

[0163] Bioactivity assay (human PBMC)

[0164] Peripheral blood mononuclear cells (PBMCs) from healthy donors or patients (respiratory diseases RESP, systemic lupus erythematosus, SLE, rheumatoid arthritis RA, inflammatory bowel disease IBD) were treated with the IL-37 variant P1AE4682, as described in Nold M. et al. (2003) [IL-18BPa: Fc cooperates with immunosuppressive drugs in human wholeblood. Biochem Pharmacol 66, 505-510] and Ellisdon, Nold-Petry et al. (2017) Science Immunology Vol. 2, No. 8, eaaj1548]. In short, PBMCs were isolated from peripheral venous blood of patients or healthy volunteers by density gradient centrifugation, as described in Nold M. et al. (2003) [IL-18BPa: Fc cooperates with immunosuppressive drugs in human wholeblood. Biochem Pharmacol 66, 505-510]. PBMCs were plated in RPMI medium containing 1% v / v human serum and 1:500 MycoZap PR (Lonza) in triplicate, and pretreated for 30 min with a medium or variant of IL-37 (concentrations from 100 pg / ml to 0.01 pg / ml), followed by stimulation with 500 μM loxolibin for 20 h. The supernatant was then analyzed for IL-6 using an Alphalisa (Perkin Elmer) according to the manufacturer's instructions. The percentage change in IL-6 inhibition by IL-37 after loxolibin administration was calculated in both healthy donor responders and patients.

[0165] Figure 4 The study showed that IL-6 secretion from freshly isolated human healthy donor and patient-derived PBMCs was inhibited upon stimulation with loxoribin. The amount of secreted IL-6 was reduced by approximately 20%–25% when a stable IL-37-Fc fusion was added at a specified concentration 30 min before the addition of 500 μM loxoribin, and cytokine levels were measured 20 h later. This demonstrates the anti-inflammatory activity of the tested variant in a variety of diseases: RESP (respiratory disorders), SLE (systemic lupus erythematosus), RA (rheumatoid arthritis), and IBD (inflammatory bowel disease).

[0166] Bioactivity assay (C57Bl / 6WT mice)

[0167] Different concentrations of P1AE3281-005, P1AE1771-016, P1AE4682, or a carrier were subcutaneously (sc) injected into C57Bl / 6WT mice. One hour later, the mice were administered 100 mg / kg loxoribin via tail vein injection (Jomar Life Research). Room temperature and humidity were continuously monitored.

[0168] Two hours after injection of loxoribin, mice were anesthetized, and serum was obtained by exsanguination of the heart into BD serum tubes. Serum mouse IL-6 was measured by Alphalisa (Perkin Elmer) and IFNα was measured by ELISA (Perkin Elmer) according to the manufacturer's instructions.

[0169] Figure 5 A and Figure 5 B shows that IL-6 secretion was inhibited in C57Bl / 6WT mice after stimulation with loxoribin. Various IL-37-Fc fusions were subcutaneously injected at specified concentrations, followed by loxoribin at a concentration of 100 mg / kg. A 40%-70% decrease in serum IL-6 after 2 hours indicates the strong anti-inflammatory activity of the tested variants.

[0170] Table 4: Sequence Overview

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] Attached Figure Description

[0182] Figure 1Extraction ion counting chromatograms of SS-linked peptides GEFLLYCDK and AQVGSWNMLECAAHPGWFIR. The linked peptides were detected at charge states 2, 3, 4, and 5, and eluted at 60.7 min.

[0183] Figure 2 Isotopic patterns of SS-linked peptides GEFLLYCDK and AQVGSWNMLECAAHPGWFIR in charge state 5.

[0184] Figure 3 : Activity in PBMCs of healthy individuals.

[0185] Figure 4 Activity in human PBMCs from healthy and patient sources.

[0186] Figure 5 A and Figure 5 B: Activity in C57Bl / 6WT mice. sequence list <110> Hofmeister Roche Ltd. Monash University <120> IL-37 fusion protein and its uses <130> AC2584 PCT S3 <150> EP 19 21 8657.5 <151> 2019-12-20 <160> 45 <170> BiSSAP 1.3.6 <210> 1 <211> 218 <212> PRT <213> Artificial sequence <220> <223> wt IL-37 <400> 1 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp 1 5 10 15 Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Gly Ser 20 25 30 Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe Val His Thr 35 40 45 Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp 50 55 60 Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val 65 70 75 80 Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser 85 90 95 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly 100 105 110 Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln 115 120 125 Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala 130 135 140 Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln 145 150 155 160 Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe 165 170 175 Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys 180 185 190 Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys 195 200 205 Ala Glu Met Ser Pro Ser Glu Val Ser Asp 210 215 <210> 2 <211> 188 <212> PRT <213> Artificial sequence <220> <223> wt IL-37 (46-218) with His-tag and C-tag <400> 2 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser His His His His His His Glu Pro Glu Ala 180 185 <210> 3 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> wt IL-37 (46-218) <400> 3 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 165 170 <210> 4 <211> 438 <212> PRT <213> Artificial sequence <220> <223> C-terminus fused to human IL-37 (46-218) on both heavy chains of the human Fc; contains cleavage sites to release IL-37 <400> 4 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser His His His His His His Glu Pro Glu Ala Gly Gly Gly Gly 180 185 190 Ser Gly Gly Gly Gly Ser Leu Glu Val Leu Phe Gln Gly Pro Gly Gly 195 200 205 Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 210 215 220 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 225 230 235 240 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 245 250 255 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 260 265 270 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 275 280 285 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 290 295 300 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 305 310 315 320 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 325 330 335 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 340 345 350 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 355 360 365 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 370 375 380 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 385 390 395 400 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 405 410 415 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 420 425 430 Ser Leu Ser Pro Gly Lys 435 <210> 5 <211> 438 <212> PRT <213> Artificial sequence <220> <223> C-terminus fused to human IL-37 (46-218) on both heavy chains of the human Fc; contains cleavage sites to release IL-37 <400> 5 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser His His His His His His Glu Pro Glu Ala Gly Gly Gly Gly 180 185 190 Ser Gly Gly Gly Gly Ser Leu Glu Val Leu Phe Gln Gly Pro Gly Gly 195 200 205 Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 210 215 220 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 225 230 235 240 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 245 250 255 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 260 265 270 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 275 280 285 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 290 295 300 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 305 310 315 320 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 325 330 335 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 340 345 350 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 355 360 365 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 370 375 380 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 385 390 395 400 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 405 410 415 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 420 425 430 Ser Leu Ser Pro Gly Lys 435 <210> 6 <211> 227 <212> PRT <213> Artificial sequence <220> <223> C-terminal fusion to human Fc on only one HC with wt IL-37 (46-218) (KiH) <400> 6 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 7 <211> 415 <212> PRT <213> Artificial sequence <220> <223> C-terminal fusion to human Fc on only one HC with wt IL-37 (46-218) (KiH) <400> 7 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 8 <211> 227 <212> PRT <213> Artificial sequence <220> <223> N-terminal fusion to human Fc wt IL-37 (46-218) (KiH) on only one HC <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 9 <211> 417 <212> PRT <213> Artificial sequence <220> <223> N-terminal fusion to human Fc wt IL-37 (46-218) (KiH) on only one HC <400> 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro 245 250 255 Lys Lys Phe Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp 260 265 270 Ser Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu 275 280 285 Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys 290 295 300 Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr 305 310 315 320 Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys 325 330 335 Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro 340 345 350 Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser 355 360 365 Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu 370 375 380 Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe 385 390 395 400 Ser Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser 405 410 415 Asp <210> 10 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Y85A, C181S, C183S, C207S IL-37 (46-218) (KiH) of human Fc fused to only one HC. <400> 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 11 <211> 415 <212> PRT <213> Artificial sequence <220> <223> Y85A, C181S, C183S, C207S IL-37 (46-218) (KiH) of human Fc fused to only one HC. <400> 11 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 12 <211> 227 <212> PRT <213> Artificial sequence <220> <223> N-terminal fusion to human Fc Y85A, C181S, C183S, C207S IL-37 (46-218) (KiH) on only one HC <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 13 <211> 417 <212> PRT <213> Artificial sequence <220> <223> N-terminal fusion to human Fc Y85A, C181S, C183S, C207S IL-37 (46-218) (KiH) on only one HC <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro 245 250 255 Lys Lys Phe Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp 260 265 270 Ser Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu 275 280 285 Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys 290 295 300 Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr 305 310 315 320 Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys 325 330 335 Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro 340 345 350 Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser 355 360 365 Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu 370 375 380 Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe 385 390 395 400 Ser Phe Gln Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser 405 410 415 Asp <210> 14 <211> 183 <212> PRT <213> Artificial sequence <220> <223> Y85A, C181S, C183S, and C207S IL-37 (46-218) with N-terminal localizer tags. <400> 14 Met Gly Gly Gly Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro 1 5 10 15 Light Light Phe Ser Ile His Asp Gln Asp His Light Val Leu Val Leu Asp 20 25 30 Ser Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu 35 40 45 Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys 50 55 60 Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr 65 70 75 80 Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys 85 90 95 Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro 100 105 110 Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser 115 120 125 Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu 130 135 140 Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe 145 150 155 160 Ser Phe Gln Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser 165 170 175 Asp Gly Ser Glu Pro Glu Ala 180 <210> 15 <211> 190 <212> PRT <213> Artificial sequence <220> <223> Y85A, C181S, C183S, and C207S IL-37 (46-218) with C-terminal localizer tags. <400> 15 Met Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe 1 5 10 15 Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn 20 25 30 Leu Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe 35 40 45 Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro 50 55 60 Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys 65 70 75 80 Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu 85 90 95 Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe 100 105 110 Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His 115 120 125 Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly 130 135 140 Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln 145 150 155 160 Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Ser 165 170 175 Leu Pro Glu Thr Gly Gly Ser Gly His His His His His 180 185 190 <210> 16 <211> 410 <212> PRT <213> Artificial sequence <220> <223> The localization enzyme conjugated to Y85A, C181S, C183S, and C207S IL-37 (46-218) from human Fc (P1AD9547) from HEK. <400> 16 Met Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe 1 5 10 15 Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn 20 25 30 Leu Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe 35 40 45 Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro 50 55 60 Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys 65 70 75 80 Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu 85 90 95 Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe 100 105 110 Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His 115 120 125 Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly 130 135 140 Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln 145 150 155 160 Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Ser 165 170 175 Leu Pro Glu Thr Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 180 185 190 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 195 200 205 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 210 215 220 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 225 230 235 240 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 245 250 255 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 260 265 270 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 275 280 285 Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 290 295 300 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 305 310 315 320 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 325 330 335 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 340 345 350 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 355 360 365 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 370 375 380 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 385 390 395 400 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 <210> 17 <211> 227 <212> PRT <213> Artificial sequence <220> <223> The localization enzyme conjugated to Y85A, C181S, C183S, and C207S IL-37 (46-218) from human Fc (P1AD9547) from HEK. <400> 17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 18 <211> 190 <212> PRT <213> Artificial sequence <220> <223> C181S, C183S, and C207S IL-37 (46-218) with C-terminal localizer tags. <400> 18 Met Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe 1 5 10 15 Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn 20 25 30 Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe 35 40 45 Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro 50 55 60 Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys 65 70 75 80 Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu 85 90 95 Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe 100 105 110 Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His 115 120 125 Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly 130 135 140 Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln 145 150 155 160 Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Ser 165 170 175 Leu Pro Glu Thr Gly Gly Ser Gly His His His His His 180 185 190 <210> 19 <211> 410 <212> PRT <213> Artificial sequence <220> <223> The localization enzyme conjugated to C181S, C183S, and C207S IL-37 (46-218) from human Fc (P1AD9547) from HEK. <400> 19 Met Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe 1 5 10 15 Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn 20 25 30 Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe 35 40 45 Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro 50 55 60 Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys 65 70 75 80 Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu 85 90 95 Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe 100 105 110 Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His 115 120 125 Pro Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly 130 135 140 Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln 145 150 155 160 Pro Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Ser 165 170 175 Leu Pro Glu Thr Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 180 185 190 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 195 200 205 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 210 215 220 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 225 230 235 240 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 245 250 255 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 260 265 270 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 275 280 285 Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 290 295 300 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 305 310 315 320 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 325 330 335 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 340 345 350 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 355 360 365 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 370 375 380 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 385 390 395 400 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 <210> 20 <211> 227 <212> PRT <213> Artificial sequence <220> <223> The localization enzyme conjugated to C181S, C183S, and C207S IL-37 (46-218) from human Fc (P1AD9547) from HEK. <400> 20 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> twenty one <211> 189 <212> PRT <213> Artificial sequence <220> <223> Y85A, C181S, C183S, and C207S IL-37 (46-218) with C-terminal localizer tags. <400> twenty one Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Ser Leu 165 170 175 Pro Glu Thr Gly Gly Ser Gly His His His His His His 180 185 <210> 22 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C119I, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 22 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 23 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C119I, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 23 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Ile Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 24 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C122L, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 24 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 25 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C122L, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 25 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Leu Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 26 <211> 227 <212> PRT <213> Artificial sequence <220> Y85A, C178A, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 26 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 27 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C178A, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 27 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Ala Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 28 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C178M, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 29 <211> 415 <212> PRT <213> Artificial sequence <220> <223> Y85A, C178M, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 29 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Met Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 30 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C178R, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 30 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 31 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C178R, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 31 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Arg Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 32 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C178T, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 33 <211> 415 <212> PRT <213> Artificial sequence <220> <223> Y85A, C178T, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 33 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Thr Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 34 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C169C, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 35 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C169C, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 35 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Cys Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Cys Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 36 <211> 227 <212> PRT <213> Artificial sequence <220> Y85A, C119I, C122L, C178T, C181S, C183S, C207S IL-37(46-218) fused to human Fc <400> 36 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 37 <211> 415 <212> PRT <213> Artificial sequence <220> <223> Y85A, C119I, C122L, C178T, C181S, C183S, C207S IL-37(46-218) fused to human Fc <400> 37 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Ile Leu Tyr Leu Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Thr Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 38 <211> 227 <212> PRT <213> Artificial Sequence <220> Y85A, C119I, S169C, C178R, C181S, C183S, C207S IL-37(46-218) fused to human Fc <400> 38 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 39 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Y85A, C119I, S169C, C178R, C181S, C183S, C207S IL-37(46-218) fused to human Fc <400> 39 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Ala Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Ile Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Cys Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Arg Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 40 <211> 227 <212> PRT <213> Artificial Sequence <220> C119I, S169C, C178R, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 40 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 41 <211> 415 <212> PRT <213> Artificial Sequence <220> C119I, S169C, C178R, C181S, C183S, C207S IL-37 (46-218) fused to human Fc <400> 41 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser 1 5 10 15 Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu 20 25 30 Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala 35 40 45 Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 50 55 60 Leu Leu Gly Val Ser Lys Gly Glu Phe Ile Leu Tyr Cys Asp Lys Asp 65 70 75 80 Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 85 90 95 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 100 105 110 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Cys Ala Ala His Pro 115 120 125 Gly Trp Phe Ile Arg Thr Ser Ser Asn Ser Asn Glu Pro Val Gly Val 130 135 140 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 145 150 155 160 Val Ser Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His 180 185 190 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 305 310 315 320 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 42 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Peptide linkers with IL-37 peptide <400> 42 Gly Gly Gly Ser 1 <210> 43 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linkers with IL-37 peptide <400> 43 Gly Gly Gly Gly Ser 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> SS-linked peptide (Cys54) <400> 44 Gly Glu Phe Leu Leu Tyr Cys Asp Lys 1 5 <210> 45 <211> 20 <212> PRT <213> Artificial sequence <220> <223> SS-linked peptide (Cys94) <400> 45 Ala Gln Val Gly Ser Trp Asn Met Leu Glu Cys Ala Ala His Pro Gly 1 5 10 15 Trp Phe Ile Arg 20

Claims

1. A fusion protein comprising an IL-37 peptide and an antibody Fc region, The amino acid sequence of the IL-37 polypeptide comprises amino acids 46 to 218 corresponding to the positions in SEQ ID NO: 1 and includes... Isoleucine at amino acid position 119, which corresponds to the position in SEQ ID NO: 1, Cysteine ​​at amino acid position 169, which corresponds to the position in SEQ ID NO: 1, Arginine at amino acid position 178, which corresponds to the position in SEQ ID NO: 1, The serine at amino acid position 181, corresponding to the position in SEQ ID NO: 1, The serine at amino acid position 183, corresponding to the position in SEQ ID NO: 1, and The serine at amino acid position 207, which corresponds to the position in SEQ ID NO: 1, and The Fc region of the antibody is the Fc region of human IgG.

2. A fusion protein comprising an IL-37 peptide and an antibody Fc region, The amino acid sequence of the IL-37 polypeptide comprises amino acids 46 to 218 corresponding to the positions in SEQ ID NO: 1 and includes... Alanine at amino acid position 85, corresponding to the position in SEQ ID NO: 1, Isoleucine at amino acid position 119, which corresponds to the position in SEQ ID NO: 1, Cysteine ​​at amino acid position 169, which corresponds to the position in SEQ ID NO: 1, Arginine at amino acid position 178, which corresponds to the position in SEQ ID NO: 1, The serine at amino acid position 181, corresponding to the position in SEQ ID NO: 1, The serine at amino acid position 183, corresponding to the position in SEQ ID NO: 1, and The serine at amino acid position 207, which corresponds to the position in SEQ ID NO: 1, and The Fc region of the antibody is the Fc region of human IgG.

3. The fusion protein according to claim 1 or 2, wherein the fusion protein is an anti-inflammatory fusion protein.

4. The fusion protein according to claim 1 or 2, wherein the Fc region of the antibody is the Fc region of human IgG1.

5. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 4.

6. A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 4.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A cell comprising a nucleic acid molecule according to claim 6 or a vector according to claim 7.

Images